CN103155785A - Establishment method for brown mushroom three-level strain propagation system - Google Patents
Establishment method for brown mushroom three-level strain propagation system Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 16
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- 238000004659 sterilization and disinfection Methods 0.000 claims description 33
- 239000002609 medium Substances 0.000 claims description 28
- 238000002360 preparation method Methods 0.000 claims description 25
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 20
- 241000894007 species Species 0.000 claims description 20
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- 238000011081 inoculation Methods 0.000 claims description 17
- 235000015099 wheat brans Nutrition 0.000 claims description 16
- 238000000855 fermentation Methods 0.000 claims description 15
- 230000004151 fermentation Effects 0.000 claims description 15
- 239000000706 filtrate Substances 0.000 claims description 12
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- 239000001963 growth medium Substances 0.000 claims description 8
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- 238000011218 seed culture Methods 0.000 claims description 8
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- 235000019634 flavors Nutrition 0.000 claims description 7
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- 238000009835 boiling Methods 0.000 claims description 5
- 235000012976 tarts Nutrition 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 abstract 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 abstract 1
- 229910052739 hydrogen Inorganic materials 0.000 abstract 1
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses an establishment method for a brown mushroom three-level strain propagation system. The establishment method for the brown mushroom three-level strain propagation system comprises three-level strain propagation of stock cultures, liquid strains and cultispecies, wherein the formula of culture mediums of the stock cultures comprises, by weight (g/L), 600g of turfy soil, 20g of glucose, 100g of wheat grains, 1.5g of magnesium sulfate, 1g of monopotassium phosphate, 2g of peptone and 1000mL of water, the formula of the liquid strains comprises, by weight (g/L), 200g of potatoes, 20g of glucose, 2g of peptone, 2g of monopotassium phosphate, 1g of magnesium sulfate and 10.01g of VB, the pH (potential of hydrogen) is natural, and the cultispecies comprise, by weight (%), 92% of wheat grains, 5% of gypsum and 3% of turfy soil. Compared with a traditional solid state three-level strain, through using the propagation system, the propagation cycle is shortened by 38 days. The establishment method for the brown mushroom three-level strain propagation system is an optimum scheme, and has a certain guiding significance to the mass industrialized production of brown mushrooms.
Description
Technical field
The invention provides the cultivation method that relates to brown mushroom, say that more specifically a kind of brown mushroom three-class strain expands numerous method.
Background technology
Brown mushroom (
Agaricus brunnescensPeck), be the rare veriety in mushroom, belong to Basidiomycetes (Basidiomycotina), Agaricales (Agaritales), Agaricus edibilis (Agaricaceae), Agaricus (
Garicus).Brown mushroom is commonly called as brown mushroom, is the Bo Ximei subspecies.This bacterial strain output is high, good mushroom quality is good, mouthfeel is fresh and tender, and its mushroom lid is the nature sepia, stem white, the fine and close consolidation of meat.All white mushroom kinds are the white mutation of brown mushroom now, that is to say that brown mushroom is the wild ancestors of white mushroom.Brown mushroom, another name white mushroom, Mushroom belong to Basidiomycetes, Agaricales, Agaricus edibilis, Agaricus.Brown mushroom is nutritious, delicious flavour, and fresh mushroom contains protein 3%-4% according to reports, fat 0.2%-0.3%, and carbohydrate 2.4%-3.8%, but digestibility reaches 70%-90%, enjoys the title of " Vegetable meat ".The amino matter acid of mushroom forms more comprehensive, especially is rich in the lysine of needed by human etc.Brown mushroom also contains the mineral elements such as abundant iron, potassium, calcium.Also contain multivitamin and the enzymes such as thiamine, vitamin b3, nicotinic acid, ascorbic acid.Owing to containing higher unsaturated fatty acid, therefore many edible mushrooms have obvious effect to reducing blood fat, to curing metastatic hepatitis, chronic hepatitis, hepatomegaly, early-phase hepatocirrhosis etc., significant curative effect is arranged all with the canned mushroom processing medicine that liquid makes of precooking.Along with people pursue natural, return the arrival that uncut jade is returned straight trend, brown mushroom is subject to pursuing of consumers in general on market, be a good commercial variety.
