CN109355200A - A kind of isolation and purification method of the small bolete mycelia of sky handle - Google Patents
A kind of isolation and purification method of the small bolete mycelia of sky handle Download PDFInfo
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Abstract
The present invention provides a kind of isolation and purification method of the small bolete mycelia of empty handle, belongs to wild mushroom hypha separation purification art, acquires the small beef liver hyphostroma of empty handle or spore carries out mycelia growth in PDA culture medium, and in Characteristic education base culture mycelia；Wherein, PDA culture medium composition includes pine stump powder, the pine stump powder the preparation method comprises the following steps: take Chinese pine or landing pine stump system, distilled water is cleaned, 25-35% tartaric acid solution, 45-50%3 are successively used, 4- methylenedioxyphenol solution and dehydrated alcohol ultrasound impregnate 3-5min, then with aseptic water washing, drying, milling is crushed, it is spare.The present invention for the first time isolates and purifies the wild small bolete mycelia of empty handle, this method is easily operated, and hypha separation success rate is high, mycelia purifying property is high, carries out bacterium colony culture using the mycelia isolated and purified, survival rate and quality are high, cultivate for the empty small bolete of handle and provide foundation.
Description
Technical field
The invention belongs to wild mushroom hypha separation purification arts, and in particular to a kind of separation of the small bolete mycelia of sky handle
Purification process.
Background technique
Empty small bolete Boletinus cavipes (Opat.) Kalchbr. of handle is Agaricales, Boletaceae, small beef liver
Pseudomonas autumn ground all living creatures or the wild mushroom grown thickly in woods.Not only edible taste is preferable but also can control lumbocrural pain, brothers for it
Numbness, grain discomfort etc..
This wild mushroom of the empty small bolete of handle is not tamed successfully so far, although widely distributed, rare numbers, with
People increase demand increasingly the understanding of edible fungi nutrition health-care effect.Especially the wild edible fungus market price is substantially
Increase, reduced year by year due to the extinctive acquisition of local people, seriously threaten the germ plasm resource of wildlife species protection and
Development and utilization.But it is found by years of researches, Boletinus wild mushroom mycelia purifying difficulty is very big, using the PDA of wide spectrum
The mycelia growth of culture medium majority separation is not strong, thus causes research that cannot continue deeper into.Therefore, introducing one kind can be more
The technology and methods that efficiently and accurately wild mushroom mycelia is separated and purified are for Boletinus wild mushroom, especially
The preservation of the empty small beef liver fungus kind of handle and consumer demand, there is great meaning.
The prior art such as Authorization Notice No. is the Chinese invention patent of 105219652 B of CN, discloses a kind of wild Bachu
The isolation and purification method of mushroom mycelium, first by wild Bachu mushroom tissue or the spore of ejection in cultivating 6-8 in PDA culture medium
It obtains heterozygosis mycelia；Then it takes heterozygosis mycelia in cultivating 7-10 days on Characteristic education base, obtains the Filamentous mycelia of single homozygosis；
The mycelia of the picking homozygosis is transferred in PDA culture medium and cultivates 6-8 days；However, the invention early period is trained using general PDA
Base is supported, is unfavorable for mycelia growth, and hypha separation success rate is low.
Summary of the invention
The purpose of the present invention is to provide a kind of isolation and purification method of the small bolete mycelia of empty handle, this method is easy to grasp
Make, hypha separation success rate is high, mycelia purifying property is high, carries out bacterium colony culture, survival rate and quality using the mycelia isolated and purified
Height is cultivated for the empty small bolete of handle and provides foundation.
The technical solution that the present invention is taken to achieve the above object are as follows:
A kind of isolation and purification method of the small bolete mycelia of sky handle, including,
Disinfection and Sterility testing are separately cultured, mycelia purifying；
Specific step is as follows:
Disinfection and Sterility testing:
It carries out disinfection to the small beef liver hyphostroma of wild sky handle or spore and carries out Sterility testing；Sterile detection method are as follows: will
Hyphostroma or spore by disinfection, which are placed in, gently rolls or is close to culture medium placement 1-2min on PDA culture medium plate, then
Hyphostroma or spore are removed, in 25-27 DEG C of constant temperature incubation of temperature, is cultivated 5-7 days, no miscellaneous bacteria grows the bacterium group after illustrating disinfection
It knits or spore surface is sterile；
It is separately cultured:
When hyphostroma or spore grow mycelia in PDA culture medium, picking mycelia under aseptic condition is inoculated in characteristic training
It supports on base plate, 25-27 DEG C of constant temperature incubation, cultivates 1-2 days, obtain dominant growth mycelia group；Mycelia is velvet-like, intermediate slightly convex, is in
Concentric circles growth, healthy and strong, the white duration is long；
Mycelia purifying:
Gained mycelia will be cultivated in 23-25 DEG C dark culture 7-10 days on pure medium, obtain single homozygous der Pilz
Silk；Mycelia pure medium is by weight percentage by glucose 2-4%, agar 2-3%, pine needle juice 25-35%, brewer's wort
20-30%, asparagus juice 15-20%, gentamicin 0.5-1% and surplus distilled water composition；Preparation method are as follows: take fresh needles,
Malt and Aloe mesophyll respectively boil filtering to get filtrate, and will pour into plate after the sterilizing of glucose, agar and distilled water, are cooled to
50 DEG C or less rear addition pine needle juices, brewer's wort, asparagus juice and gentamicin, are uniformly mixed and pour into plate extremely solidification for use；The training
Feeding base can promote Filamentous mycelia to grow and rod-shaped mycelia is inhibited to grow, to realize the separation and purifying of Filamentous mycelia.
