CN106244466A - A kind of preparation method of green muscardine fungus height cryptogam - Google Patents
A kind of preparation method of green muscardine fungus height cryptogam Download PDFInfo
- Publication number
- CN106244466A CN106244466A CN201610719429.5A CN201610719429A CN106244466A CN 106244466 A CN106244466 A CN 106244466A CN 201610719429 A CN201610719429 A CN 201610719429A CN 106244466 A CN106244466 A CN 106244466A
- Authority
- CN
- China
- Prior art keywords
- green muscardine
- parts
- muscardine fungus
- mixed liquor
- cloth
- 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.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Mycology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Mushroom Cultivation (AREA)
Abstract
The present invention provides the preparation method of a kind of green muscardine fungus height cryptogam, including: green muscardine fungus hypha fermentation liquid with through steam high temperature sterilize and cool down the nutritional solution of room temperature and mix to obtain mixed liquor in the ratio of 1:1 3, by be soaked with mixed liquor cloth or be soaked with mixed liquor sponge block load bottom have in through hole and stackable round, be placed in the fermenting cellar that temperature is 25 28 DEG C and carry out fermenting 56 days；Described nutritional solution includes that following raw material is prepared from: 1000 parts of Testa Tritici juice, 20 parts of rice flour and white sugar 40 parts；The preparation method of described Testa Tritici juice is: add 300g Testa Tritici by every 1000g water, boils 10min and crosses leaching filtrate, and the gained filtrate benefit that adds water obtains described Testa Tritici juice to 1000g.The preparation method of green muscardine fungus height cryptogam of the present invention, has that production efficiency is high, fermentation period is short, conidium and a segregative advantage of substrate, and the spore content of green muscardine fungus height cryptogam prepared by the method is 4.0 × 1010～5.0 × 1010Spore/g.
Description
Technical field
The present invention relates to biofermentation method field, particularly relate to the preparation method of a kind of green muscardine fungus height cryptogam.
Background technology
Since mid-term in 20th century, owing to chemical insecticide unrestrictedly uses, severe contamination environment and Agriculture, forestry And Animal Husbandry product,
Compromise human and livestock health, make natural ecosystems be destroyed simultaneously.Biological control is valued by the people day by day, utilizes entomiasis
Pathogenic microorganism pest control is one of important means of Biological control.Fungus insecticide is owing to having good diffusion effect, relatively
The long ability that harbors, unique integument invasion mode, and insect is not developed immunity to drugs and the feature such as the most a large amount of productions so that
Fungus insecticide has obvious advantage in the Biological control of injurious forest-insect, soil pests and sucking pest.
Green muscardine fungus is a kind of entomogenous fungi being applied to biological control of insect pests the earliest, can surpass by parasitic 8 Ge Mu30Ge section insecticides
Crossing more than 200 kind insecticides, utilize the research of green muscardine fungus pest control to exceed a century with practice, it has pathogenicity and by force, does not pollute ring
The features such as border, lasting period length, are the excellent biological preparation of a kind of sustainable pest control, are also now for the master of Biological control
Want one of fungus insecticide, Biological control has important using value.Conidium is the main one-tenth of green muscardine fungus dosage form
Point, and green muscardine fungus height cryptogam (female powder) refers to the green muscardine fungus conidial powder that the spore content through extracting, screening obtains is high, green deadlock
Bacterium height cryptogam can be as the raw material of the multiple dosage forms such as wettable powder, oil preparation, microcapsule, and purposes is quite varied.Therefore, as
What acquisition muscardine spore a large amount of, quick is basis prepared by green muscardine fungus height cryptogam.
At present, domestic and international green muscardine fungus is conidial is mainly obtained the mode using liquid-solid biphasic fermentation, will liquid
Seed is inoculated in the nutrient matrix solid dosage forms solid fermentations such as the rice of routine, Testa Tritici, Semen Maydis powder, but weak point is aobvious and easy
Seeing: in incubation, environment cytokine regulatory is complicated, is difficult to be dried, and fermentation period is long, pollution rate is high, and unstable product quality connects
Amount of planting is general 10%, and conidium mixes with solid matrix simultaneously, is not readily separated, and these factors constrain green muscardine fungus
The production application of high cryptogam and prevention effect.
As improvement, the Chinese invention patent of Publication No. CN 1212772 C discloses Metarhizium anisopliae non-woven fabrics bacterium
Bar manufacturing technique method, the method includes five road production processes, the i.e. acquirement of strain, primary inclined plane spawn culture, liquid seeds
Cultivate, liquid fermentation tank produces and the inoculated and cultured of bacterium bar.Using sterile-cloth bar as the immobilization carrier of Metarhizium anisopliae, finally
Metarhizium anisopliae non-woven fabric fungal band is directly used in Pest control, although avoid spore to a certain extent and separate with substrate difficulty
Problem, but its liquid fermentation use three grade fermemtation, fermentation time needs 8-10 days, and fermentation period is the longest, have impact on production
Efficiency, and its prepare product can only use as bacterium bar, limit its type of service, it is impossible to as separation high cryptogam
Can be as the raw material of wettable powder, oil preparation, microcapsule etc., meanwhile, its bacterium bar is relatively big, and surface area is less the most also to be limited
Make inoculum concentration, be not easy to operation.
Therefore, it is necessary to invent the preparation method of a kind of production efficiency green muscardine fungus high, segregative height cryptogam.