At present, brown mushroom is cultivated the main solid culture method that adopts, and fabrication cycle is long, adds its Production Time on the occasion of high temperature season, causes production cost to rise.And liquid culture is to adopt fluid matrix matter to ferment to carry out the production of fermented and cultured bacterial classification, its principle is: in fermentation tank or triangular flask, adopt liquid culture matrix, passing into filtrated air is stirred or is vibrated, increase the dissolved oxygen content in culture matrix, provide the mycelium respiratory metabolism needed oxygen, and control suitable external condition, make thalline grow in liquid depths breeding, obtain a large amount of mycelium or metabolite.
Because brown mushroom has market prospects preferably, but at present less to the research of its cultivation method, especially breed the time than the agaricus bisporus three-class strain longer, the present invention is intended to by to the preparation of brown liquid fungus culture and the making of cultivated species, the best that works out brown mushroom three-class strain expands numerous scheme, will have certain directive significance to the large-scale industrialized production of brown mushroom.
Summary of the invention
Technology contents disclosed by the invention is as follows:
A kind of brown mushroom three-class strain expands the method for building up of traditional font system, it is characterized in that it comprises that female kind, the three-class strain of liquid spawn, cultivated species expand numerous combining, a kind of expansion traditional font system of foundation, and its method is as follows:
(1) female preparation of planting:
1) Mother culture based formulas, i.e. turfy soil medium (g/L): turfy soil 600g, glucose 20g, wheat 100g, magnesium sulfate 1.5g, potassium dihydrogen phosphate 1g, peptone 2g, water 1000mL;
2) preparation medium: get appropriate turfy soil, wheat, add in the clear water of 1000ml and boil 20～30min, filtrate; Add magnesium sulfate, potassium dihydrogen phosphate, peptone, glucose, agar in filtrate, after medicine dissolves, supply moisture, the packing test tube;
3) sterilize: 121 ℃, sterilization 20min after sterilization finishes, takes out and puts the inclined-plane;
4) inoculation, cultivation: brown mushroom mycelium is inoculated in the middle part, inclined-plane in strict accordance with sterile working; 25 ℃ of constant temperature culture are to full packages;
5) choose assorted, preservation: the inclined-plane that microbiological contamination, growth conditions is not good will remain the inclined-plane and be placed in 4 ℃ of Refrigerator stores after removing, and will be standby;
(2) preparation of liquid spawn:
1) liquid spawn formula, i.e. seed culture medium (g/L): potato 200, glucose 20, peptone 2, potassium dihydrogen phosphate 2, magnesium sulfate 1, VB1 0.01, the pH nature; The wheat bran fermentation medium is (g/L): wheat bran 120, glucose 15, peptone 2, pH nature;
2) preparation seed liquor medium: after taking the potato processing, added water boil 30 minutes, filter, then add glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate 1, VB1 fully filters packing, 50mL/250mL triangular flask while hot after dissolving;
3) sterilization, 121 ℃, sterilization 20min;
4) preparation seed liquor: after sterilization finished, taking-up cooled, and in strict accordance with the sterile working inoculation, got 5 0.5 cm
2Fresh slant strains is inoculated in seed culture medium, and 25 ℃, the 150rpm shaking table was cultivated 7 days;
5) preparation fermentation medium: get wheat bran, add clear water to boil 20-30min, filtrate is boiled to wheat bran and is added glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate in liquid, after the medicine dissolving, adds clear water to supply moisture, packing, 100mL/250mL triangular flask;
6) sterilization: 121 ℃, sterilization 20min;
7) inoculation, cultivation: the seed liquor for preparing is moved in the 250mL triangular flask of dress fermentation medium with inoculum concentration 8%, and 25 ℃, 150rpm cultivated 7 days, namely got brown liquid fungus culture;
8) choose assorted: whether observe zymotic fluid by outward appearance every day muddy, and tart flavour is arranged, and explanation is contaminated by bacterial, and removes;
(3) making of cultivated species:
1) wheat soil medium (%): wheat 92, gypsum 5, turfy soil 3;
2) boil wheat: select the wheat of free from insect pests, pour into and boil 20～25 minutes in boiling water, and constantly stir, boil to wheat and during without the white heart, cease fire immediately wheat is pulled out, the anhydrous mark to the surface dries in the air;
3) batching: gypsum, turfy soil are added in proportion, and it is turned evenly, can bottle;
4) bottling sterilization: 121 ℃, sterilized 2～2.5 hours;
5) inoculation: with prepared liquid spawn, the inoculum concentration with 15% evenly sprays in cultivated species, requires operation in strict accordance with sterile working;
6) cultivate: postvaccinal cultivated species is placed in 25 ℃ of constant temperature culture chamber shadings cultivates, to full bottle;
7) choose assorted: cultivated species that microbiological contamination, growth conditions is not good will remain bacterial classification and save backup after removing.
