CN109122049A - A kind of more spore separators of Cordyceps militaris and method - Google Patents
A kind of more spore separators of Cordyceps militaris and method Download PDFInfo
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- CN109122049A CN109122049A CN201810882087.8A CN201810882087A CN109122049A CN 109122049 A CN109122049 A CN 109122049A CN 201810882087 A CN201810882087 A CN 201810882087A CN 109122049 A CN109122049 A CN 109122049A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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Abstract
The invention discloses a kind of more spore separators of Cordyceps militaris and methods, the present apparatus includes the cover to match each other and cylinder and limit iron wire, the structures such as bottom door, growth and maturity are chosen when being separated using the device, free of contamination Cordyceps militaris, without winning, related culture medium is out of, taking-up is inverted in separator in tissue culture box cylinder, Cordyceps militaris is set to discharge ascospore in the case where not interruption of growth, ascospore is launched after spore collects culture dish after mature sporophore, ascospore is carried out again to cut collection, this method can reduce antithetical phrase injury ascosporous in operation, it also can avoid missing the optimum growh phase for collecting spore, to obtain the vigorous spore of vitality, for subsequent culture and microscopic examination.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of more spore separators of Cordyceps militaris and method.
Background technique
Cordyceps militaris [Cordyceps militaris (L.et Fr) Link] belongs to Ascomycota, gang pyrenomycetes, Sphaerials, wheat
Angle Cordycepps, Cordyceps parasitize on the insects pupa such as Noctuidae, different from a kind of cordyceps sinensis of cordyceps sinensis, are called northern winter worm
Summer grass, pupa grass, Cordceps militaris etc..Strain separating technology mainly separation, spore separation and substrate mycelium in a organized way in production at present
Partition method etc..Tissue separation is a kind of vegetative propagation technique, refers to any a part for cutting fruit body of edible fungi or sclerotium etc.
Tissue, which is inoculated on culture medium, is trained pure mycelial method.Tissue isolation can be chosen because of the interception position of fructification,
Growth cycle is chosen, the selection of batch generates Different Results, sometimes also can be real because of influences such as personal operating experience, operation techniques
Test result.Spore separation is to sprout the method to form mycelium to obtain pure culture using the sexual spore of edible mushroom maturation.By
Strain that spore separation obtains, character is excellent, cell age is shorter, vitality is vigorous.Spore separation is divided into more spore separation and monospore point
From two kinds.Most of more spores separate mycelium obtained and are substantially capable of forming fructification, for being not easy to carry out tissue separation,
Spore is easy the mushroom sprouted, and is easier to success using the separation of more spores.It is usual in the prior art during carrying out more spore separation
Means be: Cordyceps militaris discharge spore in the period of, Cordyceps militaris is picked off from culture medium, and carry out the collection of spore.The party
Method the problem is that: 1, be easily destroyed spore during excision, influence its sprouting；2, when discharging spore for Cordyceps militaris
Phase needs the judgement of experience, is not easy to grasp；3, the Cordyceps militaris after extracing loses nutrient, and i.e. meeting dehydration is withered within a certain period of time.
Summary of the invention
It is an object of the invention to overcome in the prior art, to the deficiency of Cordyceps militaris more spores separation, a kind of Cordyceps militaris is provided
More spore separators and method, the present apparatus and method can reduce antithetical phrase injury ascosporous in operation, also can avoid missing
To the optimum growh phase that spore is collected, to obtain the vigorous spore of vitality.
The present invention is achieved by the following technical solutions:
A kind of more spore separators of Cordyceps militaris, including the knot such as the cover and cylinder that match each other and limit iron wire, bottom door
Structure, the inner barrel are provided with for accepting the limit iron wire for cultivating the culture dish for having Cordyceps militaris, and limit iron wire passes through setting
It is fixed in the aperture on barrel wall, is provided with to open or close in the horizontal direction in the bottom of cylinder and facilitates collection spore
Culture dish disengaging bottom door.
In the above-mentioned technical solutions, the quantity of the limit iron wire is 3, and three connects to form triangle.
In the above-mentioned technical solutions, the limiting iron flight lead is from 3-4cm at cartridge openings.
A kind of more spore separation methods of Cordyceps militaris, carry out according to the following steps:
Step 1: choosing growth and maturity, free of contamination Cordyceps militaris, without winning, related culture medium takes from tissue culture box
It is inverted in the cylinder of separator out, makes Cordyceps militaris naturally vertically downward for culture medium is fixed using limit iron wire；
It is placed into cylinder Step 2: the spore without nutrient is collected culture dish by bottom door, is allowed to be located at Cordyceps militaris
Vertical lower；
Step 3: separator is placed under gnotobasis, Cordyceps militaris is made to discharge son in the case where not interruption of growth
Cystospore takes out spore and collects after ascospore ejection spore described in step 2 is collected culture dish by mature sporophore
Culture dish carries out ascospore to cut collection.
In the above-mentioned technical solutions, in step 2, the spore collects culture dish by molal volume than the agar for 0.8%
And distilled water is formulated.
In the above-mentioned technical solutions, in step 3, the incubation time of Cordyceps militaris is 5-10 days.
In the above-mentioned technical solutions, it in step 3, after gathering ascospore, cuts and collects the water containing ascospore
Agar surface is attempted by PDA culture medium, is cultivated in the case where temperature is 19-23 DEG C of gnotobasis, after sprouting mycelia out, is scraped bacterium
Silk is put into preservative solution, the preservation under -4 DEG C~-70 DEG C low temperature after sealing.
In the above-mentioned technical solutions, the formula of the PDA culture medium are as follows: potato 200g, glucose 20g, agar 15~
20g, water 1000mL.
In the above-mentioned technical solutions, the preservative solution is formulated by 0.4 gram of agar is added in 30% glycerite.
The advantages and benefits of the present invention are: this method chooses growth and maturity, free of contamination Cordyceps militaris, without plucking
It takes, related culture medium makes Cordyceps militaris the not interruption of growth the case where from being taken out in the cylinder for being inverted in separator in tissue culture box
Lower release ascospore launches ascospore after spore collects culture dish after mature sporophore, then carries out to ascospore
Collection is cut, this method can reduce antithetical phrase injury ascosporous in operation, also can avoid missing to the best of spore collection
Growth period is used for subsequent culture and microscopic examination to obtain the vigorous spore of vitality.
Detailed description of the invention
Fig. 1 is separator structural schematic diagram in the present invention.
Wherein, 1 is cover, and 2 be cylinder, and 3 be limit iron wire, and 4 be bottom door, and 5 be culture medium.
It for those of ordinary skill in the art, without creative efforts, can be according to above attached
Figure obtains other relevant drawings.
Specific embodiment
In order to enable those skilled in the art to better understand the solution of the present invention, combined with specific embodiments below furtherly
Bright technical solution of the present invention.
More spores of Cordyceps militaris of the present invention separate and preservation carries out with the following method:
(1) preparation of spore collection device
The device of spore separation uses total bottle height for 14.5cm, bottleneck diameter 9.5cm, and bottom of bottle diameter is 8.3cm, has
The high-temperature resistance plastice tissue culture box of ventilating cover.Top and bottom can be opened with screw capping, and the bottle cap at the top of tissue culture box is ventilating cover, group
The bottom of training box is 6.5cm high, and diameter is 8.3cm small tissue culture box not with cover, sliceable with body bottom, for holding configuration
Good culture medium.Allow to constitute equilateral triangle around 3 points are chosen in the body position away from bottleneck 3-5cm, according to
Limit iron wire thickness 3 points punchs, iron wire is inserted in the hole, the iron wire frame an of triangle is built into, as be collection
The apparatus structure of spore.
(2) preparation of water agar
0.8% agar is added in distilled water, after being dispensed into tissue culture box, sterilizes in 121 DEG C of autoclave sterilization pots
30min。
(3) selection of more spore separation parent's fructifications
Whole box normal growth is chosen, well-grown is healthy and strong, and uninfected mature sporophore will occur on fructification head
It is put into collection device when mastoid process, this indicates that internal shell of ascus elongation is exposed, i.e., will discharge spore.It chooses
Afterwards, superclean bench will be brought into after culture bottle surface sterilization.
(4) collection of spore
Under conditions of sterile working, the related culture medium of the fructification of selection is taken out and collection device bottleneck compares one
Under, extra portion intercepts are fallen, are guaranteed on the triangle frame for not influencing to be put into collection device.The process slowly placed
In, the fructification that can be pushed down by iron wire is pushed aside with tweezers, is guaranteed that it is smoothly placed on triangle frame, is covered ventilating cover.In 20-23
It is placed 5-10 days under DEG C illumination condition, allows mature spore ejection on water agar.
(5) PDA plate culture medium and the preparation of mycelium preservative solution
PDA culture medium: potato cleans peeling, is cut into small pieces and weighs 200g, adds boiling 20min (until potato energy quilt
Glass bar is poked), juice is taken with filtered through gauze, adds 20g agar, glucose is added after agar has dissolved in heating stirring
20g is stirred evenly, and is supplied moisture again after slightly cooling down to 1000 milliliters, is dispensed sterile petri dish, 121 DEG C of autoclave sterilization pots
It is spare to be put into gnotobasis after cooling for middle sterilizing 30 minutes.
Mycelium preservative solution: the glycerite for being 30% according to institute's expense configuration concentration is added in glycerite
0.4% agar is divided in the 50ml centrifuge tube of screw capping, 121 DEG C of autoclave sterilization 30min, after cooling room temperature, chooses flowing
Property preferably uses.
(6) culture and preservation of pityrosporion ovale filament are collected
The water agar surface for taking collection to have ascospore with bacterium shovel shovel is connect in superclean bench, access prepare
PDA plate culture medium on, cultivate 5-7d under the conditions of 20-23 DEG C, be covered with mycelia to it.It is scraped in an aseptic environment with sterile shovel
Mycelia is taken, is put into mycelium preservative fluid, is sealed, concussion is wrapped with brown paper polybag a little while, is placed on -4 DEG C of preservations of refrigerator.
(7) preservation of bacteria strain activates
A pipe preservation bacterium is opened in superclean bench, is pipetted 100 μ l bacterium solutions with sterilized liquid-transfering gun and is transferred to 100ml
Training is shaken in PD fluid nutrient medium, on shaking table, cultured mycelium pellet can directly carry out out grass experiment.
The preparation of 1 Cordyceps militaris spawn of example and preservation
(1) preparation of spore collection device
High-temperature resistance plastice tissue culture box described in the above method is allowed to constitute equilateral triangle around 3 points are chosen
3 points are punched according to the thickness that thin wire is bought in market, iron wire are inserted in the hole, the iron wire of a triangle is built by shape
Frame, this is the apparatus structure for collecting spore.
(2) preparation of water agar
0.8% agar is added in distilled water, is divided in collection device 30ml, sterilizes in 121 DEG C of autoclave sterilization pots
30min。
(3) selection of more spore separation parent's fructifications
Whole box normal growth is chosen, well-grown is healthy and strong, and uninfected mature sporophore will occur on fructification head
It is put into collection device when mastoid process, this indicates that internal shell of ascus elongation is exposed, i.e., will discharge spore.It chooses
Afterwards, superclean bench will be brought into after culture bottle surface sterilization.
(4) collection of spore
Under conditions of sterile working, the related culture medium of the fructification of selection is taken out and collection device bottleneck compares one
Under, extra portion intercepts are fallen, are guaranteed on the triangle frame for not influencing to be put into collection device.Placement process is slow,
The fructification that can be pushed down by iron wire also is pushed aside with tweezers simultaneously, is finally guaranteed smoothly to be placed on triangle frame, is covered ventilating cover.
It is placed 7-10 days under 20-23 DEG C of illumination condition, allows mature spore ejection on water agar.
(5) culture and preservation of pityrosporion ovale filament are collected
Description according to the above method prepares PDA culture medium used in cultured mycelia and mycelium preservative solution.?
The water agar surface for taking collection to have ascospore with bacterium shovel shovel is connect in superclean bench, accesses the PDA plate prepared
On culture medium, 7d is cultivated under the conditions of 20-23 DEG C, is covered with mycelia to it.Mycelia is scraped in an aseptic environment with sterile shovel, is put into
In mycelium preservative fluid, sealing, concussion is wrapped with brown paper polybag a little while, is placed on -4 DEG C of preservations of refrigerator.
Example 2
Experimental period: in September, 2017
1, strain:
Cordyceps militaris (is purchased from Guangdong Jiangmen Hong Hao biotechnology company)
2, training used medium is shaken:
Peeled potatoes 250g (liquor), glucose 25g, magnesium sulfate 1.5g, potassium dihydrogen phosphate 1.2g, dipotassium hydrogen phosphate
1.8g, VB1 15mg, VB6 10mg, pH value are 6.5 or so, are settled to 1000ml, are divided in 250ml triangular flask, every bottle
100ml。
3, Cordyceps militaris produces nutrient solution:
Soya bean 50g (juicing), whole milk powder 25g, 5% magnesium sulfate 50ml, 11% potassium dihydrogen phosphate 50ml, 2%VB1
50ml, 2%VB6 50ml, are settled to 5000ml.
4, high-temperature resistance plastice band ventilating cover tissue culture box used:
Tissue culture box: bore 10cm, bottom diameter 8cm, high 8.5cm, capacity 480ml.Every box fills 30g rice and nutrition is added
Liquid 40ml sterilizes after mixing, is cooled to room temperature and is followed by bacterium.
5, the management of vegetative stage:
Rice medium after connecing bacterium was at light protected environment culture 7 days, and temperature range control is at 19-23 DEG C, humidity range control
System is in 60%-70%.During this period, culturing room will sterilize on time, if any infection cleaning in time.
6, the management in sporophore growth stage:
Mycelia is covered with entire media surface and has thorough grasp culture basal part after being protected from light culture 7 days, starts light-exposed culture, avoids
Sun light direct beam is cultivated under culturing room's natural light, if dark can open fluorescent lamp supplementary light, most suitable illumination is strong
Degree is in 550-1000lx range.After mycelia is light-exposed, culture medium meeting annesl is in crocus, and surface will form the bacterium of rice-shaped protrusion
Flower bud is further differentiated to form fructification.Sporophore growth stage, temperature range are controlled at 20-23 DEG C, and it is raw to be higher than 25 DEG C of fructifications
It is long that slowly even precocious, humidity range control keep making Interior Space air-flow in 60%-85%, fructification incubation
It is logical, culturing room's timing sterilization.
7, Culture Collection and preservation:
According to method described by example 1, separation and collection Cordyceps militaris spawn and preservation.
Example 3
Experimental period: in January, 2018
1, strain:
The made preservation of bacteria strain of example 2
2, training used medium is shaken:
Peeled potatoes 250g (liquor), glucose 25g, magnesium sulfate 1.5g, potassium dihydrogen phosphate 1.2g, dipotassium hydrogen phosphate
1.8g, VB1 15mg, VB6 10mg, pH value are 6.5 or so, are settled to 1000ml, are divided in 250ml triangular flask, every bottle
100ml。
3, Cordyceps militaris produces nutrient solution:
Soya bean 50g (juicing), whole milk powder 25g, 5% magnesium sulfate 50ml, 11% potassium dihydrogen phosphate 50ml, 2%VB1
50ml, 2%VB6 50ml, are settled to 5000ml.
4, high-temperature resistance plastice band ventilating cover tissue culture box used:
Tissue culture box: bore 10cm, bottom diameter 8cm, high 8.5cm, capacity 480ml.Every box fills 30g rice and nutrition is added
Liquid 40ml sterilizes after mixing, is cooled to room temperature and is followed by bacterium.
5, the management of vegetative stage:
Rice medium after connecing bacterium was at light protected environment culture 7 days, and temperature range control is at 19-23 DEG C, humidity range control
System is in 60%-70%.During this period, culturing room will sterilize on time, if any infection cleaning in time.
6, the management in sporophore growth stage:
Mycelia is covered with entire media surface and has thorough grasp culture basal part after being protected from light culture 7 days, starts light-exposed culture, avoids
Sun light direct beam is cultivated under culturing room's natural light, if dark can open fluorescent lamp supplementary light, most suitable illumination is strong
Degree is in 550-1000lx range.After mycelia is light-exposed, culture medium meeting annesl is in crocus, and surface will form the bacterium of rice-shaped protrusion
Flower bud is further differentiated to form fructification.Sporophore growth stage, temperature range are controlled at 20-23 DEG C, and it is raw to be higher than 25 DEG C of fructifications
It is long that slowly even precocious, humidity range control will make room air circulate in 60%-85%, fructification incubation,
If any infection cleaning in time, culturing room's timing sterilization.
7, it harvests:
Mean fresh reaches 20.3 grams/bottle after harvesting
Example 4
Experimental period: in by the end of March, 2018
1, strain:
The resulting preservation of bacteria strain of example 2
2, training used medium is shaken:
Peeled potatoes 250g (liquor), glucose 25g, magnesium sulfate 1.5g, potassium dihydrogen phosphate 1.2g, dipotassium hydrogen phosphate
1.8g, VB1 15mg, VB6 10mg, pH value are 6.5 or so, are settled to 1000ml, are divided in 250ml triangular flask, every bottle
100ml。
3, Cordyceps militaris produces nutrient solution:
Soya bean 50g (juicing), whole milk powder 25g, 5% magnesium sulfate 50ml, 11% potassium dihydrogen phosphate 50ml, 2%VB1
50ml, 2%VB6 50ml, are settled to 5000ml.
4, high-temperature resistance plastice band ventilating cover tissue culture box used:
Tissue culture box: bore 10cm, bottom diameter 8cm, high 8.5cm, capacity 480ml.Every box fills 30g rice and nutrition is added
Liquid 40ml sterilizes after mixing, is cooled to room temperature and is followed by bacterium.
5, the management of vegetative stage:
Rice medium after connecing bacterium was at light protected environment culture 7 days, and temperature range control is at 19-23 DEG C, humidity range control
System is in 60%-70%.During this period, culturing room will sterilize on time, if any infection cleaning in time.
6, the management in sporophore growth stage:
Mycelia is covered with entire media surface and has thorough grasp culture basal part after being protected from light culture 7 days, starts light-exposed culture, avoids
Sun light direct beam is cultivated under culturing room's natural light, if dark can open fluorescent lamp supplementary light, most suitable illumination is strong
Degree is in 550-1000lx range.After mycelia is light-exposed, culture medium meeting annesl is in crocus, and surface will form the bacterium of rice-shaped protrusion
Flower bud is further differentiated to form fructification.Sporophore growth stage, temperature range are controlled at 20-23 DEG C, and it is raw to be higher than 25 DEG C of fructifications
It is long that slowly even precocious, humidity range control will make room air circulate in 60%-85%, fructification incubation,
If any infection cleaning in time, culturing room's timing sterilization.
7, it harvests:
Mean fresh reaches 21.28g after harvesting
Conclusion:
By the comparison of example 2,3 and 4 results, fruiting bodies of cordyceps militaris growth phase growth characteristics, character and yield with
Difference that there are no significant between parent's fructification, so that the method for illustrating that strain is collected in the invention can retain Cordyceps militaris parent's
Hereditary capacity, and can be used as Cordyceps militaris production strain and use, the culture collection process which uses can also be protected effectively
Strain is hidden, strain quality is not influenced, can normally go out grass.
Illustrative description has been done to the present invention above, it should explanation, the case where not departing from core of the invention
Under, any simple deformation, modification or other skilled in the art can not spend the equivalent replacement of creative work equal
Fall into protection scope of the present invention.
Claims (8)
1. a kind of more spore separators of Cordyceps militaris, it is characterised in that: including the cover that matches each other and cylinder and limit iron wire,
The structures such as bottom door, the inner barrel are provided with for accepting the limit iron wire for cultivating the culture dish for having Cordyceps militaris, limit iron wire
It is fixed by the aperture being set on barrel wall, being provided in the bottom of cylinder can open or close conveniently in the horizontal direction
Collect the bottom door of the culture dish disengaging of spore.
2. the more spore separators of a kind of Cordyceps militaris according to claim 1, it is characterised in that: the quantity of the limit iron wire
It is 3, three connects to form triangle.
3. the more spore separators of a kind of Cordyceps militaris according to claim 1, it is characterised in that: the limiting iron flight lead is from cylinder
Body opening 3-4cm.
4. a kind of more spore separation methods of Cordyceps militaris, which is characterized in that carried out according to the following steps:
Step 1: choosing growth and maturity, free of contamination Cordyceps militaris, without winning, related culture medium takes out from tissue culture box
It is placed in the cylinder of separator, makes Cordyceps militaris naturally vertically downward for culture medium is fixed using limit iron wire；
It is placed into cylinder Step 2: the spore without nutrient is collected culture dish by bottom door, is allowed to hanging down positioned at Cordyceps militaris
Straight lower section；
Step 3: separator is placed under gnotobasis, Cordyceps militaris is made to discharge ascus spore in the case where not interruption of growth
Son takes out spore and collects culture after ascospore ejection spore described in step 2 is collected culture dish by mature sporophore
Ware carries out ascospore to cut collection.
5. the more spore separation methods of a kind of Cordyceps militaris according to claim 4, it is characterised in that: in step 2, the spore
Collect culture dish by molal volume than for 0.8% agar and distilled water be formulated.
6. the more spore separation methods of a kind of Cordyceps militaris according to claim 4, it is characterised in that: in step 3, Cordyceps militaris
Incubation time is 5-10 days.
7. the more spore separation methods of a kind of Cordyceps militaris according to claim 4, it is characterised in that: in step 3, gathering
It after ascospore, cuts and collects the water agar surface containing ascospore, be attempted by PDA culture medium, be 19-23 DEG C in temperature
It is cultivated under gnotobasis, after sprouting mycelia out, scraping mycelia is put into preservative solution, after sealing under -4 DEG C~-70 DEG C low temperature
Preservation.
8. the more spore separation methods of a kind of Cordyceps militaris according to claim 4, it is characterised in that: the PDA culture medium is matched
Side are as follows: potato 200g, glucose 20g, 15~20g of agar, water 1000mL.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117063780A (en) * | 2023-10-17 | 2023-11-17 | 四川朕源生物科技有限公司 | Cordyceps sinensis ascospore harvesting culture medium and harvesting method thereof |
| CN117063780B (en) * | 2023-10-17 | 2023-12-26 | 四川朕源生物科技有限公司 | Cordyceps sinensis ascospore harvesting culture medium and harvesting method thereof |
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