Summary of the invention
The technical problem to be solved is: the preparation of a kind of production efficiency green muscardine fungus high, segregative height cryptogam
Method.
In order to solve above-mentioned technical problem, the technical solution used in the present invention is:
The present invention provides the preparation method of a kind of green muscardine fungus height cryptogam, including: green muscardine fungus hypha fermentation liquid is high with through steam
Temperature sterilizing also cools down the nutritional solution of room temperature and mixes to obtain mixed liquor in the ratio of 1:1-3, will be soaked with the cloth of mixed liquor or is soaked with mixed
Close and have in through hole and stackable round bottom the sponge block loading of liquid, be placed in the fermenting cellar that temperature is 25-28 DEG C
Carry out fermentation 5-6 days；
Described nutritional solution includes that following raw material is prepared from: 1000 parts of Testa Tritici juice, 20 parts of rice flour and white sugar 40
Part；
The preparation method of described Testa Tritici juice is: add 300g Testa Tritici by every 1000g water, boils 10min and crosses leaching filtrate, institute
The filtrate benefit that adds water obtains described Testa Tritici juice to 1000g.
Described cloth can be arbitrary fabric fibre block, and the geotextile cloth being preferably, specification is 700～1000g/
m2, described sponge block is PVA high density water-absorbing sponge.
The beneficial effects of the present invention is: it is raw as green muscardine fungus that (1) is soaked with the cloth of mixed liquor or sponge block by employing
Producing and produce the immobilization carrier of spore, compared with the nutrient matrix such as traditional rice, Testa Tritici, Semen Maydis powder, rate of drying is fast, spore easily divides
From, and owing to the volume of cloth or sponge block is little, surface area makes greatly its inoculum concentration, and (its inoculum concentration is cultivated for tradition equal-volume greatly
2.5-5 times of base inoculum concentration), the average fermentation cycle is 5-6 days, 7-10 days of relatively conventional solid substrate cultivation, substantially reduces
Incubation time, therefore, substantially increases production efficiency and the constant product quality of green muscardine fungus height cryptogam；(2) divide due to green muscardine fungus
Raw spore is the raw spore of gas, in incubation, owing to there is passage in round intermediate layer, keeps the ventilatory effect in intermediate layer
Good, substrate height≤3cm in addition, beneficially spore produce, and in incubation, whole round is airtight, in cloth or sponge block
Moisture scatter and disappear hardly, so also can guarantee that high humidity required during green muscardine fungus mycelial growth without Regulate Environment humidity
Degree, simplifies production technology；(3) use fabric cloth or sponge block to produce as green muscardine fungus and produce the immobilization carrier of spore, convenient receipts
Operation during collection spore；(4) above-mentioned nutritional solution is added at green muscardine fungus hypha fermentation liquid, it is ensured that be inoculated into the bacterium on cloth or sponge block
Silk regrowth and the nutrition of product spore, increase breeding coefficient simultaneously, increase the conidial product of green muscardine fungus on cloth or sponge block
Amount.
Detailed description of the invention
By describing the technology contents of the present invention in detail, being realized purpose and effect, it is explained below in conjunction with embodiment.
The design of most critical of the present invention is: will be soaked with cloth or the sponge block generation of green muscardine fungus fluid medium and nutritional solution
Produce and the immobilization carrier of product spore, cloth or sponge block as green muscardine fungus for nutrient matrixes such as conventional rice, Testa Tritici, Semen Maydis powder
Surface area big, green muscardine fungus is at cloth or sponge block fast-growth and produces spore, then muscardine spore is sieved into green muscardine fungus
High cryptogam.
The present invention provides the preparation method of a kind of green muscardine fungus height cryptogam, including: green muscardine fungus hypha fermentation liquid is high with through steam
Temperature sterilizing also cools down the nutritional solution of room temperature and mixes to obtain mixed liquor in the ratio of 1:1-3, will be soaked with the cloth of mixed liquor or is soaked with mixed
Close and have in through hole and stackable round bottom the sponge block loading of liquid, be placed in the fermenting cellar that temperature is 25-28 DEG C
Carry out fermentation 5-6 days；Described nutritional solution includes that following raw material is prepared from: 1000 parts of Testa Tritici juice, 20 parts of rice flour and white
Saccharum Sinensis Roxb. 40 parts；The preparation method of described Testa Tritici juice is: add 300g Testa Tritici by every 1000g water, boils 10min and crosses leaching filtrate,
The gained filtrate benefit that adds water obtains described Testa Tritici juice to 1000g.
Described cloth can be arbitrary fabric fibre block, and the geotextile cloth being preferably, specification is 700～1000g/
m2, described sponge block is PVA high density water-absorbing sponge.