In order to determine best culture matrix, the present invention has carried out a large amount of experimental research work, take the lead at last having created the three-class strain multiplication technique system of female kind of (slant strains) → original seed (liquid spawn) → cultivated species (wheat kind) in brown mushroom expansion is numerous.Adopt this expansion traditional font system than traditional solid-state three-class strain, expanding numerous cycle has shortened 38 days.
It is method for building up that bacterial classification disclosed by the invention expands the traditional font, is a kind of foundation of innovation system.Because tradition is separately carried out mother's kind, original seed, three grades of kinds, the present invention combines the three, and innovative point is changing traditional wheat original seed into the liquid original seed, and the growth cycle of bacterial classification has been shortened in the foundation of this individual system greatly, and strain quality is excellent.
The main excellent characteristics of brown mushroom MC441 bacterial strain that the present invention selects are:
1. this bacterial strain mushroom cap surface is the nature sepia, mushroom handle, bacterial context white; Good mushroom quality, the fruit body rounding, the mushroom lid is thick, solid, not concave crown; Delicious flavour, mouthfeel is fresh and tender, long fresh-keeping period.Mycelial growth rate is fast.Output is high, under the same conditions, does sth. in advance 2-3 days fruitings than white mushroom kind, and mushroom tide is neat, output reach the 9-10 kilogram/square.3. the fruiting temperature range is wide, and especially the low temperature ability is stronger than conventional white mushroom, and under 5 ℃-8 ℃, yield increased 2796 is high by 70%.Culture technique is simple and easy, the culturing raw material source is wide.Its culture material formula is take the paddy and wheat grass as primary raw material, and composts or fertilisers of cultivating and fermentation reactor system method are identical with conventional mushroom.
The good effect that brown mushroom three-class strain expanding propagation method disclosed by the invention compared with prior art has is:
(1) bacterial classification is purer, and liquid spawn produces through screening, purifies, and belongs to pure culture.
(2) cycle shorter, adopt liquid spawn to cover with cultivation bag from the production of hybrid seeds to the mycelia and only need 30 days.
(3) pollute still less, sprouted in 24 hours after the employing liquid-spawn inoculation, bacterial classification sprouting speed surpasses varied bacteria growing speed.
(4) fruiting neatly goes on the market early, adopts the brown mushroom of liquid spawn plantation, and fruiting is neat, can shift to an earlier date listing in 2 months.
Embodiment
Below in conjunction with embodiment, the present invention is described, the scheme of embodiment described here, do not limit the present invention, one of skill in the art can make improvements and change according to spirit of the present invention, described these improvement and variation all should be considered as within the scope of the invention, and scope of the present invention and essence are limited by claim.Wherein brown mushroom MC441 draws from Fujian Province's mushroom externally on sale.
Embodiment 1
Brown mushroom three-class strain expands the foundation of traditional font system
1.1 female preparation of planting:
(1) Mother culture based formulas, i.e. turfy soil medium (g/L): turfy soil 600g, glucose 20g, wheat 100g, magnesium sulfate 1.5g, potassium dihydrogen phosphate 1g, peptone 2g, water 1000mL.
(2) preparation medium.Get appropriate turfy soil, wheat, add in a certain amount of clear water and boil 20～30min, with 4 layers of filtered through gauze, get filtrate.Add magnesium sulfate, potassium dihydrogen phosphate, peptone, glucose, agar in filtrate, after medicine dissolves, supply moisture, the packing test tube.
(3) sterilization.121 ℃, sterilization 20min.After sterilization finishes, take out and put the inclined-plane.
(4) inoculation, cultivation.In strict accordance with sterile working, brown mushroom mycelium is inoculated in the middle part, inclined-plane.25 ℃ of constant temperature culture are to full packages.
(5) choose assorted, preservation.The inclined-plane that microbiological contamination, growth conditions is not good will remain the inclined-plane and be placed in 4 ℃ of Refrigerator stores after removing, and will be standby.
1.2 the preparation of liquid spawn:
(1) liquid spawn formula, i.e. seed culture medium (g/L): potato 200, glucose 20, peptone 2, potassium dihydrogen phosphate 2, magnesium sulfate 1, VB1 0.01, the pH nature.The wheat bran fermentation medium is (g/L): wheat bran 120, glucose 15, peptone 2, pH nature.
(2) preparation seed liquor medium.Take appropriate potato, clean peeling, trachoma, chopping adds suitable quantity of water and boils half an hour, filtered through gauze, then add glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate 1, VB1, abundant filtered through gauze while hot after dissolving, packing, 50mL/250mL triangular flask.
(3) sterilization.121 ℃, sterilization 20min.
(4) preparation seed liquor.After sterilization finished, taking-up cooled.In strict accordance with the sterile working inoculation, get 5 0.5 cm
2Fresh slant strains is inoculated in seed culture medium, and 25 ℃, the 150rpm shaking table was cultivated 7 days.
(5) preparation fermentation medium.Get appropriate wheat bran, add a certain amount of clear water to boil 20-30min, with 4 layers of filtered through gauze, get filtrate.Boil to wheat bran and add glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate in liquid, after the medicine dissolving, add a certain amount of clear water to supply moisture, packing, 100mL/250mL triangular flask.
(6) sterilization.121 ℃, sterilization 20min.
(7) inoculation, cultivation.The seed liquor for preparing is moved in the 250mL triangular flask of dress fermentation medium with inoculum concentration 8%, and 25 ℃, 150rpm cultivated 7 days, namely got brown liquid fungus culture.
(8) choose assorted.Whether observe zymotic fluid by outward appearance every day muddy, and tart flavour is arranged, and explanation is contaminated by bacterial, and removes.After fermentation ends, 1. outward appearance is observed, the zymotic fluid clarification, and a fairly large number of diameter 2-3mm mycelium pellet is arranged at the bottom, or more mycelia is arranged in the zymotic fluid thickness, has illustrated that polysaccharide produces, and has the distinctive fragrant of mushroom, without tart flavour.2. microscopic examination, whether the zymotic fluid that takes a morsel with sarranine or violet staining, has living contaminants at oily Microscopic observation.The normal liquid spawn of fermenting is continued to employ.
1.3 the making of cultivated species:
(1) wheat soil medium (%): wheat 92, gypsum 5, turfy soil 3.
(2) boil wheat.Select the wheat of free from insect pests, pour into and boil 20～25 minutes in boiling water, during boiling drag for the floating foreign material such as wheat is flat, and constantly stir, boil to wheat and during without the white heart, cease fire immediately wheat is pulled out, must guard against the wheat epidermis is boiled brokenly.Anhydrous mark dries in the air in the cement flooring to the surface.
(3) batching.Gypsum, turfy soil are added in proportion, and it is turned evenly, can bottle.
(4) bottling sterilization.121 ℃, sterilized 2～2.5 hours.
(5) inoculation.With prepared liquid spawn, the inoculum concentration with 15% evenly sprays in cultivated species, requires operation in strict accordance with sterile working.
(6) cultivate.Postvaccinal cultivated species is placed in 25 ℃ of constant temperature culture chamber shadings cultivates, to full bottle.
(7) choose assorted.The cultivated species that microbiological contamination, growth conditions is not good will remain bacterial classification and save backup after removing.
Embodiment 2
A kind of brown mushroom three-class strain expands the method for building up of traditional font system
(1) female preparation of planting:
1) Mother culture based formulas, i.e. turfy soil medium (g/L): turfy soil 600g, glucose 20g, wheat 100g, magnesium sulfate 1.5g, potassium dihydrogen phosphate 1g, peptone 2g, water 1000mL;
2) preparation medium: get appropriate turfy soil, wheat, add in the clear water of 1000ml and boil 30min, filtrate; Add magnesium sulfate, potassium dihydrogen phosphate, peptone, glucose, agar in filtrate, after medicine dissolves, supply moisture, the packing test tube;
3) sterilize: 121 ℃, sterilization 20min after sterilization finishes, takes out and puts the inclined-plane;
4) inoculation, cultivation: brown mushroom mycelium is inoculated in the middle part, inclined-plane in strict accordance with sterile working; 25 ℃ of constant temperature culture are to full packages;
5) choose assorted, preservation: the inclined-plane that microbiological contamination, growth conditions is not good will remain the inclined-plane and be placed in 4 ℃ of Refrigerator stores after removing, and will be standby;
(2) preparation of liquid spawn:
1) liquid spawn formula, i.e. seed culture medium (g/L): potato 200, glucose 20, peptone 2, potassium dihydrogen phosphate 2, magnesium sulfate 1, VB1 0.01, the pH nature; The wheat bran fermentation medium is (g/L): wheat bran 120, glucose 15, peptone 2, pH nature;
2) preparation seed liquor medium: after taking the potato processing, added water boil 30 minutes, filter, then add glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate 1, VB1 fully filters packing, 50mL/250mL triangular flask while hot after dissolving;
3) sterilization, 121 ℃, sterilization 20min;
4) preparation seed liquor: after sterilization finished, taking-up cooled, and in strict accordance with the sterile working inoculation, got 5 0.5 cm
2Fresh slant strains is inoculated in seed culture medium, and 25 ℃, the 150rpm shaking table was cultivated 7 days;
5) preparation fermentation medium: get wheat bran, add clear water to boil 20min, filtrate is boiled to wheat bran and is added glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate in liquid, after the medicine dissolving, adds clear water to supply moisture, packing, 100mL/250mL triangular flask;
6) sterilization: 121 ℃, sterilization 20min;
7) inoculation, cultivation: the seed liquor for preparing is moved in the 250mL triangular flask of dress fermentation medium with inoculum concentration 8%, and 25 ℃, 150rpm cultivated 7 days, namely got brown liquid fungus culture;
8) choose assorted: whether observe zymotic fluid by outward appearance every day muddy, and tart flavour is arranged, and explanation is contaminated by bacterial, and removes;
(3) making of cultivated species:
1) wheat soil medium (%): wheat 92, gypsum 5, turfy soil 3;
2) boil wheat: select the wheat of free from insect pests, pour into and boil 20～25 minutes in boiling water, and constantly stir, boil to wheat and during without the white heart, cease fire immediately wheat is pulled out, the anhydrous mark to the surface dries in the air;
3) batching: gypsum, turfy soil are added in proportion, and it is turned evenly, can bottle;
4) bottling sterilization: 121 ℃, sterilized 2.5 hours;
5) inoculation: with prepared liquid spawn, the inoculum concentration with 15% evenly sprays in cultivated species, requires operation in strict accordance with sterile working;
6) cultivate: postvaccinal cultivated species is placed in 25 ℃ of constant temperature culture chamber shadings cultivates, to full bottle;
7) choose assorted: cultivated species that microbiological contamination, growth conditions is not good will remain bacterial classification and save backup after removing.
Claims (1)
1. a brown mushroom three-class strain expands the method for building up of traditional font system, it is characterized in that it comprises that female kind, the three-class strain of liquid spawn, cultivated species expand numerous combining, a kind of expansion traditional font system of foundation, and its method is as follows:
(1) female preparation of planting:
1) Mother culture based formulas, i.e. turfy soil medium (g/L): turfy soil 600g, glucose 20g, wheat 100g, magnesium sulfate 1.5g, potassium dihydrogen phosphate 1g, peptone 2g, water 1000mL;
2) preparation medium: get appropriate turfy soil, wheat, add in the clear water of 1000ml and boil 20～30min, filtrate; Add magnesium sulfate, potassium dihydrogen phosphate, peptone, glucose, agar in filtrate, after medicine dissolves, supply moisture, the packing test tube;
3) sterilize: 121 ℃, sterilization 20min after sterilization finishes, takes out and puts the inclined-plane;
4) inoculation, cultivation: brown mushroom mycelium is inoculated in the middle part, inclined-plane in strict accordance with sterile working; 25 ℃ of constant temperature culture are to full packages;
5) choose assorted, preservation: the inclined-plane that microbiological contamination, growth conditions is not good will remain the inclined-plane and be placed in 4 ℃ of Refrigerator stores after removing, and will be standby;
(2) preparation of liquid spawn:
1) liquid spawn formula, i.e. seed culture medium (g/L): potato 200, glucose 20, peptone 2, potassium dihydrogen phosphate 2, magnesium sulfate 1, VB1 0.01, the pH nature; The wheat bran fermentation medium is (g/L): wheat bran 120, glucose 15, peptone 2, pH nature;
2) preparation seed liquor medium: after taking the potato processing, added water boil 30 minutes, filter, then add glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate 1, VB1 fully filters packing, 50mL/250mL triangular flask while hot after dissolving;
3) sterilization, 121 ℃, sterilization 20min;
4) preparation seed liquor: after sterilization finished, taking-up cooled, and in strict accordance with the sterile working inoculation, got 5 0.5 cm
2Fresh slant strains is inoculated in seed culture medium, and 25 ℃, the 150rpm shaking table was cultivated 7 days;
5) preparation fermentation medium: get wheat bran, add clear water to boil 20-30min, filtrate is boiled to wheat bran and is added glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate in liquid, after the medicine dissolving, adds clear water to supply moisture, packing, 100mL/250mL triangular flask;
6) sterilization: 121 ℃, sterilization 20min;
7) inoculation, cultivation: the seed liquor for preparing is moved in the 250mL triangular flask of dress fermentation medium with inoculum concentration 8%, and 25 ℃, 150rpm cultivated 7 days, namely got brown liquid fungus culture;
8) choose assorted: whether observe zymotic fluid by outward appearance every day muddy, and tart flavour is arranged, and explanation is contaminated by bacterial, and removes;
(3) making of cultivated species:
1) wheat soil medium (%): wheat 92, gypsum 5, turfy soil 3;
2) boil wheat: select the wheat of free from insect pests, pour into and boil 20～25 minutes in boiling water, and constantly stir, boil to wheat and during without the white heart, cease fire immediately wheat is pulled out, the anhydrous mark to the surface dries in the air;
3) batching: gypsum, turfy soil are added in proportion, and it is turned evenly, can bottle;
4) bottling sterilization: 121 ℃, sterilized 2～2.5 hours;
5) inoculation: with prepared liquid spawn, the inoculum concentration with 15% evenly sprays in cultivated species, requires operation in strict accordance with sterile working;
6) cultivate: postvaccinal cultivated species is placed in 25 ℃ of constant temperature culture chamber shadings cultivates, to full bottle;
7) choose assorted: cultivated species that microbiological contamination, growth conditions is not good will remain bacterial classification and save backup after removing.
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