CN104126415B - Radix Astragali stalk and stilbene head solid compound criteria material and produce the method for Xingbao mushroom with it - Google Patents
Radix Astragali stalk and stilbene head solid compound criteria material and produce the method for Xingbao mushroom with it Download PDFInfo
- Publication number
- CN104126415B CN104126415B CN201410405340.2A CN201410405340A CN104126415B CN 104126415 B CN104126415 B CN 104126415B CN 201410405340 A CN201410405340 A CN 201410405340A CN 104126415 B CN104126415 B CN 104126415B
- Authority
- CN
- China
- Prior art keywords
- mushroom
- temperature
- bag
- radix astragali
- parts
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Mushroom Cultivation (AREA)
Abstract
The invention discloses a kind of Radix Astragali stalk and stilbene head solid compound criteria material and produce the method for Xingbao mushroom with it, produce Xingbao mushroom there is lack of raw materials to solve and existing method produces the problem that Xingbao mushroom yields poorly.The method comprises pleurotus eryngii quel strains purifying and prepared by rejuvenation, pleurotus eryngii liquid strain, complex solid composts or fertilisers of cultivating is prepared and planting almond abalone mushroom step.The present invention is combined into the solid compound criteria material of suitable Growth of Pleurotus eryngii Radix Astragali stalk and agricultural crop straw, is that cultivated species produces Xingbao mushroom with pleurotus eryngii liquid strain.For people provide the becteriums product of high-quality while protection of the environment, good society, economy, ecological benefits can be produced.Be conducive to the industry restructuring of Edible Fungi enterprise, increase new product, make mushroom industry sustainable development.
Description
Technical field
The invention belongs to Edible Fungi field, be specifically related to a kind of Radix Astragali stalk and stilbene head solid compound criteria material and produce the method for Xingbao mushroom with it.
Background technology
Xingbao mushroom is the high-quality edible mushroom of double nutritive value, medical value and economic worth and one, and there is lack of raw materials, culture technique falls behind the development limiting Xingbao mushroom industry to produce Xingbao mushroom.
Prior art discloses the method utilizing Chinese medicine slag to produce edible mushroom, but Chinese medicine slag composition is complicated, pharmaceutical ingredient content is low, and cultivation effect is undesirable.
Summary of the invention
The object of this invention is to provide a kind of Radix Astragali stalk and stilbene head solid compound criteria material, produce the Xingbao mushroom problem that there is lack of raw materials to solve.
Another object of the present invention is to provide a kind of method of Radix Astragali stalk and stilbene head solid compound criteria material production Xingbao mushroom, to solve the problem that existing method production Xingbao mushroom yields poorly.
Technical solution of the present invention is as follows: a kind of Radix Astragali stalk and stilbene head solid compound criteria material, it with Radix Astragali stalk and stilbene head for major ingredient, with maize straw, corncob is auxiliary material, add water by following raw material and make, Radix Astragali stalk and 25.0 parts ~ 35.0 parts, Radix Astragali stilbene head, maize straw and corncob 25.0 parts ~ 35.0 parts, cotton seed hulls 15.0 parts ~ 25.0 parts, wood chip 25.0 parts ~ 35.0 parts, 10.0 parts ~ 15.0 parts, wheat bran, corn flour 2.0 parts ~ 3.0 parts, analysis for soybean powder 1.0 parts ~ 3.0 parts, 1 part, gypsum, 1 part, lime, dipotassium hydrogen phosphate 0.05 part-0.15 part, potassium dihydrogen phosphate 0.05 part-0.15 part, 0.05 part-0.15 part, magnesium sulfate, material-water ratio is 1:1.25-1.50, the weight ratio of Radix Astragali stalk and Radix Astragali stilbene head is 1:1, the weight ratio of maize straw and corncob is 1:1.
Produce the method for Xingbao mushroom with above-mentioned Radix Astragali stalk and stilbene head solid compound criteria material, comprise the following steps: A, pleurotus eryngii quel strains purifying and rejuvenation, prepared by B, pleurotus eryngii liquid strain, C, planting almond abalone mushroom; Wherein steps A pleurotus eryngii quel strains purifying and rejuvenation process as follows:
A. the protoplast formation of Xingbao mushroom and purifying: by Xingbao mushroom (
pleurotuseryngii) dicaryon mycelium of (buying in Chinese Academy of Medical Sciences's biological study institute's biotechnology room) be inoculated in be covered with glassine paper ring add on rich PDA medium, cultivate 4-5 days for 20-28 DEG C; When mycelia covers with glassine paper ring, glassine paper ring proceeds to together with mycelium in the mixed enzyme solution of 4-6ml cellulase and glusulase by sterile working, enzymolysis 6-10h in the shaking table of 32-36 DEG C of temperature, 350-450rpm rotating speed; Enzymolysis liquid concentration is the MgSO of 0.5-0.7 mole
47H
2o solution dilution, MgSO
47H
2the consumption of O solution is 2 times of enzymolysis liquid volume, filters; Filtrate is centrifugal with 2400-2600rpm rotating speed; Get middle level liquid and add MgSO
4solution makes purification protoplast suspension; Get protoplast suspension and add PDA culture fluid, in Tissue Culture Flask at 22-25 DEG C of temperature quiescent culture; The protoplast liquid medium that PDA culture fluid of learning from else's experience cultivates 46-50h is coated with the flat board having comprehensive PDA culture medium, cultivates, cultivate about 8-12 days at 22-25 DEG C of temperature, and when regenerating bacterium colony and occurring, tube is cultivated immediately, and mycelia carries out microscopy after covering with test tube; Mycelium is monocaryon X without clamp connection, and what have clamp connection expands cultivation for dicaryon Y, dicaryon Y, preserves;
B. monocaryon mating is cultivated: the monocaryon X obtained by the step a of two pseudo copulations is inoculated on the flat board with comprehensive PDA culture medium at a distance of about 0.5cm, is inverted and cultivates 5-7d under 25 DEG C of dark conditions; 5-7d after two bacterium colonies intersect, gets the mycelium of two bacterium colony intersections and outer rim thereof, forwards in the flat board with comprehensive PDA culture medium and cultivate, microexamination after mycelium germination 3d, chooses the nucleated mycelium with clamp connection and expands cultivation, preserve;
C. fruiting experiment: make fruiting experiment with the dicaryon new strains that the nucleated mycelium mating that step a expands dicaryon Y and the step b expansion cultivation of cultivating obtains; Evaluating selection, be separated desirable fruit body individuality tissue, double-core is stablized, and plants, save backup, claim Xingbao mushroom one-level kind using the bacterial classification of biological character and stable yield as production is female.
Preferably, the mass percent concentration of the mixed enzyme solution of described step a cellulase and glusulase is 1.0%.
Preferably, the process that in described step a, preparation purification protoplast suspension gets middle level liquid is, gets the MgSO that concentration that middle level liquid 2.0ml adds 3-5ml is 0.5-0.7 mole
4solution shakes up, centrifugal, washing, repeated washing like this 2 times; Finally get the MgSO that concentration that middle level liquid 2.0ml adds 8.0ml is 0.5-0.7 mole
4solution makes purification protoplast suspension.
Preferably, getting protoplast suspension quiescent culture process in described step a is, gets protoplast suspension 2.0ml and adds 7-9ml and cultivate containing the PDA culture fluid of 0.4 mole of sucrose; Then get protoplast liquid medium 0.2ml and be coated with the flat board having comprehensive PDA culture medium or the flat board with comprehensive PDA culture medium be coated with containing 0.4 mole of sucrose.
