CN107771613A - A kind of naked umbrella novel bacterial of yellow pleat and its cultural method and purposes - Google Patents
A kind of naked umbrella novel bacterial of yellow pleat and its cultural method and purposes Download PDFInfo
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Abstract
The present invention relates to a kind of novel bacterial and its artificial cultivation method, more particularly to a kind of naked umbrella novel bacterial of yellow pleat and its cultural method and purposes, the naked umbrella novel bacterial of yellow pleat is the naked umbrella Gymnopilus luteofolius HMGIM X120086 of yellow pleat, and deposit number is CCTCC No：M2017212.The cultural method will be included after separating parent species, prepare original seed, make production kind, after production kind is inoculated into planting material, carry out cultivation management.By inventor's repetition test research, grope to have obtained the culture medium for cultivating and cultural method for being suitable for the naked umbrella novel bacterial of yellow pleat of the present invention, can successfully realize the artificial cultivation of the naked umbrella of yellow pleat.
Description
Technical field
The present invention relates to a kind of novel bacterial and its artificial cultivation method, more particularly to a kind of naked umbrella novel bacterial of yellow pleat and its cultivation
Training and purposes.
Background technology
It is reported that it is about 150000 kinds to currently exist in tellurian macro fungi, known is no more than
10%, outside the medicinal fungus such as edible mushroom, ganoderma lucidum, cordyceps sinensis such as our known mushroom, agaric, flat mushrooms, also have substantial amounts of
Urgently researcher goes to disclose and utilized the affluent resources of macro fungi.
China's wild mushroom species is various, and most wild mushrooms can be gathered and eaten by people, and some species are
Poisonous, it is impossible to it is directly edible, but medicine of the toxin therein as human or animal can be extracted, or as agrochemical
Developed.Some wild mushroom species fructifications are hard, can not be consumed directly by humans, but can use them to produce various
Useful enzyme, for producing cellulose or being degraded to organic matter.Some species are being total to for the trees such as pine tree, birch, robur
Raw bacterium, sapling can be bred using them, improve the cultivation survival rate of seedling.
Although wild mushroom is large number of, current energy artificial cultivation is only accounted for less than 3.4%, and the overwhelming majority is all in natural life
Long status, wild mushroom domestication research still have very big potentiality, wherein being no lack of fine quality, have edible or medical value species.
Carry out the domestication work of wild mushroom, can not only increase market edible and medical fungi species, promote China's edible and medical fungi industry development, it is right
Increase farmers' income, promote agricultural development, promote building Socialist New all significant.
Gymnopilus Gymnopilus is to belong to Agaricales (Agaricales), the one of Strophariaceae (Strophariaceae)
Class macro fungi, many kinds have toxicity in the category, gymnopilus spectabi1is Gymnopilus spectabilis that China has reported,
Green and brown naked umbrella Gymnopilus aeruginosus, naked umbrella Gymnopilus liquieiriae of striped, the yellow naked umbrella of storage
Yellow naked umbrella Gymnopilus picrina of Gymnopilus penetrans, China fir etc. are poisonous species, and Poisoning Types are mostly
Gastroenteritis type or neuropsychosis type.Research shows that naked umbrella contains psilocybin, and it is a kind of Nervous toxicity for having neural hallucinogenic action
Element, the mushroom containing psilocybin have used centuries in some religious rites of the original inhabitant on the ground such as Mexico.Psilocybin
Toxicological effect mechanism currently there are no and understand fully completely, it is generally recognized that it initially enters blood circulation, then reaches nervous system, so
Activate 5-hydroxytryptamine receptor afterwards and cause innervation.Psilocybin mainly acts on autonomic nerve, cause nervous excitation,
Unreal, serious appearance tachycardia, pupil amplification, dysuria are caused, but typically will not threat to life.
Studied in Gymnopilus more is gymnopilus spectabi1is.Gymnopilus spectabi1is (Gymnopilus spectabilis (Fr.)
Sing.), Chinese is commonly called as " laugh bacterium ", is distributed widely in all over the world.It is reported that gymnopilus spectabi1is is a kind of poisonous mushroom, by mistake
It can cause that head eye is dim-sighted, such as drunk person's instability of gait after food, the lighter is extremely frivolous to laugh, and severe one stupor is even dead.It is in addition, orange naked
Bitter principle in umbrella also has nervous excitation and antitumor action.From the last century 70's, people are begun to orange naked
Destroying angel chemical composition is studied, and is found that many structures novelties and the compound with certain physiological function.Zeng You
Research report shows that the yellow naked umbrella Gymnopilus luteofolius of pleat are that one kind is hopeful to carry out lumbering factory polluted soil
Biodegradable fungi.
In terms of domestication, Bao Haiying et al. realizes the domesticating and cultivating to the bacterial strain first, the results showed that：Gymnopilus spectabi1is
Can be in wheat bran, sawdust, gypsum, lime, and do not add and grow fructification on the compost of carbendazim, from being inoculated into the existing former base time
For 6 months.Gymnopilus spectabi1is is a kind of fungus that can realize artificial cultivation, but because of factor, the mesh such as the cycle is long, fruiting body yield is low
It is preceding not yet to carry out commercialization cultivation.
In view of the poisonous kind of Gymnopilus is more, and the research to the naked umbrella of yellow pleat at present is almost blank, for its open country
Raw strain studied in terms of domestication's research and function, there is provided the strain and cultural method of the yellow naked umbrella of pleat, for open country
The application of raw edible mushroom has positive function and significance.
The content of the invention
The deficiency for more than, the present invention provide a kind of naked umbrella novel bacterial of yellow pleat and its cultivation and purposes.