Knowable to foregoing description, the beneficial effects of the present invention is: be soaked with cloth or the sponge block of mixed liquor by employing
Produce and produce the immobilization carrier of spore, compared with the nutrient matrix such as traditional rice, Testa Tritici, Semen Maydis powder, rate of drying as green muscardine fungus
Hurry up, spore easily separated, and owing to the volume of cloth or sponge block is little, surface area makes greatly its inoculum concentration, and big (its inoculum concentration is for passing
2.5-5 times of system equal-volume culture medium inoculated amount)；The average fermentation cycle is 5-6 days, the 7-10 that relatively conventional solid substrate is cultivated
My god, substantially reduce incubation time.Therefore, production efficiency and the constant product quality of green muscardine fungus height cryptogam are substantially increased；By
It is the raw spore of gas in green muscardine fungus conidium, in incubation, owing to there is through hole in round intermediate layer, keeps the logical of intermediate layer
Gas is effective, in addition substrate height≤3cm, and beneficially spore produces, and in incubation, whole round is airtight, cloth or sea
Moisture in continuous block scatters and disappears hardly, thus required during also can guarantee that green muscardine fungus mycelial growth without Regulate Environment humidity
High humility, simplify production technology；Use cloth or sponge block to produce as green muscardine fungus and produce the immobilization carrier of spore, convenient receipts
Operation during collection spore；Above-mentioned nutritional solution is added, it is ensured that be inoculated into the mycelia on cloth or sponge block at green muscardine fungus hypha fermentation liquid
Regrowth and the nutrition of product spore, increase breeding coefficient simultaneously, increase the conidial yield of green muscardine fungus on cloth or sponge block.
Further, the cultivation of described green muscardine fungus hypha fermentation liquid: by the examination filling slant medium of green muscardine fungus strain
Pipe is Secondary Culture, activation under the conditions of 24-26 DEG C, when the chamfered surface of described slant medium covers with spore in sterile working
In platform, access through steam high temperature sterilize from a small amount of spore of inclined-plane picking and the lawn of described slant medium and be cooled to room temperature
In fluid medium；Postvaccinal fluid medium is at vibration 25 ± 1 DEG C of constant temperature culture 60-72h of case, thickness mycelium to be grown up to
After i.e. obtain described green muscardine fungus hypha fermentation liquid.
Further, described fluid medium includes that following raw material is prepared from: 1000 parts of water, Semen Maydis powder 20
Part, 20 parts of rice flour and white sugar 40 parts.
Seen from the above description, the beneficial effects of the present invention is: using Semen Maydis powder, rice flour and white sugar as liquid culture
Base, preparation is simple, and raw material is inexpensive, wide material sources, reduces production cost.
Further, described cloth or a length of 4-6cm of sponge block, width is 2-4cm, and height is 0.3-0.5cm.
Seen from the above description, the beneficial effects of the present invention is: the length of cloth or sponge block is the least, relatively
For simply with long cloth or strip sponge, surface area is bigger, can cultivate more green muscardine funguss at the same space, improve space
Utilization rate, thus sporulation quantity increases, and then the spore that vibrosieve is obtained is more, i.e. makes green muscardine fungus in same space
Conidial output increased.
Further, be soaked with described in mixed liquor cloth or described in be soaked with mixed liquor the preparation method of sponge block be: will
Cloth described in 10-15 block or described sponge block are put in mixed liquor described in 100mL, take out and be soaked with mixing described in i.e. obtaining after 20-60S
The cloth of liquid or described in be soaked with the sponge block of mixed liquor.
Seen from the above description, the beneficial effects of the present invention is: described cloth or sponge block are directly immersed in mixed liquor,
Quickly and easily complete inoculation, again nutritional solution has been adsorbed onto on cloth or sponge block, has met green muscardine fungus growth and produce spore
Need.
Further, the geotextile cloth that described cloth is preferably, specification is 700～1000g/m2, described sponge
Block is PVA high density water-absorbing sponge.
Seen from the above description, the beneficial effects of the present invention is: described geotextile cloth or the water suction of described high density
The moisture retention of sponge block is good, in addition the sealing of round, it is possible to reduce humidity during green muscardine fungus mycelial growth and product spore
Regulation and control.
The detailed description of the invention of the present invention has:
The preparation method of a kind of green muscardine fungus height cryptogam, comprises the steps:
The cultivation of 1 green muscardine fungus hypha fermentation liquid
1.1 strains: produce the green muscardine fungus that spore is good.General with 4 DEG C of inclined-plane cryopreservation.
1.2 slant strains: in advance by green muscardine fungus strain test tube slant Secondary Culture under the conditions of 25 ± 1 DEG C, work as inclined-plane
Can use when surface covers with spore.Slant medium is potato dextrose agar (PDA) general in microorganism culturing
Culture medium.
1.3 fluid mediums: Semen Maydis powder 20 parts by weight, 20 parts of rice flour and white sugar 40 parts, after mix homogeneously, add
1000 parts of water, obtains fluid medium, loads 200mL culture medium, steam high temperature sterilize at 121 DEG C in each 500mL triangular flask
30min, standby after cooling.
1.4 inoculations: in aseptic operating platform, from strain inclined plane, a small amount of spore of picking and lawn access aforesaid liquid cultivation
In base.
1.5 shaking tables are cultivated: shaken cultivation 60-72h in 25 ± 1 DEG C of constant-temperature shaking incubators, after growing up to thickness mycelium
It is green muscardine fungus hypha fermentation liquid.
The inoculated and cultured of 2 truffle
The preparation method of 2.1 nutritional solutions: add 300g Testa Tritici by every 1000g water, boils 10min and crosses leaching filtrate, gained
Filtrate adds water to mend and obtains described Testa Tritici juice to 1000g, and 1000 parts of described Testa Tritici juice, 20 parts of rice flour and white sugar 40 parts are mixed to obtain institute
Stating nutritional solution, the triangular flask of each 500mL loads nutritional solution described in 200mL, steam high temperature sterilize 30min at 121 DEG C, cooling
The most standby.
2.2 cloths or sponge block sterilization treatment: by a length of 4-6cm, width is 2-4cm, and height is the soil of 0.3-0.5cm
(specification is 700～1000g/m to work fabric panel2) or PVA high density water-absorbing sponge packaged enter plastic bag, under the conditions of 121 DEG C
Steam high temperature sterilize 30min.
2.3 inoculations: in the closed room carrying out space sterilizing in advance, by described green muscardine fungus hypha fermentation liquid and through steam
High temperature sterilize cool down the nutritional solution of room temperature to mix in the ratio of 1:1-3, stir to obtain mixed liquor, i.e. inoculum concentration be 25%-50%,
Above-mentioned for the 10-15 block cloth through the process of steam high temperature sterilize or sponge block are put into 100mL mixed liquor, and every 5S stirs mixed once
Cloth in liquid or sponge block, after 20-60S, until cloth or sponge block absorb saturated after, take out described in be soaked with the cloth of mixed liquor
Block or described in be soaked with the sponge block of mixed liquor.
2.4 cultivate: by the described cloth being soaked with mixed liquor or described in be soaked with mixed liquor sponge block load through surface
The bottom of sterilization have in through hole and stackable plastics fermenting case (outside dimension: 480 × 355 × 170mm, inside dimension: 445 ×
320 × 165mm), proceed to fermenting cellar immediately and cultivate, be placed in the fermenting cellar that temperature is 25-28 DEG C and carry out fermentation 5-6 days.
Potato dextrose agar described in step 1.2 (PDA) culture medium, concrete formula is: the Rhizoma Solani tuber osi containing 100g (is boiled
Filter), 10g glucose, 20g agar and water 1000g；
Nutritional solution described in step 2.3, concrete formula is: 1000 parts of Testa Tritici juice, 20 parts of rice flour and white sugar 40 parts, institute
The preparation method stating Testa Tritici juice is: add 300g Testa Tritici by every 1000g water, boils 10min and crosses leaching filtrate, and gained filtrate adds water
Benefit obtains described Testa Tritici juice to 1000g；
Cloth described in step 2.3 or a length of 4-6cm of sponge block, width is 2-4cm, and height is 0.3-0.5cm.
Embodiment one
The preparation method of the green muscardine fungus height cryptogam of the present embodiment, comprises the steps:
The cultivation of 1 green muscardine fungus hypha fermentation liquid
1.1 strains: green muscardine fungus (MaZPTR-01) bacterial strain, Fujian Academy of Forestry's forest conservation institute preserves,
For known bacterial strain.
1.2 slant strains: in advance by green muscardine fungus strain test tube slant Secondary Culture, activation under the conditions of 25 ± 1 DEG C, when
Can use when chamfered surface covers with spore.Slant medium is potato dextrose agar general in microorganism culturing
(PDA) culture medium.
1.3 fluid mediums: Semen Maydis powder 20 parts by weight, 20 parts of rice flour and white sugar 40 parts, after mix homogeneously, add
1000 parts of water, obtains fluid medium, prepares this fluid medium 2L, loading 200mL culture medium in each 500mL triangular flask, and 121
Steam high temperature sterilize 30min at DEG C, standby after cooling.
1.4 inoculations: in aseptic operating platform, from strain inclined plane, a small amount of spore of picking and lawn access aforesaid liquid cultivation
In base.
1.5 shaking tables are cultivated: shaken cultivation 60h in 25 ± 1 DEG C of constant-temperature shaking incubators, are after growing up to thickness mycelium
Green muscardine fungus hypha fermentation liquid.
The inoculated and cultured of 2 truffle
The preparation method of 2.1 nutritional solutions: add 300g Testa Tritici by every 1000g water, boils 10min and crosses leaching filtrate, gained
Filtrate adds water to mend and obtains described Testa Tritici juice to 1000g, and 1000 parts of described Testa Tritici juice, 20 parts of rice flour and white sugar 40 parts are mixed to obtain institute
State nutritional solution, the triangular flask of each 500mL loads nutritional solution described in 200mL, and at 121 DEG C, steam high temperature sterilize 30min, cold
Rear standby.
2.2 cloths or sponge block sterilization treatment: by a length of 4cm, width is 2cm, and height is the geotextile cloth of 0.3cm
Block or high density water-absorbing sponge packaged enter polypropylene plastics pocket (being resistant to moist heat sterilization), steam high temperature sterilize under the conditions of 121 DEG C
30min。
2.3 inoculations: in the closed room carrying out space sterilizing in advance, by described green muscardine fungus hypha fermentation liquid and through steam
High temperature sterilize cool down the nutritional solution of room temperature to mix in the ratio of 1:1, stir to obtain mixed liquor, i.e. inoculum concentration be 50%, by 10 pieces
The above-mentioned cloth through the process of steam high temperature sterilize or sponge block put into 100mL mixed liquor, and every 5S stirs the cloth in mixed once liquid
Block or sponge block, after 20S, until cloth or sponge block absorb saturated after, take out described in be soaked with the cloth of mixed liquor or described leaching
There is the sponge block of mixed liquor.
2.4 cultivate: by the described cloth being soaked with mixed liquor or described in be soaked with mixed liquor sponge block load through surface
The bottom of sterilization have in through hole and stackable plastics fermenting case (outside dimension: 480 × 355 × 170mm, inside dimension: 445 ×
320 × 165mm), proceed to fermenting cellar immediately and cultivate, be placed in the fermenting cellar that temperature is 25-28 DEG C and carry out fermenting 5 days.