Preferably, to the wild empty small beef liver hyphostroma of handle using the auxiliary disinfection of 35-50% alcoholic solution ultrasonic wave, ultrasound
Wave temperature is 20-22 DEG C, power 20-35W, action time 5-7min；Then aseptically, using through flame calcination
The knife blade crossed is longitudinal sectional in the middle part of stem or cap, tears, a length of 0.3-0.5cm, the width of picking cap and stem intersection
For the tissue block of 0.1-0.3cm；Ultraviolet disinfection is carried out to spore: using wavelength for the ultraviolet light of 225-300nm, in temperature 20-
22 DEG C, sterilize 10-15s under envionmental humidity 60-65%；The wild small beef liver hyphostroma selection stem of sky handle and cap engaging portion
Bacterial context, which is easy disinfection thoroughly first is that living contaminants are few, second is that active growth, is conducive to the culture fast fast-growing of mycelia
It is long；To progress ultrasonic wave auxiliary disinfection is organized, alcoholic solution can be helped to infiltrate in tissue surface and shallow-layer and pass through concussion fastly
Speed kills miscellaneous bacteria, and eliminates miscellaneous bacteria in the remaining possibility of tissue surface, so that disinfection is thorough；Spore is sterilized using ultraviolet light,
It is safely and fast and not damaged to spore cell.
Preferably, wiping surface to isolated tissue block 60-70% cotton ball soaked in alcohol, and tissue 1- is encased with alcohol swab
2min；So that tissue obtains a degree of wetting (still having certain degree of hardness), guarantees uprightly to be inserted into culture medium, be convenient for
The tissue site of insertion obtains genesis analysis in the medium, just causes the growth rate of different distributing position mycelia in this way
Difference improves the success rate of hypha separation convenient for selecting the mycelia of dominant growth and being separated.
Preferably, when carrying out cultural hypha to hyphostroma: by isolated tissue block in the PDA culture medium of improvement
5-7 days acquisition mycelia of upper 21-25 DEG C of dark culture；It organizes bacterial context and improves the culture weight ratio of PDA culture medium to be 1:50-85；Tool
Body method are as follows: tissue block is uprightly inserted into the middle and lower part position of culture medium, the position 1/6-1/5 is made to expose media surface；Insertion
Tissue site obtain genesis analysis in the medium, the nutritional ingredient and oxygen obtained makes a difference, and can be formed in this way
Different distributing position mycelial growth rates is different, raw convenient for selecting advantage by the benign competition environment of building mycelia growth
Long mycelia is simultaneously separated, and the success rate of hypha separation is improved；
Preferably, when carrying out cultural hypha using bacterium spore: by sporangium fritter, first using wavelength for 250-290nm
Ultraviolet light irradiate 5-8min, be then inoculated in 5-7 days acquisition mycelia of 21-25 DEG C of dark culture in the PDA culture medium of improvement；It is wild
The inoculum concentration of the spore sterile water of the empty small bolete of handle is 0.01-0.03%；
Preferably, the PDA culture medium of improvement includes: potato full-powder 35-40% by weight percentage, cotton seed hull
1-2%, glucose 5-8%, agar 3-5%, source area hayashishita extract 15-20%, pine stump powder 6-9%, vitamin B 0.1-
0.3%, distilled water surplus, pH nature；Source area hayashishita extract and pine stump powder are subjected to PDA culture medium improvement, can be used as bacterium
The activation factor that silk is sprouted can be symbiotic environment known to mycelium germination growth creation, reduce mycelia and adapt to germination and growth institute
The time needed, accelerate the growth rate of mycelia；Mycelia can also be supplemented and grow required nutriment, effective microorganisms etc.,
It may advantageously facilitate mycelia growth, improve tissue cultures survival rate and increase mycelium morphology factor；
Preferably, Characteristic education based formulas forms are as follows: medium matrix is vermiculite: wheat: cavings=1:1-2:1, liquid
Body is the MRD for diluting 3-4 times；PH value is 5-6；The Characteristic education base can provide mycelia and grow the nutrition such as the optimum source C, the source N
Source accelerates mycelia growth, improves mycelium morphology factor, is conducive to purify for hypha separation and lay the groundwork.
Preferably, prepared by source area hayashishita extract: the acquisition small bolete place of production fertile soil 100-200g of wild empty handle,
It ferments in the test tube equipped with liquid broth medium of sterilizing, sufficiently oscillation mixes and is placed on 35-38 DEG C of constant temperature
Shaking table shake culture 36-48h is repeated 2-3 times；The culture solution fermented 2500-3500r/min is centrifuged 3-5min, is taken
Clear liquid is centrifuged 5-8min with 8000-9500r/min, takes 0.1-0.5 μm of filtering with microporous membrane degerming of supernatant, 2-4 DEG C of preservation
It is spare；Tunning first removes the biggish impurity of particle through low-speed centrifugal, and then high speed centrifugation obtains extract, contains in the extract
There are many beneficial microbe for originating in the small bolete growth of air-ground handle, such as nitrogen-fixing bacteria, bacillus, streptomycete, these growths
Beneficial bacterium bolete small for empty handle, which is cultivated, has growth promotion, resistance harmful bacteria and other effects；In addition to this, also contain in extract
There are other nutritional ingredients of the small bolete growth of wild empty handle, as indoor medium additives, can be improved bacterial strain training
Feeding survival rate and culture efficiency.