The present invention reaches above-mentioned purpose by following scheme：
In the first aspect of the present invention, there is provided a kind of yellow naked umbrella novel bacterial of pleat, the novel bacterial is in Linzhi Area of Tibet color season
Draw mountain lookout terrace to collect, be the naked umbrella novel bacterial of yellow pleat by morphology and molecular biology identification, by fructification portion
Divide and carry out the isolated original strain of tissue, be named as the naked umbrella Gymnopilus luteofolius HMGIM- of yellow pleat
X120086, being preserved in China typical culture collection center on April 27th, 2017, (abbreviation CCTCC, address are：Wuhan City
Hongshan District Bayi Road Wuhan University), deposit number is CCTCC No：M2017212.
Its ITS molecular sequences is determined through molecular biology, with the naked umbrella Gymnopilus luteofolius similitudes of yellow pleat
Up to 99%, by Morphological Identification, its gross feature and microscopic features and the naked umbrella Gymnopilus luteofolius of yellow pleat
Unanimously, the naked umbrella Gymnopilus luteofolius of yellow pleat are accredited as.
The yellow naked umbrella Gymnopilus luteofolius HMGIM-X120086 of pleat have following morphological feature：Fructification is small
Type is gradually open and flat to median size, bacteria cover diameter 1.5-5cm, convex lens shape, later stage；Cap surface sticks, initial stage aubergine to bronzing,
Lilac red in the middle part of later stage, edge gradually become shallower as, and powder yellow is presented.Bacterial context is thin, and initial stage is lavender, and the later stage becomes yellow, taste
Hardship, smell unobvious.Lamella growing straight to slightly prolonging life, initial stage cinnamon, later stage yellowish-brown, pleat breadth is close.It is raw in stem, it is long
1.51.5-3.5cm, thick 0.3-0.8cm, waits thick or gradually thick downwards, and closely homochromy with cap surface, base portion color is deeper, collarium with
The lower fine striga of tool, middle reality.Collarium is upper, cellulosic, easy to fall off.
Basidiospore ellipse is to oval, and 5.5-7 × 4-5 μm, germ pore is small, and wall is thin, smooth, and it is in shallow rust to meet KOH solution
Brown is to brown of becoming rusty；Spore print rust brown.Load club-like, 17-23 × 4-5.5 μm, have 2-4 basidiospore stigma, be about 2.5
μm, wall is thin, smooth, and it is light yellow to meet the presentation of KOH solution inclusion.Cheilocyst is grown thickly, 25-40 × 8-10 μm, and Duo is spun in plan
For shape to nearly club-like, wall is thin, smooth, meets KOH solution and is clear to band brown.Lamella trama parallel type, about 10 μm of hyphal diameter, wall
It is thin, it is smooth, meet KOH solution and be clear to band yellow.2.5-5 μm of pileipellis hyphal diameter, wall is thin, smooth, and it is saturating to meet KOH solution
It is bright.Bacterial context hyphal cell slightly expands, and 6.5-8.5 μm of diameter, wall is thin, smooth, meets KOH solution and is clear to band yellow, tool lock shape connection
Close.
The yellow naked umbrella Gymnopilus luteofolius of pleat are recorded in 1875 by Charles Horton Peck earliest,
Agaricus luteofolius are named as, it are classified as to one kind of Agaricus, Pier Andrea Saccardo will within 1887
It is renamed as Pholiota luteofolius, belongs to Pholiota.Nineteen fifty-one mycologist Rolf, Singer was named as
Existing name.
The yellow naked umbrella Gymnopilus luteofolius of pleat are often referred to as the naked umbrella of yellow lamella, are distributed widely in close
On the withered broad leaf tree and coniferous tree of grove.July Mo is betided in the east coast of North America, West Coast then betides winter.It has
There are become rusty orange spore print and bitter taste.It, which contains, causes unreal psilocybin.
The scholar Tu Liguer that the country has research to this thinks Gymnopilus luteofolius still according to microscopic feature
Yellow pleat squama umbrella Pholiota luteofolia (Pk.) Sacc should be named as, is Heilungkiang with proposing its distribution in China, Russia
Ross (the Far East Area) is also distributed (2005).Think being mainly characterized by for this kind:Cap convex lens shape, surface stick, and middle part is pale purple
Red, edge is gradually shallow, in powder yellow；Stem is closely homochromy with capping；Basidiospore side is Kidney bean shape；Facial cystidium is in middle swollen
Big plan spindle.This kind was once placed in Gymnopilus (Gymnopilus Karst.) by Singer R., as Gymnopilus
luteofolius(Pk.)Sing..But the main distinction of Gymnopilus and Pholiota is the state of basidiospore wall:Gymnopilus carries on a shoulder pole spore
Sub- wall is coarse, has tubercle, without germ pore；The basidiospore wall of Pholiota is smooth, germ pore with or without.
Inventor's observational study finds that the basidiospore wall-like state of the strain of the present invention is more likely to Pholiota, therefore temporarily by this kind
It is put into Pholiota, and is used as domestic studies.
According to Laura Guzma ' n-Da ' valos etc. result of study, the traditional classification of Gymnopilus and the knot of Molecular Identification
Fruit can not fit like a glove, in view of the viewpoint that Laura Guzma ' n-Da ' valos are illustrated is incorporated on Indexfungorum
Current name, inventor will its be named as Gymnopilus luteofolius.
A kind of the second aspect of the present invention, there is provided above-mentioned yellow naked umbrella novel bacterial (CCTCC No of pleat：M2017212 people)
Work cultural method, as a kind of Rare edible fungus, the success of its artificial cultivation, there is important economic value and social value.
A kind of above-mentioned yellow naked umbrella novel bacterial (CCTCC No of pleat：M2017212 cultural method), it is main to include separation parent species
Afterwards, prepare original seed, prepare production kind, cultivation management will be carried out after production kind is inoculated into planting material, the cultivation management it is main
Step includes：After 20-25 DEG C of lucifuge culture 30-35 days, mycelia maturation, it is placed at 20-25 DEG C of shading, then through 10-15 days, bacterium
Silk is completed latter stage of ripening, and control temperature is between 18-22 DEG C, relative air humidity 85-95%, intensity of illumination about 200-300lux,
Daily illumination 10-12 hours, carbon dioxide content is in below 1200ppm, until cap is open and flat, harvesting, wherein, the planting material
Composition includes：Wood chip 55-58%, cotton seed hulls 31-33%, wheat bran 8-10%, CaCO31-2%, the water content of the planting material
60-65%.
A kind of preferable cultural method, it is main to include after separating parent species, original seed is prepared, production kind is prepared, by life
After production kind is inoculated into planting material, cultivation management is carried out, the key step of the cultivation management includes：In 25 DEG C of lucifuge culture 30-
35 days, after mycelia maturation, be placed at 25 DEG C of shadings, then through 10-15 days, mycelia was completed latter stage of ripening, control temperature at 18-22 DEG C,
Between relative air humidity 85-90%, intensity of illumination about 200-300lux, daily illumination 12 hours, carbon dioxide content exists
Below 1200ppm, until cap is open and flat, harvesting, wherein, the planting material composition includes：Wood chip 55-58%, cotton seed hulls 31-
33%th, wheat bran 8-10%, CaCO31-2%, the water content 60-65% of the planting material.
It is further preferred that the key step of the cultivation management includes：
(1) in 20-25 DEG C of lucifuge culture 30-35 days, now mycelia covers with planting material substantially；
(2) after mycelia maturation, it is placed at 20-25 DEG C of shading, then through 10-15 days, mycelia completed latter stage of ripening；
(3) temperature is controlled between 18-22 DEG C, relative air humidity 85-95%, intensity of illumination about 200-300lux, often
Illumination 10-12 hours day, carbon dioxide content is in below 1200ppm, until cap is open and flat, harvesting.
Preferably, the separation mother culture media that the separation parent species process uses includes following components in percentage by weight：
Potato 20%, glucose 2%, agar 2%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.15%, vitamin B1 are micro, and remaining is
Water.
Preferably, the step of preparative separation mother culture media includes：The component for separating mother culture media is mixed, it is molten
Test tube is dispensed after solution agar, high-temperature sterilization, obtains separating mother culture media.
It is further preferred that the high-temperature sterilization includes：In 0.11MPa atmospheric pressure, 121 DEG C of HTHP moist heat sterilizations
30min。
Preferably, the separation parent species step includes：The strain of sterile working access tissue separation, is placed in 25 DEG C of incubators
Middle constant temperature light culture, after mycelia covers with mother culture media such as mother culture media inclined-plane after can then be transferred.
It is further preferred that the separation parent species step includes：The strain of sterile working access tissue separation, is placed in 25 DEG C
Constant temperature light culture in incubator, after mycelia covers with mother culture media such as mother culture media inclined-plane after can then be transferred；Its
In, the separation mother culture media that the separation parent species process uses includes following components in percentage by weight：Potato 20%, Portugal
Grape sugar 2%, agar 2%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.15%, vitamin B1 are micro, and remaining is water.
Preferably, the original seed material for preparing original seed includes following components in percentage by weight：Sorghum 75-79%, millet
16-20%, CaCO31-2%, the water content 55-60% of the original seed material.
Preferably, described the step of preparing original seed material, includes：Sorghum is soaked through water, such as soaked overnight, mix in proportion
Enter millet, calcium carbonate, be fitted into transparent polypropylene strain bag, install material after sack set on plastic hoop, buckle supporting lid,
Obtain original seed material.
It is further preferred that the transparent polypropylene strain bag is the resistant to elevated temperatures transparent polypropylene strains of 13cm × 25cm
Bag, convert into per packed siccative 60-80g.
It is further preferred that original seed will be prepared after the original seed Bag Material high-temperature sterilization.
It is further preferred that the high-temperature sterilization step includes：In 0.11MPa atmospheric pressure, 121 DEG C of HTHPs are damp and hot goes out
Bacterium 30min.
It is further preferred that described the step of preparing original seed, includes：Into original seed material, sterile working accesses parent species, during inoculation
Ensure in parent species material block embedment original seed material, vaccinated original seed is placed in constant temperature light culture in 25 DEG C of incubators, it is full to treat that mycelia eats
Can then original seed be used as to transfer after material.
It is further preferred that described the step of preparing original seed, includes：Into original seed material, sterile working accesses parent species, during inoculation
Ensure in parent species material block embedment original seed material, vaccinated original seed is placed in constant temperature light culture in 25 DEG C of incubators, it is full to treat that mycelia eats
Can then original seed be used as to transfer after material, the step probably needs 15d or so；The original seed material for preparing original seed includes following weight
The component of percentage：Sorghum 75-79%, millet 16-20%, CaCO31-2%, the water content 55-60% of the original seed material.
Preferably, the production kind material for preparing production kind includes following components in percentage by weight：Cotton seed hulls 87-
89%th, wheat bran 10-12%, CaCO31-2%；Or cotton seed hulls 48-52%, wood chip 25-30%, wheat bran 18-20%, calcium carbonate 1-
2%th, calcium sulfate 1-2%, the water content of the production kind material is 55-60%.
Preferably, described the step of preparing production kind material, includes：Cotton seed hulls is soaked through water, preferably steep it is wet overnight, by than
Example is mixed into wood chip, wheat bran, calcium carbonate, is fitted into transparent polypropylene strain bag, is burrowed after installing material with small wood in Bag Material, hole
A bag bottom is deep to, then the plastic hoop on sack set, buckles supporting lid, obtains production kind Bag Material.
Preferably, the transparent polypropylene strain bag is the resistant to elevated temperatures transparent polypropylene strain bags of 13cm × 25cm, is converted into
Per packed siccative 250-300g.
It is further preferred that production kind will be prepared after the production kind Bag Material high-temperature sterilization.
It is further preferred that the high-temperature sterilization includes：In 0.147MPa atmospheric pressure, 128 DEG C of HTHP moist heat sterilizations
90min。
It is further preferred that described the step of preparing production kind, includes：Into production kind material, sterile working access original seed, connects
Ensure that original seed material block is embedded in production kind material during kind, the vaccinated kind that produces is placed in constant temperature light culture in 25 DEG C of incubators, treated
Mycelia can then be used as production kind switching after eating full material.
It is further preferred that described the step of preparing production kind, includes：Into production kind material, sterile working access original seed, connects
Ensure that original seed material block is embedded in production kind material during kind, the vaccinated kind that produces is placed in constant temperature light culture in 25 DEG C of incubators, treated
Mycelia, which eats, can then be used as production kind switching after full material, it is described the step for probably need 30d or so；The life for preparing production kind
Production kind material includes following components in percentage by weight：Cotton seed hulls 87-89%, wheat bran 10-12%, CaCO31-2%；Or cotton seed hulls
48-52%, wood chip 25-30%, wheat bran 18-20%, calcium carbonate 1-2%, calcium sulfate 1-2%, the water content of the production kind material
For 55-60%.
Preferably, the planting material includes following components in percentage by weight：Wood chip 55-58%, cotton seed hulls 31-33%,
Wheat bran 8-10%, CaCO31-2%, the water content 60-65% of the planting material.
Preferably, the step of preparation planting material includes：Cotton seed hulls is soaked through water, preferably steep it is wet overnight, in proportion
Wood chip, wheat bran, calcium carbonate are mixed into, is fitted into transparent polypropylene strain bag, is burrowed after installing material with small wood in Bag Material, hole is deep
To bag bottom, then the plastic hoop on sack set, buckles supporting lid, obtains planting material.
Preferably, the transparent polypropylene strain bag is the resistant to elevated temperatures transparent polypropylene strain bags of 17cm × 35cm, is converted into
Per packed siccative 450-500g.
Preferably, described the step of production kind is inoculated into planting material, includes：Production kind sterile working is transferred to planting material
In, subsequently carry out cultivation management.
In a preferred embodiment, naked umbrella novel bacterial (the CCTCC No of the yellow pleat：M2017212 cultural method),
Including：
1st, parent species are separated：The strain of sterile working access tissue separation, is placed in constant temperature light culture in 25 DEG C of incubators, treats bacterium
It can then be transferred after behind the full mother culture media such as mother culture media inclined-plane of filament length；
2nd, original seed is prepared：Into original seed material, sterile working accesses parent species, ensures during inoculation in parent species material block embedment original seed material,
Vaccinated original seed is placed in constant temperature light culture in 25 DEG C of incubators, can then be used as original seed to transfer after mycelia eats full material, it is described
Step probably needs 15d or so；
3rd, production kind is prepared：The sterile working access original seed into production kind material, the embedment production of original seed material block is ensured during inoculation
In kind material, vaccinated production kind is placed in constant temperature light culture in 25 DEG C of incubators, can then be used as and produce after mycelia eats full material
Kind switching, it is described the step for probably need 30d or so；
4th, production kind is inoculated into planting material：Production kind sterile working is transferred in planting material, cultivation management；
5th, cultivation management, including：
(1) in 20-25 DEG C of lucifuge culture 30-35 days, now mycelia covers with planting material substantially；
(2) after mycelia maturation, it is placed at 20-25 DEG C of shading, then through 10-15 days, mycelia completed latter stage of ripening；
(3) temperature is controlled between 18-22 DEG C, relative air humidity 85-95%, intensity of illumination about 200-300lux, often
Illumination 10-12 hours day, carbon dioxide content is in below 1200ppm, until cap is open and flat, harvesting.
In another preferred embodiment, naked umbrella novel bacterial (the CCTCC No of the yellow pleat：M2017212 cultivation side)
Method, in addition to including above method step, in addition to culture medium is as follows：
The separation mother culture media that the separation parent species process uses includes following components in percentage by weight：Potato
20%th, glucose 2%, agar 2%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.15%, vitamin B1 are micro, and remaining is water；
The original seed material for preparing original seed includes following components in percentage by weight：Sorghum 75-79%, millet 16-20%,
CaCO31-2%, the water content 55-60% of the original seed material；
The production kind material for preparing production kind includes following components in percentage by weight：Cotton seed hulls 87-89%, wheat bran
10-12%, CaCO31-2%；Or cotton seed hulls 48-52%, wood chip 25-30%, wheat bran 18-20%, calcium carbonate 1-2%, calcium sulfate
1-2%, the water content of the production kind material is 55-60%；
The planting material composition includes：Wood chip 55-58%, cotton seed hulls 31-33%, wheat bran 8-10%, CaCO31-2%, institute
State the water content 60-65% of planting material.
In another preferred embodiment, naked umbrella novel bacterial (the CCTCC No of the yellow pleat：M2017212 cultivation side)
Method, including：
1st, parent species are separated：The strain of sterile working access tissue separation, is placed in constant temperature light culture in 25 DEG C of incubators, treats bacterium
It can then be transferred after behind the full mother culture media such as mother culture media inclined-plane of filament length；Wherein, the separation parent species process uses
Separation mother culture media include following components in percentage by weight：Potato 20%, glucose 2%, agar 2%, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.3%, magnesium sulfate 0.15%, vitamin B1 are micro, and remaining is water；
2nd, original seed is prepared：Into original seed material, sterile working accesses parent species, ensures during inoculation in parent species material block embedment original seed material,
Vaccinated original seed is placed in constant temperature light culture in 25 DEG C of incubators, can then be used as original seed to transfer after mycelia eats full material, it is described
Step probably needs 15d or so；The original seed material for preparing original seed includes following components in percentage by weight：Sorghum 75-79%,
Millet 16-20%, CaCO31-2%, the water content 55-60% of the original seed material；
3rd, production kind is prepared：The sterile working access original seed into production kind material, the embedment production of original seed material block is ensured during inoculation
In kind material, vaccinated production kind is placed in constant temperature light culture in 25 DEG C of incubators, can then be used as and produce after mycelia eats full material
Kind switching, it is described the step for probably need 30d or so；The production kind material for preparing production kind includes following percentage by weight
Component：Cotton seed hulls 87-89%, wheat bran 10-12%, CaCO31-2%；Or cotton seed hulls 48-52%, wood chip 25-30%, wheat bran
18-20%, calcium carbonate 1-2%, calcium sulfate 1-2%, the water content of the production kind material is 55-60%；
4th, production kind is inoculated into planting material：Production kind sterile working is transferred in planting material, cultivation management；The cultivation
Material composition includes：Wood chip 55-58%, cotton seed hulls 31-33%, wheat bran 8-10%, CaCO31-2%, the water content of the planting material
60-65%；
5th, cultivation management, including：
(1) in 20-25 DEG C of lucifuge culture 30-35 days, now mycelia covers with planting material substantially；
(2) after mycelia maturation, it is placed at 20-25 DEG C of shading, then through 10-15 days, mycelia completed latter stage of ripening；
(3) temperature is controlled between 18-22 DEG C, relative air humidity 85-95%, intensity of illumination about 200-300lux, often
Illumination 10-12 hours day, carbon dioxide content is in below 1200ppm, until cap is open and flat, harvesting.
Using the present invention cultural method cultivated so that the present invention in the naked umbrella novel bacterial of yellow pleat cultivation period compared with
It is short, it can be completed within about 2 months from fruit body development is inoculated into, in the culture medium (kind material) used based on cotton seed hulls, wood chip is
It is auxiliary, different from the culture medium for cultivating (kind is expected) for belonging to other kinds such as gymnopilus spectabi1is together and cultural method of prior art.Especially
Ground, in the cultivation of the naked umbrella of yellow pleat, the preparation of culture medium and temperature humidity during parent species separation, original seed making and cultivation management
The control of the conditions such as illumination has important influence for its quick fruiting.
Compared with prior art, domestication naked umbrella novel bacterial (the CCTCC No of novel bacterial Huang pleat of the invention that not only succeed：
M2017212), and the artificial cultivation cycle is short, it is only necessary to which 1-2 months are a phase, have important economic value and promote and anticipate
Justice.
In the third aspect of the present invention, the present invention also provides a kind of above-mentioned yellow naked umbrella novel bacterial CCTCC No of pleat：
The naked umbrella CCTCC No of M2017212 or yellow pleats：Purposes of the M2017212 extracts in prevention and/or tumor is prepared.
Preferably, the tumour includes breast cancer.
Preferably, breast cancer caused by the breast cancer behaviour malignant breast cancer cells (MT-1).
Preferably, the naked umbrella CCTCC No of the yellow pleat：M2017212 extracts are extractive with organic solvent, further preferably
For esters extract, such as ethyl acetate extract.
In a preferred embodiment, there is provided a kind of yellow naked umbrella CCTCC No of pleat：M2017212 ethyl acetate extracts
Purposes of the thing in prevention and/or tumor is prepared, tumour include breast cancer, such as antitumor cell MT-1.
By inventor's repetition test research, grope to have obtained the cultivation for being suitable for the naked umbrella novel bacterial of yellow pleat of the present invention
Culture medium and cultural method, it can successfully realize the artificial cultivation of the naked umbrella of yellow pleat.And confirm that its ethyl acetate extract etc. exists
The remarkable result of anti-tumor aspect.
Brief description of the drawings
Fig. 1 is the naked umbrella novel bacterial spore sterogram of yellow pleat that the cultivation of embodiment 1 obtains.
Fig. 2 is the naked umbrella novel bacterial figure of yellow pleat of the field acquisition of embodiment 2.
Embodiment
The present invention is further described below in conjunction with specific embodiment.
Embodiment 1
First, naked umbrella novel bacterial (the CCTCC No of yellow pleat：M2017212 cultural method), culture medium are as follows：
1st, mother culture media
Formula：Potato 20%+ glucose 2%+ agar 2%+ potassium dihydrogen phosphate 0.3%+ magnesium sulfate 0.15%+ vitamins
B1 is micro, 1000 milliliters of water.
2nd, original seed material
Formula：Sorghum 75-79%, millet 16-20%, CaCO31-2%, water content 55-60%.
3rd, production kind material
Formula 1：Cotton seed hulls 87-89%, wheat bran 10-12%, CaCO31-2%, water content 55-60%.
Or；
Formula 2：Cotton seed hulls 48-52%+ wood chip 25-30%+ wheat bran 18-20%+ calcium carbonate 1-2%+ calcium sulfate 1-2%,
Water content 55-60%.
4th, planting material
Wood chip 55-58%, cotton seed hulls 31-33%, wheat bran 8-10%, CaCO31-2%, water content 60-65%, pH are natural.
2nd, naked umbrella novel bacterial (the CCTCC No of yellow pleat：M2017212 cultural method), step are as follows：
1st, parent species are separated：
Mother culture media is made by formula, test tube is dispensed after dissolving agar, in 0.11MPa atmospheric pressure, 121 DEG C of HTHPs
Moist heat sterilization 30min, take out the strain of cooling sterile working access tissue separation.
Be placed in constant temperature light culture in 25 DEG C of incubators, after mycelia covers with inclined-plane after can then be transferred.
2nd, original seed is prepared：
The sorghum of ratio, wet overnight through bubble needed for weighing, and is mixed into millet, calcium carbonate in proportion, loads 13cm × 25cm
In resistant to elevated temperatures transparent polypropylene strain bag, convert into per packed siccative 60-80g.Install material after sack set on plastic hoop, buckle
Supporting lid, produce the original seed made a Bag Material.In 0.11MPa atmospheric pressure, 121 DEG C of HTHP moist heat sterilization 30min,
Sterile working accesses parent species after taking out cooling.Ensure during inoculation in parent species material block embedment original seed material.Vaccinated original seed is placed in
Constant temperature light culture in 25 DEG C of incubators, (general 15 days or so) can then use access cultivating bag as original seed after mycelia eats full material
In.
3rd, production kind is prepared：
The cotton seed hulls of ratio, wet overnight through bubble needed for weighing, and is mixed into wood chip, wheat bran, calcium carbonate in proportion, loads 13cm
In the resistant to elevated temperatures transparent polypropylene strain bags of × 25cm, convert into per packed siccative 250-300g.Small wood is used in bag after installing material
Burrowed in material, hole is deep to a bag bottom, and then the plastic hoop on sack set, buckles supporting lid, produce the production made a kind
Bag Material.
In 0.147MPa atmospheric pressure, 128 DEG C of HTHP moist heat sterilization 90min, sterile working access is former after taking out cooling
Kind.Ensure during inoculation in original seed material block embedment production kind material.Vaccinated production kind is placed in constant temperature in 25 DEG C of incubators secretly to train
Support, (general 30d or so) then can be used as production kind to use in access cultivating bag after mycelia eats full material.
4th, production kind is inoculated into planting material：
For the manufacturing process of cultivation material bag with kind, but formula difference used is produced, sack used is 17cm × 35cm high temperature resistants
Transparent polypropylene strain bag.Equivalent every packed siccative 450-500g.Production kind is inoculated into planting material.
5th, cultivation management：
(1) in 20-25 DEG C of lucifuge culture 30-35 days, now mycelia covers with planting material substantially；
(2) after mycelia maturation, it is placed at 20-25 DEG C of shading, then through 10-15 days, mycelia completed latter stage of ripening；
(3) temperature is controlled between 18-22 DEG C, relative air humidity 85-95%, intensity of illumination about 200-300lux, often
Illumination 10-12 hours day, carbon dioxide content is in below 1200ppm, until cap is open and flat, harvesting.
3rd, it is as follows to cultivate result：
1st, fruiting phase：Fructification occurs to cap is open and flat to take about 20 days from former base.
2nd, yield：1st damp fructification about 47.9g/ bags (fresh weight).
3rd, obtained fructification character is cultivated：Fructification is small-sized gradually open and flat to median size, cap convex lens shape, later stage；Bacterium
Cap surface sticks, and lilac red, edge gradually becomes shallower as, and powder yellow is presented.Bacterial context is thin, and initial stage is lavender, and the later stage becomes yellow, taste
Hardship, smell unobvious.Lamella growing straight is to slightly prolonging life, yellowish-brown, and pleat breadth is close.It is raw in stem, have cilium shape squama below collarium
Piece, middle reality.Collarium is upper, cellulosic, easy to fall off.As shown in Figure 1.
Compared with wild state, the strain is changed into the state of growing thickly, Neng Goupei after domestication from the raw state of list of collection
Raise more fructification, and fructification can be grown on Bag Material, yield increase, the stem of fructification increases, embody compared with
For typical fructification feature.
The strain idenfication of embodiment 2
The wild fructification of strain of the invention that Mount Sejila lookout terrace collects in Linzhi Area of Tibet, such as Fig. 2 institutes
Show, its pure culture is obtained by tissue isolation, it is separately sampled to domestication fructification, obtain its lamella and stem tissue low-temperature
(40 DEG C) drying, using liquid nitrogen grinding, using Ezup pillar fungal genomic DNA extraction agent boxes, carry out carrying for DNA genomes
Take, obtained DNA solution.
Pass through fungi ribosomes intergenic region universal primer ITS1/ITS4 (primer 1ITS1:
TCCGTAGGTGAACCTGCGG, primer 2 ITS4:TCCTCCGCTTATTGATATGC, carry out ITS-PCR experiments, PCR reaction solutions
Forming (totally 50 μ l) is：
Reaction condition is：
94 DEG C of reaction 5min；94 DEG C of reaction 1min, 55 DEG C of reaction 1min, 72 DEG C of reaction 1min, 30 circulate；72 DEG C of reactions
10min。
Obtained PCR extension products are sequenced, obtained ITS sequence carries out sequence B last with GenBank, finds and Huang
The naked umbrella Gymnopilus luteofolius similitudes of pleat are up to 99%.
Further, combining form is identified, the strain gross feature and microscopic features and the naked umbrella Gymnopilus of yellow pleat
Luteofolius descriptions are consistent, and qualification result is the naked umbrella Gymnopilus luteofolius of yellow pleat.
The anti-tumor function of embodiment 3 determines
1st, the preparation of ethyl acetate extract
The bacterial strain of the present invention is subjected to liquid fermentation, collects 60 degree of drying, the mycelium second of drying after mycelium filters
Acetoacetic ester soak time can ultrasonic extraction after 10 hours, ultrasonic 100min, extraction is secondary, merges Secondary Organic phase, will collect
After good organic phase is filtered, the liquid that suction filtration obtains is rotated to dripless state, low-temperature preservation (4 with Rotary Evaporators
DEG C) standby.
2nd, tumor cell culture
Enter pedestrian's malignant breast cancer cells with the DMEM nutrient solutions containing penicillin, streptomysin and 10% hyclone (FBS)
(MT-1) cultivate.By the DMEM cell suspension inoculations containing 10%FBS in 24 hole tissue culturing plates, cell concentration is 0.5 ×
105/ hole.Culture is placed in 37 DEG C, 5%CO2Incubator for tissue culture in cultivate 4 hours it is stand-by.
3rd, tumour cell experiment is suppressed
Mycelial ethyl acetate extract is dissolved in DMSO, adds culture medium, is made into 50mg/mL mother liquor,
Mother liquor is diluted to 50 respectively with the DMEM nutrient solutions containing 10% hyclone (FBS) after filtering, 100,200,400,800,
1600 μ g/ml sample solution, carefully sucks the cell culture fluid in 96 well culture plates, be separately added into final concentration be respectively 50,
100th, 200,400,800,1600 μ g/ml sample solution, 37 DEG C, 5%CO are subsequently placed in2Culture 48 is small in incubator for tissue culture
When.Control group is containing only PBS and culture medium.
After culture terminates, cell is collected, with platform phenol indigo plant 1：1 mixing carries out refusing the dyeing of dye method.Dead cell is dyed to blueness, living
Cell is uncolored because there is complete cell membrane.Viable count is determined with cell counter.Each experiment is in triplicate.
4th, result
Counted using the special statistical softwares of SPSS.16, calculate the naked umbrella CCTCC M 2017212 of yellow pleat of various concentrations
Ethyl acetate extract is to Vitro Tumor breast cancer cell MT-1 inhibiting rate, using paired t-test, P<0.05 has conspicuousness
Difference.It is 50 μ g/mL that IC50, which is calculated, by experimental data, and concrete outcome is as shown in table 1.
Table 1：The yellow naked ethyl acetate extracts of umbrella CCTCC M 2017212 of pleat are for Vitro Tumor breast cancer cell MT-1's
Inhibitory action experimental result
It was found from the data of table 1, suppression breast cancer cell MT-1 effect is very strong outside the yellow naked umbrella body of pleat, due to the second of ganoderma lucidum
The IC50 values of acetoacetic ester extract are between 200-300 μ g/ml, and the IC50 of its ethyl acetate is in 50 μ g/ml.Experiment card
Bright, the effect that the yellow naked alcohol extracts of umbrella CCTCC M 2017212 of pleat suppress breast cancer cell MT-1 in vitro is to have to open clearly
One wild varieties of hair prospect.
It is described above, it is only the preferable specific embodiment of the present invention, but protection scope of the present invention is not limited thereto,
Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its
Design is subject to equivalent substitution or change, should all cover within the scope of the present invention.
Claims (10)
1. a kind of naked umbrella novel bacterial of yellow pleat, the yellow naked umbrella novel bacterial of pleat is the naked umbrella Gymnopilus luteofolius of yellow pleat
HMGIM-X120086, deposit number are CCTCC No：M2017212.
A kind of 2. above-mentioned yellow naked umbrella novel bacterial CCTCC No of pleat：M2017212 cultural method, it is characterised in that methods described bag
Include after separation parent species, prepare original seed, prepare production kind, after production kind is inoculated into planting material, carry out cultivation management, the cultivation
The key step of training management includes：After 20-25 DEG C of lucifuge culture 30-35 days, mycelia maturation, it is placed at 20-25 DEG C of shading, then
Through 10-15 days, mycelia was completed latter stage of ripening, and control temperature is between 18-22 DEG C, relative air humidity 85-95%, and intensity of illumination is about
200-300lux, daily illumination 10-12 hours, carbon dioxide content is in below 1200ppm, until cap is open and flat, harvesting, its
In, the planting material composition includes：Wood chip 55-58%, cotton seed hulls 31-33%, wheat bran 8-10%, CaCO31-2%, the cultivation
Train the water content 60-65% of material.
3. cultural method according to claim 2, it is characterised in that the key step of the cultivation management includes：
(1) in 20-25 DEG C of lucifuge culture 30-35 days, now mycelia covers with planting material substantially；
(2) after mycelia maturation, it is placed at 20-25 DEG C of shading, then through 10-15 days, mycelia completed latter stage of ripening；
(3) temperature is controlled between 18-22 DEG C, relative air humidity 85-95%, intensity of illumination about 200-300lux, per daylight
According to 10-12 hours, carbon dioxide content is in below 1200ppm, until cap is open and flat, harvesting.
4. cultural method according to claim 2, it is characterised in that the separation parent species step includes：Sterile working connects
Enter the strain of tissue separation, be placed in constant temperature light culture in 25 DEG C of incubators, treat that mycelia covers with mother culture media such as Mother culture
It can then be transferred after behind base inclined-plane, wherein, the separation mother culture media that the separation parent species process uses includes following weight
The component of percentage：Potato 20%, glucose 2%, agar 2%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.15%, vitamin
B1 is micro, and remaining is water.
5. cultural method according to claim 2, it is characterised in that described the step of preparing original seed includes：To original seed material
Middle sterile working accesses parent species, ensures in parent species material block embedment original seed material during inoculation, vaccinated original seed is placed in 25 DEG C of cultures
Constant temperature light culture in case, can then original seed be used as to transfer after mycelia eats full material；The original seed material for preparing original seed includes following heavy
Measure the component of percentage：Sorghum 75-79%, millet 16-20%, CaCO31-2%, the water content 55-60% of the original seed material.
6. cultural method according to claim 2, it is characterised in that described the step of preparing production kind includes：To production
Sterile working access original seed in kind of material, ensures during inoculation in original seed material block embedment production kind material, vaccinated production kind is placed in
Constant temperature light culture in 25 DEG C of incubators, production kind switching can be then used as after mycelia eats full material；The production for preparing production kind
Kind material includes following components in percentage by weight：Cotton seed hulls 87-89%, wheat bran 10-12%, CaCO31-2%；Or cotton seed hulls
48-52%, wood chip 25-30%, wheat bran 18-20%, calcium carbonate 1-2%, calcium sulfate 1-2%, the water content of the production kind material
For 55-60%.
7. cultural method according to claim 2, it is characterised in that described the step of production kind is inoculated into planting material wraps
Include：Production kind sterile working is transferred in planting material, follow-up cultivation management.
8. cultural method according to claim 2, it is characterised in that including：
1st, parent species are separated：The strain of sterile working access tissue separation, is placed in constant temperature light culture in 25 DEG C of incubators, treats that mycelia grows
It can then be transferred after behind full mother culture media such as mother culture media inclined-plane；
2nd, original seed is prepared：Into original seed material, sterile working accesses parent species, ensures during inoculation in parent species material block embedment original seed material,
The original seed of inoculation is placed in constant temperature light culture in 25 DEG C of incubators, can then be used as original seed to transfer after mycelia eats full material；
3rd, production kind is prepared：The sterile working access original seed into production kind material, original seed material block embedment production kind material is ensured during inoculation
In, vaccinated production kind is placed in constant temperature light culture in 25 DEG C of incubators, can then be used as production kind to turn after mycelia eats full material
Connect；
4th, production kind is inoculated into planting material：Production kind sterile working is transferred in planting material, cultivation management；
5th, cultivation management, including：
(1) in 20-25 DEG C of lucifuge culture 30-35 days, now mycelia covers with planting material substantially；
(2) after mycelia maturation, it is placed at 20-25 DEG C of shading, then through 10-15 days, mycelia completed latter stage of ripening；
(3) temperature is controlled between 18-22 DEG C, relative air humidity 85-95%, intensity of illumination about 200-300lux, per daylight
According to 10-12 hours, carbon dioxide content is in below 1200ppm, until cap is open and flat, harvesting.
9. the naked umbrella novel bacterial CCTCC No of yellow pleat described in claim 1：The naked umbrella novel bacterial CCTCC No of M2017212 or yellow pleats：
Purposes of the M2017212 extracts in prevention and/or tumor is prepared, the tumour preferably include breast cancer.
10. purposes according to claim 9, it is characterised in that the extract is ethyl acetate extract.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108770592A (en) * | 2018-05-10 | 2018-11-09 | 河北师范大学 | A kind of cultural method of lemon squama umbrella cultivation culture medium and lemon squama umbrella |
| CN109618809A (en) * | 2018-12-13 | 2019-04-16 | 广东省微生物研究所（广东省微生物分析检测中心） | Naematoloma fasciculare novel bacterial and its artificial cultivation method and application |
| CN109906877A (en) * | 2018-12-24 | 2019-06-21 | 广东省微生物研究所（广东省微生物分析检测中心） | One kind sticking up squama mushroom novel bacterial and its domesticating cultivation method and application |
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| CN102630481A (en) * | 2012-04-11 | 2012-08-15 | 广东省微生物研究所 | Cultivation method for oospore oudemansiella mucida |
| CN103749151A (en) * | 2013-12-26 | 2014-04-30 | 广东省微生物研究所 | Artificial cultivation method of Amauroderma rude |
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| CN102630481A (en) * | 2012-04-11 | 2012-08-15 | 广东省微生物研究所 | Cultivation method for oospore oudemansiella mucida |
| CN103749151A (en) * | 2013-12-26 | 2014-04-30 | 广东省微生物研究所 | Artificial cultivation method of Amauroderma rude |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108770592A (en) * | 2018-05-10 | 2018-11-09 | 河北师范大学 | A kind of cultural method of lemon squama umbrella cultivation culture medium and lemon squama umbrella |
| CN109618809A (en) * | 2018-12-13 | 2019-04-16 | 广东省微生物研究所（广东省微生物分析检测中心） | Naematoloma fasciculare novel bacterial and its artificial cultivation method and application |
| CN109906877A (en) * | 2018-12-24 | 2019-06-21 | 广东省微生物研究所（广东省微生物分析检测中心） | One kind sticking up squama mushroom novel bacterial and its domesticating cultivation method and application |
| CN109906877B (en) * | 2018-12-24 | 2020-12-01 | 广东省微生物研究所（广东省微生物分析检测中心） | Lentinula edodes new strain and domestication cultivation method and application thereof |
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