Unveiling the Secrets of Cultivating Cordyceps militaris Mushroom: Achieving Maximum Yield Through Expert Techniques
- Neeraj Kumar
- Nov 14, 2024
- 9 min read
Updated: Nov 16, 2024
Cordyceps militaris cultivation is a fascinating journey that combines science and passion. Known for its impressive health benefits, this mushroom is not just another addition to your diet; it has the potential to boost energy, enhance athletic performance, and support overall wellness. In this guide, we will explore effective techniques to cultivate Cordyceps militaris, ensuring maximum yield and quality in your harvest.
Obtaining a pure culture of Cordyceps militaris
The process of cordyceps militaris cultivation starts by acquiring a high-quality pure culture of this mushroom. The appearance of colonies on agar plates can differ based on the strain, the composition of the media, the incubation conditions and illumination (especially, development of carotenoid). Regardless of the origin and colony morphology, it is essential to initially test the culture for yield before advancing to large-scale production.
Composition of Seed Culture media for Cordyceps militaris
Preparation of Liquid Spawn of Cordyceps militaris
Among the wide array of liquid media available for preparing seed culture or liquid spawn of Cordyceps militaris, the one yielding higher spore count shall be preferred. Also note that the rate of agitation also greatly affect the quality of liquid spawn.
The terms 'Seed Culture Media' and 'Liquid Spawn' being discussed here have identical chemical composition; differ in terms of scale of production and end use.
The composition of Seed Culture media for Cordyceps militaris or Liquid Spawn media of our lab is given below-
Ingredients | % Composition | grams / liter |
|---|---|---|
Glucose | 1.50 | 15.00 |
Peptone | 0.50 | 5.00 |
Yeast Extract | 0.25 | 2.50 |
KH2PO4 | 0.30 | 3.00 |
K2HPO4 | 0.10 | 1.00 |
MgSO4.7H2O | 0.05 | 0.50 |
NaCl | 0.05 | 0.50 |
We have also tested another seed culture media for Cordyceps militaris, a modification of Carilli-Pacioni media [ref: https://doi.org/10.1016/S0007-1536(77)80013-2 ]. It also yields nearly identical results with an advantage that this media need not be filtered before use. Composition of modified Carilli-Pacioni media is-
Ingredients | % Composition | grams / liter |
Soyatone | 2.00 | 20.0 |
Glycerol | 5.00 | 50.0 |
MgSO4.7H2O | 0.05 | 0.50 |
Preparation of Seed Culture Media for Cordyceps militaris
Prepare the required quantity of the seed culture media.
Dispense in suitable glassware or culture vessel, secure the mouth with cotton plug or better substitute.
Sterilize by autoclaving at 121.0 degree C for 15-20 minutes.
Allow it to cool to room temperature.
Inoculate with a small piece of agar block from the pure culture plate [or, liquid inoculum as available.] under aseptic conditions.
Incubate at 25 deg. C, 180-220 RPM for 4-7 days. Note: The time for full growth will vary depending on the quantity of inoculum used.
Use fresh or store at 4-8 deg. C for a maximum of 30 days if to be used later.
Filtration of the Liquid Spawn
An important factor for successfully cultivating the Keeda Jadi mushroom, Cordyceps militaris, is a high-quality liquid spawn. Unlike most other mushroom varieties, it is recommended that the liquid spawn for Cordyceps militaris mainly contains spores rather than mycelia. While a liquid spawn with a higher concentration of mycelia can still be effective, the optimal outcome is achieved with a higher proportion of spores.
When mycelial lumps are predominant in the liquid spawn, there forms a Yellow Mycelia Coat on top. This "yellow mycelia coat" consumes a significant amount of nutrients at the beginning and delays formation of primordia. Though the yellow mycelia coat gradually decays revealing the late-formed primordia in around 7-15 days, it reduces the yield as well as prolongs the cultivation cycle.
Sometimes aerial hyphae may also emerge along with the fruiting body that further adversely affects the yield and quality of the mushroom.
The mycelia lumps in the liquid spawn can simply be filtered through sterile cotton, 10 micron cotton felt, 200-1000 micron SS304 sieve or any other suitable aid.
The final liquid spawn ready for use shall have minimal mycelia lumps in it.
Liquid Supplement for Fruiting Media (LSFM) for Cordyceps militaris
The liquid supplement for fruiting media is a nutrient rich supplement to the basal media (rice, silk worm pupae, etc.). Since rice is principally carbohydrate, this supplementation is required for optimal growth of Cordyceps militaris.