Preferably, the detailed process of described step B is as follows:
(1) secondary solid fermentation pedigree seed culture medium is made: graininess wood chip and wheat bran are sieved, select the particle between 0.425-0.850mm to be raw material; By the formula mixed material of secondary solid fermentation pedigree seed culture medium, stir, make water content between 60-65%; Loaded by secondary solid fermentation pedigree seed culture medium in the seed bottle of 750ml, compress, flatten, every bottled wet feed is about 400-450g or siccative 180-240g, covers tampon, under 0.13mpa-0.15mpa pressure, and sterilizing 80-100min;
The weight proportion of above-mentioned secondary solid fermentation pedigree seed culture medium is: wood chip 35.0 parts ~ 45.0 parts, 40.0 parts ~ 55.0 parts, wheat bran, corn flour 2.0 parts ~ 6.0 parts, analysis for soybean powder 1.0 parts ~ 5.0 parts, dipotassium hydrogen phosphate 0.05 part-0.15 part, potassium dihydrogen phosphate 0.05 part-0.15 part, 0.05 part-0.15 part, magnesium sulfate; Material-water ratio is 1:1.25-1.50;
(2) cultivation of Xingbao mushroom secondary solid fermentation original seed: above-mentioned Xingbao mushroom one-level kind is inoculated in the Xingbao mushroom secondary solid fermentation pedigree seed culture medium of step (1), cultivates at 23-25 DEG C of temperature, mycelia cover with after;
(3) liquid spawn makes: be dispensed into by Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii in triangular flask and (fill Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii 300ml in 500ml triangular flask, the bottled middle dress Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii 600ml of 1000ml triangle); Cover tampon, at 0.13mpa-0.14mpa sterilized under pressure 30min; When medium temperature is down to below 30 DEG C, the Xingbao mushroom secondary solid fermentation original seed (access 2 inoculation spoon Xingbao mushroom secondary solid fermentation original seeds in 500ml triangular flask, access 4 inoculation spoon Xingbao mushroom secondary solid fermentation original seeds in 1000ml triangular flask) that access step (2) obtains; Finished triangular flask is 23-28 DEG C of temperature, and under 120-180rpm rotating speed, oscillator vibrates cultivates 3-8 days, becomes pleurotus eryngii liquid strain;
The weight proportion of above-mentioned Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii is: wheat bran 2.0% ~ 5.0%, corn flour 1.0% ~ 3.0%, analysis for soybean powder 1.0% ~ 2.0%, peptone 0.10% ~ 0.2%, dipotassium hydrogen phosphate 0.05%-0.15%, potassium dihydrogen phosphate 0.05%-0.15%, magnesium sulfate 0.05%-0.10%, and surplus is water; Manufacturing process is by wheat bran, corn flour, analysis for soybean powder liquor, and filter, the aqueous solution of filtrate and all the other components merges, and supplying moisture content becomes Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii;
When the pleurotus eryngii liquid strain amount of production is if desired larger, can liquid bacterial culture device (fermentation tank) be directly used to produce;
In fermentation tank, inject Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii by operational procedure, sterilizing, inoculate when cooling to below 30 DEG C;
(4) inoculate: Xingbao mushroom secondary solid fermentation original seed is aseptically smashed to pieces, be incorporated to and fill in the 1000ml sterilizing triangular flask of 800ml sterile water, shake up, under the protection of Alcohol Flame, to entering in fermentation tank, inoculum concentration is that 100L medium meets secondary solid fermentation original seed 2-5L;
(5) cultivate: under the condition of 24-26 DEG C of temperature, tank pressure 0.02-0.04mpa, incubation time 3-8d, through after the assay was approved, is pleurotus eryngii liquid strain.
Preferably, the detailed process of described step D is as follows:
(1) bacterium bag cooling: by the Radix Astragali stalk of claim 1 and the sterilizing of stilbene head solid compound criteria material, is placed in the cooling of clean place by pocket; In the overall process of pack sterilizing cooling, pocket is preferably contained in Turnround basket, and minimizing is moved, and reduces pollution rate;
(2) liquid-spawn inoculation: when pocket temperature is cooled to below 28 DEG C, can be placed in inoculating hood or inoculation indoor inoculation; Conventional method is sterilized, strict with sterile working inoculation, upright fruiting one inoculation, and wall-fruiting two is inoculated, and thin excellent long bag accumbency fruiting plays three inoculations in bag body homonymy; Each vaccination inoculating gun moves into pleurotus eryngii liquid strain 5.0ml, sealing;
(3) hair tube reason: after inoculation, moves in time in prior culturing room of cleaning sterilization and sends out bacterium; Groundwork between bacteria developing period is temperature control, wet down, ventilation, shading, turning and removing miscellaneous bacteria; Culturing room's temperature should control between 20-26 DEG C, and initial stage temperature is advisable with 25 DEG C; After material temperature in bacterium bag rises, room temperature should be turned down to 20-25 DEG C; In whole bacterium stage, material temperature does not all exceed 28 DEG C; Material temperature is too high, gently then produces heat evil and hinders a bacterium, affect later stage output; Heavy then burn bacterium, cause cultivating unsuccessfully;
Sending out the bacterium stage, shading is answered by culturing room, and remain dark, relative air humidity controls within 70%, frequent ventilation, keeps air fresh; Every 10d turning once, make bacterium bag send out bacterium even, pick contaminated bacteria bag simultaneously; For accelerating mycelial growth, the bacterium bag of jag sealing should be dazzling ventilative at mycelia front cover and after material feeding 2-3cm; When temperature is higher, insect protected should be noted; Under adapt circumstance condition, mycelia generally can cover with bacterium bag through 40d;
(4) urge flower bud: after mycelia covers with bag, continue to cultivate 10d, accumulate more nutrient; When mercury dropped to 10-18 DEG C, just can carry out urging flower bud to manage; In order to make its fruiting neat and consistent, first mycelium stimulation process can be carried out; Specific practice is: scrape off the old bacterial classification in sack top layer with little spoon, and sack charge level is flattened; The mycelium stimulation time generally can carry out after mycelia purseful, and because also needing the mycelia recovery time of 10d after mycelium stimulation, bacterium bag during this period can be ripe further;
If have the formation of former base in the middle part of sack or bacterium bag, then no longer carry out mycelium stimulation; Directly can enter management of producing mushroom; Will carry out moisturizing management after mycelium stimulation, with plastic film, sack be covered, or cover collar extension seals by paper using; About cross 10d, when charge level grows new white aerial hyphae again, work of water sprinkling for better material moisture can urge flower bud; During urging flower bud, temperature controls between 10-18 DEG C, and lower than 8 DEG C or be all difficult to form former base higher than 20 DEG C, the best urges Lei Wendu to be 10-15 DEG C;
During urging flower bud, air humidity should maintain 90%-95%, and increasing scattered light stimulates, and adds forced ventilation, keeps air fresh; Through 3-6d, can occur white former base, former base is differentiated to form mushroom flower bud further;
(5) management of producing mushroom: when former base is differentiated to form 1-2cm small mushroom bud, should open the management that bag carries out the fruiting phase in time; Open bag too early, former base be difficult to be formed or fruiting irregular; Open bag excessively slow, fruit body is own grows up in bag, can form misshapen mushroom, and time serious, fruit body can be rotted in atrophy; Folding under outside for sack plastic film turnup, to higher than charge level 2-3cm, is exposed small mushroom bud;
If young mushroom is overstocked, suitably can dredge flower bud, eliminate bad deposit excellent; Short every bag, bag stays 3-4, and 1-3 piece is stayed in the every cave of long bag, and to keep mushroom good shape, greatly individual, commodity rate is high; In the fruiting stage, must be noted that the regulation and control of temperature, humidity, illumination and ventilation, could high yield be obtained.
Gansu is the important traditional Chinese medicine original producton location of China and main product ground, and herb resource overlay area is wide, resource category is many, cultivated area is large, and output is high, and special advantage is fairly obvious, and genuine species the advantage is outstanding.In recent years, continue to increase the guiding to Chinese Medicine Industry and support, the existing 2540 kinds of traditional Chinese medicinal materials assortment resources of the whole province, long-term tame kind has more than 350 to plant, large authentic medicinal herbs has kind more than 30, and especially the major product output such as Radix Angelicae Sinensis, Radix Codonopsis, Huang (red) stilbene, rheum officinale, Radix Glycyrrhizae of Gansu real estate accounts for 70% to 95% of the whole nation.Through sustainable development in recent years, begin to take shape the Chinese Medicine Industry system of certain scale, become the important component part of the whole province's medication economics, there is stronger advantages for development and broad mass market prospect.Cultivated area is large, and output is high.
The development of Chinese herbal medicine industry have accumulated a large amount of stalks, the first-class biomass material of stilbene, arbitrarily stacks, rots, burns, cause the problems such as environmental pollution, the wasting of resources.
Radix Astragali stalk and stilbene head contain the nutrient components such as a certain amount of polysaccharide, saponin(e, flavonoids isoreactivity composition and a large amount of raw fiber, crude fat, starch, Thick many candies.Very abundant at astragalus cultivation area resource, can Substitute For Partial culturing raw material Xinbao mushroom culturing.The present invention is combined into the solid compound criteria material of suitable Growth of Pleurotus eryngii Radix Astragali stalk and agricultural crop straw, is that cultivated species produces Xingbao mushroom with pleurotus eryngii liquid strain.For people provide the becteriums product of high-quality while protection of the environment, good society, economy, ecological benefits can be produced.Be conducive to the industry restructuring of Edible Fungi enterprise, increase new product, make mushroom industry sustainable development.
Show that effect of the present invention is as follows through experiment in cultivation: 1. reduce costs: every bag reduces costs 0.35 yuan.Concrete accounting is as follows: every packed siccative 1.0 jin, water content 64%, wet feed 2.7 jin, cost 1.75 yuan.Replace 20% with Radix Astragali acrial part, 0.35 Yuan ∕ bag can be saved.2. shorten Mycelium culture time 8-10 days: utilizing pleurotus eryngii liquid strain to cultivate the mycelia purseful time is 30-40 days, and the solid spawn inoculation mycelium purseful time is 40-48 days.3. improve biological efficiency 10%-20%: utilize the inventive method to produce Xingbao mushroom every bag and produce Xingbao mushroom fruit body 7-8 two, and traditional cultivation Xingbao mushroom every bag produces Xingbao mushroom fruit body 5-6 two.4. Xingbao mushroom unique flavor; The Xingbao mushroom produced with Radix Astragali stalk, stilbene head solid compound criteria material is with the peculiar taste of the Radix Astragali, and mouthfeel is better.
Embodiment
The following examples can further illustrate the present invention, but do not limit the present invention in any way.
Embodiment 1. prepares Radix Astragali stalk and stilbene head solid compound criteria material, according to following weight weighing:
Radix Astragali stalk and Radix Astragali stilbene head 35.0kg, maize straw and corncob 25.0kg, cotton seed hulls 15.0kg, wood chip 25.0kg, wheat bran 15.0kg, corn flour 3.0kg, analysis for soybean powder 3.0kg, gypsum 1kg, lime 1kg, dipotassium hydrogen phosphate 0.15kg, potassium dihydrogen phosphate 0.15kg, magnesium sulfate 0.15kg, material-water ratio is 1:1.50, the weight ratio of Radix Astragali stalk and Radix Astragali stilbene head is 1:1, and the weight ratio of maize straw and corncob is 1:1.
Manufacturing process is: pulverized by various raw material, and Radix Astragali stalk, Radix Astragali stilbene head, maize straw, maize cob meal are broken into 3-5mm size; Weigh respectively, the first dry mixing of water-fast raw material is even, the more fully mixing that adds water, (as comparatively large in the material such as weed tree sawdust, corncob particle, heap of first prewetting is vexed, mixes after fully suctioning moisture with other material again); Water-soluble raw material potassium dihydrogen phosphate, magnesium sulfate are admixed in material after being dissolved in water, pack sterilizing; Sterilization time requires the sterilized under pressure 2h at 0.15MPa, or lower 100 DEG C of normal pressure maintains 12-14h.
Embodiment 2. prepares Radix Astragali stalk and stilbene head solid compound criteria material, according to following weight weighing:
Radix Astragali stalk and Radix Astragali stilbene head 25.0kg, maize straw and corncob 35.0kg, cotton seed hulls 25.0kg, wood chip 35.0kg, wheat bran 10.0kg, corn flour 2.0kg, analysis for soybean powder 1.0kg, gypsum 1kg, lime 1kg, dipotassium hydrogen phosphate 0.05kg, potassium dihydrogen phosphate 0.05kg, magnesium sulfate 0.05kg, material-water ratio is 1:1.25, the weight ratio of Radix Astragali stalk and Radix Astragali stilbene head is 1:1, and the weight ratio of maize straw and corncob is 1:1.Manufacturing process is with embodiment 1.
Embodiment 3. prepares Radix Astragali stalk and stilbene head solid compound criteria material, according to following weight weighing:
Radix Astragali stalk and Radix Astragali stilbene head 30.0kg, maize straw and corncob 30.0kg, cotton seed hulls 20.0kg, wood chip 30.0kg, wheat bran 12.0kg, corn flour 2.5kg, analysis for soybean powder 2.0kg, gypsum 1kg, lime 1kg, dipotassium hydrogen phosphate 0.10kg, potassium dihydrogen phosphate 0.10kg, magnesium sulfate 0.10kg, material-water ratio is 1:1.35, the weight ratio of Radix Astragali stalk and Radix Astragali stilbene head is 1:1, and the weight ratio of maize straw and corncob is 1:1.Manufacturing process is with embodiment 1.
The preparation process of embodiment 4. Xingbao mushroom one-level kind is as follows:
A. the protoplast formation of Xingbao mushroom and purifying: by Xingbao mushroom (
pleurotuseryngii) dicaryon mycelium be inoculated in be covered with glassine paper ring add on rich PDA medium, cultivate 4 days for 20 DEG C; When mycelia covers with glassine paper ring, sterile working by glassine paper ring together with mycelium proceed to 4ml mass percent concentration be 1.0% cellulase and glusulase mixed enzyme solution in, at 32 DEG C of temperature, enzymolysis 6h in the shaking table of 350rpm rotating speed; Enzymolysis liquid concentration is the MgSO of 0.5 mole
47H
2o solution dilution, MgSO
47H
2the consumption of O solution is 2 times of enzymolysis liquid volume, then uses aperture G
3number core frit; Filtrate is with the centrifugal 15min of 2400rpm rotating speed; Get middle level liquid 2.0ml, the concentration adding 3ml is the MgSO of 0.5 mole
4solution shakes up, centrifugal, washing, repeated washing like this 2 times; Finally get the MgSO that concentration that middle level liquid 2.0ml adds 8.0ml is 0.5 mole
4solution makes purification protoplast suspension; Get protoplast suspension 2.0ml, add 7ml containing the PDA culture fluid of 0.4 mole of sucrose, in Tissue Culture Flask at 22 DEG C of temperature quiescent culture; The protoplast liquid medium 0.2ml that PDA culture fluid of learning from else's experience cultivates 46h is coated with the flat board having comprehensive PDA culture medium or the flat board with comprehensive PDA culture medium be coated with containing 0.4 mole of sucrose, cultivate at 22 DEG C of temperature, cultivate about 8 days, when regenerating bacterium colony and occurring, tube is cultivated immediately, after mycelia covers with test tube, carries out microscopy, microscopy process is cut into the glassine paper sheet of about 8 × 5mm for getting, sterilizing in empty ware.By tweezers gripping a slice glassine paper sheet under sterile working, have in the test tube of mycelia in length and touch mycelia, then put on slide glass and add water and cover plate microscopy.Mycelium is monocaryon X without clamp connection, and what have clamp connection expands cultivation for dicaryon Y, dicaryon Y, preserves;
B. monocaryon mating is cultivated: the monocaryon X obtained by the step a of two pseudo copulations is inoculated on the flat board with comprehensive PDA culture medium at a distance of about 0.5cm, is inverted and cultivates 5d under 25 DEG C of dark conditions; 5d after two bacterium colonies intersect, gets the mycelium of two bacterium colony intersections and outer rim thereof, forwards in the flat board with comprehensive PDA culture medium and cultivate, microexamination after mycelium germination 3d, chooses the nucleated mycelium with clamp connection and expands cultivation, preserve;
C. fruiting experiment: make fruiting experiment with the dicaryon new strains that the nucleated mycelium mating that step a expands dicaryon Y and the step b expansion cultivation of cultivating obtains; Evaluating selection, be separated desirable fruit body individuality tissue, double-core is stablized, and plants, save backup, claim Xingbao mushroom one-level kind using the bacterial classification of biological character and stable yield as production is female.
The preparation process of embodiment 5. Xingbao mushroom one-level kind is as follows:
A. the protoplast formation of Xingbao mushroom and purifying: by Xingbao mushroom (
pleurotuseryngii) dicaryon mycelium be inoculated in be covered with glassine paper ring add on rich PDA medium, cultivate 5 days for 28 DEG C; When mycelia covers with glassine paper ring, sterile working by glassine paper ring together with mycelium proceed to 6ml mass percent concentration be 1.0% cellulase and glusulase mixed enzyme solution in, at 36 DEG C of temperature, enzymolysis 10h in the shaking table of 450rpm rotating speed; Enzymolysis liquid concentration is the MgSO of 0.7 mole
47H
2o solution dilution, MgSO
47H
2the consumption of O solution is 2 times of enzymolysis liquid volume, then uses aperture G
3number core frit; Filtrate is with the centrifugal 15min of 2600rpm rotating speed; Get middle level liquid 2.0ml, the concentration adding 5ml is the MgSO of 0.7 mole
4solution shakes up, centrifugal, washing, repeated washing like this 2 times; Finally get the MgSO that concentration that middle level liquid 2.0ml adds 8.0ml is 0.7 mole
4solution makes purification protoplast suspension; Get protoplast suspension 2.0ml, add 9ml containing the PDA culture fluid of 0.4 mole of sucrose, in Tissue Culture Flask at 25 DEG C of temperature quiescent culture; The protoplast liquid medium 0.2ml that PDA culture fluid of learning from else's experience cultivates 50h is coated with the flat board having comprehensive PDA culture medium or the flat board with comprehensive PDA culture medium be coated with containing 0.4 mole of sucrose, cultivate at 25 DEG C of temperature, cultivate about 12 days, when regenerating bacterium colony and occurring, tube is cultivated immediately, after mycelia covers with test tube, carries out microscopy, microscopy process is cut into the glassine paper sheet of about 8 × 5mm for getting, sterilizing in empty ware.By tweezers gripping a slice glassine paper sheet under sterile working, have in the test tube of mycelia in length and touch mycelia, then put on slide glass and add water and cover plate microscopy.Mycelium is monocaryon X without clamp connection, and what have clamp connection expands cultivation for dicaryon Y, dicaryon Y, preserves;
B. monocaryon mating is cultivated: the monocaryon X obtained by the step a of two pseudo copulations is inoculated on the flat board with comprehensive PDA culture medium at a distance of about 0.5cm, is inverted and cultivates 7d under 25 DEG C of dark conditions; 7d after two bacterium colonies intersect, gets the mycelium of two bacterium colony intersections and outer rim thereof, forwards in the flat board with comprehensive PDA culture medium and cultivate, microexamination after mycelium germination 3d, chooses the nucleated mycelium with clamp connection and expands cultivation, preserve;
C. fruiting experiment: make fruiting experiment with the dicaryon new strains that the nucleated mycelium mating that step a expands dicaryon Y and the step b expansion cultivation of cultivating obtains; Evaluating selection, be separated desirable fruit body individuality tissue, double-core is stablized, and plants, save backup, claim Xingbao mushroom one-level kind using the bacterial classification of biological character and stable yield as production is female.
The preparation process of embodiment 6. pleurotus eryngii liquid strain is as follows:
(1) secondary solid fermentation pedigree seed culture medium is made: graininess wood chip and wheat bran are sieved, select the particle between 0.425mm to be raw material; By the formula mixed material of secondary solid fermentation pedigree seed culture medium, stir, make water content between 60%; Loaded by secondary solid fermentation pedigree seed culture medium in the seed bottle of 750ml, compress, flatten, every bottled wet feed is about 400g or siccative 180g, covers tampon, under 0.13mpa pressure, and sterilizing 80min;
The weight proportion of above-mentioned secondary solid fermentation pedigree seed culture medium is: wood chip 35.0kg, wheat bran 40.0kg, corn flour 2.0kg, analysis for soybean powder 1.0kg, dipotassium hydrogen phosphate 0.05kg, potassium dihydrogen phosphate 0.05kg, magnesium sulfate 0.05kg; Material-water ratio is 1:1.25;
(2) cultivation of Xingbao mushroom secondary solid fermentation original seed: the Xingbao mushroom one-level kind that embodiment 4 is obtained is inoculated in the Xingbao mushroom secondary solid fermentation pedigree seed culture medium of step (1), cultivates at 23 DEG C of temperature, mycelia cover with after;
(3) liquid spawn makes: be dispensed into by Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii in triangular flask and (fill Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii 300ml in 500ml triangular flask); Cover tampon, at 0.13mpa sterilized under pressure 30min; When medium temperature is down to below 30 DEG C, the Xingbao mushroom secondary solid fermentation original seed (accessing 2 inoculation spoon Xingbao mushroom secondary solid fermentation original seeds in 500ml triangular flask) that access step (2) obtains; Finished triangular flask is 23 DEG C of temperature, and under 120rpm rotating speed, oscillator vibrates cultivates 3 days, becomes pleurotus eryngii liquid strain;
The weight proportion of above-mentioned Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii is: wheat bran 2.0%, corn flour 1.0%, analysis for soybean powder 1.0%, peptone 0.10%, dipotassium hydrogen phosphate 0.05%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.05%, and surplus is water; Manufacturing process is by wheat bran, corn flour, analysis for soybean powder liquor, and filter, the aqueous solution of filtrate and all the other components merges, and supplying moisture content becomes Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii;
When the pleurotus eryngii liquid strain amount of production is if desired larger, can liquid bacterial culture device (fermentation tank) be directly used to produce; In fermentation tank, inject Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii by operational procedure, sterilizing, inoculate when cooling to below 30 DEG C;
(4) inoculate: Xingbao mushroom secondary solid fermentation original seed is aseptically smashed to pieces, be incorporated to and fill in the 1000ml sterilizing triangular flask of 800ml sterile water, shake up, under the protection of Alcohol Flame, to entering in fermentation tank, inoculum concentration is that 100L medium meets secondary solid fermentation original seed 2L;
(5) cultivate: under the condition of 24 DEG C of temperature, tank pressure 0.02mpa, incubation time 3d, through after the assay was approved, is pleurotus eryngii liquid strain.
The preparation process of embodiment 7. pleurotus eryngii liquid strain is as follows:
(1) secondary solid fermentation pedigree seed culture medium is made: graininess wood chip and wheat bran are sieved, select the particle between 0.850mm to be raw material; By the formula mixed material of secondary solid fermentation pedigree seed culture medium, stir, make water content between 65%; Loaded by secondary solid fermentation pedigree seed culture medium in the seed bottle of 750ml, compress, flatten, every bottled wet feed is about 450g or siccative 240g, covers tampon, under 0.15mpa pressure, and sterilizing 100min;
The weight proportion of above-mentioned secondary solid fermentation pedigree seed culture medium is: wood chip 45.0kg, wheat bran 55.0kg, corn flour 6.0kg, analysis for soybean powder 5.0kg, dipotassium hydrogen phosphate 0.15kg, potassium dihydrogen phosphate 0.15kg, magnesium sulfate 0.15kg; Material-water ratio is 1:1.25-1.50;
(2) cultivation of Xingbao mushroom secondary solid fermentation original seed: the Xingbao mushroom one-level kind that embodiment 5 is obtained is inoculated in the Xingbao mushroom secondary solid fermentation pedigree seed culture medium of step (1), cultivates at 25 DEG C of temperature, mycelia cover with after;
(3) liquid spawn makes: be dispensed into by Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii (the bottled middle dress Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii 600ml of 1000ml triangle) in triangular flask; Cover tampon, at 0.14mpa sterilized under pressure 30min; When medium temperature is down to below 30 DEG C, the Xingbao mushroom secondary solid fermentation original seed (accessing 4 inoculation spoon Xingbao mushroom secondary solid fermentation original seeds in 1000ml triangular flask) that access step (2) obtains; Finished triangular flask is 28 DEG C of temperature, and under 180rpm rotating speed, oscillator vibrates cultivates 8 days, becomes pleurotus eryngii liquid strain;
The weight proportion of above-mentioned Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii is: wheat bran 5.0%, corn flour 3.0%, analysis for soybean powder 2.0%, peptone 0.2%, dipotassium hydrogen phosphate 0.15%, potassium dihydrogen phosphate 0.15%, magnesium sulfate 0.10%, and surplus is water; Manufacturing process is by wheat bran, corn flour, analysis for soybean powder liquor, and filter, the aqueous solution of filtrate and all the other components merges, and supplying moisture content becomes Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii;
(4) inoculate: Xingbao mushroom secondary solid fermentation original seed is aseptically smashed to pieces, be incorporated to and fill in the 1000ml sterilizing triangular flask of 800ml sterile water, shake up, under the protection of Alcohol Flame, to entering in fermentation tank, inoculum concentration is that 100L medium meets secondary solid fermentation original seed 5L;
(5) cultivate: under the condition of 26 DEG C of temperature, tank pressure 0.04mpa, incubation time 8d, through after the assay was approved, is pleurotus eryngii liquid strain.
The method of Xingbao mushroom produced by embodiment 8. Radix Astragali stalk, stilbene head solid compound criteria material, and detailed process is as follows:
(1) bacterium bag cooling: by Radix Astragali stalk obtained for embodiment 3 and the sterilizing of stilbene head solid compound criteria material, is placed in the cooling of clean place by pocket; In the overall process of pack sterilizing cooling, pocket is preferably contained in Turnround basket, and minimizing is moved, and reduces pollution rate;
(2) liquid-spawn inoculation: when pocket temperature is cooled to below 28 DEG C, can be placed in inoculating hood or inoculation indoor inoculation; Conventional method is sterilized, strict with sterile working inoculation, upright fruiting one inoculation, and wall-fruiting two is inoculated, and thin excellent long bag accumbency fruiting plays three inoculations in bag body homonymy; Each vaccination inoculating gun moves into pleurotus eryngii liquid strain 5.0ml, sealing;
(3) hair tube reason: after inoculation, moves in time in prior culturing room of cleaning sterilization and sends out bacterium; Groundwork between bacteria developing period is temperature control, wet down, ventilation, shading, turning and removing miscellaneous bacteria; Culturing room's temperature should control at 25 DEG C;
Sending out the bacterium stage, shading is answered by culturing room, and remain dark, relative air humidity controls within 70%, frequent ventilation, keeps air fresh; Every 10d turning once, make bacterium bag send out bacterium even, pick contaminated bacteria bag simultaneously; For accelerating mycelial growth, the bacterium bag of jag sealing should be dazzling ventilative at mycelia front cover and after material feeding 3cm; When temperature is higher, insect protected should be noted; Under adapt circumstance condition, mycelia generally can cover with bacterium bag through 40d;
(4) urge flower bud: after mycelia covers with bag, continue to cultivate 10d, accumulate more nutrient; When mercury dropped to 18 DEG C, just can carry out urging flower bud to manage; In order to make its fruiting neat and consistent, first mycelium stimulation process can be carried out; Specific practice is: scrape off the old bacterial classification in sack top layer with little spoon, and sack charge level is flattened; The mycelium stimulation time generally can carry out after mycelia purseful, and because also needing the mycelia recovery time of 10d after mycelium stimulation, bacterium bag during this period can be ripe further; If have the formation of former base in the middle part of sack or bacterium bag, then no longer carry out mycelium stimulation; Directly can enter management of producing mushroom; Will carry out moisturizing management after mycelium stimulation, with plastic film, sack be covered, or cover collar extension seals by paper using; About cross 10d, when charge level grows new white aerial hyphae again, work of water sprinkling for better material moisture can urge flower bud; During urging flower bud, temperature controls at 18 DEG C; During urging flower bud, air humidity should maintain 95%, and increasing scattered light stimulates, and adds forced ventilation, keeps air fresh; Through 6d, can occur white former base, former base is differentiated to form mushroom flower bud further;
(5) management of producing mushroom: when former base is differentiated to form 1-2cm small mushroom bud, should open the management that bag carries out the fruiting phase in time; Open bag too early, former base be difficult to be formed or fruiting irregular; Open bag excessively slow, fruit body is own grows up in bag, can form misshapen mushroom, and time serious, fruit body can be rotted in atrophy; Folding under outside for sack plastic film turnup, to higher than charge level 2-3cm, is exposed small mushroom bud;
If young mushroom is overstocked, suitably can dredge flower bud, eliminate bad deposit excellent; Short every bag, bag stays 3-4, and 1-3 piece is stayed in the every cave of long bag, and to keep mushroom good shape, greatly individual, commodity rate is high; In the fruiting stage, must be noted that the regulation and control of temperature, humidity, illumination and ventilation, could high yield be obtained;
5.1 temperature adjustings: temperature should remain on 18 DEG C, under this temperature condition, sporophore growth is grown normal healthy and strong; The Xingbao mushroom kind of different ecological type, also different to the adaptability of temperature, should specifically kind specifically treat during actual cultivation; Under normal circumstances, when temperature is higher, sporophore growth is fast, and mushroom body is little, easy parachute-opening, not pure white, poor quality; Therefore, in fruit body development process, if temperature is higher, should in conjunction with water spray, night ventilation carry out cooling process earthward; As temperature is on the low side, should suitably close the doors and windows, outdoor planting thickeies covering, or adopts way of manually heating, and improves cultivating chamber temperature;
5.2 humidity regulation: moisture and moisture management extremely important for the Differentiation and development of fruit body; Initial stage relative air humidity will remain on 90%; After bacteria cover diameter grows to 2-3cm, suitably can turn down, control 88%, be conducive to preventing damage by disease and insect from occurring; When temperature is high, air humidity lower than 80% time, should suitable spray cooling humidification, but the main way with light spray, diligent spray is toward ground and space water spray; Note trying not water to be sprayed onto on mushroom body, in order to avoid cause fruit body yellow atrophy, bacterial infection time serious and perish, affect the seed output and quality of fruit body; 2-3d before gathering, in order to extend the freshness date after adopting, relative air humidity controls to be advisable 85%; After a damp mushroom is terminated, the dehydration of bacterium bag is more, and available water flood and bowssening are to the moisturizing of bacterium bag;
5.3 Illumination adjustings: the fruit body initiation and development stage all needs scattered light, intensity of illumination is advisable like 1000lx; Illumination is excessively weak, easily forms mushroom without a head; Illumination is excessively strong, the easy desiccation of fruit body, and color is not white, and commodity declines;
5.4 air conditionings: fruit body development demands stronger ventilation amount, remain that in mushroom room, air is fresh; When room temperature higher (more than 18 DEG C), when humidity is bigger than normal, more to note adding forced ventilation, avoid the hot and humid fruit body that causes to rot and damage by disease and insect harm; Ventilation should with temperature control damping unified management, accomplishing can well-ventilated, can keep again good temperature, humidity environment;
(6) gather and to manage with change of tide: when cap is open and flat, spore not yet launches, bacteria cover diameter is consistent with stem or when being slightly smaller than stem, will gather in time; First damp mushroom should clear up charge level after gathering in time, and cut off the water bacteria 5d, then regulates the condition such as epidemic disaster and ventilation in mushroom room; To be separated by 14d, the second damp mushroom of also can gathering; The output of Xingbao mushroom mainly concentrates on the first damp mushroom, accounts for more than 70% of gross yield, and a second damp mushroom shape is little, and stem is short, yields poorly; A damp mushroom therefore factory culture is only gathered; If manage proper, total biologicak efficiency that bag is planted can reach 60%; As by the bacterium bag of a damp mushroom of gathering de-bag soil covering culture again, the output of two damp mushrooms can be significantly improved.
The method of Xingbao mushroom produced by embodiment 9. Radix Astragali stalk, stilbene head solid compound criteria material, and detailed process is as follows:
(1) bacterium bag cooling: by Radix Astragali stalk obtained for embodiment 1 and the sterilizing of stilbene head solid compound criteria material, is placed in the cooling of clean place by pocket; In the overall process of pack sterilizing cooling, pocket is preferably contained in Turnround basket, and minimizing is moved, and reduces pollution rate;
(2) liquid-spawn inoculation: when pocket temperature is cooled to below 28 DEG C, can be placed in inoculating hood or inoculation indoor inoculation; Conventional method is sterilized, strict with sterile working inoculation, upright fruiting one inoculation, and wall-fruiting two is inoculated, and thin excellent long bag accumbency fruiting plays three inoculations in bag body homonymy; Each vaccination inoculating gun moves into pleurotus eryngii liquid strain 5.0ml, sealing;
(3) hair tube reason: after inoculation, moves in time in prior culturing room of cleaning sterilization and sends out bacterium; Groundwork between bacteria developing period is temperature control, wet down, ventilation, shading, turning and removing miscellaneous bacteria; Culturing room's temperature should control between 20 DEG C, and initial stage temperature is advisable with 25 DEG C; After material temperature in bacterium bag rises, room temperature should be turned down to 20 DEG C; In whole bacterium stage, material temperature does not all exceed 28 DEG C; Material temperature is too high, gently then produces heat evil and hinders a bacterium, affect later stage output; Heavy then burn bacterium, cause cultivating unsuccessfully;
Sending out the bacterium stage, shading is answered by culturing room, and remain dark, relative air humidity controls within 70%, frequent ventilation, keeps air fresh; Every 10d turning once, make bacterium bag send out bacterium even, pick contaminated bacteria bag simultaneously; For accelerating mycelial growth, the bacterium bag of jag sealing should be dazzling ventilative at mycelia front cover and after material feeding 2cm; When temperature is higher, insect protected should be noted; Under adapt circumstance condition, mycelia generally can cover with bacterium bag through 40d;
(4) urge flower bud: after mycelia covers with bag, continue to cultivate 10d, accumulate more nutrient; When mercury dropped to 10 DEG C, just can carry out urging flower bud to manage; In order to make its fruiting neat and consistent, first mycelium stimulation process can be carried out; Specific practice is: scrape off the old bacterial classification in sack top layer with little spoon, and sack charge level is flattened; The mycelium stimulation time generally can carry out after mycelia purseful, and because also needing the mycelia recovery time of 10d after mycelium stimulation, bacterium bag during this period can be ripe further;
If have the formation of former base in the middle part of sack or bacterium bag, then no longer carry out mycelium stimulation; Directly can enter management of producing mushroom; Will carry out moisturizing management after mycelium stimulation, with plastic film, sack be covered, or cover collar extension seals by paper using; About cross 10d, when charge level grows new white aerial hyphae again, work of water sprinkling for better material moisture can urge flower bud; During urging flower bud, temperature controls at 10 DEG C;
During urging flower bud, air humidity should maintain 90%, and increasing scattered light stimulates, and adds forced ventilation, keeps air fresh; Through 3d, can occur white former base, former base is differentiated to form mushroom flower bud further;
(5) management of producing mushroom: when former base is differentiated to form 1-2cm small mushroom bud, should open the management that bag carries out the fruiting phase in time; Open bag too early, former base be difficult to be formed or fruiting irregular; Open bag excessively slow, fruit body is own grows up in bag, can form misshapen mushroom, and time serious, fruit body can be rotted in atrophy; Folding under outside for sack plastic film turnup, to higher than charge level 2-3cm, is exposed small mushroom bud; If young mushroom is overstocked, suitably can dredge flower bud, eliminate bad deposit excellent; Short every bag, bag stays 3-4, and 1-3 piece is stayed in the every cave of long bag, and to keep mushroom good shape, greatly individual, commodity rate is high; In the fruiting stage, must be noted that the regulation and control of temperature, humidity, illumination and ventilation, could high yield be obtained;
5.1 temperature adjustings: temperature should remain on 15 DEG C, under this temperature condition, sporophore growth is grown normal healthy and strong; The Xingbao mushroom kind of different ecological type, also different to the adaptability of temperature, should specifically kind specifically treat during actual cultivation; Under normal circumstances, when temperature is higher, sporophore growth is fast, and mushroom body is little, easy parachute-opening, not pure white, poor quality; Therefore, in fruit body development process, if temperature is higher, should in conjunction with water spray, night ventilation carry out cooling process earthward; As temperature is on the low side, should suitably close the doors and windows, outdoor planting thickeies covering, or adopts way of manually heating, and improves cultivating chamber temperature;
5.2 humidity regulation: moisture and moisture management extremely important for the Differentiation and development of fruit body; Initial stage relative air humidity will remain on 90%; After bacteria cover diameter grows to 2-3cm, suitably can turn down, control 85%, be conducive to preventing damage by disease and insect from occurring; When temperature is high, air humidity lower than 80% time, should suitable spray cooling humidification, but the main way with light spray, diligent spray is toward ground and space water spray; Note trying not water to be sprayed onto on mushroom body, in order to avoid cause fruit body yellow atrophy, bacterial infection time serious and perish, affect the seed output and quality of fruit body; 2d before gathering, in order to extend the freshness date after adopting, relative air humidity controls to be advisable 85%; After a damp mushroom is terminated, the dehydration of bacterium bag is more, and available water flood and bowssening are to the moisturizing of bacterium bag;
5.3 Illumination adjustings: the fruit body initiation and development stage all needs scattered light, intensity of illumination is advisable like 500lx; Illumination is excessively weak, easily forms mushroom without a head; Illumination is excessively strong, the easy desiccation of fruit body, and color is not white, and commodity declines;
5.4 air conditionings: fruit body development demands stronger ventilation amount, remain that in mushroom room, air is fresh; When room temperature higher (more than 18 DEG C), when humidity is bigger than normal, more to note adding forced ventilation, avoid the hot and humid fruit body that causes to rot and damage by disease and insect harm; Ventilation should with temperature control damping unified management, accomplishing can well-ventilated, can keep again good temperature, humidity environment;
(6) gather and to manage with change of tide: when cap is open and flat, spore not yet launches, bacteria cover diameter is consistent with stem or when being slightly smaller than stem, will gather in time; First damp mushroom should clear up charge level after gathering in time, and cut off the water bacteria 4d, then regulates the condition such as epidemic disaster and ventilation in mushroom room; To be separated by 14d, the second damp mushroom of also can gathering; The output of Xingbao mushroom mainly concentrates on the first damp mushroom, accounts for more than 70% of gross yield, and a second damp mushroom shape is little, and stem is short, yields poorly; A damp mushroom therefore factory culture is only gathered; If manage proper, total biologicak efficiency that bag is planted can reach 50%-60%; As by the bacterium bag of a damp mushroom of gathering de-bag soil covering culture again, the output of two damp mushrooms can be significantly improved.
Embodiment 10. difference from Example 8 is to adopt the obtained Radix Astragali stalk of embodiment 2 and stilbene head solid compound criteria material to produce Xingbao mushroom.
Claims (8)
1. Radix Astragali stalk and a stilbene head solid compound criteria material, is characterized in that: it for major ingredient with Radix Astragali stalk and stilbene head, with maize straw, corncob for auxiliary material, adds water by following raw material and make,
Radix Astragali stalk and 25.0 parts ~ 35.0 parts, Radix Astragali stilbene head, maize straw and corncob 25.0 parts ~ 35.0 parts, cotton seed hulls 15.0 parts ~ 25.0 parts, wood chip 25.0 parts ~ 35.0 parts, 10.0 parts ~ 15.0 parts, wheat bran, corn flour 2.0 parts ~ 3.0 parts, analysis for soybean powder 1.0 parts ~ 3.0 parts, 1 part, gypsum, 1 part, lime, dipotassium hydrogen phosphate 0.05 part-0.15 part, potassium dihydrogen phosphate 0.05 part-0.15 part, 0.05 part-0.15 part, magnesium sulfate, material-water ratio is 1:1.25-1.50, the weight ratio of Radix Astragali stalk and Radix Astragali stilbene head is 1:1, and the weight ratio of maize straw and corncob is 1:1.
2. produce the method for Xingbao mushroom with Radix Astragali stalk described in claim 1 and stilbene head solid compound criteria material, it is characterized in that it comprises the following steps:
A, pleurotus eryngii quel strains purifying and rejuvenation,
Prepared by B, pleurotus eryngii liquid strain,
C, planting almond abalone mushroom;
Wherein steps A pleurotus eryngii quel strains purifying and rejuvenation process as follows:
A. the protoplast formation of Xingbao mushroom and purifying: by Xingbao mushroom (
pleurotuseryngii) dicaryon mycelium be inoculated in be covered with glassine paper ring add on rich PDA medium, cultivate 4-5 days for 20-28 DEG C; When mycelia covers with glassine paper ring, glassine paper ring proceeds to together with mycelium in the mixed enzyme solution of 4-6ml cellulase and glusulase by sterile working, enzymolysis 6-10h in the shaking table of 32-36 DEG C of temperature, 350-450rpm rotating speed; Enzymolysis liquid concentration is the MgSO of 0.5-0.7 mole
47H
2o solution dilution, MgSO
47H
2the consumption of O solution is 2 times of enzymolysis liquid volume, filters; Filtrate is centrifugal with 2400-2600rpm rotating speed; Get middle level liquid and add MgSO
4solution makes purification protoplast suspension; Get protoplast suspension and add PDA culture fluid, in Tissue Culture Flask at 22-25 DEG C of temperature quiescent culture; The protoplast liquid medium that PDA culture fluid of learning from else's experience cultivates 46-50h is coated with the flat board having comprehensive PDA culture medium, cultivates at 22-25 DEG C of temperature, cultivates 8-12 days, and when regenerating bacterium colony and occurring, tube is cultivated immediately, and mycelia carries out microscopy after covering with test tube; Mycelium is monocaryon X without clamp connection, and what have clamp connection expands cultivation for dicaryon Y, dicaryon Y, preserves;
B. monocaryon mating is cultivated: the monocaryon X obtained by the step a of two pseudo copulations is inoculated on the flat board with comprehensive PDA culture medium at a distance of about 0.5cm, is inverted and cultivates 5-7d under 25 DEG C of dark conditions; 5-7d after two bacterium colonies intersect, gets the mycelium of two bacterium colony intersections and outer rim thereof, forwards in the flat board with comprehensive PDA culture medium and cultivate, microexamination after mycelium germination 3d, chooses the nucleated mycelium with clamp connection and expands cultivation, preserve;
C. fruiting experiment: make fruiting experiment with the dicaryon new strains that the nucleated mycelium mating that step a expands dicaryon Y and the step b expansion cultivation of cultivating obtains; Evaluating selection, be separated desirable fruit body individuality tissue, double-core is stablized, and plants, save backup, claim Xingbao mushroom one-level kind using the bacterial classification of biological character and stable yield as production is female.
3. the method for Xingbao mushroom produced by Radix Astragali stalk according to claim 2 and stilbene head solid compound criteria material, it is characterized in that: the mass percent concentration of the mixed enzyme solution of described step a cellulase and glusulase is 1.0%.
4. the method for Xingbao mushroom produced by Radix Astragali stalk according to claim 2 and stilbene head solid compound criteria material, it is characterized in that: the process that in described step a, preparation purification protoplast suspension gets middle level liquid is, get the MgSO that concentration that middle level liquid 2.0ml adds 3-5ml is 0.5-0.7 mole
4solution shakes up, centrifugal, washing, repeated washing like this 2 times; Finally get the MgSO that concentration that middle level liquid 2.0ml adds 8.0ml is 0.5-0.7 mole
4solution makes purification protoplast suspension.
5. the method for Xingbao mushroom produced by Radix Astragali stalk according to claim 2 and stilbene head solid compound criteria material, it is characterized in that: getting protoplast suspension quiescent culture process in described step a is, get protoplast suspension 2.0ml and add 7-9ml and cultivate containing the PDA culture fluid of 0.4 mole of sucrose; Then get protoplast liquid medium 0.2ml and be coated with the flat board having comprehensive PDA culture medium or the flat board with comprehensive PDA culture medium be coated with containing 0.4 mole of sucrose.
6. the method for Xingbao mushroom produced by Radix Astragali stalk according to claim 2 and stilbene head solid compound criteria material, it is characterized in that the detailed process of described step B is as follows:
(1) secondary solid fermentation pedigree seed culture medium is made: graininess wood chip and wheat bran are sieved, select the particle between 0.425-0.850mm to be raw material; By the formula mixed material of secondary solid fermentation pedigree seed culture medium, stir, make water content between 60-65%; Loaded by secondary solid fermentation pedigree seed culture medium in the seed bottle of 750ml, compress, flatten, every bottled wet feed 400-450g or siccative 180-240g, covers tampon, under 0.13mpa-0.15mpa pressure, and sterilizing 80-100min;
The weight proportion of above-mentioned secondary solid fermentation pedigree seed culture medium is: wood chip 35.0 parts ~ 45.0 parts, 40.0 parts ~ 55.0 parts, wheat bran, corn flour 2.0 parts ~ 6.0 parts, analysis for soybean powder 1.0 parts ~ 5.0 parts, dipotassium hydrogen phosphate 0.05 part-0.15 part, potassium dihydrogen phosphate 0.05 part-0.15 part, 0.05 part-0.15 part, magnesium sulfate; Material-water ratio is 1:1.25-1.50;
(2) cultivation of Xingbao mushroom secondary solid fermentation original seed: the Xingbao mushroom one-level kind in claim 1 is inoculated in the Xingbao mushroom secondary solid fermentation pedigree seed culture medium of step (1), cultivates at 23-25 DEG C of temperature, mycelia cover with after;
(3) liquid spawn makes: be dispensed into by Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii in triangular flask, fill Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii 300ml in 500ml triangular flask, the bottled middle dress Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii 600ml of 1000ml triangle; Cover tampon, at 0.13mpa-0.14mpa sterilized under pressure 30min; When medium temperature is down to below 30 DEG C, the Xingbao mushroom secondary solid fermentation original seed that access step (2) obtains, access 2 inoculation spoon Xingbao mushroom secondary solid fermentation original seeds in 500ml triangular flask, in 1000ml triangular flask, access 4 inoculation spoon Xingbao mushroom secondary solid fermentation original seeds; Finished triangular flask is 23-28 DEG C of temperature, and under 120-180rpm rotating speed, oscillator vibrates cultivates 3-8 days, becomes pleurotus eryngii liquid strain;
The weight proportion of above-mentioned Cultivation Medium of Liquid Fungus Seeds of Pleurotus Eryngii is: wheat bran 2.0% ~ 5.0%, corn flour 1.0% ~ 3.0%, analysis for soybean powder 1.0% ~ 2.0%, peptone 0.10% ~ 0.2%, dipotassium hydrogen phosphate 0.05%-0.15%, potassium dihydrogen phosphate 0.05%-0.15%, magnesium sulfate 0.05%-0.10%, and surplus is water;
(4) inoculate: Xingbao mushroom secondary solid fermentation original seed is aseptically smashed to pieces, be incorporated to and fill in the 1000ml sterilizing triangular flask of 800ml sterile water, shake up, under the protection of Alcohol Flame, to entering in fermentation tank, inoculum concentration is that 100L medium meets secondary solid fermentation original seed 2-5L;
(5) cultivate: under the condition of 24-26 DEG C of temperature, tank pressure 0.02-0.04mpa, incubation time 3-8d, through after the assay was approved, is pleurotus eryngii liquid strain.
7. the method for Xingbao mushroom produced by Radix Astragali stalk according to claim 2 and stilbene head solid compound criteria material, it is characterized in that: the detailed process of described step C is as follows:
(1) bacterium bag cooling: by the Radix Astragali stalk of claim 1 and the sterilizing of stilbene head solid compound criteria material, is placed in the cooling of clean place by pocket; In the overall process of pack sterilizing cooling, pocket is contained in Turnround basket, and minimizing is moved, and reduces pollution rate;
(2) liquid-spawn inoculation: when pocket temperature is cooled to below 28 DEG C, can be placed in inoculating hood or inoculation indoor inoculation; Conventional method is sterilized, strict with sterile working inoculation, upright fruiting one inoculation, and wall-fruiting two is inoculated, and thin excellent long bag accumbency fruiting plays three inoculations in bag body homonymy; Each vaccination inoculating gun moves into pleurotus eryngii liquid strain 5.0ml, sealing;
(3) hair tube reason: after inoculation, moves in time in prior culturing room of cleaning sterilization and sends out bacterium; Groundwork between bacteria developing period is temperature control, wet down, ventilation, shading, turning and removing miscellaneous bacteria; Culturing room's temperature should control between 20-26 DEG C, initial stage temperature 25 DEG C; After material temperature in bacterium bag rises, room temperature should be turned down to 20-25 DEG C; In whole bacterium stage, material temperature does not all exceed 28 DEG C; Material temperature is too high, gently then produces heat evil and hinders a bacterium, affect later stage output; Heavy then burn bacterium, cause cultivating unsuccessfully;
Sending out the bacterium stage, shading is answered by culturing room, and remain dark, relative air humidity controls within 70%, frequent ventilation, keeps air fresh; Every 10d turning once, make bacterium bag send out bacterium even, pick contaminated bacteria bag simultaneously; For accelerating mycelial growth, the bacterium bag of jag sealing should be dazzling ventilative at mycelia front cover and after material feeding 2-3cm; When temperature is higher, insect protected should be noted; Under adapt circumstance condition, mycelia can cover with bacterium bag through 40d;
(4) urge flower bud: after mycelia covers with bag, continue to cultivate 10d, accumulate more nutrient; When mercury dropped to 10-18 DEG C, just can carry out urging flower bud to manage;
In order to make its fruiting neat and consistent, first mycelium stimulation process can be carried out; Specific practice is: scrape off the old bacterial classification in sack top layer with little spoon, and sack charge level is flattened; The mycelium stimulation time can carry out after mycelia purseful, and because also needing the mycelia recovery time of 10d after mycelium stimulation, bacterium bag during this period can be ripe further;
If have the formation of former base in the middle part of sack or bacterium bag, then no longer carry out mycelium stimulation; Directly can enter management of producing mushroom; Will carry out moisturizing management after mycelium stimulation, with plastic film, sack be covered, or cover collar extension seals by paper using; About cross 10d, when charge level grows new white aerial hyphae again, work of water sprinkling for better material moisture can urge flower bud; During urging flower bud, temperature controls between 10-18 DEG C, lower than 8 DEG C or be all difficult to form former base higher than 20 DEG C;
During urging flower bud, air humidity should maintain 90%-95%, and increasing scattered light stimulates, and adds forced ventilation, keeps air fresh; Through 3-6d, can occur white former base, former base is differentiated to form mushroom flower bud further;
(5) management of producing mushroom: when former base is differentiated to form 1-2cm small mushroom bud, should open the management that bag carries out the fruiting phase in time; Open bag too early, former base be difficult to be formed or fruiting irregular; Open bag excessively slow, fruit body is own grows up in bag, can form misshapen mushroom, and time serious, fruit body can be rotted in atrophy; Folding under outside for sack plastic film turnup, to higher than charge level 2-3cm, is exposed small mushroom bud; If young mushroom is overstocked, suitably dredge flower bud, eliminate bad deposit excellent; Short every bag, bag stays 3-4, and 1-3 piece is stayed in the every cave of long bag, and to keep mushroom good shape, greatly individual, commodity rate is high; In the fruiting stage, must be noted that the regulation and control of temperature, humidity, illumination and ventilation.
8. the method for Xingbao mushroom produced by Radix Astragali stalk according to claim 7 and stilbene head solid compound criteria material, it is characterized in that: described step (4) urges the best in flower bud to urge Lei Wendu to be 10-15 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410405340.2A CN104126415B (en) | 2014-08-18 | 2014-08-18 | Radix Astragali stalk and stilbene head solid compound criteria material and produce the method for Xingbao mushroom with it |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410405340.2A CN104126415B (en) | 2014-08-18 | 2014-08-18 | Radix Astragali stalk and stilbene head solid compound criteria material and produce the method for Xingbao mushroom with it |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104126415A CN104126415A (en) | 2014-11-05 |
| CN104126415B true CN104126415B (en) | 2016-04-13 |
Family
ID=51799468
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410405340.2A Expired - Fee Related CN104126415B (en) | 2014-08-18 | 2014-08-18 | Radix Astragali stalk and stilbene head solid compound criteria material and produce the method for Xingbao mushroom with it |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104126415B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104429606A (en) * | 2014-12-05 | 2015-03-25 | 丽水学院 | Culture method of pleurotus eryngii |
| CN104584874A (en) * | 2015-02-15 | 2015-05-06 | 邬金梅 | Pleurotus eryngii cultivating method |
| CN106434357A (en) * | 2015-08-11 | 2017-02-22 | 天津市庆祥农业科技有限公司 | Preparation process of edible fungus solid liquefaction strains |
| CN106962018B (en) * | 2017-03-29 | 2021-01-05 | 徐州康汇百年食品有限公司 | Method for cultivating pleurotus eryngii by burdock solid fermentation |
| CN107083337A (en) * | 2017-06-13 | 2017-08-22 | 上海市农业科学院 | The preparation method and protoplast of a kind of edible mushroom protoplast |
| CN107926483A (en) * | 2017-12-26 | 2018-04-20 | 张掖金泰现代农业科技有限责任公司 | A kind of cultivation compost of radix astragali mushroom and the preparation method of radix astragali mushroom |
| CN108450235A (en) * | 2017-12-28 | 2018-08-28 | 山东常生源菌业有限公司 | A kind of Pleurotus nebrodensis production method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101391920A (en) * | 2008-10-31 | 2009-03-25 | 山西太岳食用菌有限公司 | Culture medium for producing selenium-enriched pleurotus eryngii |
| CN101597192A (en) * | 2008-06-05 | 2009-12-09 | 姚淑先 | Chinese herb culture medium, its preparation method and culturing edible fungi |
| CN102951969A (en) * | 2012-11-28 | 2013-03-06 | 菏泽普恩药业有限公司 | Preparation method of edible fungus culture medium |
| CN103449896A (en) * | 2013-07-11 | 2013-12-18 | 华中农业大学 | Culture material for cultivating pleurotus eryngii, and preparation method thereof |
| CN103771994A (en) * | 2012-10-18 | 2014-05-07 | 王永成 | Mushroom culture medium containing traditional Chinese medicine and preparation method of mushroom culture medium |
| CN103875457A (en) * | 2014-04-16 | 2014-06-25 | 山西奥格姆农业科技有限公司 | Production method for improving fruiting quality of bagged cultivated pleurotus eryngii |
-
2014
- 2014-08-18 CN CN201410405340.2A patent/CN104126415B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597192A (en) * | 2008-06-05 | 2009-12-09 | 姚淑先 | Chinese herb culture medium, its preparation method and culturing edible fungi |
| CN101391920A (en) * | 2008-10-31 | 2009-03-25 | 山西太岳食用菌有限公司 | Culture medium for producing selenium-enriched pleurotus eryngii |
| CN103771994A (en) * | 2012-10-18 | 2014-05-07 | 王永成 | Mushroom culture medium containing traditional Chinese medicine and preparation method of mushroom culture medium |
| CN102951969A (en) * | 2012-11-28 | 2013-03-06 | 菏泽普恩药业有限公司 | Preparation method of edible fungus culture medium |
| CN103449896A (en) * | 2013-07-11 | 2013-12-18 | 华中农业大学 | Culture material for cultivating pleurotus eryngii, and preparation method thereof |
| CN103875457A (en) * | 2014-04-16 | 2014-06-25 | 山西奥格姆农业科技有限公司 | Production method for improving fruiting quality of bagged cultivated pleurotus eryngii |
Non-Patent Citations (3)
| Title |
|---|
| 中药药渣在杏鲍菇生产中的应用;佘红等;《山东农业科学》;20091231(第06期);第50-51页 * |
| 利用黄芪和甘草茎叶栽培鸡腿菇技术;柴月明;《甘肃农业科技》;20111231(第10期);第60-61页 * |
| 玉米秸秆黄芪茎叶栽培姬菇技术;刘爱军;《食用菌》;20121231(第05期);第65页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104126415A (en) | 2014-11-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104126415B (en) | Radix Astragali stalk and stilbene head solid compound criteria material and produce the method for Xingbao mushroom with it | |
| CN102786333B (en) | Phellinus igniarius bag cultivation medium and method for cultivating phellinus igniarius sporophore by same | |
| CN103598010B (en) | Original ecological imitative wild cultivation method for inonotus sanghuang | |
| CN102318504B (en) | Method for collecting spore powder by cultivating ganoderma with layered rack type stereoscopic bag material | |
| CN104041330B (en) | Ganoderma tsugae imitates wild juggle cultivation method | |
| CN102318505B (en) | Ganoderma lucidum potted landscape cultivation method | |
| CN106489542B (en) | A kind of Ganoderma tsugae quick-growing cultivation method | |
| CN102351609B (en) | Culture material for black fungus cultivation | |
| CN105940960B (en) | A kind of soil covering culture method of interplanting ganoderma lucidum in tea gardens | |
| CN101578945A (en) | Cultivating method of tuckahoe | |
| CN101455161B (en) | North semi-clinker open type pasania fungus production method | |
| CN1961652A (en) | Application of mycorrhizal fungi in large-sclae plantation of dendrobium stem | |
| CN101743849A (en) | Pleurotus citrinopileatus composite optimization culturing method | |
| CN103583225A (en) | Method for cultivating good-quality high-yield pleurotus geesteranus by means of cassava stems | |
| CN105794438A (en) | Gastrodia elata cultivation method | |
| CN107371785A (en) | A kind of method of artificial cultivation hickory chick comprehensive utilization | |
| CN101874453B (en) | Culture method of snow white mushroom strains and sporocarps | |
| CN103650919A (en) | Full-sun cultivation method of black fungus | |
| CN105474994A (en) | Original-ecology wild-cultivation-simulated method of ganodorma lucidum | |
| CN106034749A (en) | Crop rotation cultivating method of ganoderma lucidum and pleurotus ostreatus | |
| CN106856984A (en) | A kind of Hydnum tree and its cultural method | |
| CN105237139A (en) | Preparation and culturing method of tree branch bacterial strain of pleurotus eryngii and hypsizygus marmoreus | |
| CN107409743A (en) | A kind of artificial generation material pseudo-wild cultivating method of rainbow conk | |
| CN101514116A (en) | Formula for culture medium for culturing mushrooms with bagasse | |
| CN103229664A (en) | Method for planting sweet lucid ganoderma by usage of uncrushed tea seed shells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160413 Termination date: 20170818 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |