Chapter 2 - 3 - 3

CHAPTER 2.
MUSHROOM SPAWN
PRODUCTION TECHNOLOGY
CONTENT
2.1. Overview of mushroom spawn production
2.2. Isolate and preserve mushroom cultures
2.3. Spawn production techniques
2.4. Procedure for spawn production of some
species
CHAPTER 2. MUSHROOM SPAWN
 Some basic concepts:
 pure culture: isolated from fruiting body tissue, single
or multi-spore and then inoculated on agar media
supplemented with nutrient.
 Pure culture is used to produce mother culture. 1
tube of pure culture could produce 30 tubes of
mother culture.
 Normally, after propagating commercial spawn,1
tube of pure culture could be used to cultivate 30-40
tons material substrate. Thus, pure culture need to
be “pure” (not contaminate) and reach standards

 Mother culture: is sub cultured from pure
culture and inoculated on agar medium
supplemented with nutrition.
 Mother culture could be used to produce
mother spawn.1 tube of mother culture can
be used to produce 2 bottles (300g/bottle) of
master spawn.

 Master spawn: is sub cultured from mother
culture and inoculated on seed/stick
substrate media.
 master spawn could be used to produce
commercial spawn (cultivation spawn). 1
bottle of master spawn can produce 15 kg of
commercial spawn (equally 30 bags)

 Commercial spawn (cultivation spawn):is sub
cultured from master spawn
 Commercial spawn is used as spawn to
cultivate.
Mushroom spawn production technology
60-100
tons of
900- 1500 kg substrate
of commercial
60- 100 spawn
bottles of
30-50 tubes master
of mother spawn
culture
1 tube of
pure
culture
7

 The required conditions for mushroom spawn
production:
 Infrastructure :
 Media preparation room
 Mushroom Inoculation room
 Spawn Incubation room
 Spawn storage room
 Storage room (tool, materials and chemical).

 The required conditions for mushroom spawn
production:
 Tool, Equipment:
 Autoclave
 Clean bench
 Incubator
 Refrigerator
 Air conditioner
 Others…

2.2.1. Isolation
 Prepare the necessary steps to isolate:
 Step1. Select fruiting body
 Step 2. Pure culture media preparation
 Step 3. Prepare room, tool and equipment for
isolation
 Step 4. Isolate and Select pure culture
 Step 5. Prepare required conditions for pur
culture incubation
2.2.1. Isolation
 Step1. Collect and select gene source:
 Collect fruiting body from nature
 Determining the collected sample season: Based on
the biological characteristics of the mushroom,
researcher should determine the suitable time for
sample collection;
 For example, To collect straw mushroom samples,
you must collect in the hot season with high
temperature. However, for Lingzhi mushroom
samples collection, you have to go in the rainy
season with a range temperature of 25-30ºC
2.2.1. Isolation
 Ecological conditions: Before samples collection, the
region with many samples should be determined.
 Example: Lingzhi mushroom often grows in Son
Dong, Quang Nam limestone forest; straw
mushroom or button mushroom often grow in region
where many straws are decomposed.
2.2.1. Isolation
 Observe the morphology, characteristics, condition
recording when sampling ...
2.2.1. Isolation
 Collect fruiting body from cultivation:
- Pure culture: select fruiting body which have typical
characteristics of the species, balanced, uninfected.
Based on the aim of research, researcher can select
fruiting body, example: to obtain strains which resistance
disease, researcher should collect fruiting body grown
from infected population.

2.2.1. Isolation
 Step2-3. prepare media, room, tool for isolation:
Medium 1 (Czap medium)
 Sucrose: 30g;
 NaNO3 : 2g;
 KH2PO4: 1g;
 MgSO4 *7H2O: 0.5g;
 FeSO4*7H2O: 0.01g;
 KCl: 0.5g;
 Agar : 20g;
 Water: 1000ml
 pH: 6 - 6,5
2.2.1. Isolation
Medium 2 (Agaricus medium)
 Potato: 200g;
 Pepton: 2g;
 Na2HPO4: 2g;
 MgSO4 *7H2O: 0.5g;
 Glucose: 20g;
 Agar: 20g;
 Water: 1000ml
 pH: 6 - 6,5
2.2.1. Isolation
Medium 3 (Raper medium
 Yeast extract: 2g;
 Pepton: 2g;
 KH2PO4: 0.46g;
 K2HPO4: 1g;
 MgSO4 *7H2O: 0.5g;
 Glucose: 20g;
 Agar: 20g
 Water: 1000ml
 pH: 6 - 6,5
2.2.1. Isolation
Medium 4 (Organic medium)
 Potato: 250g;
 Bean sprouts: 200g
 Mushroom fresh: 100g
 Glucose: 20g;
 Agar: 20g
 Water: 1000ml
 pH: 6 - 6,5

2.2.1. Isolation
 Step 4. Isolation techniques
 Isolate from fruiting body tissue
 Isolated from spore
 Isolated from fruiting body
tissue
2.2.1. Isolation
 Step 4. Isolation techniques
 Isolate spore
 Select fresh, healthy and uninfected mature
fruiting body grown.
 Cut cap of mushroom and keep on sterilized petri
at 30-35º C for 2-4h.
2.2.1. Isolation
 Step 4. Isolation techniques:
 Isolate spore
 Spores on the petri dish are mixed with 10ml sterile
water and diluted until a concentration of 50 spores
/ml. Spore counting is carried out by using
Hemocytometer.
 The spores are spreaded onto PGA medium,
incubated at ideal temperature until small colonies
appear.

2.2.1. Isolation techniques
 B5. Preparing conditions for pure culture :
 Ensuring the best conditions for temperature,
humidity, light, air for mycelial growth

2.2.2. preserve mushroom cultures
 After culture grow throughout media and
establish total colonisation, if not used, it
must be preserved to prevent aging
 Selecting cultures reach standards (grow
well, pure, not containminated)
2.3. mushroom spawn production methods

 Solid spawn:
 Spawns are cultured on PGA media
 Grain spawn
 Stalk spawn
 Sawdust spawn
 Compost spawn
 Liquid spawn: spawns are cultured on liquid
medium
Mushroom spawn production methods
Solid spawn Liquid spawn
Spawn Grain Stalk Sawdust Compost

on PGA spawn spawn spawn spawn
Spawns are cultured in
media liquid medium

 Solid spawns:
 Spawns are cultured on PGA media
 PGA media supplemented with essential nutrients is
used for pure culture production
Pure culture spawn was sub sultured

Pure culture
 Solid spawn:
 Grain spawn
 Grain spawn were used as mother culture
and commercial spawn
master spawn
 Diagram of master spawn production process
Required Soaking Boiling bottled Sterilization

Material (2) (3) (4) (5)
(1)
Production Incubation Inoculation

(8) (7) (6)
2.4. Protocol for the spawn production of some

mushrooms
2.4.1. Protocol for master spawn production
Material:
 Choose the type of paddy with low
amylopectin content, from the previous crop,
no chemical, less bulky, uniform
 Glass bottles with capacity of 500 - 600ml
 CaCO3 powder
 Others
Question
Why should we choose paddy with low amylopectin
content?
When paddy with high amylopectin content is
boiled, the grains are easy to stick together into
blocks, there is no porosity, obstructing the growth
of mycelia.
mushrooms
(1) Calculation: suitable amount of paddy soaked, usually
1kg dry paddy will correspond to 1.6-1.8 kg of boiled one.
 (2) Soaking:
 Washes in sufficient water to remove straw particles,
soil debris and undesirable seed of grasses, weeds.
 Washed paddies are soaked in sufficient water from 8
to14 hours.
 After soaking, clean the sour smell.
QUESTION
Why should we soak paddy before boiling?
If we do not soak paddy, the required time for boiling will be

extended;
Water does not penetrate into the grain of rice, so when it boils
outside it is crushed but the inner core is still alive
mushrooms
(3) Boiling:
 Pour water into paddy until above 20 cm of surface,
heat up the fire.
 When water reaches the boiling point for 10-15
minutes, paddies are cracked, then turn down fire,
occasionally stir.
 When more than 80% of paddy is hatched (1/2 -2/3 of
the rice grain is hatched but not crushed and break
out the rice grain, there is no white core in the
middle when rice grain is broken out).
mushrooms
 (3) Boiling:
 Pick up the paddy to the basket and keep on

the shelf to drain the water, brush the thin
before the fan to evaporate the steam quickly.
 When the paddy is cold, the surface of the
paddy husk is dry, adding light powder.
 Humidity 60- 62%.
mushrooms
(4) Mixing light powder (CaCO3) and package
 Use 1-1.5% light powder compared to the
amount of boiled paddy.
 Mix the light powder when paddy has cooled
 Based on the nature of the boiled paddy, the
required amount of light powder can be
reduced or increased.
Question
The role of CaCO3?

- increase porosity;
- Keep and release water if necessary;
2.4. Protocol for the spawn production of

some mushrooms
(4) Phối trộn bột nhẹ và đóng chai
 Bottled: About 300 gam prepared
substrate is filled in bottles (substrate
should not be filled full in bottles).
 Clean mouth of Bottles before plugged
with cotton.
mushrooms
( 5) Sterilization
 Sterilization: Autoclave is used to sterilize (Before
autoclaving, autoclave should be checked carefully)
 Autoclaving at 121°C for 120 minutes (since the
pressure is reached).
 Note:
 temperature and time for sterilization are not ensured,
→ contamination;
 If temperature is very high, substrate will be
caramened.
mushrooms
( 5) Sterilization
 Notes:
 If high pressure is maintained for long time →
nutrition is decomposed
 Boiled paddy should be autoclaved as soon as
possible, not to exceed 12 hours because it is
susceptible to infection by microorganisms.
 After autoclaving, media is cooled and transfer
into clean room.
mushrooms
Protocol for master spawn production
(6) Inoculation
 Spawn preparation.
 Inoculation room and clean bench: inoculation room
should be cleaned and aired. Before inoculation, UV-
light need to be turned on. After that, turn off UV-light
and turn on the fan for 30 minutes.
 Prepare sufficient tools: Tray, implant, alcohol lamp,
cotton and sterilized wipes, non-erasable pen,
notebook.
mushrooms
(6) Inoculation
 Spawn preparation:
 Before inoculation, pure culture spawn should
be checked carefully, not contamination;
 Based on the characterization of strain, the rate
of inoculation is differenced;
 Normally, 1 tube of pure culture spawn is used
to inoculate 2-3 bottles. If strain grows slowly,
the rate of inoculation should be increased.

some mushrooms
(7) Incubation
 Place the inoculated bottles on the scaffold.
the spawn should be kept in the middle of
the bottle.
 The room is clean, airy. Inoculated bottles
should be kept at the appropriate
temperature for each type of mushroom.
mushrooms
 (7) Incubation
 Selection:
 After inoculation 1-2 days, contaminated bottles
should be selected and removed.
 For slow growing strain, spawn should be
checked periodically every 3 days;
 For fast growing strain, it is necessary to check
daily.

mushrooms
2.4.2. Protocol for commercial spawn production
Material:
 Plain paddy
 Cassava stalk
 Sawdust
 Mixed substrates: paddy + sawdust, sawdust
+ husk, …

some mushrooms
2.4.2. Protocol for commercial spawn
production
 Commercial spawn production using
paddy as substrate:
 Similar to produce master spawn.
 Can be applied to most of the currently
cultivated mushrooms in our country.
2.3. Mushroom spawn production methods

Solid spawn:
 Spawn is cultured on stalk
 Cassava stems, soft wood stems ..…
 When mushrooms are cultivated on
sawdust or crushed materials.
 Farms used in large quantities.
Spawn cultured on cassava stalk
mushrooms
 Protocol for commercial spawn production using cassava
stalk :
Cassav Soaking Boili packing Auto

a stalk with lime ng (4) calvi
(1) water (2) (3) ng
(5)
Contanminated Incubation Inoculatio
selection (7) n(6)
(8)

mushrooms
 Protocol for commercial spawn production using cassava
stalk :
 (1) cassava stalk: dry, size ( 12- 15 cm in length; 2cm in
width), not moldy.
 (2) Soaking with lime water: Prepare lime water with
pH=12, cassava stalks are soaked for 8 -14h. Then,
wash 2-3 times until the cassava stalk turns bright yellow.
 (3) Boiling
 (4) Packing

some mushrooms
production
 Commercial spawn production using sawdust:
 (4) Autoclaving: 150 minutes with pressure 1,3-1,5at.
 (5) Inoculation
 (6) Incubation.

mushrooms
 Protocol for commercial spawn production using
sawdust:
 Used to cultivate ear mushroom, shiitake mushroom, ,
lingzhi mushroom on logs by punching method
Dry Composted Packing Sterilization

sawdust(1) sawdust (3) (4)
(2)
Containminated Incubation Inoculation(5
selection (6) )
(7)

mushrooms
 Protocol for commercial spawn production using
sawdust:
 (1) dry sawdust: Rubber, Bodhi ...
 (2) composted sawdust: sawdust is moisten by using lime
water with pH = 12 and pasteurized for 5-10 days. On
sawdust is turned and adjusted moisture on 4th- 5th day.
 (3) Packing: substrate is supplemented with 20% rice
bran and corn bran +1% light powder. Weight per bag is
0.4-0.5kg.

some mushrooms
production
 Spawn cultured on composed substrate:
 used for species with short growth period.
 Species are cultivated in bad conditions.
2.3. Các phương pháp nhân giống nấm
 Liquid spawn:
a. Concept of liquid spawn

- Liquid spawn is spawn cultured in liquid medium.
- Spawns are cultured in different types of bottles through
the process of stirring constantly which help the pellet to be
in contact with nutrition and oxygen to grow well in liquid
medium.
Liquid spawn was cultured in small bioreactor
Liquid spawn was cultured in big bioreactor
spawn was cultured in liquid medium using shaking machine
Liquid spawn:
b. Factors effecting on liquid culture spawn process
Nutrients
- Cacbon: Cacbon is one of main components of mycelial
fungi cells and basic element in the production of
mycelium. Cacbon sources such as cellulose,
hemicellulose, lignin, starch etc. are used for the growth
of mycelim
CHAPTER 2. MUSHROOM SPAWN PRODUCTION
TECHNOLOGY
 Liquid spawn:
Nutrients
- Nitrogen: is a vital component of proteins, nucleic
acids, cell structure and cytoplasm.
Nitrogen sources are often used for the growth of
mycelim that is organic nitrogen.
TECHNOLOGY
 Liquid spawn:
Nutrients
C/N: Carbon to nitrogen ratio is important
Different strains required different ratio. C/N also is related to
growth period.
Normally, the most appropriate C/N ratio for growth and
fruiting body formation period is 20:1 and 30:1 - 40:1, respectively.
TECHNOLOGY
 Liquid spawn:
Nutrients
Minerals such as phosphorus, sulfur, potassium,
magnesim, calcium… are essential elements for the
growth of mycelium
- Phosphorus and sulfur are principal components of
nucleic acid and protein, respectively
TECHNOLOGY
 Liquid spawn:
Nutrients
- Minerals :
- Required component of many enzymes
- Tham gia vào quá trình chuyển hóa năng lượng
- Duy trì tính thấm của tế bào
- Cofactor in enzyme function
TECHNOLOGY
 Liquid spawn:
Nutrients
Micro-mineral elements
- In order to growth, mushroom require essential
micronutrients such as: Fe, Zn, Mn, Cu ...
- Generally, micronutrient concentration which required
for the growth of mushroom is 0,001 - 0,5 ppm.
TECHNOLOGY
 Liquid spawn:
Nutrients
. Vitamin
- can act both as catalysts and participants in the
chemical reaction
- The most prominent function of the vitamins is to serve
as cofactor (co-enzyme) for enzyme activity.
TECHNOLOGY
 Liquid spawn:
Nutrients
. Vitamin
Several mushrooms can produce vitamin on simple
medium. However, for growth, some mushroom species
require medium supplemented with vitamin
TECHNOLOGY
 Liquid spawn:
* Physical factors
* Temperature
- temperature is the most important factor that determines
the rate of growth, fruiting body formation, spore
germination etc...and influences enzyme-catalyzed
reactions.
- Like other living organisms, temperature has a
significant influence on growth and development of
mushroom
TECHNOLOGY
 Liquid spawn:
* Physical factors
* Temperature
At certain temperature range, an increase in temperature will
increase enzyme activity, and therefore, the growth of mushroom
increase
Beyond a certain point of higher temperature, slow growth takes
place and damages the microorganisms by denaturing enzymes,
transport carriers and other proteins. The plasma membrane also is
disrupted as lipid bilayer simply melts and the damage is such an
extent that it cannot be repaired.
TECHNOLOGY
 Liquid spawn:
* Physical factors
* Temperature
-When the temperature rises too high, microorganisms could not
grow. Because of high temperature, enzymes Due to the high
temperature will cause the denaturation of the enzyme, the
membrane is damaged, leading to inhibition of growth.
- At very low temperature, membranes solidify. Both the
function and cell structure is greatly affected. The growth of
microorganisms is stopped.
TECHNOLOGY
 Liquid spawn:
* Physical factors
* Light: Mushrooms are not capable of photosynthesis.
However, at different stages of growth, mushrooms
require different light.
- The stage of mycelial growth, mushrooms do not need
light but in the mature stage, certain scattered light is
required.
TECHNOLOGY
 Liquid spawn:
* Physical factors
• Humidity:
- The mushroom mycelium only grows when there is enough
moisture in the substrate cultivation.
- Water is a substance that dissolves essential nutrients for the life
of mushrooms.
- Water helps the chemical reactions such as hydrolysis, oxidation
occurs more positively.
- Water causes swelling and softening of hard substances
TECHNOLOGY
 Liquid spawn:
* Physical factors
* Air
-Oxygen has a very important effect on mushroom
because oxygen is needed for the respiration of
mushroom.
-Depending on mushroom species and the stage growth,
the demand for oxygen varies
TECHNOLOGY
 Liquid spawn:
* Physical factors
* Air
-During the fruiting boy formation stage, oxygen demand
is lower than the mycelial growth stage. However, after
the fruiting body formation stage, the respiratory process
rises and oxygen demand soars.
-The concentration of carbon dioxide also affects the
respiration of mushroom.
TECHNOLOGY
 Liquid spawn:
* Physical factors
• Air: Based on fungal susceptibility to carbon dioxide,
mushroom can be divided into two types:
+ Type sensitive to CO2: increased CO2 concentration has caused
great harm to mature fruiting bodies stage (button mushroom,
lingzhi mushroom, ear mushroom, shiitake mushroom, ...).
+ Non-sensitive type: when the concentration of CO2 increases, it
has little effect on mature of fruiting bodies stage (needles
mushroom, oyster mushroom, ...)
TECHNOLOGY
 Liquid spawn:
* Physical factors
*pH: Most of mushrooms are suitable in a pH
range between 5-7.
pH affects enzyme activity, on the solubility of
compounds in the environment; therefore the pH
of the medium has a great influence on the growth
of mycelium.
TECHNOLOGY
 Liquid spawn:
* Physical factors
*pH: Wood-decay fungus, especially the parasitic fungi
on plants, prefer acidic or acidic environments about 5, 6
or even 4 (lingzhi mushroom, ear mushoom).
Mushroom species grow on soils that are rich in humus or
on rice straw prefer neutral or slightly alkaline pH (7-
7.5).
TECHNOLOGY
 Liquid spawn:
* Physical factors
Oxygen is essential to the life of any living organism.
In liquid spawn production, dissolved oxygen levels in
the medium are indispensable factors.
Studies suggest that the speed of shaking and aeration is
the only basis for providing oxygen to mushroom growth
in a liquid medium.
TECHNOLOGY
 Liquid spawn:
c. Protocol of liquid spawn culture
* Introduce steps in protocol (diagram)
Spawn production using traditional technology in Vietnam
Pure culture Mother culture

15 -35 days
20-65 days
106 - 116 days

Fruting body Master spawn
Commercial spawn
55-100 days 15 -65 days

Liquid spawn production technology
Pure culture Mother cultures

3-4 days
3-4 days
Giống nấm trung gian
Commercial spawn
42- 46 days 3-4 days

Compare advantages and disadvantages of
two spawn production methods
Liquid spawn Solid spawn
Short cycle Long cycle
Age of spawn is uniform; spawn can Age of spawn varies; compare with liquid
growth well after inoculation spawn, solid spawn can not growth well
Saving labor and packaging Costly labor and packaging
Fruiting body forms uniformly Fruiting body can not form uniformly
Advantage in industrialization of Suitable for small –scale family production
mushroom production
Application of many fields: Limited applications in other fields.

manufacturing pharmaceuticals, spices,
drinks……
Difficult to transport and store Easy to transport and store
expensive equipments, very strict and Inexpensive equipments, simple steps
synchronized steps
Stages Solid spawn Liquid spawn
Pure culture
Inoculate on PGA medium supplemented with nutrition Inoculate on PGA medium supplemented with nutrition
Mother culture
Spawn is cultured in liquid medium on shaking machine (volume of

Spawn is cultured in medium supplemented with agar spawn is from 100 to 500ml)
Master spawn
Spawn is cultured in liquid medium with aeration

Spawn is cultured on grain (volume of spawn is from 2000 to 5000 ml)
Commercial
spawn
Spawn is cultured in nutrient medium in a bioreactor with aeration
Spawn is cultured on combine substrate and stirring (volume of spawn is from 50 – 100 lít)
Mother culture was sub-
cultured on grain medium
Mother liquid spawn was

ub-cultured on grain
medium
Solid and liquid spawn was sub-cultured on grain medium (6 days)

Alternation of liquid spawn and solid spawn production
15-20 days 3-4 days
Mother spawn 15-20 days Pure culture 3-4 days

Mother spawn
3-4 days
Master spawn Master sapwn
15-20 days
Commercial spawn 42-45 days

58-60 days Commerical spawn
Total cycle: 51 – 57 days

Total cycle: 106 - 116 days
TECHNOLOGY
 Liquid spawn:
Some notices during liquid spawn production
1. Select suitable equipment
2. Understand principle of equitments
3. Understand biological characteristics of mushroom spawn
4. Know the contamination test method, the phenomenon of pilling
the pillet during incubation.
5. Understand the target, scale, level of the object will use spawn
6. Understand plan to use the spawn

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CHAPTER 2.

2.1. Overview of mushroom spawn production

2.1. Overview of mushroom spawn production

2.1. Overview of mushroom spawn production

2.1. Overview of mushroom spawn production

2.1. Overview of mushroom spawn production

2.2. Isolate and preserve mushroom cultures

2.2. Isolate and preserve mushroom cultures

2.2. Isolate and preserve mushroom cultures

2.2. Isolate and preserve mushroom cultures

2.2. Isolate and preserve mushroom cultures

2.3. mushroom spawn production methods

Solid spawn Liquid spawn

Spawn Grain Stalk Sawdust Compost

2.3. mushroom spawn production methods

Pure culture spawn was sub sultured

Required Soaking Boiling bottled Sterilization

Production Incubation Inoculation

2.4. Protocol for the spawn production of some

Why should we soak paddy before boiling?

If we do not soak paddy, the required time for boiling will be

 Pick up the paddy to the basket and keep on

The role of CaCO3?

2.4. Protocol for the spawn production of

2.4. Protocol for the spawn production of

2.4. Protocol for the spawn production of some

2.4. Protocol for the spawn production of

2.3. Mushroom spawn production methods

Cassav Soaking Boili packing Auto

2.4. Protocol for the spawn production of some

2.4. Protocol for the spawn production of

2.4. Protocol for the spawn production of some

Dry Composted Packing Sterilization

2.4. Protocol for the spawn production of some

2.4. Protocol for the spawn production of

a. Concept of liquid spawn

Pure culture Mother culture

106 - 116 days

55-100 days 15 -65 days

Pure culture Mother cultures

Giống nấm trung gian

42- 46 days 3-4 days

Short cycle Long cycle

Application of many fields: Limited applications in other fields.

Spawn is cultured in liquid medium on shaking machine (volume of

Spawn is cultured in liquid medium with aeration

Mother liquid spawn was

Solid and liquid spawn was sub-cultured on grain medium (6 days)

15-20 days 3-4 days

Mother spawn 15-20 days Pure culture 3-4 days

Commercial spawn 42-45 days

Total cycle: 51 – 57 days

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