The composition of Liquid Supplement for Fruiting Media for Cordyceps militaris being used at our facility is-
Ingredients | % Composition | grams / liter |
Glucose | 1.00 | 10.00 |
Sucrose | 1.40 | 14.00 |
Soyatone | 1.00 | 10.00 |
Yeast Extract | 1.00 | 10.00 |
Malt extract | 1.00 | 10.00 |
MgSO4.7H2O | 0.05 | 0.50 |
KH2PO4 | 0.30 | 3.00 |
K2HPO4 | 0.10 | 1.00 |
Ca(NO3)2 | 0.10 | 1.00 |
Calculations for starting Cordyceps militaris cultivation
Wondering the relative quantity of rice as the basal media, liquid supplement for fruiting media and inoculum could be a nightmare for beginners. We have standardized this work for reproducible, successful cultivation of Cordyceps militaris at our facility.
Standardized quantity of Rice = 0.52 g per square cm of culturable area
Standardized quantity of LSFM = 1.6 mL per g Rice
Standardized quantity of Inoculum = 0.14 mL per g Rice
A +/- 5% deviation in the above values generally does not affect the outcomes.
A Sample calculation is presented here for a culture vessel (round glass bottle) of inner diameter of 10 cm. So, the culturable area, i.e. area of the bottom of this is equal to (Pi*(r)^2 = 3.14 x (10.0 cm / 2)^2 = 78.50 cm^2
Once the culturable area is calculated, the rest values are calculated accordingly-
Diameter of Bottle (Culture Vessel) = | 10.00 cm |
Culturable Area = area of bottle's base | 3.14 x (10.0 cm/2)^2 = 78.50 sq.cm |
Standardized Qty of Rice = | 0.52 g / sq.cm |
So, Quantity of Rice grains per bottle = | (0.52 g / cm^2) x 78.50 cm^2 = 40.82 g |
Standardized Qty of LSFM = | 1.60 mL / g Rice |
So, Quantity of LSFM per bottle = | (1.60 mL / g Rice) x 40.82 g = 65.31 mL |
Standardized Qty of Inoculum = | 0.14 mL / sq.cm |
So, Quantity of Inoculum per bottle = | (0.14 mL / cm^2) x 78.5 cm^2 = 10.99 mL |
Let, number of Culture Vessels (bottle) = | 100 |
Total quantity of Rice = | (40.82 g/bottle) x 100 bottles = 4082.0 g |
Total quantity of LSFM = | (65.31 mL/bottle)x100 bottles = 6531 mL |
Total quantity of Inoculum = | (11.0 mL/bottle)x100 bottle = 1100.0 mL |
Standard observed yield = | 0.0611 g sq.cm |
Standard observed yield per bottle = | (0.0611 g/cm^2) x 78.50 cm^2 = 4.80 g |
Expected Yield for 100 bottles = | (4.80g/bottle) x 100 bottle = 480.0 g |
Basal Media for Cordyceps militaris mushroom cultivation
Rice is the most popular basal media for cultivation of Cordyceps militaris mushroom. Among several types of rice varieties and forms available, any PARA-BOILED rice variety can be used.
Preparation of Fruiting Media for Cordyceps militaris mushroom cultivation
Clean rice in potable water and let it soak for around 30-60 minutes. Let the weigh to dry rice grains is 1.00 kg.
Collect the rice in muslin cloth or other cotton cloth and let is stand for another 15-30 minutes. Note the weight of wet rice grains. Let it be 1.50 kg. Note: the weight of wet rice must be take just before filling it in bottle.
We need to add 40.82 g rice (dry) per bottle. Required quantity of wet rice = (1.50 kg / 1.00 kg) x 40.82 g = 61.23 gram. So, fill a pre-cleaned bottle with 61.23 wet rice.
Add 65.31 mL liquid supplement of fruiting media (Fruiting media = Basal media + LSFM) to each bottle. Seal the cap (must be fitted with aeration port).
Sterilize the bottles by autoclaving at 121.0 deg. C for 15-20 minutes. Note: The sterilization time may vary depending on total load in the autoclave.
Allow it to cool to room temperature. It can be stored at 20 - 25.0 deg. C for around a week, beyond that there would be significant loss of moisture from bottles.
Inoculation of sterile fruiting media of Cordyceps militaris mushroom
Aseptically add around 11.0 mL of the liquid spawn (seed culture) to each bottle.
Gently swirl the bottle for even distribution of the inoculum through the grain surface.
Incubation conditions for Cordyceps militaris mushroom cultivation
The incubation conditions for Cordyceps militaris requires an orchestrated control of temperature, humidity, illumination or lighting and air exchanges.
The whole incubation period shall preferably be controlled using suitable temperature-humidity controller. Attach air conditioner and humidifier of suitable capacity to the controller.
A room heater would be needed to be installed in cold regions. It must be controlled with a temperature controller, too.
Illumination or lighting periodicity can be achieved using digital electronic timer and controller.
Install HEPA filter of suitable capacity for air exchanges.
There are several tweaks to optimize this coordinated controls simultaneously with reducing the cost (up to 30-40%) but are beyond the scope of this article at this moment.
Incubate the inoculated bottles under following incubation conditions (phase / stage wise)-
Different Growth Phases of Cordyceps militaris Cultivation
Each growth phase of Cordyceps militaris cultivation has its unique feature. These standard features can be used for monitoring the growth of this mushroom. The precise control of the incubation conditions is crucial for obtaining the standard or better outcomes at the end of respective cultivation phase.
I. Mycelia run under Dark:
The first incubation stage facilitates mycelial growth through the fruiting media by incubating the inoculated culture vessels under dark for the first 4 days in dark. Look for white, nearly smooth, cottony mycelial growth at the top surface of fruiting media.
II. Carotenoid Development:
Carotenoid development in Cordyceps militaris takes around 4 days after the mycelial run phase. It is a light dependent process. Illuminate for 10-12 hrs per day. Look for the development of carotenoid pigmentation/ yellow-orange coloration of the mycelia.
Note:
1. Mycelial bumps are also observed during this phase. Formation of tiny mycelial bumps (approx. size of a lentil grain) is a light-independent process as it occurs even under dark if the mycelial run phase is allowed to run for 2-3 more days.
2. A granular mycelial texture with uniform orange colored mycelial surface marks the completion of carotenoid development phase.
III. Primordia Induction:
The third phase, primordia induction, is the most critical phase. If this phase goes well, the rest of the growth pattern is relatively stable and unaffected by incubation conditions to a large extent.
However, primordia induction fails when temperature is lower than 14 degC (as observed during Cultivation Batch 2, started on 1st Jan 2024).
Effect of higher temperatures has not been recorded so far because of lack of such incidences. But, do not increase the temperature above 25 deg C.
The incubation conditions may remain unchanged for the next 2-3 weeks. There is not a clear-cut endpoint to this phase as it is in continuation with the fruiting body development phase. For sake of simplicity, primordia induction phase is the time for appearance of pinheads throughout the culture vessel, generally 1 week time after carotenoid development phase is sufficient.
IV. Development of Stroma/ Fruiting Body:
The primordia grow in length and diameter during this phase. It shall continue for around 4-5 weeks. The end of this phase is marked by gradual change in the morphology of the head/tip of the fruiting body from roughly pointed to CLUB (SWOLLEN) shaped.
The end of this phase may not be synchronized among the strains as well as culture vessels in the incubation chamber. Once CLUB morphology appears, switch to the next phase, maturation, of cultivation.
V. Maturation of Stroma/ Fruiting Body:
During this phase, the fruiting body exhibit CLUB shaped heads. As time proceeds, the CLUB shape becomes more and more apparent. It shall continue for 1-2 more weeks but must carefully be monitored for SPORULATION Phase. The fruiting body must be harvested before the first sign of sporulation.
Harvesting: The fruiting body is harvested under hygiene conditions at the end of incubation period. The harvested production is sent to the Processing & Value Addition unit.
Drying Cordyceps militaris fruiting body or mushroom
The fruiting body of C. militaris has approximate 85% moisture content, which makes is susceptible for microbial contamination and subsequent deterioration at room temperature.
There are several methods for drying Cordyceps. Options include air drying, vacuum drying, using a dehydrator, or oven drying at low temperatures (below 60°C or 140°F). Properly drying your mushrooms not only prevents spoilage but also preserves their health benefits. Well-dried mushrooms have a shelf life of over a year when stored correctly.
At our lab, we do an initial drying at even lower temperature 33-38 deg C to reduce the moisture content of fresh fruiting body from 85% to 15-17%. The partially dried mushroom is then dried at 52-54 deg C to the final moisture content of 5%. The whole drying process is carried out under aseptic conditions in combination of vacuum as required.
The morphology of dried fruiting body i.e. Cordyceps militaris mushroom is the characteristic of each strain. There may be some pro and cons of each strain. However, the cultivation protocol generally remains the same.
Storage
The dried Cordyceps mushroom shall preferably be vacuum packed in food-grade, water-resistant bags. Store at minus (-)20 deg C until used. The finally dried mushroom (moisture content of 5% or less), vacuum packed in water-impermeable bags or pouches can be stored for several years at (-)20.0 deg C.
Your Path to Success
By implementing these techniques, you can enhance your Cordyceps militaris cultivation journey. From selecting the right substrate to maintaining the ideal growing conditions, each step is crucial for success.
With careful monitoring and intentional actions, you can enjoy a robust harvest while contributing to the growing interest in mushroom cultivation. Embracing this journey can be rewarding for both seasoned growers and newcomers alike. Happy cultivating!
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