Preferably, pine stump powder is powder made of Chinese pine or landing pine stump system；The preparation method comprises the following steps: taking Chinese pine or landing pine stump
System, distilled water are cleaned, and 25-35% tartaric acid solution, 45-50%3,4- methylenedioxyphenol solution and dehydrated alcohol are successively used
Ultrasound impregnates 3-5min, then with aseptic water washing, drying, crushes milling, spare；Tartaric acid and 3,4- methylenedioxyphenol
Special presence kill the bacterial organisms on root system surface and surface layer, and in root system surface shape first is that carry out disinfection to pine tree root system
At one layer of faintly acid protective film, pollution risk is reduced；Meanwhile be conducive to improve the histiocytic permeability of root system, reinforce its with
The fusion of nutrient media components is formed advantageous so that media environment is more nearly the growing environment of the small bolete of wild empty handle
Imitative field grown condition, it is thus possible to improve the survival rate of the small beef liver hyphostroma culture of empty handle, mycelia is promoted to be formed.
The obtained Filamentous mycelia of purifying is subjected to Characteristic education base and expands culture, 26-28 DEG C culture 7-10 days, handle of having leisure is small
Bolete bacterium colony: colony diameter reaches 80-85mm, and bacterium colony surface is velvet-like, and it is taupe that center is slightly convex, and surrounding is white, in concentric
Circle growth, dense sturdy, non-pigment secretion；It can be seen that the isolation and purification method of the empty small bolete mycelia of handle through the invention,
Have great importance to the empty small beef liver fungus kind of handle is cultivated.
The invention has the benefit that
1) present invention for the first time separates the wild small bolete mycelia of empty handle；
2) culture medium that the present invention isolates and purifies the small bolete of empty handle is improved, and PDA culture medium is first used in such as separation,
Characteristic education base is used again；It is added to the small bolete source area hayashishita extract of wild empty handle and pine stump powder in PDA culture medium, can make
For the activation factor of mycelium germination, known symbiotic environment can be created for mycelium germination growth, reduce mycelia and adapt to sprout life
The long required time, accelerate the growth rate of mycelia；Mycelia can also be supplemented and grow required nutriment, effective microorganisms
Deng, may advantageously facilitate mycelia growth, improve tissue cultures survival rate and increase mycelium morphology factor；Characteristic education base can provide mycelia
The nutrient sources such as the optimum source C, the source N are grown, accelerates mycelia growth, improves mycelium morphology factor, are conducive to as hypha separation purifying
It lays the groundwork；
3) in pine stump powder preparation of the present invention, tartaric acid and 3, the special presence of 4- methylenedioxyphenol, first is that pine tree
Root system carries out disinfection, and kills the bacterial organisms on root system surface and surface layer, and form one layer of faintly acid protective film, drop on root system surface
Low pollution risk；Meanwhile being conducive to improve the histiocytic permeability of root system, reinforce its merging with nutrient media components, so that
Media environment is more nearly the growing environment of the small bolete of wild empty handle, forms advantageous imitative field grown condition, thus energy
Enough survival rates for improving the small beef liver hyphostroma culture of empty handle, promote mycelia to be formed；
4) when the present invention carries out cultural hypha separation using hyphostroma, tissue block is uprightly inserted into the middle and lower part position of culture medium
In, so that the position 1/6-1/5 is exposed media surface；The tissue site of insertion obtains genesis analysis in the medium, obtains
Nutritional ingredient and oxygen make a difference, and can form different distributing position mycelial growth rate differences in this way, pass through and construct bacterium
The benign competition environment of silk growth improves the success rate of hypha separation convenient for selecting the mycelia of dominant growth and being separated.
Present invention employs above-mentioned technical proposals to provide model essay, compensates for the deficiencies in the prior art, design is reasonable, operation side
Just.
Specific embodiment
Present invention is further described in detail with reference to embodiments:
Embodiment 1:
A kind of isolation and purification method of the small bolete mycelia of sky handle, specifically includes the following steps:
(1) disinfection of the small beef liver bacteria strain of wild empty handle and Sterility testing
It carries out disinfection to the small beef liver hyphostroma of wild sky handle or spore；35% wine is used to the wild empty small beef liver hyphostroma of handle
The auxiliary disinfection of smart solution ultrasonic wave, ultrasonic temperature is 20 DEG C, power 20W, action time 5min；Then in aseptic condition
Under, it is longitudinal sectional in the middle part of stem or cap using the knife blade through flame calcination, tear, picking cap and stem intersection
A length of 0.4cm, the tissue block that width is 0.2cm；Ultraviolet disinfection is carried out to spore: using wavelength for the ultraviolet light of 225nm, in temperature
20 DEG C, the lower disinfection 10s of envionmental humidity 60%；The bacterium of wild sky the handle small selection of beef liver hyphostroma stem and cap engaging portion
Meat, for the tissue site first is that living contaminants are few, easy disinfection is thorough, second is that active growth, is conducive to culture mycelia fast-growth；It is right
Tissue carries out ultrasonic wave auxiliary disinfection, and alcoholic solution can be helped to infiltrate in tissue surface and shallow-layer and pass through concussion and quickly killed
Miscellaneous bacteria, and miscellaneous bacteria is eliminated in the remaining possibility of tissue surface, so that disinfection is thorough；Spore is sterilized using ultraviolet light, safety is fast
It is fast and not damaged to spore cell；
Surface is wiped to 60% cotton ball soaked in alcohol of isolated tissue block, and encases tissue 1min with alcohol swab；So that tissue
A degree of wetting (still there is certain degree of hardness) is obtained, guarantees uprightly to be inserted into culture medium, the organization department facilitated insertion into
Position obtains genesis analysis in the medium, and the growth rate for just causing different distributing position mycelia in this way is different, convenient for choosing
It selects the mycelia of dominant growth and is separated, improve the success rate of hypha separation；
