Abstract
Cordyceps militaris is a fungus with numerous therapeutic properties that has gained worldwide popularity due to its potential health benefits. The fruiting body of this mushroom is highly expensive and takes a longer time to produce, making mycelial a sustainable and cost-effective alternative. The study investigates and optimizes cultural and nutritional conditions to maximize mycelial biomass. The initial optimization was done by the conventional single-factor approach, followed by Plackett–Burman design to screen the most significant variables, with yeast extract, temperature, and glucose being the most significant, contributing 11.58%, 49.74%, and 27.98%, respectively, in mycelial biomass production. These variables were then optimized using response surface methodology (RSM) based on central composite design (CCD). The study observed that temperature and glucose had the highest impact on mycelial biomass, with p-values of 0.0128 and 0.0191, respectively. Under the optimized conditions, temperature 20 °C, glucose 2.5% (w/v), and yeast extract 0.8% (w/v), the maximal yield of mycelial biomass reached 547 ± 2.09 mg/100 mL, which was 1.95-fold higher than the yield in the basal medium. These findings suggest that optimizing the cultural and nutritional conditions can enhance mycelial biomass production of Cordyceps militaris, offering a sustainable and cost-effective source of this valuable fungus.
Keywords: Cordyceps militaris, Mycelial growth, Plackett–Burman Design, Response Surface Methodology, Central Composite Design
Introduction
In recent years, medicinal mushrooms have gained significant importance and popularity among the public and the scientific community. According to the World Health Organization, over 75 percent of the world's population uses traditional medicine, including mushrooms, herbs, and other remedies, to treat various diseases such as breast cancer (12%) [1, 2], liver disease (21%) [3], HIV (22%) [4], asthma (24%) [5], and rheumatologic diseases (26%) [6]. The prevalence of side effects linked to synthetic drugs has led a significant portion of the population to opt for herbal remedies as a preferred choice, aiming to minimize the risk of adverse reactions.
The Cordyceps militaris is a fungus belonging to the order Hypocreales and family Cordycipitaceae. Cordyceps is known by various popular names, including Dong Chong Xia Cao (China), Yarsagumba, Jeebanbuti, Sanjivani (Nepal), Keeda Jadi, Himalayan Viagra (India), Tochukaso (Japan) and Tong ch'ug ha ch'o (Korea). The Cordyceps genus has been in existence since 2000 BC [7], and the name Cordyceps is derived from the combination of two Latin words, cord, and ceps, which mean club and head, respectively. Cordyceps has been used for centuries as a tonic and dietary supplement [8]. More than 680 species of Cordyceps are known to exist on six continents, in different climates, and habitats [9]. However, cultivating Cordyceps fruiting bodies can take several months, making it impractical for large-scale production. Nevertheless, the submerged fermentation of Cordyceps mycelial biomass is a more efficient alternative that can increase the production of mycelial biomass in a smaller space and in a shorter time with less contamination risk. In recent times, through the application of advanced scientific techniques, researchers have successfully extracted various bioactive molecules from the Cordyceps militaris mycelial. These molecules have demonstrated remarkable various therapeutic properties [10]. To enhance these therapeutic properties of the mycelial, it is imperative to increase the mycelial biomass.
While numerous reports have explored the optimization of culture media for the production of the fruiting body, there is a notable scarcity of literature regarding submerged fermentation of Cordyceps militaris specifically for mycelial biomass production. A significant gap persists in the existing literature, lacking comprehensive reports that definitively identify the key factors responsible for enhancing mycelial biomass.
To achieve maximum biomass, it is crucial to optimize the various cultural and nutritional conditions as they play a vital role in synthesizing bioactive metabolites within the mycelial [10]. The conventional single-factor optimization approach is inadequate, as result a statistical approach becomes essential to optimize the nutrient components and other environmental factors effectively to meet the commercial demand of mycelial biomass. Two highly effective biostatistical tools for optimizing the biological process are the Plackett–Burman design (PBD) and response surface methodology (RSM) [11, 12]. The Plackett–Burman design is a fractional factorial design that is suitable for screening a large number of factors using a small number of experiments. Plackett–Burman design helps to screen the most significant factors that affect mycelial biomass. However, to study the relationship between these significant factors and mycelial biomass, more complex experimental designs, such as response surface methodology (RSM), RSM includes a series of experiments that are designed based on a statistical design of experiments (DOE) approach, such as a central composite design (CCD). The CCD allows for the investigation of the effects of the key factors on mycelial biomass yield at different levels, also allowing to create a second-order quadratic model [13]. This mathematical model can describe the relationship between the significant variables and the mycelial biomass yield [14]. Understanding such relationships plays a vital role in achieving maximum mycelial biomass.
The study involves investigating various cultural and nutritional conditions with the aim of maximizing mycelial biomass. In the initial optimization stage, a conventional approach was used where only one factor was considered at a time. The optimization was followed by a Plackett–Burman design (PBD), and then Response Surface Methodology (RSM) was used to understand the relationship between the significant variables and their effects on mycelial biomass production.
Material & methods
Chemicals and microbial strain
The chemicals utilized in the study were purchased from Hi-Media Limited, Mumbai, India. A strain of Cordyceps militaris was obtained from Thanvi Biotech, Bengaluru, India. The culture was revived and is being maintained on potato dextrose agar (PDA).
Seed culture preparation
To initiate the experiment, a spore suspension of Cordyceps militaris spores was inoculated onto petri plates containing potato dextrose agar (PDA). The plates were then incubated at 20 °C for seven days. After this period, mycelial discs measuring 5 mm were extracted using a sterile cork borer and placed into 250 mL Erlenmeyer flasks. The flasks were then incubated in a rotary shaker incubator (110 rpm) at 20 °C for three days, while containing 100 mL of basal medium (peptone 0.5%, glucose 1.5%, K2HPO4 0.1%, KH2PO4 0.3%, NaCl 0.05%, MgSO4 0.05%).
Optimization of temperature
Cordyceps militaris was cultured in six 250 mL Erlenmeyer flasks containing 100 mL of basal medium and a 6% inoculum to investigate the effect of temperature on mycelial biomass. The flasks were incubated for ten days under static conditions at different temperatures ranging from 15 °C to 35 °C with intervals of 5 °C.
Optimization of pH
To investigate the impact of pH on mycelial biomass, nine 250 mL Erlenmeyer flasks were prepared, each containing 100 mL of basal medium with a 6% (v/v) inoculum size. The pH levels were adjusted to 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, and 8 using 1N HCl or 1N NaOH. The flasks were then kept at 20 °C under static conditions for ten days.
Optimization of inoculum size
To optimize the inoculum size, six 250 mL Erlenmeyer flasks were prepared, each containing 100 mL of basal medium with a pH of 5.5 and a different inoculum size (2%, 4%, 6%, 8%, 10%, and 12% v/v). The flasks were then incubated under static conditions at 20 °C for ten days to study the effect of inoculum size on mycelial biomass.
Optimization of carbon source
To determine the most effective carbon source, seven Erlenmeyer flasks (250 mL) were used, each containing 100 mL of basal medium with a pH of 5.5 and 1.5% (w/v) of one of seven different carbon sources: fructose, glucose, sucrose, galactose, maltose, starch, and lactose. After inoculating the culture, the flasks were incubated under static conditions at 20 °C for ten days. The carbon source that resulted in the highest mycelial biomass was selected and further tested to determine its optimal concentration requirement.
Optimization of nitrogen source
To evaluate the most effective nitrogen source six Erlenmeyer flasks (250 mL) containing 100 mL of the basal medium of pH 5.5 were used to test the six different nitrogen sources: organic (beef extract, casein, peptone, and yeast extract) and inorganic (ammonium sulfate and sodium nitrate) at a concentration of 0.5% (w/v). After inoculating the culture, the flasks were kept in a static condition at 20 °C for ten days. The nitrogen source that resulted in the highest mycelial biomass was selected and further tested to determine its optimal concentration requirement.
Plackett–Burman design
The effects of each component on biomass synthesis were investigated at high ( +) and low (-) experimental levels in 12 different series of experiments (Table 1). These experiments were conducted in 250 mL Erlenmeyer flasks containing 100 mL Plackett–Burman media. The experiments were performed in triplicate, and the average biomass production of mycelial (mg/100 mL) was used as the response. The response surface method based on the central composite design was used for further optimization.
Table 1.
The experimental variables at different levels for the production of Cordyceps militaris mycelial biomass using the Plackett–Burman design. Each variable was tested at two levels, high ( +) and low (-)
| Symbol code |
Variable | Units | Experimental levels Lower Higher | |
|---|---|---|---|---|
| (-) | ( +) | |||
| I | Temperature | °C | 20 | 25 |
| II | pH | - | 5.5 | 6 |
| III | Inoculum size | % (v/v) | 8 | 10 |
| IV | Glucose | % (w/v) | 2.5 | 5 |
| V | Incubation Period | Day’s | 8 | 10 |
| VI | Yeast Extract | % (w/v) | 0.8 | 1 |
| VII | KH2PO4 | % (w/v) | 0.3 | 0.5 |
| VIII | K2HPO4 | % (w/v) | 0.1 | 0.2 |
| IX | MgSO4 | % (w/v) | 0.05 | 0.1 |
| X | NaCl | % (w/v) | 0.05 | 0.1 |
| XI | Dummy 1 | - | - | - |
Central composite design analysis
The study investigated the impact of glucose, yeast extract, and temperature on mycelial biomass production, using response surface methodology. Table 3 shows the coded and actual values for each parameter, and the 3D graphs visually depict the interactions among the three components. The experiments were conducted in 250 mL conical flasks with 100 mL of media, and twenty tests were performed. After ten days of incubation at 20 °C, the biomass was collected from the flasks, and the average mycelial biomass (mg/100 mL) from all the experiments was used as the response.
Table 3.
ANOVA results of Plackett–Burman experimental design for mycelial biomass production
| Symbol code | Variables | Sum of Square (SS) | p-value | Contribution % |
|---|---|---|---|---|
| I | Temperature | 26885.30 | 0.0179 | 49.75 |
| II | pH | 1925.33 | 0.0668 | 3.56 |
| III | Inoculum size | 2080.33 | 0.0642 | 3.84 |
| IV | Glucose | 15123 | 0.0239 | 27.98 |
| V | Incubation Period | 26.33 | 0.1772 | 0.48 |
| VI | Yeast Extract | 6256.33 | 0.0371 | 11.58 |
| VII | KH2PO4 | 147 | 0.2317 | 0.27 |
| VIII | K2HPO | 481.33 | 0.1321 | 0.89 |
| IX | MgSO4 | 768 | 0.1051 | 1.42 |
| X | NaCl | 96.33 | 0.2800 | 0.18 |
Contribution % indicates how much each variable has contributed to the mycelial biomass production of Cordyceps militaris
Harvesting of dry mycelial
After incubation, the mycelia were separated from the culture medium by decanting and filtering through Whatman #4 filter paper [14]. The mycelia were then washed twice with double distilled water to remove any residual culture medium. This step is crucial as it can impact the accuracy of mycelial biomass measurement. After washing, the mycelia were dried in an oven at a constant temperature of 45 °C overnight. Drying the mycelia at a constant temperature is important to obtain a consistent dry weight, which can be used as a reliable measure of mycelial biomass. Consistent dry weight allows for accurate comparisons between different experimental runs and helps to eliminate any potential variability due to differences in moisture content.
Statistical analysis
To ensure reproducibility, all experiments were conducted in triplicate. The data generated from the response surface methodology and Plackett–Burman design were analyzed using analysis of variance (ANOVA) [15]. The statistical program Design Expert 12.0.0 was used to improve the accuracy and robustness of the statistical analysis. This software provides tools for analyzing experimental data, such as ANOVA and regression analysis, which enables a more detailed and comprehensive analysis of the results.
Results & discussion
Optimization of temperature, pH, and inoculum size
Temperature and pH are crucial factors that directly impact cellular morphology and metabolism, [16, 17]. The findings study demonstrated that the maximum dry mycelial biomass was achieved at 20 °C, which is consistent with prior research on the growth of basidiomycetes in liquid culture during mycelial development [18]. Furthermore, the study determined that the optimal initial pH was 5.5, in agreement with the general observation that Cordyceps militaris and many other mushrooms thrive best in a slightly acidic environment [19]. To identify the ideal inoculum size, Cordyceps militaris was cultivated at various inoculum volumes ranging from 2–12% (v/v). The results revealed that the optimum inoculum volume was 8% (v/v), resulting in a dry mycelial biomass of 315 ± 2.29 mg/100 mL, as depicted in Fig. 1 (A, B, and C.)
Fig. 1.
The variations in mycelial biomass resulting from the influence of temperature A, pH B, and inoculum size C
Optimization of carbon and nitrogen source
This study examined the effects of different carbon sources on the mycelial growth of Cordyceps militaris. Various carbon sources were added to a basal medium at a concentration of 1.5% (w/v) and incubated under static conditions for ten days. The results demonstrated that the medium containing glucose produced the highest amount of mycelial biomass, with 317 ± 3.10 mg/100 mL, followed by sucrose and starch, which yielded 299 ± 2.52 mg/100 mL and 286 ± 4.13 mg/100 mL of mycelial biomass, respectively, as illustrated in Fig. 2(A).
Fig. 2.
A: Effect of carbon source on dry mycelial biomass. B: Effect of nitrogen source on dry mycelial biomass
In this study, the effects of various nitrogen sources on the mycelial growth of Cordyceps militaris were investigated. Different nitrogen sources were added to a basal medium at a concentration of 0.5% (w/v) and incubated under static conditions for ten days. The findings revealed that yeast extract as the nitrogen source produced the highest mycelial biomass of 323 ± 2.65 mg/100 mL, followed by peptone and beef extract with 303 ± 3.85 mg/100 mL and 274 ± 4.11 mg/100 mL of mycelial biomass, respectively.
This study aimed to determine the optimal concentrations of glucose and yeast extract for the growth and development of Cordyceps militaris mycelial by varying their concentrations from 0.5 to 5.0% (w/v) and 0.1 to 1.0% (w/v), respectively, in the medium. The results revealed that the highest mycelial biomass of 317 ± 1.64 mg/100 mL was obtained at a glucose concentration of 2.5%, while the highest mycelial biomass of 329 ± 2.02 mg/100 mL was achieved at a yeast extract concentration of 0.8% as shown in Fig. 3.
Fig. 3.
Effect of glucose A and yeast extract B on mycelial biomass
Plackett–Burman design
The Plackett–Burman experimental design methodology was utilized to identify the crucial parameters affecting the mycelial biomass of Cordyceps militaris. In this study, the impact of ten factors was assessed through 12 series of experiments. Table 2 presents a comprehensive experimental design and the results of each experiment conducted.
Table 2.
Plackett–Burman experimental design for screening important variables for biomass production of Cordyceps militaris. Each variable was tested at two levels, low (-) and high ( +), and the effect of each variable on biomass production was examined in 12 experimental series
| Run | Coded Values | Biomass (mg/100 mL) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| I | II | III | IV | V | VI | VII | VIII | IX | X | XI | ||
| 1 | 1 | 1 | -1 | 1 | 1 | 1 | -1 | -1 | -1 | 1 | -1 | 251 ± 1.80 |
| 2 | -1 | 1 | 1 | -1 | 1 | 1 | 1 | -1 | -1 | -1 | 1 | 433 ± 2.00 |
| 3 | 1 | -1 | 1 | 1 | -1 | 1 | 1 | 1 | -1 | -1 | -1 | 312 ± 3.12 |
| 4 | -1 | 1 | -1 | 1 | 1 | -1 | 1 | 1 | 1 | -1 | -1 | 316 ± 1.73 |
| 5 | -1 | -1 | 1 | -1 | 1 | 1 | -1 | 1 | 1 | 1 | -1 | 497 ± 2.20 |
| 6 | -1 | -1 | -1 | 1 | -1 | 1 | 1 | -1 | 1 | 1 | 1 | 392 ± 2.50 |
| 7 | 1 | -1 | -1 | -1 | 1 | -1 | 1 | 1 | -1 | 1 | 1 | 310 ± 2.30 |
| 8 | 1 | 1 | -1 | -1 | -1 | 1 | -1 | 1 | 1 | -1 | 1 | 357 ± 2.78 |
| 9 | 1 | 1 | 1 | -1 | -1 | -1 | 1 | -1 | 1 | 1 | -1 | 321 ± 2.64 |
| 10 | -1 | 1 | 1 | 1 | -1 | -1 | -1 | 1 | -1 | 1 | 1 | 351 ± 3.60 |
| 11 | 1 | -1 | 1 | 1 | 1 | -1 | -1 | -1 | 1 | -1 | 1 | 270 ± 1.73 |
| 12 | -1 | -1 | -1 | -1 | -1 | -1 | -1 | -1 | -1 | -1 | -1 | 400 ± 1.32 |
I: Temperature (°C), II: pH, III: Inoculum size (% v/v), IV: Glucose (% w/v), V: Incubation period (days), VI: Yeast Extract (% w/v), VII: KH2PO4 (% w/v), VIII: K2HPO4 (% w/v), IX: MgSO4 (% w/v), X: NaCl (% w/v), XI: Dummy variable
All variables were screened at a 95% confidence level, and Table 3 displays the p-value and percentage contribution of each variable. The p-value is an indicator of the significance of a factor in influencing the outcome of interest, with smaller p-values indicating greater significance. A p-value of less than 0.05 is considered statistically significant at a 95% confidence level. The results demonstrated that yeast extract, temperature, and glucose were the top three factors that contributed the most to mycelial biomass, with contributions of 11.58%, 49.74%, and 27.98%, respectively. The high R2 value of 0.9996 indicates that the model fits the data well and provides a reasonable explanation for the outcome of interest with statistical significance. These three variables had the most substantial impact on the mycelial biomass and were likely the most critical factors to consider in further analysis.
Central composite design
The CCD was employed to assess the impact of yeast extract, temperature, and glucose, which were selected using the Plackett–Burman design. In total, twenty experimental runs were conducted to investigate the effects and interactions of these critical factors on the mycelial biomass of Cordyceps militaris. Tables 3 and 4 represent the effects of the three independent variables in coded patterns. As shown in Table 5, the observed values represent the actual results of the experiments, whereas the predicted values are the values expected based on the statistical model used in the analysis.
Table 4.
Central composite experimental design variables at different levels. (Actual and Coded values)
| Variables | Units | Experimental values | ||||
|---|---|---|---|---|---|---|
| -2 | -1 | 0 | 1 | 2 | ||
| Temperature | (°C) | 11.59 | 15 | 20 | 25 | 28.41 |
| Glucose | (% w/v) | 1.66 | 2 | 2.5 | 3 | 3.34 |
| Yeast Extract | (% w/v) | 0.63 | 0.7 | 0.8 | 0.9 | 0.97 |
-2, − 1,0, + 1, + 2- coded values of each variable
Table 5.
Central composite experimental design with observed and predicted responses
| Runs | Variables | Biomass (mg/100 mL) | |||
|---|---|---|---|---|---|
| Temperature | Glucose | Yeast Extract | Observed | Predicted | |
| 1 | 0 | 0 | 0 | 547 ± 2.09 | 533.94 |
| 2 | 0 | 0 | 0 | 537 ± 2.75 | 533.94 |
| 3 | 1 | -1 | 1 | 298 ± 1.80 | 296.08 |
| 4 | 0 | 0 | 0 | 539 ± 2.66 | 533.94 |
| 5 | 0 | 0 | 2 | 317 ± 1.73 | 323.54 |
| 6 | 0 | 0 | 0 | 531 ± 2.02 | 533.94 |
| 7 | -1 | -1 | 1 | 329 ± 2.86 | 326.83 |
| 8 | 0 | 2 | 0 | 307 ± 1.51 | 320.1 |
| 9 | 0 | 0 | 0 | 525 ± 2.29 | 533.94 |
| 10 | 0 | 0 | -2 | 301 ± 1.90 | 308.04 |
| 11 | 0 | 0 | 0 | 527 ± 2.30 | 533.94 |
| 12 | 1 | 1 | -1 | 363 ± 2.59 | 355.56 |
| 13 | -2 | 0 | 0 | 304 ± 1.84 | 321.19 |
| 14 | 2 | 0 | 0 | 358 ± 2.82 | 354.39 |
| 15 | 0 | -2 | 0 | 289 ± 2.22 | 289.49 |
| 16 | -1 | 1 | -1 | 293 ± 2.65 | 285.32 |
| 17 | -1 | 1 | 1 | 295 ± 2.91 | 279.03 |
| 18 | 1 | -1 | -1 | 265 ± 3.10 | 271.36 |
| 19 | -1 | -1 | -1 | 306 ± 2.93 | 295.12 |
| 20 | 1 | 1 | 1 | 341 ± 1.84 | 342.28 |
The surface response for mycelial biomass production was investigated using central composite design (CCD). Factors including temperature (A), glucose (B), yeast extract (C), and their interactions (AB, BC, AC, A2, B2, and C2) were examined as a function of a quadratic model. The statistical importance of the quadratic model was estimated using analysis of variance (ANOVA). The results of the ANOVA analysis are presented in Table 6, which shows that the model terms A, B, AB, BC, A2, B2, and C2 had p-values less than 0.05, indicating that they were statistically significant. This suggests that these factors and their interactions significantly impacted the mycelial biomass. Among the three independent variables, temperature (A) and glucose (B) were the most significant, with p-values of 0.0128 and 0.0191, respectively. However, yeast extract, initially considered significant based on the Plackett–Burman design, was found to be non-significant in the CCD with a p-value of 0.1882. These results demonstrate that temperature and glucose had a greater impact on the mycelial biomass than yeast extract (C).
Table 6.
ANOVA for the experimental outcomes of the CCD quadratic model for mycelial biomass
| Symbol code |
Sum of Squares (SS) |
F-value | p-value |
|---|---|---|---|
| Temperature (A) | 1330.88 | 9.16 | 0.0128 |
| B-Glucose (B) | 1130.83 | 7.78 | 0.0191 |
| Yeast Extract (C) | 289.78 | 1.99 | 0.1882 |
| AB | 4418 | 30.41 | 0.0003 |
| AC | 24.5 | 0.1686 | 0.69 |
| BC | 722 | 4.97 | 0.0499 |
| A2 | 69311.26 | 477.02 | < 0.0001 |
| B2 | 94594.27 | 651.02 | < 0.0001 |
| C2 | 85730.66 | 590.02 | < 0.0001 |
| Lack of Fit | 222.34 | 3.26 | 0.1105 |
It has been observed that the interaction between independent variables plays a crucial role in mycelial biomass production. The CCD analysis results showed that the interactions between temperature and glucose (AB) and glucose and yeast extract (BC) were statistically significant, with p-values of 0.0003 and 0.0499, respectively. The lack of fit of the model, as evidenced by the p-values of 0.1105 and 0.0001, and the f-values and lack of fit (165.53 and 3.26) demonstrated that the model was highly statistically significant. Therefore, the analysis results are reliable, and the model can be used to predict biomass production. The following second-order polynomial equation can be used to indicate the anticipated response for biomass production (R):
where, the temperature is represented by A, glucose by B, and yeast extract by C.
The relationships between the three factors, namely temperature, glucose, and yeast extract, and their cumulative impact on the mycelial biomass synthesis of Cordyceps militaris were visualized using 3D contour plots and response surface plots. These plots provide valuable insights into the significant reciprocal interactions between the independent variables.
The contour plot’s elliptical pattern suggests that there are significant interactions between the independent variables [19]. Additionally, the 3D response surface and contour plots can help identify the optimal values for the independent variables that result in maximum mycelial biomass synthesis, as shown in Fig. 4. The temperature-glucose (AB) and yeast extract-glucose (CB) interactions are depicted in the 3D surface plots and contour plots, respectively.
Fig. 4.
(a) 3D Response Plot and (b) Contour Plot, showing interaction between temperature and glucose (A—B) and yeast extract and glucose (C—B)
The graphical representations provide a clear understanding of how the independent variables interact with each other and influence the synthesis of mycelial biomass in Cordyceps militaris. By analyzing the relationships between these factors, the study can determine the ideal conditions that would lead to maximum mycelial biomass production.
To validate the model, Cordyceps militaris was cultivated using the optimal variable levels determined, namely temperature at 20 °C, yeast extract at 0.8% (w/v), and glucose at 2.5% (w/v). The maximum mycelial biomass obtained was found to be 547 mg/100 mL, which is highly correlated to the expected value of 534 mg/100 mL of the model, with a difference of only 2.3%. This close similarity between the actual and predicted results indicates that the independent variables, i.e., temperature, yeast extract, and glucose, have a significant impact on the mycelial biomass production of Cordyceps militaris. The high correlation between the actual and predicted results suggests that the response surface methodology was effective in determining the optimal conditions for mycelial biomass production. These findings are consistent with previous studies that have identified temperature, yeast extract, and glucose as important factors for mycelial growth. Furthermore, the close agreement between the actual and predicted results indicates that the model can predict the mycelial biomass production of Cordyceps militaris under different conditions.
There are limited number of studies on the statistical optimization of mycelial biomass production in Cordyceps militaris. One study employed the response surface approach to optimize various nitrogen and carbon sources for the synthesis of exopolysaccharides and mycelial biomass, identifying peptone and glucose as the best nitrogen and carbon sources, respectively [20]. Another study aimed to improve the cultural mechanism for synthesizing exopolysaccharides and mycelial biomass by Cordyceps militaris C738 and found that the optimal pH range was between 6 and 9 and the ideal temperature range for mycelial biomass production was 20–25 °C, resulting in a 1.95-fold increase in biomass yield in the basal medium [21]. Other studies have explored the use of various elements and mixtures for synthesizing targeted metabolites such as cordycepin [22, 23], exopolysaccharides [24, 25], and adenosine [26]. Previous research on this medicinal mushroom has predominantly focused on the fruiting body, with less emphasis on mycelial biomass. Nonetheless, recent studies have shown that mycelial biomass has the potential to be a source of bioactive compounds [27–30]. One such study investigated the therapeutic properties of mycelial, finding that Cordyceps militaris mycelial has similar properties to the fruiting body, including antioxidant, anti-angiogenic, and anti-inflammatory properties [31]. Thus, it is crucial to understand the factors affecting mycelial growth and production. This study provides valuable insights into optimizing mycelial biomass production by examining a range of variables and utilizing statistical techniques.
Conclusions
In the existing scientific literature, a noticeable gap persists in research exclusively focused on statistically optimizing the mycelial biomass of Cordyceps militaris. Traditional methodologies have proven insufficient in identifying the key factors driving mycelial biomass growth. This study aimed to address this gap by identifying critical variables and establishing optimal cultural and nutritional conditions conducive to enhancing mycelial biomass production in Cordyceps militaris. To achieve this, a comprehensive approach involving single-factor experiments, Plackett–Burman designs, and central composite designs was employed. The findings underscore the pivotal role played by cultural conditions and medium constituents in governing mycelial growth. Notably, a significant decrease in biomass was observed at temperatures exceeding the designated optimal range. The investigation revealed Cordyceps militaris's utilization of diverse nitrogen and carbon sources, with a particular preference for yeast extract and glucose as the preferred carbon and nitrogen sources in the experimental context. It is imperative to emphasize the significance of a statistical approach in optimizing mycelial biomass production. Both Plackett–Burman and central composite designs identified temperature (maintained at 20 °C) and glucose concentration (2.5% w/v) as the most influential variables impacting biomass production. The robustness of the model was supported by remarkably low p-values (< 0.0001) and a lack of fit value (0.1105). The implications of this study extend beyond Cordyceps militaris mycelial fermentation processes. The insights gained hold promise for practical applications in the production of bioactive compounds, not limited to Cordyceps but also extendable to other medicinal mushrooms.
Acknowledgements
The author would like to express his gratitude to the L J School of Applied Sciences, L J University Ahmedabad, for equipping us with the resources we required to complete our study.
Declarations
Conflict of interest
All authors declare no conflict of interest.
Footnotes
Responsible Editor: Julio Santos
Publisher's Note
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