COMMERCIAL

CULTIVATION OF
CORDYCEP MILITARIS

2020
TECHNICAL MANUAL

FEBRUARY 12

HIMANG INDUSTRIES
Authored by: SRIVATSAVA RAJAGOPALAN

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Table of Contents
Commercial Cultivation of Cordyceps Militaries..........................................................3
Introduction ............................................................................................................4
Spatial Needs for Cordyceps Culture .......................................................................6
List of Equipment Needed .......................................................................................6
List of consumables Needed....................................................................................7
Operational procedures ..........................................................................................7
Preparation of Culture media ..............................................................................8
Inoculation of Culture media ...............................................................................9
Preparation of Growing media ..........................................................................11
Inoculation of Growing media ...........................................................................13
Growing Room Activities.......................................................................................13
Fumigation of Growing Rooms and Lab .............................................................13
Spawn Run / Dark Phase ...................................................................................13
Growing Phase / Bright Phase ...........................................................................14
Harvest and Post-harvest ......................................................................................14
Harvest .............................................................................................................14
Post-harvest ......................................................................................................14

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Commercial Cultivation of Cordyceps
Militaries

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 Introduction

This is a technical guide for Cordyceps cultivators. It assumes the reader to possess no
prior knowledge of cordyceps but however to have a flair for culture of cordyceps
militaries. This report details out the end to end requirement and processes involved in
the culture of cordyceps. I have tried my best to keep it simple and understandable
however some prior study about the need and usage of equipment used through a
Google search by the reader might help him/her understand things better.

Cordyceps militaries is a fungi which is a nucleoside of Adenosine. Consumption of

Cordyceps increases the level of adenosine tri phosphate in the blood. Adenosine tri
phosphate is popularly known as molecular unit of currency. It reinforces the
mitochondria which is otherwise known as the power house of human cells there by
increasing the amount of energy available to drive many processes in the cell.

Also Cordyceps induces apoptosis in the human body, Apoptosis is otherwise known as
programmed cell death. This programmed cell death is as important as programmed
cell birth and growth to the human body. But why is it so important?

Because if this (Apoptosis) doesn’t occur as designed it gives raise to unwanted cell
growth, when fortified with free radicles these eventually mature as cancer.

However care needs to be taken in avoiding cordyceps by those who suffer from
Autoimmune disease or bleeding disorder because cordyceps is proven to boost
immunity and reduce the speed of clotting. If you are scheduled for a surgery it would
be best to stop consuming cordyceps at least 2 weeks before surgery. Cordyceps have
been clinically proven to be excellent regulators and controllers of thyroid and many
other hormones. Cordyceps is known to treat type 2 diabetes by activating the hormone
and by mimicking the action of insulin. Also it has been observed from research paper
published by Soo Jung Parka,#, Hyun-Jin Jangb,#, In-Hu Hwangc on the 20th November
2017 in Natural Product Communications 2018 vol 13 No 4 pages 465 to 470 that
Cordyceps militaris Extract Inhibits the NF-κB pathway and Induces Apoptosis through
MKK7-JNK Signaling Activation in TK-10 Human Renal Cell Carcinoma
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Spatial Needs for Cordyceps Culture

A cordyceps culture facility needs to address the following minimal requirements for
the produce to be economically viable ( Economies of Scale)
1. Growing Room : Should be at least 100 SqFt ( 10 ft * 10 ft)
2. Culture Room : Should be at least 100 SqFt ( 10 ft * 10 Ft)
3. Kitchen : for at least 50sqft with space for a Stove and Autoclave
4. Processing Room : For at least 50 Sqft
This set up is good enough for a production capacity of 60 Kg/annum on a 60 days
harvest cycle.

List of Equipment Needed

1. Laminar Air flow
2. Auto clave – Basic or Automatic
3. Hot air oven for Drying
4. Lab shaker ( Should hold at least 8 conical flasks)
5. Air conditioner 1.5 TR * 3 (Two for the growing room on primary and standby
mode, One for the Sterile processing room) Preferably LG 1.5 TR Dual Inverter.
6. Humidifier 11 liters or More ( Should provide 75 % to 90 % Rh)
7. Angle racks for growing stay ( Depending on Room size)
8. LED Strip light Pink Color
9. Thermal Insulation : Cross type Link Poly Ethylene ( XLPE) ( 8 mm) , Brand –
Supreme
10.Domestic RO purifier – 1
11.Growing containers 3.5 inches by 6 inches tall round ( Depending on number of
racks and space) Glass or PP Only ( Must be Autoclavable / Microwavable )
12.Conical flasks 1000 ml – 30 Nos
13.Beaker 500 Ml – 2 nos
14.Petridis ( If needed)
15.One liter cylindrical Measurement Jar
16.Electric Weighing scale – 1 No
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List of consumables Needed
1. 10 Ml Syringes – 10 Nos
2. Disposable Gloves ( At least One dozen) .
3. Sterile Gown ( At least 4 Nos)
4. Masks – at least 2 dozens
5. Sterile Foot ware / Shoes

Operational procedures
The operational stages of Cordyceps involves 3 stages
1. Preparation of Culture media
2. Preparation of the Growing media
3. Inoculation
4. Incubation
5. Growing & Fruiting
6. Harvest
7. Processing.

Note :
Growing media preparation and culture media preparation are independent activities
they can be done in parallel, There is no dependency of the culture media preparation
on the Growing media preparation, However the Growing media requires the culture to
be available at least before the inoculation stage (Explained in item 6 of Step 3 under the
heading preparation of growing container). Always ensure that you have sufficient
quantities of culture and culture media before you prepare the growing media.

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Preparation of Culture media

The above diagram shows the arrangement of the lab wares that needs to be followed
for the preparation of culture media and its inoculation inside the Laminar Airflow. To
prepare the culture media perform the following steps
1. Have 1000 ml sterilized conical flasks ( 7 numbers Ready)
2. Details provided in this heading pertains to per 1000 ml of culture media or
per conical flask, you need to compute quantities in accordance with the number
of conical flasks of culture media you intend to produce.
3. Perform items 1 to 11 explained under the heading “Preparation of potato fresh
starch ( Step 1)”
4. Measure 800 Ml of pure RO treated water in a beaker
5. Transfer it to a clean sterilized blending vessel.
6. Add the fresh potato starch obtained from item 3(in this heading) to the blending
vessel.
7. To this mixture add 30 grams of Dextrose Anhydrous while stirring constantly.
8. To this add 5 grams of peptone and stir constantly.
9. To this add 5 grams of yeast extract and stir constantly.
10.To this add 0.5 grams of Magnesium sulfide and still constantly.
11.Take a 1000 ml conical flask and pour only 600ml of the above mixture in to it.
12.Close the Conical flask with tightly packed and rolled nonabsorbent surgical
grade cotton (As its lid).
13.Autoclave it for 30 minutes @ 121 degrees centigrade at 15 PSI.

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 14.Cool it in a sterile zone ( inside the Laminar air flow ) for 8 Hours
15.Expose it to UV radiation for 15 minutes prior to inoculation.

Inoculation of Culture media

1. In case of Liquid culture (Which is what we would be doing) use 10 Ml of
inoculant (Culture) for every Conical flask.
2. Inoculation of culture media needs to the done inside the laminar air flow.
3. Ensure that the laminar flow is switched on and UV is on for at least 15 minutes
before the inoculation.
4. Please cleanse your hands with hospital spirit or hand sanitizer
5. Also PLEASE ENSURE THAT THE UV LAMP IS SWITCHED OFF IN THE LAMINAR
FLOW WHEN THE MOTHER CULTURE IS BROUGHT IN TO THE LAMINAR FLOW. IF
NOT THE ENTIRE MOTHER CULTURE WOULD DIE
6. Put on your disposable sterile working glove.
7. Go to the changing area
8. Put on your sterile Robe.
9. Put on your sterile foot ware
10.Put on your sterile head cap
11.Put on your face mask.
12.Go in to the growing area and bring the existing Mother culture (F1)
13.Ensure that your Laminar flow has been running for at least 15 minutes with the
UV ON before you commence.
14.Ensure that the Laminar flow fan and light are running but UV IS SWITHCED OFF
before you place the mother culture in the Laminar Air Flow.
15.Light a spirit lamp on the laminar flow.
16.Take a Sterile 10 ml syringe.
17.Have the autoclaved culture media ( The conical flasks from item 12 to 15 in the
previous step Preparation of Culture media) handy inside the laminar flow.
18.Transfer required amount of mother culture in to a sterilized beaker kept in the
laminar flow and replace it back in the growing room.
19.Open the Culture media conical flask’s cotton cock.
20.Draw 10 ml from the beaker which has the mother culture using the syringe.

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21. Inoculate the culture media by injecting the syringe directly in to the conical
flask, the stream should not hit the side walls of the conical flask and must fall
straight in to the middle of the bottom of conical flask.
22.Douse the lower end of cotton closing cock of the conical flask in the spirit lamp
fire and seal the inoculated conical flask with it immediately.
23.Repeat items 20 to 22 for that many number of conical flasks of culture media
that you might have.
24.Ensure that once inoculated these flasks are still kept in the sterile zone.
25.Once done, take these inoculated conical flasks and place them on the rotary
shaker placed in the growing room.
26.Switch on the rotary shaker and set it to 120 rpm. Ensure that the indoor
temperature is 18 degrees centigrade with a relative humidity of 75% ( Which it
be as it is placed in the growing room).
27.Once done return back to the changing room, remove your sterile apron, sterile
head cap, sterile footwear and gloves and place them in the UV closet.
28.Change back in to your regular working attire
29.The culture would be ready by the end of the 7th day and looks like the one
shown in the following image ( Pearly beads)

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Preparation of Growing media

The diagram above shows the layout to be followed while preparing the growing media
for inoculation inside the laminar airflow. The first step in the entire process is to
prepare the growing media. The growing media is comprised of the following elements
/items
1. Potato based fresh starch
2. Dextrose Anhydrous ( Glucose)
3. Peptone
4. Yeast extract
5. Magnesium Sulfate
6. Growing substrate – The following could be used

a. Brown Rice – Matta Rice

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 b. Wheat husk
Preparation of potato fresh starch (Step 1)
Perform the following steps to prepare the potato fresh starch
1. Take 200 grams of cleanly washed potato
2. Slice them thin like wafer chips ( with the skin on , do not de skin )
3. Measure 1 liter of RO treated Water in a measuring jar.
4. Transfer it to a stainless steel boiling vessel with lid.
5. Add the sliced potatoes to the vessel.
6. Close the lid of the vessel.
7. Switch on the stove.
8. Simmer it and let it boil for 15minutes.
9. After 15 minutes, Switch off the stove and let it cool to the room temperature.
10.Once cooled to the room temperature open the lid of the boiling vessel.
11.Use a fine filter and filter the boiled starch in to a clean and sterilized vessel.
Preparation of Nutrition Growing Media (Step 2).
1. Measure 800 Ml of clean RO treated water in the measuring Jar.
2. Pour it in to a clean sterilized mixing container.
3. Add the 200 ml of filtered fresh potato starch obtained from step 1 to this while
stirring constantly and gently.
4. Add 20 Grams of Dextrose (Glucose) to this and stir well.
5. Add 10 Grams of Peptone to this and stir well.
6. Add 5 Grams of Yeast extract to this and stir well.
7. Add 50 mg ( Mill grams ) powdered multivitamin tablets to this and stir well.
8. Keep stirring until all the solids added are completely dissolved and there are no
visible lumps.

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 9. The nutrition growing media is now ready.
Inoculation of Growing media
1. Take 70 grams of Brown rice in the growing container
2. Add 120 ml of the growing nutrition media prepared in step 2 to the container
3. Close the lid and Seal it tight
4. Autoclave it for 30 minutes at 121 degrees centigrade at 15 PSI .
5. Let it cool over night
6. Inoculate this box with the culture within 24 hours (of autoclaving).
7. Place this container in the Darkness zone (Inside the growing room).
8. Subsequent procedures for growing are explained in the section Growing Room
Activities and starts with the Spawn run or the Dark Phase.

Growing Room Activities

Fumigation of Growing Rooms and Lab
The first step in the growing room activity before seeding the culture on or after the
completion of complete harvest is to fumigate the growing rooms with and the Lab
with KmNO4 and Formalin for 2 days , Have the doors and windows open ensure that
the mother culture is stored in a safe place un affected by fumigation.

Spawn Run / Dark Phase

This is a crucial phase for the mycelium to grow , in this phase
1. The inoculated growing media boxes (from steps 1 to 8 under Inoculation of
Growing media) are placed in the growing racks as shown in the placement chart
in the appendix.
2. Once arranged they are covered with a sterile black cloth or a black colored
polyethylene sheet to cut off any light that might fall on the growing jar.
3. Ensure that the ambient temperature is 18 degrees centigrade and the relative
humidity is 75 %
4. The growing phase would be for 15 days commencing from the date of
placement in the dark room. In some cases it might take up to 20 days for the
mycelium to grow.

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Growing Phase / Bright Phase

1. From the 15th day or the 20th day (depending on the growth of the mycelium)
expose the grow boxes to light of maximum of not more than 300 lumens of Lux,
This can vary from 100 lux to 300 lux for 16 hours a day , the lower the light
intensity the higher the percentage of Cordycepin and the higher the light
intensity the Higher the percentage of Adenosine in the fruiting body .
2. Fruiting would Start by the 30th to the 35th Day and visible pin heads would

appear as shown here

3. Inspect the grow boxes daily and isolate those boxes that might have calcareous
porium ( A white colored pathogen for Cordyceps) it looks like this
4. << Insert image here >>
Harvest and Post-harvest
Cordyceps militaries start growing from the 30th or 35th day to the 60th or 65th day, The
militaries grow tall and strong with a long and slender fruiting body. When their Corpus
reached the lid of the growing jar it is identified as a signal for harvest .
Harvest
1. Fetch the growing containers from the growing room to the processing lab.
2. Open the containers.
3. Gently pluck out the Cordyceps militaries colonies.
4. Do not scrape till the growing media base.
5. Empty the growing base media in to a separate plastic bag.
Post-harvest
1. Put the harvested cordyceps on a sterile tray and place it in the oven.
2. Switch the oven on and set a drying temperature of 60 degrees centigrade ,
3. Let it dry for 2 hours ,

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4. Keep inspecting the cordyceps once every 30 minutes and toss them around so
that their other sides also dries off.
5. Inspect the moisture content using a moisture measurement meter, it should be
less than 3 %.
6. Remove it , place it in the Laminar flow with the UV lamp, Allow it to cool down
to room temperature , vacuum seal it and pack it in PVC pouches .

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