Sterile detection method are as follows: by Jing Guo Xiao Du hyphostroma or spore be placed on PDA culture medium plate gently roll or
It is close to culture medium and places 1min, then remove hyphostroma or spore, in 25 DEG C of constant temperature incubations of temperature, cultivates 5 days, no miscellaneous bacteria grows
Hyphostroma or spore surface after illustrating disinfection is sterile；
(2) hypha separation culture
When hyphostroma or spore grow mycelia in PDA culture medium, picking mycelia under aseptic condition is inoculated in characteristic training
It supports on base plate, 25 DEG C of constant temperature incubations, cultivates 1 day, obtain dominant growth mycelia group；Mycelia is velvet-like, intermediate slightly convex, is in concentric circles
Growth, healthy and strong, the white duration is long；
Above-mentioned PDA culture medium composition includes: potato full-powder 35% by weight percentage, cotton seed hull 1%, glucose
5%, agar 3%, source area hayashishita extract 15%, pine stump powder 6%, vitamin B 0.1%, distilled water surplus, pH nature；It will
Source area hayashishita extract and pine stump powder carry out PDA culture medium improvement, can be used as the activation factor of mycelium germination, can be mycelia
Germination and growth creates known symbiotic environment, reduces the time needed for mycelia adapts to germination and growth, accelerates the growth rate of mycelia；
Mycelia can also be supplemented and grow required nutriment, effective microorganisms etc., may advantageously facilitate mycelia growth, improve tissue training
It forms motility rate and increases mycelium morphology factor；
Wherein, source area hayashishita extract preparation process are as follows: the acquisition small bolete place of production fertile soil 100g of wild empty handle, in
It ferments in the test tube equipped with liquid broth medium of sterilizing, sufficiently oscillation, which mixes, is placed on 35 DEG C of constant-temperature table shakes
Culture 36h is swung, is repeated 2 times；The culture solution fermented 2500r/min is centrifuged 3min, supernatant is taken to be centrifuged with 8000r/min
5min takes 0.1 μm of filtering with microporous membrane degerming of supernatant, and 2 DEG C save backup；Tunning first removes particle through low-speed centrifugal
Biggish impurity, then high speed centrifugation obtains extract, containing having there are many small bolete growth of source area sky handle in the extract
Beneficial microorganism, such as nitrogen-fixing bacteria, bacillus, streptomycete, these growth beneficial bacterium boletes small for empty handle, which are cultivated to have, to be promoted
Growth, resistance harmful bacteria and other effects；In addition to this, other nutrition in extract also containing the wild empty small bolete growth of handle
Ingredient can be improved the survival rate and culture efficiency of strain culturing as indoor medium additives；
Pine stump powder is powder made of Chinese pine or landing pine stump system；The preparation method comprises the following steps: taking Chinese pine or landing pine stump system, distilled water
It cleans, successively impregnates 3min with 25% tartaric acid solution, 45%3,4- methylenedioxyphenol solution and dehydrated alcohol ultrasound, so
Afterwards with aseptic water washing, drying, milling is crushed, it is spare；Tartaric acid and 3, the special presence of 4- methylenedioxyphenol, first is that right
Pine tree root system carries out disinfection, and kills the bacterial organisms on root system surface and surface layer, and form one layer of faintly acid protection on root system surface
Film reduces pollution risk；Meanwhile being conducive to improve the histiocytic permeability of root system, reinforce its merging with nutrient media components,
So that media environment is more nearly the growing environment of the small bolete of wild empty handle, advantageous imitative field grown condition is formed, because
And can be improved the survival rate of the small beef liver hyphostroma culture of sky handle, promote mycelia to be formed；
Characteristic education based formulas composition are as follows: medium matrix is vermiculite: wheat: cavings=1:1:1, and liquid is 3 times of dilution
MRD；PH value is 5；The Characteristic education base can provide mycelia and grow the nutrient sources such as the optimum source C, the source N, accelerate mycelia growth,
Mycelium morphology factor is improved, is conducive to purify for hypha separation and lay the groundwork；
When carrying out cultural hypha to hyphostroma: by isolated tissue block in 21 DEG C dark culture 5 days in PDA culture medium
Obtain mycelia；It organizes bacterial context and improves the culture weight ratio of PDA culture medium to be 1:50；Method particularly includes: tissue block is uprightly inserted
Enter in the middle and lower part position of culture medium, 1/5 position is made to expose media surface；The tissue site of insertion obtains longitudinal direction in the medium
Distribution, the nutritional ingredient and oxygen obtained make a difference, and can form different distributing position mycelial growth rates so not
Together, by the benign competition environment of building mycelia growth, convenient for selecting the mycelia of dominant growth and being separated, mycelia point is improved
From success rate；When carrying out cultural hypha using bacterium spore: by sporangium fritter, wavelength first being used to shine for the ultraviolet light of 250nm
5min is penetrated, 21 DEG C of dark cultures, 5 days acquisition mycelia in PDA culture medium are then inoculated in；The spore of the small bolete of wild sky handle is sterile
The inoculum concentration of water is 0.01%；
It takes and grows most vigorous mycelia block in being dripped on the coverslip for having distilled water or 2%KOH solution in advance, close the lid glass
Piece observes mycelia situation under the microscope；It was found that hypha form is mainly filiform, it is contaminated with a small amount of rod-shaped；
(3) mycelia purifies
Gained mycelia will be separately cultured in 23 DEG C dark culture 7 days on pure medium, obtain the Filamentous mycelia of single homozygosis；
Mycelia pure medium is by weight percentage by glucose 2%, agar 2%, pine needle juice 25%, brewer's wort 20%, asparagus juice
15%, gentamicin 0.5% and surplus distilled water composition；Preparation method are as follows: fresh needles, malt and Aloe mesophyll is taken to carry out respectively
It boils and filters to get filtrate, plate will be poured into after the sterilizing of glucose, agar and distilled water, pine needle is added after being cooled to 50 DEG C or less
Juice, brewer's wort, asparagus juice and gentamicin are uniformly mixed and pour into plate extremely solidification for use；The culture medium can promote der Pilz
Silk grows and rod-shaped mycelia is inhibited to grow, to realize the separation and purifying of Filamentous mycelia.
The Filamentous mycelia that purifying is obtained carries out Characteristic education base and expands culture, and 26 DEG C are cultivated 7 days, the small bolete of handle of having leisure
Bacterium colony: colony diameter reaches 80mm, and bacterium colony surface is velvet-like, and it is taupe that center is slightly convex, and surrounding is white, is grown in concentric circles, dense
It is close sturdy, non-pigment secretion；It can be seen that the isolation and purification method of the empty small bolete mycelia of handle through the invention, empty to cultivating
The small beef liver fungus kind of handle has great importance.
Embodiment 2:
A kind of isolation and purification method of the small bolete mycelia of sky handle, specifically includes the following steps:
(1) disinfection of the small beef liver bacteria strain of wild empty handle and Sterility testing
It carries out disinfection to the small beef liver hyphostroma of wild sky handle or spore；45% wine is used to the wild empty small beef liver hyphostroma of handle
The auxiliary disinfection of smart solution ultrasonic wave, ultrasonic temperature is 21 DEG C, power 32W, action time 6min；Then in aseptic condition
Under, it is longitudinal sectional in the middle part of stem or cap using the knife blade through flame calcination, tear, picking cap and stem intersection
A length of 0.4cm, the tissue block that width is 0.2cm；Ultraviolet disinfection is carried out to spore: using wavelength for the ultraviolet light of 280nm, in temperature
21 DEG C, the lower disinfection 12s of envionmental humidity 60%；
Surface is wiped to 65% cotton ball soaked in alcohol of isolated tissue block, and encases tissue 1min with alcohol swab；So that tissue
A degree of wetting (still there is certain degree of hardness) is obtained, guarantees uprightly to be inserted into culture medium, the organization department facilitated insertion into
Position obtains genesis analysis in the medium, and the growth rate for just causing different distributing position mycelia in this way is different, convenient for choosing
It selects the mycelia of dominant growth and is separated, improve the success rate of hypha separation；
Sterile detection method are as follows: by Jing Guo Xiao Du hyphostroma or spore be placed on PDA culture medium plate gently roll or
It is close to culture medium and places 1min, then remove hyphostroma or spore, in 26 DEG C of constant temperature incubations of temperature, cultivates 6 days, no miscellaneous bacteria grows
Hyphostroma or spore surface after illustrating disinfection is sterile；
(2) hypha separation culture
When hyphostroma or spore grow mycelia in PDA culture medium, picking mycelia under aseptic condition is inoculated in characteristic training
It supports on base plate, 26 DEG C of constant temperature incubations, cultivates 2 days, obtain dominant growth mycelia group；Mycelia is velvet-like, intermediate slightly convex, is in concentric circles
Growth, healthy and strong, the white duration is long；
Above-mentioned PDA culture medium composition includes: potato full-powder 38% by weight percentage, cotton seed hull 1.3%, grape
Sugar 6.8%, agar 4.3%, source area hayashishita extract 16%, pine stump powder 8%, vitamin B 0.2%, distilled water surplus, pH
It is natural；
Wherein, source area hayashishita extract preparation process are as follows: the acquisition small bolete place of production fertile soil 100g of wild empty handle, in
It ferments in the test tube equipped with liquid broth medium of sterilizing, sufficiently oscillation mixes and is placed on 36.5 DEG C of constant-temperature tables
Shake culture 42h, is repeated 3 times；By the culture solution fermented with 3000r/min be centrifuged 4min, take supernatant 9000r/min from
Heart 6min takes 0.3 μm of filtering with microporous membrane degerming of supernatant, and 3 DEG C save backup；
Pine stump powder is powder made of Chinese pine or landing pine stump system；The preparation method comprises the following steps: taking Chinese pine or landing pine stump system, distilled water
It cleans, successively impregnates 5min with 30% tartaric acid solution, 48%3,4- methylenedioxyphenol solution and dehydrated alcohol ultrasound, so
Afterwards with aseptic water washing, drying, milling is crushed, it is spare；
Characteristic education based formulas composition are as follows: medium matrix is vermiculite: wheat: cavings=1:2:1, and liquid is 4 times of dilution
MRD；PH value is 5.2；The Characteristic education base can provide mycelia and grow the nutrient sources such as the optimum source C, the source N, accelerate mycelia raw
It is long, mycelium morphology factor is improved, is conducive to purify for hypha separation and lay the groundwork；
When carrying out cultural hypha to hyphostroma: by isolated tissue block in 23 DEG C dark culture 6 days in PDA culture medium
Obtain mycelia；It organizes bacterial context and improves the culture weight ratio of PDA culture medium to be 1:76；Method particularly includes: tissue block is uprightly inserted
Enter in the middle and lower part position of culture medium, 1/5 position is made to expose media surface；The tissue site of insertion obtains longitudinal direction in the medium
Distribution, the nutritional ingredient and oxygen obtained make a difference, and can form different distributing position mycelial growth rates so not
Together, by the benign competition environment of building mycelia growth, convenient for selecting the mycelia of dominant growth and being separated, mycelia point is improved
From success rate；When carrying out cultural hypha using bacterium spore: by sporangium fritter, wavelength first being used to shine for the ultraviolet light of 280nm
6min is penetrated, 23 DEG C of dark cultures, 6 days acquisition mycelia in PDA culture medium are then inoculated in；The spore of the small bolete of wild sky handle is sterile
The inoculum concentration of water is 0.02%；
It takes and grows most vigorous mycelia block in being dripped on the coverslip for having distilled water or 2%KOH solution in advance, close the lid glass
Piece observes mycelia situation under the microscope；It was found that hypha form is mainly filiform, it is contaminated with a small amount of rod-shaped；
(3) mycelia purifies
Gained mycelia will be separately cultured in 24 DEG C dark culture 8 days on pure medium, obtain the Filamentous mycelia of single homozygosis；
Mycelia pure medium is by weight percentage by glucose 3%, agar 2%, pine needle juice 30%, brewer's wort 25%, asparagus juice
18%, gentamicin 0.7% and surplus distilled water composition；Preparation method are as follows: fresh needles, malt and Aloe mesophyll is taken to carry out respectively
It boils and filters to get filtrate, plate will be poured into after the sterilizing of glucose, agar and distilled water, pine needle is added after being cooled to 50 DEG C or less
Juice, brewer's wort, asparagus juice and gentamicin are uniformly mixed and pour into plate extremely solidification for use；The culture medium can promote der Pilz
Silk grows and rod-shaped mycelia is inhibited to grow, to realize the separation and purifying of Filamentous mycelia.
The Filamentous mycelia that purifying is obtained carries out Characteristic education base and expands culture, and 27 DEG C are cultivated 9 days, the small bolete of handle of having leisure
Bacterium colony: colony diameter reaches 83.5mm, and bacterium colony surface is velvet-like, and it is taupe that center is slightly convex, and surrounding is white, grows in concentric circles,
It is dense sturdy, non-pigment secretion；It can be seen that the isolation and purification method of the empty small bolete mycelia of handle through the invention, to cultivation
The empty small beef liver fungus kind of handle has great importance.
Embodiment 3:
(1) disinfection of the small beef liver bacteria strain of wild empty handle and Sterility testing
It carries out disinfection to the small beef liver hyphostroma of wild sky handle or spore；50% wine is used to the wild empty small beef liver hyphostroma of handle
The auxiliary disinfection of smart solution ultrasonic wave, ultrasonic temperature is 22 DEG C, power 35W, action time 7min；Then in aseptic condition
Under, it is longitudinal sectional in the middle part of stem or cap using the knife blade through flame calcination, tear, picking cap and stem intersection
A length of 0.4cm, the tissue block that width is 0.2cm；Ultraviolet disinfection is carried out to spore: using wavelength for the ultraviolet light of 300nm, in temperature
22 DEG C, the lower disinfection 15s of envionmental humidity 65%；
Surface is wiped to 70% cotton ball soaked in alcohol of isolated tissue block, and encases tissue 1min with alcohol swab；So that tissue
A degree of wetting (still there is certain degree of hardness) is obtained, guarantees uprightly to be inserted into culture medium, the organization department facilitated insertion into
Position obtains genesis analysis in the medium, and the growth rate for just causing different distributing position mycelia in this way is different, convenient for choosing
It selects the mycelia of dominant growth and is separated, improve the success rate of hypha separation；
Sterile detection method are as follows: by Jing Guo Xiao Du hyphostroma or spore be placed on PDA culture medium plate gently roll or
It is close to culture medium and places 2min, then remove hyphostroma or spore, in 27 DEG C of constant temperature incubations of temperature, cultivates 7 days, no miscellaneous bacteria grows
Hyphostroma or spore surface after illustrating disinfection is sterile；
(2) hypha separation culture
When hyphostroma or spore grow mycelia in PDA culture medium, picking mycelia under aseptic condition is inoculated in characteristic training
It supports on base plate, 27 DEG C of constant temperature incubations, cultivates 2 days, obtain dominant growth mycelia group；Mycelia is velvet-like, intermediate slightly convex, is in concentric circles
Growth, healthy and strong, the white duration is long；
Above-mentioned PDA culture medium composition includes: potato full-powder 40% by weight percentage, cotton seed hull 2%, glucose
8%, agar 5%, source area hayashishita extract 20%, pine stump powder 9%, vitamin B 0.3%, distilled water surplus, pH nature；
Wherein, source area hayashishita extract preparation process are as follows: the acquisition small bolete place of production fertile soil 200g of wild empty handle, in
It ferments in the test tube equipped with liquid broth medium of sterilizing, sufficiently oscillation, which mixes, is placed on 38 DEG C of constant-temperature table shakes
Culture 48h is swung, is repeated 3 times；The culture solution fermented 3500r/min is centrifuged 5min, supernatant is taken to be centrifuged with 9500r/min
8min takes 0.5 μm of filtering with microporous membrane degerming of supernatant, and 4 DEG C save backup；
Pine stump powder is powder made of Chinese pine or landing pine stump system；The preparation method comprises the following steps: taking Chinese pine or landing pine stump system, distilled water
It cleans, successively impregnates 5min with 35% tartaric acid solution, 50%3,4- methylenedioxyphenol solution and dehydrated alcohol ultrasound, so
Afterwards with aseptic water washing, drying, milling is crushed, it is spare；
Characteristic education based formulas composition are as follows: medium matrix is vermiculite: wheat: cavings=1:2:1, and liquid is 4 times of dilution
MRD；PH value is 6；The Characteristic education base can provide mycelia and grow the nutrient sources such as the optimum source C, the source N, accelerate mycelia growth,
Mycelium morphology factor is improved, is conducive to purify for hypha separation and lay the groundwork；
When carrying out cultural hypha to hyphostroma: by isolated tissue block in 25 DEG C dark culture 7 days in PDA culture medium
Obtain mycelia；It organizes bacterial context and improves the culture weight ratio of PDA culture medium to be 1:85；Method particularly includes: tissue block is uprightly inserted
Enter in the middle and lower part position of culture medium, 1/5 position is made to expose media surface；The tissue site of insertion obtains longitudinal direction in the medium
Distribution, the nutritional ingredient and oxygen obtained make a difference, and can form different distributing position mycelial growth rates so not
Together, by the benign competition environment of building mycelia growth, convenient for selecting the mycelia of dominant growth and being separated, mycelia point is improved
From success rate；When carrying out cultural hypha using bacterium spore: by sporangium fritter, wavelength first being used to shine for the ultraviolet light of 290nm
8min is penetrated, 25 DEG C of dark cultures, 7 days acquisition mycelia in PDA culture medium are then inoculated in；The spore of the small bolete of wild sky handle is sterile
The inoculum concentration of water is 0.03%；
It takes and grows most vigorous mycelia block in being dripped on the coverslip for having distilled water or 2%KOH solution in advance, close the lid glass
Piece observes mycelia situation under the microscope；It was found that hypha form is mainly filiform, it is contaminated with a small amount of rod-shaped；
(3) mycelia purifies
Gained mycelia will be separately cultured in 25 DEG C dark culture 10 days on pure medium, obtain single homozygous der Pilz
Silk；Mycelia pure medium is by weight percentage by glucose 4%, agar 3%, pine needle juice 35%, brewer's wort 30%, aloe
Juice 20%, gentamicin 1% and surplus distilled water composition；Preparation method are as follows: fresh needles, malt and Aloe mesophyll is taken to carry out respectively
It boils and filters to get filtrate, plate will be poured into after the sterilizing of glucose, agar and distilled water, pine needle is added after being cooled to 50 DEG C or less
Juice, brewer's wort, asparagus juice and gentamicin are uniformly mixed and pour into plate extremely solidification for use；The culture medium can promote der Pilz
Silk grows and rod-shaped mycelia is inhibited to grow, to realize the separation and purifying of Filamentous mycelia.
The Filamentous mycelia that purifying is obtained carries out Characteristic education base and expands culture, and 28 DEG C are cultivated 10 days, the small beef liver of handle of having leisure
Bacterium bacterium colony: colony diameter reaches 81mm, and bacterium colony surface is velvet-like, and it is taupe that center is slightly convex, and surrounding is white, grows in concentric circles,
It is dense sturdy, non-pigment secretion；It can be seen that the isolation and purification method of the empty small bolete mycelia of handle through the invention, to cultivation
The empty small beef liver fungus kind of handle has great importance.
Comparative example 1:
Source area hayashishita extract and pine stump powder, rest part and embodiment 2 complete one are free of in the PDA culture medium of improvement
It causes.
Comparative example 2:
Tartaric acid and 3,4- methylenedioxyphenol, rest part and embodiment 2 complete one is not used in the preparation of pine stump powder
It causes.
Comparative example 3:
When mycelia is separately cultured, Characteristic education base, rest part and embodiment 2 are not only utilized using PDA culture medium
It is completely the same.
Comparative example 4:
When using hyphostroma block culture, it is entirely laterally unfolded to immerse in culture medium, rest part and embodiment 2 are complete
Unanimously.
Embodiment 4:
The small bolete bacterium colony culture of empty handle, the life that different details of operation causes bacterium colony different are carried out using the method for the present invention
Long situation, as shown in table 1；
In table 1, +++ indicate that growing way is strong；++ indicate that growing way is stronger；+ indicate that growing way is weak；
It is described by table 1 it is found that first carrying out cultural hypha separation using the method for the present invention: successively being cultivated using the PDA of improvement
Base and Characteristic education base；Then mycelia purifying is carried out；Mycelia after purification is finally subjected to bacterium colony culture；Wherein, improvement PDA training
Support base, Characteristic education base and mycelia pure medium have effects that respective formula composition and, play a role jointly, culture institute
The bacterium colony quality of the small bolete of handle of having leisure is preferable.
The prior art of routine techniques dawn known to those skilled in the art in above-described embodiment, therefore herein no longer in detail
It repeats.
The above embodiments are only used to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this field
Member can also make a variety of changes and modification without departing from the spirit and scope of the present invention.Therefore, all equivalent
Technical solution also belong to scope of the invention, scope of patent protection of the invention should be defined by the claims.
Claims (9)
1. a kind of isolation and purification method of the small bolete mycelia of sky handle, it is characterised in that: acquire the small beef liver hyphostroma of empty handle or spore
Son carries out cultural hypha in PDA culture medium；The PDA culture medium composition includes pine stump powder；The preparation method of the pine stump powder
Are as follows: it takes Chinese pine or landing pine stump system, distilled water to clean, successively uses 25-35% tartaric acid solution, 45-50%3,4- methylene-dioxy
Phenol solution and dehydrated alcohol ultrasound impregnate 3-5min, then with aseptic water washing, drying, crush milling, spare.
2. a kind of isolation and purification method of the small bolete mycelia of empty handle according to claim 1, it is characterised in that: described
PDA culture medium composition includes: potato full-powder 35-40% by weight percentage, cotton seed hull 1-2%, glucose 5-8%, fine jade
Rouge 3-5%, source area hayashishita extract 15-20%, pine stump powder 6-9%, vitamin B 0.1-0.3%, distilled water surplus, pH are natural.
3. a kind of isolation and purification method of the small bolete mycelia of empty handle according to claim 2, it is characterised in that: the original
Place of production hayashishita extract is the preparation method comprises the following steps: acquire the small bolete place of production fertile soil 100-200g of wild empty handle, in being equipped with for sterilizing
It ferments in the test tube of liquid broth medium, sufficiently oscillation mixes and is placed on 35-38 DEG C of constant-temperature table shake culture
36-48h is repeated 2-3 times；The culture solution fermented 2500-3500r/min is centrifuged 3-5min, takes supernatant 8000-
9500r/min is centrifuged 5-8min, takes 0.1-0.5 μm of filtering with microporous membrane degerming of supernatant, 2-4 DEG C saves backup.
4. a kind of isolation and purification method of the small bolete mycelia of empty handle according to claim 1, it is characterised in that: including such as
Lower step:
Disinfection and Sterility testing: it carries out disinfection to the small beef liver hyphostroma of wild sky handle or spore and carries out Sterility testing；Without bacterial examination
Survey method are as follows: by Jing Guo Xiao Du hyphostroma or spore be placed in gently roll or be close on PDA culture medium plate culture medium place
1-2min then removes hyphostroma or spore, in 25-27 DEG C of constant temperature incubation of temperature, cultivates 5-7 days, no miscellaneous bacteria, which grows, to be illustrated
Hyphostroma or spore surface after disinfection is sterile；
Be separately cultured: when hyphostroma or spore grow mycelia in PDA culture medium, picking mycelia under aseptic condition is inoculated in
On Characteristic education base plate, 25-27 DEG C of constant temperature incubation is cultivated 1-2 days, obtains dominant growth mycelia group；Mycelia is velvet-like, and centre is slightly
It is convex, it is grown in concentric circles, healthy and strong, the white duration is long；
Mycelia purifying: will be separately cultured gained mycelia in 23-25 DEG C dark culture 7-10 days on pure medium, obtain single pure
Plying shape mycelia；Mycelia pure medium is by weight percentage by glucose 2-4%, agar 2-3%, pine needle juice 25-35%, wheat
Bud juice 20-30%, asparagus juice 15-20%, gentamicin 0.5-1% and surplus distilled water composition.
5. a kind of isolation and purification method of the small bolete mycelia of empty handle according to claim 4, it is characterised in that: described right
The wild sky small beef liver hyphostroma of handle is 20-22 DEG C, power using the auxiliary disinfection of 35-50% alcoholic solution ultrasonic wave, ultrasonic temperature
For 20-35W, action time 5-7min；Then aseptically, using the knife blade through flame calcination from stem or
Cap middle part is longitudinal sectional, tears, a length of 0.3-0.5cm of picking cap and stem intersection, the tissue block that width is 0.1-0.3cm；
Ultraviolet disinfection is carried out to spore: using wavelength for the ultraviolet light of 225-300nm, in 20-22 DEG C of temperature, envionmental humidity 60-
65% lower disinfection 10-15s.
6. a kind of isolation and purification method of the small bolete mycelia of empty handle according to claim 4, it is characterised in that: described right
When hyphostroma carries out cultural hypha: by isolated tissue block in 21-25 DEG C dark culture 5-7 days in the PDA culture medium of improvement
Obtain mycelia；It organizes bacterial context and improves the culture weight ratio of PDA culture medium to be 1:50-85；Method particularly includes: tissue block is upright
It is inserted into the middle and lower part position of culture medium, the position 1/6-1/5 is made to expose media surface；The tissue site of insertion obtains in the medium
To genesis analysis.
7. a kind of isolation and purification method of the small bolete mycelia of empty handle according to claim 4, it is characterised in that: described right
When bacterium spore carries out cultural hypha: by sporangium fritter, first using wavelength to irradiate 5-8min for the ultraviolet light of 250-290nm, so
It is inoculated in 5-7 days acquisition mycelia of 21-25 DEG C of dark culture in the PDA culture medium of improvement afterwards；The spore of wild sky handle small bolete without
The inoculum concentration of bacterium water is 0.01-0.03%.
8. a kind of isolation and purification method of the small bolete mycelia of empty handle according to claim 4, it is characterised in that: the spy
Color culture medium prescription composition are as follows: medium matrix is vermiculite: wheat: cavings=1:1-2:1, and liquid is the MRD for diluting 3-4 times；pH
Value is 5-6.
9. a kind of isolation and purification method of the small bolete mycelia of empty handle according to claim 4, it is characterised in that: will purify
Obtained Filamentous mycelia carries out Characteristic education base and expands culture, 26-28 DEG C culture 7-10 days, the small bolete bacterium colony of handle of having leisure: bacterium
Diameter is fallen up to 80-85mm, bacterium colony surface is velvet-like, and it is taupe that center is slightly convex, and surrounding is white, is grown in concentric circles, dense thick
It is strong, non-pigment secretion.
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