3 cultivation results
Be soaked with described in treating mixed liquor cloth or described in be soaked with mixed liquor the surface of sponge block cover with the mitogenetic spore of green muscardine fungus
Son, tunning is placed at shady and cool ventilation naturally dry pulvis grossus (or in 28-30 DEG C of baking oven dry 12-24h, aqueous
Rate≤10%), then use 200 mesh vibrosieves to sieve, during screening, the discharging opening of vibrosieve is closed, treat in culture medium
Spore powder opens discharging opening after having sieved, and prepares green muscardine fungus height cryptogam；Weigh prepared green muscardine fungus height cryptogam agent 5g at random, use
0.03% tween 80 solution is diluted, and blood counting chamber counts, and the average spore content of described green muscardine fungus height cryptogam is 5.0 ×
1010Spore/g.
Embodiment two
The preparation method of the green muscardine fungus height cryptogam of the present embodiment, comprises the steps:
The cultivation of 1 green muscardine fungus hypha fermentation liquid
1.1 strains: green muscardine fungus (MaYTTR-04) bacterial strain, Fujian Academy of Forestry's forest conservation institute preserves,
For known bacterial strain.
1.2 slant strains: in advance by green muscardine fungus strain test tube slant Secondary Culture, activation under the conditions of 25 ± 1 DEG C, when
Can use when chamfered surface covers with spore.Slant medium is potato dextrose agar general in microorganism culturing
(PDA) culture medium.
1.3 fluid mediums: Semen Maydis powder 20 parts by weight, 20 parts of rice flour and white sugar 40 parts, after mix homogeneously, add
1000 parts of water, obtains fluid medium, prepares this fluid medium 4L, loading 200mL culture medium in each 500mL triangular flask, and 121
Steam high temperature sterilize 50min at DEG C, standby after cooling.
1.4 inoculations: in aseptic operating platform, from strain inclined plane, a small amount of spore of picking and lawn access aforesaid liquid cultivation
In base.
1.5 shaking tables are cultivated: shaken cultivation 72h in 25 ± 1 DEG C of constant-temperature shaking incubators, are after growing up to thickness mycelium
Green muscardine fungus hypha fermentation liquid.
The inoculated and cultured of 2 truffle
The preparation method of 2.1 nutritional solutions: add 300g Testa Tritici by every 1000g water, boils 10min and crosses leaching filtrate, gained
Filtrate adds water to mend and obtains described Testa Tritici juice to 1000g, and 1000 parts of described Testa Tritici juice, 20 parts of rice flour and white sugar 40 parts are mixed to obtain institute
Stating nutritional solution, the triangular flask of each 500mL loads nutritional solution described in 200mL, steam high temperature sterilize 50min at 121 DEG C, cooling
The most standby.
2.2 cloths or sponge block sterilization treatment: by a length of 6cm, width is 4cm, and height is the geotextile cloth of 0.5cm
Block or high density water-absorbing sponge packaged enter polypropylene plastics pocket (being resistant to moist heat sterilization), steam high temperature sterilize under the conditions of 121 DEG C
50min。
2.3 inoculations: in the closed room carrying out space sterilizing in advance, by described green muscardine fungus hypha fermentation liquid and through steam
High temperature sterilize cool down the nutritional solution of room temperature to mix in the ratio of 1:3, stir to obtain mixed liquor, i.e. inoculum concentration be 25%, by 15 pieces
The above-mentioned cloth through the process of steam high temperature sterilize or sponge block put into 100mL mixed liquor, and every 5S stirs the cloth in mixed once liquid
Block or sponge block, after 60S, until cloth or sponge block absorb saturated after, take out described in be soaked with the cloth of mixed liquor or described leaching
There is the sponge block of mixed liquor,
2.4 cultivate: by the described cloth being soaked with mixed liquor or described in be soaked with mixed liquor sponge block load through surface
The bottom of sterilization have in through hole and stackable plastics fermenting case (outside dimension: 480 × 355 × 170mm, inside dimension: 445 ×
320 × 165mm), proceed to fermenting cellar immediately and cultivate, be placed in the fermenting cellar that temperature is 25-28 DEG C and carry out fermenting 6 days.
3 cultivation results
Be soaked with described in treating mixed liquor cloth or described in be soaked with mixed liquor the surface of sponge block cover with the mitogenetic spore of green muscardine fungus
Son, tunning is placed at shady and cool ventilation naturally dry pulvis grossus (or in 28-30 DEG C of baking oven dry 12-24h, aqueous
Rate≤10%), then use 200 mesh vibrosieves to sieve, during screening, the discharging opening of vibrosieve is closed, treat in culture medium
Spore powder opens discharging opening after having sieved, and prepares green muscardine fungus height cryptogam；Weigh prepared green muscardine fungus height cryptogam agent 5g at random, use
0.03% tween 80 solution is diluted, and blood counting chamber counts, and the average spore content of described green muscardine fungus height cryptogam is 4.5 ×
1010Spore/g.
Embodiment three
The preparation method of the green muscardine fungus height cryptogam of the present embodiment, comprises the steps:
The cultivation of 1 green muscardine fungus hypha fermentation liquid
1.1 strains: green muscardine fungus (MaFZ-01) bacterial strain, Fujian Academy of Forestry's forest conservation institute preserves, for
Known bacterial strain.
1.2 slant strains: in advance by green muscardine fungus strain test tube slant Secondary Culture, activation under the conditions of 25 ± 1 DEG C, when
Can use when chamfered surface covers with spore.Slant medium is potato dextrose agar general in microorganism culturing
(PDA) culture medium.
1.3 fluid mediums: Semen Maydis powder 20 parts by weight, 20 parts of rice flour and white sugar 40 parts, after mix homogeneously, add
1000 parts of water, obtains fluid medium, prepares this fluid medium 3L, loading 200mL culture medium in each 500mL triangular flask, and 121
Steam high temperature sterilize 40min at DEG C, standby after cooling.
1.4 inoculations: in aseptic operating platform, from strain inclined plane, a small amount of spore of picking and lawn access aforesaid liquid cultivation
In base.
1.5 shaking tables are cultivated: shaken cultivation 66h in 25 ± 1 DEG C of constant-temperature shaking incubators, are after growing up to thickness mycelium
Green muscardine fungus hypha fermentation liquid.
The inoculated and cultured of 2 truffle
The preparation method of 2.1 nutritional solutions: add 300g Testa Tritici by every 1000g water, boils 10min and crosses leaching filtrate, gained
Filtrate adds water to mend and obtains described Testa Tritici juice to 1000g, and 1000 parts of described Testa Tritici juice, 20 parts of rice flour and white sugar 40 parts are mixed to obtain institute
Stating nutritional solution, the triangular flask of each 500mL loads nutritional solution described in 200mL, steam high temperature sterilize 40min at 121 DEG C, cooling
The most standby.
2.2 cloths or sponge block sterilization treatment: by a length of 5cm, width is 3cm, and height is the geotextile cloth of 0.4cm
Block or high density water-absorbing sponge packaged enter polypropylene plastics pocket (being resistant to moist heat sterilization), steam high temperature sterilize under the conditions of 121 DEG C
40min。
2.3 inoculations: in the closed room carrying out space sterilizing in advance, by described green muscardine fungus hypha fermentation liquid and through steam
High temperature sterilize cool down the nutritional solution of room temperature to mix in the ratio of 1:2, stir to obtain mixed liquor, i.e. inoculum concentration be 33.3%, by 12
The above-mentioned cloth through the process of steam high temperature sterilize of block or sponge block put into 100mL mixed liquor, and every 5S stirs in mixed once liquid
Cloth or sponge block, after 40S, until cloth or sponge block absorb saturated after, take out described in be soaked with the cloth of mixed liquor or described
It is soaked with the sponge block of mixed liquor,
2.4 cultivate: by the described cloth being soaked with mixed liquor or described in be soaked with mixed liquor sponge block load through surface
The bottom of sterilization have in through hole and stackable plastics fermenting case (outside dimension: 480 × 355 × 170mm, inside dimension: 445 ×
320 × 165mm), proceed to fermenting cellar immediately and cultivate, be placed in the fermenting cellar that temperature is 25-28 DEG C and carry out fermenting 5.5 days.
3 cultivation results
Be soaked with described in treating mixed liquor cloth or described in be soaked with mixed liquor the surface of sponge block cover with the mitogenetic spore of green muscardine fungus
Son, tunning is placed at shady and cool ventilation naturally dry pulvis grossus (or in 30-35 DEG C of baking oven dry 12-24h, aqueous
Rate≤10%), then use 200 mesh vibrosieves to sieve, during screening, the discharging opening of vibrosieve is closed, treat in culture medium
Spore powder opens discharging opening after having sieved, and prepares green muscardine fungus height cryptogam；Weigh prepared green muscardine fungus height cryptogam agent 5g at random, use
0.03% tween 80 solution is diluted, and blood counting chamber counts, and the average spore content of described green muscardine fungus height cryptogam is 4.8 ×
1010Spore/g.
In sum, the preparation method of the green muscardine fungus height cryptogam that the present invention provides, the cloth of mixed liquor it is soaked with by employing
Or sponge block produces as green muscardine fungus and produces the immobilization carrier of spore, compared with the nutrient matrix such as traditional rice, Testa Tritici, Semen Maydis powder,
Rate of drying is fast, spore is easily separated, and owing to the volume of cloth or sponge block is little, surface area makes greatly its inoculum concentration, and (it connects greatly
The amount of kind is 2.5-5 times of tradition equal-volume culture medium inoculated amount), the average fermentation cycle is 5-6 days, and relatively conventional solid substrate is cultivated
7-10 days, substantially reduce incubation time, therefore, the production efficiency and the product quality that substantially increase green muscardine fungus height cryptogam are steady
Fixed；Owing to green muscardine fungus conidium is the raw spore of gas, in incubation, owing to there is through hole in round intermediate layer, keep middle
The ventilatory effect of layer is good, substrate height≤3cm in addition, and beneficially spore produces, and in incubation, whole round is airtight, cloth
Moisture in block or sponge block scatters and disappears hardly, so also can guarantee that green muscardine fungus mycelial growth process without Regulate Environment humidity
High humility needed in, simplifies production technology；Use cloth or sponge block to produce as green muscardine fungus and produce the immobilization carrier of spore,
Operation during convenient collection spore；Above-mentioned nutritional solution is added, it is ensured that be inoculated on cloth or sponge block at green muscardine fungus hypha fermentation liquid
Regeneration of Mycelium length and produce the nutrition of spore, increase the conidial yield of green muscardine fungus on cloth or sponge block simultaneously.
The foregoing is only embodiments of the invention, not thereby limit the scope of the claims of the present invention, every utilize this
The equivalents that bright description is made, or directly or indirectly it is used in relevant technical field, the most in like manner it is included in this
In bright scope of patent protection.
Claims (6)
1. the preparation method of a green muscardine fungus height cryptogam, it is characterised in that including: green muscardine fungus hypha fermentation liquid with through steam high temperature
Sterilizing also cools down the nutritional solution of room temperature and mixes to obtain mixed liquor in the ratio of 1:1-3, will be soaked with the cloth of mixed liquor or is soaked with mixing
The sponge block of liquid has in through hole and stackable round bottom loading, and is placed in the fermenting cellar that temperature is 25-28 DEG C
Row fermentation 5-6 days；
Described nutritional solution includes that following raw material is prepared from: 1000 parts of Testa Tritici juice, 20 parts of rice flour and white sugar 40 parts；
The preparation method of described Testa Tritici juice is: add 300g Testa Tritici by every 1000g water, boils 10min and crosses leaching filtrate, and gained is filtered
The liquid benefit that adds water obtains described Testa Tritici juice to 1000g.
The preparation method of a kind of green muscardine fungus height cryptogam the most according to claim 1, it is characterised in that described green muscardine fungus mycelia
The cultivation of fermentation liquid: green muscardine fungus strain is used test tube Secondary Culture, the activation under the conditions of 24-26 DEG C filling slant medium,
When the chamfered surface of described slant medium covers with spore in aseptic operating platform, from the inclined-plane picking of described slant medium
A small amount of spore and lawn access through steam high temperature sterilize and be cooled to room temperature fluid medium in；Postvaccinal fluid medium
In 25 ± 1 DEG C of constant temperature culture 60-72h of constant temperature oscillation case, after growing up to thickness mycelium, i.e. obtain described green muscardine fungus hypha fermentation liquid.
The preparation method of a kind of green muscardine fungus height cryptogam the most according to claim 2, it is characterised in that described fluid medium
It is prepared from including following raw material: 1000 parts of water, Semen Maydis powder 20 parts, 20 parts of rice flour and white sugar 40 parts.
The preparation method of a kind of green muscardine fungus height cryptogam the most according to claim 1, it is characterised in that described cloth or sponge
The a length of 4-6cm of block, width is 2-4cm, and height is 0.3-0.5cm.
The preparation method of a kind of green muscardine fungus height cryptogam the most according to claim 4, it is characterised in that described in be soaked with mixed liquor
Cloth or described in be soaked with mixed liquor the preparation method of sponge block be: cloth described in 10-15 block or described sponge block are put into
In mixed liquor described in 100mL, take out after 20-60S be soaked with described in i.e. obtaining mixed liquor cloth or described in be soaked with the sponge of mixed liquor
Block.
The preparation method of a kind of green muscardine fungus height cryptogam the most according to claim 1, it is characterised in that comprise the steps:
Step 1, by green muscardine fungus strain with filling test tube Secondary Culture, activation under the conditions of 24-26 DEG C of slant medium, work as institute
State when the chamfered surface of slant medium covers with spore in aseptic operating platform, a small amount of from the inclined-plane picking of described slant medium
Spore and lawn access through steam high temperature sterilize and be cooled to room temperature fluid medium in；Postvaccinal fluid medium is shaking
Swing 25 ± 1 DEG C of constant temperature culture 60-72h of case, after growing up to thickness mycelium, i.e. obtain described green muscardine fungus hypha fermentation liquid；
Described slant medium is potato dextrose agar, and described potato dextrose agar is by weight
It is prepared from including following raw material: 100 portions of Rhizoma Solani tuber osis, 10 parts of glucoses and 20 parts of agar and 1000 parts of water；Described liquid
Body culture medium includes that following raw material is prepared from: 1000 parts of water, Semen Maydis powder 20 parts, 20 parts of rice flour and white sugar 40 parts；
Step 2, by described green muscardine fungus hypha fermentation liquid with through steam high temperature sterilize the nutritional solution ratio by 1:1-3 that cools down room temperature
Example mixes to obtain mixed liquor, puts into 10-15 block cloth or the sponge block through steam high temperature sterilize in mixed liquor described in 100mL
In, after 20-60S described in be soaked with mixed liquor cloth or described in be soaked with the sponge block of mixed liquor, by the described mixed liquor that is soaked with
Cloth or described in be soaked with mixed liquor sponge block load bottom have in through hole and stackable round, being placed in temperature is
The fermenting cellar of 25-28 DEG C carries out fermentation 5-6 days；
Described nutritional solution includes that following raw material is prepared from: 1000 parts of Testa Tritici juice, 20 parts of rice flour and white sugar 40 parts, institute
The preparation method stating Testa Tritici juice is: add 300g Testa Tritici by every 1000g water, boils 10min and crosses leaching filtrate, and gained filtrate adds water
Benefit obtains described Testa Tritici juice to 1000g；Described cloth or a length of 4-6cm of sponge block, width is 2-4cm, and height is 0.3-
0.5cm。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610719429.5A CN106244466B (en) | 2016-08-25 | 2016-08-25 | A kind of preparation method of the high cryptogam of green muscardine fungus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610719429.5A CN106244466B (en) | 2016-08-25 | 2016-08-25 | A kind of preparation method of the high cryptogam of green muscardine fungus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106244466A true CN106244466A (en) | 2016-12-21 |
| CN106244466B CN106244466B (en) | 2019-07-19 |
Family
ID=57595364
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610719429.5A Active CN106244466B (en) | 2016-08-25 | 2016-08-25 | A kind of preparation method of the high cryptogam of green muscardine fungus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106244466B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446829A (en) * | 2017-09-18 | 2017-12-08 | 广东省林业科学研究院 | A kind of green muscardine fungus pocket type solid culture technique |
| CN109090145A (en) * | 2018-08-04 | 2018-12-28 | 普定县顺和水果苗木种植有限公司 | A kind of chafer naturally kills preparation and preparation method thereof |
| CN110819544A (en) * | 2019-12-19 | 2020-02-21 | 琼台师范学院 | Preparation method of metarhizium anisopliae high spore powder |
| CN111944703A (en) * | 2020-08-24 | 2020-11-17 | 琼台师范学院 | Method for preparing metarhizium anisopliae conidium microcapsule emulsion |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1419823A (en) * | 2002-11-20 | 2003-05-28 | 安徽林苑虫草研究所 | Process for producing Metarrhizium anisopliae non-woven fabric ribbon |
| CN104911111A (en) * | 2015-06-24 | 2015-09-16 | 南京林业大学 | Metarhizium anisopliae solid culture method |
| CN204897895U (en) * | 2015-06-24 | 2015-12-23 | 福建省林业科学研究院 | Fermenting installation of green muscardine fungus fungus strip |
-
2016
- 2016-08-25 CN CN201610719429.5A patent/CN106244466B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1419823A (en) * | 2002-11-20 | 2003-05-28 | 安徽林苑虫草研究所 | Process for producing Metarrhizium anisopliae non-woven fabric ribbon |
| CN104911111A (en) * | 2015-06-24 | 2015-09-16 | 南京林业大学 | Metarhizium anisopliae solid culture method |
| CN204897895U (en) * | 2015-06-24 | 2015-12-23 | 福建省林业科学研究院 | Fermenting installation of green muscardine fungus fungus strip |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446829A (en) * | 2017-09-18 | 2017-12-08 | 广东省林业科学研究院 | A kind of green muscardine fungus pocket type solid culture technique |
| CN109090145A (en) * | 2018-08-04 | 2018-12-28 | 普定县顺和水果苗木种植有限公司 | A kind of chafer naturally kills preparation and preparation method thereof |
| CN110819544A (en) * | 2019-12-19 | 2020-02-21 | 琼台师范学院 | Preparation method of metarhizium anisopliae high spore powder |
| CN111944703A (en) * | 2020-08-24 | 2020-11-17 | 琼台师范学院 | Method for preparing metarhizium anisopliae conidium microcapsule emulsion |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106244466B (en) | 2019-07-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102964178B (en) | Composite microbial bactericide, and preparation method and application thereof | |
| CN102533579B (en) | Culture medium for producing spores, and optimizing process and application thereof | |
| CN106244466B (en) | A kind of preparation method of the high cryptogam of green muscardine fungus | |
| CN104212724B (en) | Beaueria bassaria (Balsamo) Vuillemin bacterial strain GZGY-1-3 and application thereof | |
| CN101659933A (en) | Antagonistic bacteria preventing and removing continuous cropping tomato bacterial wilt and microbial organic fertilizer thereof | |
| CN104263655B (en) | Beaueria bassaria(Balsamo)Vuillemin SCWJ-2 strain and application thereof | |
| CN101502271B (en) | Method for producing fungal granular formulation for preventing and treating pest | |
| CN110343017A (en) | Polyglutamic acid gel microspherical microbial inoculum and application thereof | |
| CN105918127B (en) | A kind of method that DSE- plant symbiosis systems are established based on DSE fast-propagations | |
| CN106497831A (en) | A kind of preventing and treating Phytophthora nicotianae disease composite bacteria agent capable and its preparation method and application | |
| CN103843583A (en) | Industrialized production method of green cordyceps militaris | |
| CN104054513A (en) | Cordyceps militaris strain cultivation method | |
| CN107711891A (en) | A kind of method prevented and treated matrimony vine root rot and extremely set | |
| CN105948831B (en) | A method of biological and ecological methods to prevent plant disease, pests, and erosion fertilizer is produced using brewex's grains | |
| CN103333851A (en) | Method of producing conidiospore by solid fermentation of trichoderma harzianum | |
| CN104911111B (en) | A kind of Metarhizium anisopliae solid culture method | |
| CN102286385B (en) | Method for carrying out solid culturing on beauveria bassiana by stages | |
| CN107950288A (en) | A kind of planting technique of straw mushroom | |
| CN105112302B (en) | One plant is intended Metarhizium Strains and its application of the spine longicorn with pathogenicity to Laos | |
| CN106350457B (en) | A kind of preparation method of the high cryptogam of muscardine | |
| CN109020637A (en) | A method of organic fertilizer is produced using stalk | |
| CN105439657A (en) | Preparation method for strawberry dedicated anti-continuous cropping biological organic fertilizer | |
| CN102960369B (en) | Preparation method of granular paecilomyces lilacinus biological nematocide | |
| CN107446829A (en) | A kind of green muscardine fungus pocket type solid culture technique | |
| CN108277167A (en) | A kind of method of biological control Phytophthora capsici disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |