CN106244466B - A kind of preparation method of the high cryptogam of green muscardine fungus - Google Patents
A kind of preparation method of the high cryptogam of green muscardine fungus Download PDFInfo
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- CN106244466B CN106244466B CN201610719429.5A CN201610719429A CN106244466B CN 106244466 B CN106244466 B CN 106244466B CN 201610719429 A CN201610719429 A CN 201610719429A CN 106244466 B CN106244466 B CN 106244466B
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Abstract
The present invention provides a kind of preparation method of high cryptogam of green muscardine fungus, including: green muscardine fungus hypha fermentation liquid mixes to obtain mixed liquor in the ratio of 1:1-3 with the nutrient solution through steam high-temperature sterilization and cooling room temperature, the cloth for being soaked with mixed liquor or the sponge block for being soaked with mixed liquor, which are fitted into bottom, to be had in through-hole and stackable round, is placed in the fermenting cellar that temperature is 25-28 DEG C and is carried out fermentation 5-6 days；The nutrient solution includes that following raw material is prepared: 1000 parts of wheat bran juice, 20 parts of rice flour and 40 parts of white granulated sugar；The wheat bran juice the preparation method comprises the following steps: filter to take filtrate by 10min in every 1000g water plus 300g wheat bran, is boiled, gained filtrate adds water to mend to 1000g to obtain the wheat bran juice.The preparation method of the high cryptogam of green muscardine fungus of the present invention, has the advantages that short high production efficiency, fermentation period, conidium and matrix are segregative, and the spore content of the high cryptogam of green muscardine fungus of this method preparation is 4.0 × 1010~5.0 × 1010Spore/g.
Description
Technical field
The present invention relates to biofermentation method field more particularly to a kind of preparation methods of the high cryptogam of green muscardine fungus.
Background technique
Since 20th century mid-terms, since chemical insecticide unlimitedly uses, have seriously polluted the environment with Agriculture, forestry And Animal Husbandry product,
Human and livestock health is compromised, while destroys natural ecosystems.Biological control is increasingly valued by the people, and utilizes entomiasis
Pathogenic microorganism pest control is one of the important means of biological control.Fungus insecticide due to good diffusion effect, compared with
Long harbors ability, unique integument invasion mode, and the features such as do not develop drug resistance pest with easy mass production, so that
Fungus insecticide has apparent advantage in the biological control of injurious forest-insect, soil pests and sucking pest.
Green muscardine fungus is a kind of entomogenous fungi for being applied to biological control of insect pests earliest, and the parasitic 8 Ge Mu30Ge section insects of energy are super
More than 200 insects are crossed, have exceeded a century using the research and practice of green muscardine fungus pest control, it is strong with pathogenicity, do not pollute ring
The features such as border, long lasting period is a kind of excellent biological agent of sustainable pest control, and the current master for being used for biological control
One of fungus insecticide is wanted, there is important application value in biological control.Conidium be green muscardine fungus dosage form it is main at
Point, and the high cryptogam of green muscardine fungus (female powder) refers to the green muscardine fungus conidial powder high by the spore content for extracting, sieving, green deadlock
The high cryptogam of bacterium can be used as the raw material of a variety of dosage forms such as wettable powder, finish, microcapsule formulations, and purposes is very extensive.Therefore, such as
How largely, it is quickly obtained the basis that muscardine spore is the high cryptogam preparation of green muscardine fungus.
It is mainly obtained in such a way that liquid consolidates biphasic fermentation currently, green muscardine fungus is conidial both at home and abroad, i.e., by liquid
Seed is inoculated in the nutrient matrixes solid dosage forms solid fermentation such as conventional rice, wheat bran, corn flour, but shortcoming is aobvious and easy
See: environment cytokine regulatory is complicated in incubation, is not easy drying, and fermentation period is long, and pollution rate is high, and unstable product quality connects
Kind amount is generally 10%, while conidium mixes with solid matrix, is not readily separated, these factors constrain green muscardine fungus
The production application and control efficiency of high cryptogam.
As an improvement, the Chinese invention patent of 1212772 C of Publication No. CN discloses Metarhizium anisopliae non-woven fabrics bacterium
Manufacturing technique method, this method include five production processes, the i.e. acquirement of strain, primary inclined plane Spawn incubation, liquid seeds
The inoculated and cultured of culture, liquid fermentation tank production and bacterium item.Using sterile cloth as the immobilization carrier of Metarhizium anisopliae, finally
Metarhizium anisopliae non-woven fabric fungal band is directly used in control of insect, is separated although avoiding spore to a certain extent with matrix difficulty
The problem of, but its liquid fermentation uses three grade fermemtation, fermentation time needs 8-10 days, and fermentation period is very long, affects production
Efficiency, and most product made from it can only be used as bacterium item, limit its use form, it cannot be as the high cryptogam of separation
It can be used as the raw material of wettable powder, finish, microcapsule formulations etc., meanwhile, bacterium item is larger, and surface area is smaller also to be limited to a certain degree
Inoculum concentration has been made, it is also not easy to operate.
Therefore, it is necessary to invent a kind of high production efficiency, the preparation method of the high cryptogam of segregative green muscardine fungus.
Summary of the invention
The technical problems to be solved by the present invention are: the preparation of a kind of high production efficiency, the high cryptogam of segregative green muscardine fungus
Method.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention are as follows:
The present invention provides a kind of preparation method of high cryptogam of green muscardine fungus, comprising: green muscardine fungus hypha fermentation liquid with through steam height
Temperature sterilizes and the nutrient solution of cooling room temperature mixes to obtain mixed liquor in the ratio of 1:1-3, by the cloth for being soaked with mixed liquor or is soaked with mixed
The sponge block for closing liquid, which is fitted into bottom, to be had in through-hole and stackable round, is placed in the fermenting cellar that temperature is 25-28 DEG C
Carry out fermentation 5-6 days；
The nutrient solution includes that following raw material is prepared: 1000 parts of wheat bran juice, 20 parts of rice flour and white granulated sugar 40
Part；
The wheat bran juice adds 300g wheat bran the preparation method comprises the following steps: pressing in every 1000g water, boil 10min and filter to take filtrate, institute
Filtrate adds water to mend to 1000g to obtain the wheat bran juice.
The cloth can be arbitrary fabric fibre block, geotextile cloth preferably, and specification is 700~1000g/
m2, the sponge block is PVA high density water-absorbing sponge.
The beneficial effects of the present invention are: (1) it is by using the cloth or sponge block that are soaked with mixed liquor as green muscardine fungus raw
The immobilization carrier of spore is produced and produces, compared with the nutrient matrixes such as traditional rice, wheat bran, corn flour, rate of drying is fast, spore easily divides
From and small in size due to cloth or sponge block, surface area makes greatly its inoculum concentration, and big (its inoculum concentration is the isometric culture of tradition
2.5-5 times of base inoculum concentration), the average fermentation period is 5-6 days, compared with 7-10 days of conventional solid matrix culture, is substantially reduced
Therefore incubation time substantially increases the production efficiency and stable product quality of the high cryptogam of green muscardine fungus；(2) due to green muscardine fungus point
Spore is given birth to as the raw spore of gas, in incubation, since round middle layer has venthole, keeps the ventilatory effect of middle layer
Good, matrix height≤3cm, is conducive to spore generation in addition, and entire round is closed in incubation, in cloth or sponge block
Moisture hardly scatter and disappear, so also can guarantee high humidity needed for green muscardine fungus mycelia growth course without Regulate Environment humidity
Degree, simplifies production technology；(3) immobilization carrier for being produced and being produced spore as green muscardine fungus using fabric cloth or sponge block, facilitates receipts
It is operated when collecting spore；(4) above-mentioned nutrient solution is added in green muscardine fungus hypha fermentation liquid, it is ensured that the bacterium being inoculated on cloth or sponge block
Silk regrowth and the nutrition for producing spore, while increasing breeding coefficient, increase the conidial production of green muscardine fungus on cloth or sponge block
Amount.
Specific embodiment
To explain the technical content, the achieved purpose and the effect of the present invention in detail, it is explained below in conjunction with embodiment.
The most critical design of the present invention is: the cloth or the sponge block generation that green muscardine fungus fluid nutrient medium and nutrient solution will be soaked with
The immobilization carrier of spore, cloth or sponge block are produced and produced as green muscardine fungus for nutrient matrixes such as conventional rice, wheat bran, corn flour
Surface area it is big, green muscardine fungus is in cloth or sponge block fast-growth and produces spore, and muscardine spore is then sieved into green muscardine fungus
High cryptogam.
The present invention provides a kind of preparation method of high cryptogam of green muscardine fungus, comprising: green muscardine fungus hypha fermentation liquid with through steam height
Temperature sterilizes and the nutrient solution of cooling room temperature mixes to obtain mixed liquor in the ratio of 1:1-3, by the cloth for being soaked with mixed liquor or is soaked with mixed
The sponge block for closing liquid, which is fitted into bottom, to be had in through-hole and stackable round, is placed in the fermenting cellar that temperature is 25-28 DEG C
Carry out fermentation 5-6 days；The nutrient solution includes that following raw material is prepared: 1000 parts of wheat bran juice, 20 parts of rice flour and white
40 parts of granulated sugar；The wheat bran juice adds 300g wheat bran the preparation method comprises the following steps: pressing in every 1000g water, boil 10min and filter to take filtrate,
Gained filtrate adds water to mend to 1000g to obtain the wheat bran juice.
The cloth can be arbitrary fabric fibre block, geotextile cloth preferably, and specification is 700~1000g/
m2, the sponge block is PVA high density water-absorbing sponge.
As can be seen from the above description, the beneficial effects of the present invention are: by using the cloth or sponge block for being soaked with mixed liquor
The immobilization carrier that spore is produced and produced as green muscardine fungus, compared with the nutrient matrixes such as traditional rice, wheat bran, corn flour, rate of drying
Fastly, spore is easily separated, and small in size due to cloth or sponge block, and surface area makes greatly its inoculum concentration, and big (its inoculum concentration is passes
2.5-5 times of isometric culture medium inoculated amount of uniting)；The average fermentation period is 5-6 days, compared with the 7-10 of conventional solid matrix culture
It, substantially reduces incubation time.Therefore, the production efficiency and stable product quality of the high cryptogam of green muscardine fungus are substantially increased；By
It is the raw spore of gas in green muscardine fungus conidium, in incubation, since round middle layer has through-hole, keeps the logical of middle layer
Gas effect is good, in addition matrix height≤3cm, is conducive to spore generation, and entire round is closed in incubation, cloth or sea
Moisture in continuous block hardly scatters and disappears, so needed for also can guarantee in green muscardine fungus mycelia growth course without Regulate Environment humidity
High humility, simplify production technology；The immobilization carrier for being produced and being produced spore as green muscardine fungus using cloth or sponge block, facilitates receipts
It is operated when collecting spore；Above-mentioned nutrient solution is added in green muscardine fungus hypha fermentation liquid, it is ensured that the mycelia being inoculated on cloth or sponge block
Regrowth and the nutrition for producing spore, while increasing breeding coefficient, increase the conidial yield of green muscardine fungus on cloth or sponge block.
Further, the culture of the green muscardine fungus hypha fermentation liquid: by the examination for filling slant medium of green muscardine fungus strain
Pipe secondary culture, activation under the conditions of 24-26 DEG C, when the chamfered surface of the slant medium covers with spore in sterile working
In platform, through steam high-temperature sterilization and it is cooled to room temperature from the access of a small amount of spore of inclined-plane picking and lawn of the slant medium
In fluid nutrient medium；Fluid nutrient medium after inoculation is in oscillation 25 ± 1 DEG C of constant temperature incubation 60-72h of case, wait grow up to sticky mycelium
Afterwards up to the green muscardine fungus hypha fermentation liquid.
Further, the fluid nutrient medium includes that following raw material is prepared: 1000 parts of water, corn flour 20
Part, 20 parts of rice flour and 40 parts of white granulated sugar.
Seen from the above description, the beneficial effects of the present invention are: using corn flour, rice flour and white granulated sugar as Liquid Culture
Base is prepared simply, and raw material is inexpensive, from a wealth of sources, reduces production cost.
Further, the length of the cloth or sponge block is 4-6cm, width 2-4cm, is highly 0.3-0.5cm.
Seen from the above description, the beneficial effects of the present invention are: the length of cloth or sponge block is smaller, relatively
In simply with for long cloth or strip sponge, surface area is bigger, space can be improved in the more green muscardine fungus of the same space culture
Utilization rate, so that sporulation quantity increases, so that the spore that vibrosieve obtains is more, i.e., so that green muscardine fungus in same space
Conidial output increased.
Further, the cloth for being soaked with mixed liquor or the sponge block for being soaked with mixed liquor the preparation method comprises the following steps: will
Cloth described in 10-15 block or the sponge block are put into the mixed liquor described in 100mL, take out after 20-60S and are soaked with mixing up to described
The cloth of liquid or the sponge block for being soaked with mixed liquor.
Seen from the above description, the beneficial effects of the present invention are: the cloth or sponge block are directly immersed in mixed liquor,
Quick and easy completion inoculation, and nutrient solution has been adsorbed on cloth or sponge block, green muscardine fungus growth is met and has produced spore
It needs.
Further, the geotextile cloth that the cloth is preferably, specification are 700~1000g/m2, the sponge
Block is PVA high density water-absorbing sponge.
Seen from the above description, the beneficial effects of the present invention are: the geotextile cloth or high density water suctions
The moisture retention of sponge block is good, in addition the sealing of round, it is possible to reduce humidity during the growth of green muscardine fungus mycelia and production spore
Regulation.
A specific embodiment of the invention has:
A kind of preparation method of the high cryptogam of green muscardine fungus, includes the following steps:
The culture of 1 green muscardine fungus hypha fermentation liquid
1.1 strains: the good green muscardine fungus of spore is produced.Generally with 4 DEG C of inclined-plane cryo-conservation.
1.2 slant strains: in advance by green muscardine fungus strain test tube slant under the conditions of 25 ± 1 DEG C secondary culture, work as inclined-plane
Surface can be used when covering with spore.Slant medium is potato dextrose agar (PDA) general in microculture
Culture medium.
1.3 fluid nutrient mediums: 20 parts of corn flour by weight, 20 parts of rice flour and 40 parts of white granulated sugar are added after mixing
1000 parts of water, fluid nutrient medium is obtained, 200mL culture medium, steam high-temperature sterilization at 121 DEG C are packed into each 500mL triangular flask
30min, it is spare after cooling.
1.4 inoculations: in aseptic operating platform, a small amount of spore of picking and lawn access aforesaid liquid culture from strain inclined plane
In base.
1.5 shaking table cultures: shaken cultivation 60-72h in 25 ± 1 DEG C of constant-temperature shaking incubators, after growing up to sticky mycelium
As green muscardine fungus hypha fermentation liquid.
The inoculated and cultured of 2 fungus blocks
The preparation method of 2.1 nutrient solutions: it by 300g wheat bran is added in every 1000g water, boils 10min and filters to take filtrate, gained
Filtrate adds water to mend to 1000g to obtain the wheat bran juice, 1000 parts of the wheat bran juice, 20 parts of rice flour and 40 parts of white granulated sugar are mixed to obtain institute
Nutrient solution is stated, the triangular flask of each 500mL is packed into nutrient solution described in 200mL, and the steam high-temperature sterilization 30min at 121 DEG C is cooling
It is spare afterwards.
2.2 cloths or sponge block sterilization treatment: it is 4-6cm, width 2-4cm by length, is highly the soil of 0.3-0.5cm
(specification is 700~1000g/m to work fabric panel2) or PVA high density water-absorbing sponge it is packaged enter polybag, under the conditions of 121 DEG C
Steam high-temperature sterilization 30min.
2.3 inoculations: in the closed room for carrying out space sterilizing in advance, by the green muscardine fungus hypha fermentation liquid and through steam
High-temperature sterilization and the nutrient solution of cooling room temperature are mixed in the ratio of 1:1-3, stir to obtain mixed liquor, i.e. inoculum concentration is 25%-50%,
The above-mentioned cloth or sponge block handled through steam high-temperature sterilization of 10-15 block is put into 100mL mixed liquor, every 5S stirs mixed once
Cloth or sponge block in liquid after 20-60S, after cloth or sponge block absorb saturation, take out to obtain the cloth for being soaked with mixed liquor
Block or the sponge block for being soaked with mixed liquor.
2.4 cultures: the cloth for being soaked with mixed liquor or the sponge block loading for being soaked with mixed liquor are had been subjected into surface
The bottom of disinfection have in through-hole and stackable plastics fermenting case (outside dimension: 480 × 355 × 170mm, inside dimension: 445 ×
320 × 165mm), it is transferred to fermenting cellar culture immediately, is placed in the fermenting cellar that temperature is 25-28 DEG C and carries out fermentation 5-6 days.
Potato dextrose agar described in step 1.2 (PDA) culture medium, specific formula are as follows: potato containing 100g (is boiled
Filtering), 10g glucose, 20g agar and water 1000g；
Nutrient solution described in step 2.3, specific formula are as follows: 1000 parts of wheat bran juice, 20 parts of rice flour and 40 parts of white granulated sugar, institute
State wheat bran juice by every 1000g water plus 300g wheat bran the preparation method comprises the following steps: boiling 10min and filtering to take filtrate, gained filtrate adds water
It mends to 1000g and obtains the wheat bran juice；
The length of cloth described in step 2.3 or sponge block is 4-6cm, width 2-4cm, is highly 0.3-0.5cm.
Embodiment one
The preparation method of the high cryptogam of the green muscardine fungus of the present embodiment, includes the following steps:
The culture of 1 green muscardine fungus hypha fermentation liquid
1.1 strains: green muscardine fungus (MaZPTR-01) bacterial strain, forest conservation research institute, Fujian Academy of Forestry save,
For known bacterial strain.
1.2 slant strains: in advance by green muscardine fungus strain test tube slant under the conditions of 25 ± 1 DEG C secondary culture, activation, when
Chamfered surface can be used when covering with spore.Slant medium is potato dextrose agar general in microculture
(PDA) culture medium.
1.3 fluid nutrient mediums: 20 parts of corn flour by weight, 20 parts of rice flour and 40 parts of white granulated sugar are added after mixing
1000 parts of water, fluid nutrient medium is obtained, prepares fluid nutrient medium 2L, loading 200mL culture medium in each 500mL triangular flask, 121
Steam high-temperature sterilization 30min at DEG C, it is spare after cooling.
1.4 inoculations: in aseptic operating platform, a small amount of spore of picking and lawn access aforesaid liquid culture from strain inclined plane
In base.
1.5 shaking table cultures: shaken cultivation 60h in 25 ± 1 DEG C of constant-temperature shaking incubators, after growing up to sticky mycelium as
Green muscardine fungus hypha fermentation liquid.
The inoculated and cultured of 2 fungus blocks
The preparation method of 2.1 nutrient solutions: it by 300g wheat bran is added in every 1000g water, boils 10min and filters to take filtrate, gained
Filtrate adds water to mend to 1000g to obtain the wheat bran juice, 1000 parts of the wheat bran juice, 20 parts of rice flour and 40 parts of white granulated sugar are mixed to obtain institute
Nutrient solution is stated, the triangular flask of each 500mL is packed into nutrient solution described in 200mL, and steam high-temperature sterilization 30min, cold at 121 DEG C
But spare after.
2.2 cloths or sponge block sterilization treatment: it is 4cm, width 2cm by length, is highly the geotextile cloth of 0.3cm
Block or high density water-absorbing sponge it is packaged enter polypropylene plastics pocket (being resistant to moist heat sterilization), the steam high-temperature sterilization under the conditions of 121 DEG C
30min。
2.3 inoculations: in the closed room for carrying out space sterilizing in advance, by the green muscardine fungus hypha fermentation liquid and through steam
High-temperature sterilization and the nutrient solution of cooling room temperature are mixed in the ratio of 1:1, stir to obtain mixed liquor, i.e. inoculum concentration is 50%, by 10 pieces
The above-mentioned cloth handled through steam high-temperature sterilization or sponge block are put into 100mL mixed liquor, and every 5S stirs the cloth in mixed once liquid
Block or sponge block after 20S, after cloth or sponge block absorb saturation, take out to obtain the cloth for being soaked with mixed liquor or the leaching
There is the sponge block of mixed liquor.
2.4 cultures: the cloth for being soaked with mixed liquor or the sponge block loading for being soaked with mixed liquor are had been subjected into surface
The bottom of disinfection have in through-hole and stackable plastics fermenting case (outside dimension: 480 × 355 × 170mm, inside dimension: 445 ×
320 × 165mm), it is transferred to fermenting cellar culture immediately, is placed in the fermenting cellar that temperature is 25-28 DEG C and carries out fermentation 5 days.
3 cultivation results
Surface to the cloth for being soaked with mixed liquor or the sponge block for being soaked with mixed liquor covers with the mitogenetic spore of green muscardine fungus
Son, by tunning be placed at shady and cool ventilation naturally dry dust base (or 12-24h is dried in 28-30 DEG C of baking oven, it is aqueous
Rate≤10%), it is then sieved, is closed the discharge port of vibrating screen when screening, on culture medium using 200 mesh vibrating screens
Conidia powder opens discharge port after having sieved, and the high cryptogam of green muscardine fungus is made；The high cryptogam agent 5g of green muscardine fungus obtained is weighed at random, is used
0.03% Tween-80 solution is diluted, and blood counting chamber counts, and the average spore content of the high cryptogam of green muscardine fungus is 5.0 ×
1010Spore/g.
Embodiment two
The preparation method of the high cryptogam of the green muscardine fungus of the present embodiment, includes the following steps:
The culture of 1 green muscardine fungus hypha fermentation liquid
1.1 strains: green muscardine fungus (MaYTTR-04) bacterial strain, forest conservation research institute, Fujian Academy of Forestry save,
For known bacterial strain.
1.2 slant strains: in advance by green muscardine fungus strain test tube slant under the conditions of 25 ± 1 DEG C secondary culture, activation, when
Chamfered surface can be used when covering with spore.Slant medium is potato dextrose agar general in microculture
(PDA) culture medium.
1.3 fluid nutrient mediums: 20 parts of corn flour by weight, 20 parts of rice flour and 40 parts of white granulated sugar are added after mixing
1000 parts of water, fluid nutrient medium is obtained, prepares fluid nutrient medium 4L, loading 200mL culture medium in each 500mL triangular flask, 121
Steam high-temperature sterilization 50min at DEG C, it is spare after cooling.
1.4 inoculations: in aseptic operating platform, a small amount of spore of picking and lawn access aforesaid liquid culture from strain inclined plane
In base.
1.5 shaking table cultures: shaken cultivation 72h in 25 ± 1 DEG C of constant-temperature shaking incubators, after growing up to sticky mycelium as
Green muscardine fungus hypha fermentation liquid.
The inoculated and cultured of 2 fungus blocks
The preparation method of 2.1 nutrient solutions: it by 300g wheat bran is added in every 1000g water, boils 10min and filters to take filtrate, gained
Filtrate adds water to mend to 1000g to obtain the wheat bran juice, 1000 parts of the wheat bran juice, 20 parts of rice flour and 40 parts of white granulated sugar are mixed to obtain institute
Nutrient solution is stated, the triangular flask of each 500mL is packed into nutrient solution described in 200mL, and the steam high-temperature sterilization 50min at 121 DEG C is cooling
It is spare afterwards.
2.2 cloths or sponge block sterilization treatment: it is 6cm, width 4cm by length, is highly the geotextile cloth of 0.5cm
Block or high density water-absorbing sponge it is packaged enter polypropylene plastics pocket (being resistant to moist heat sterilization), the steam high-temperature sterilization under the conditions of 121 DEG C
50min。
2.3 inoculations: in the closed room for carrying out space sterilizing in advance, by the green muscardine fungus hypha fermentation liquid and through steam
High-temperature sterilization and the nutrient solution of cooling room temperature are mixed in the ratio of 1:3, stir to obtain mixed liquor, i.e. inoculum concentration is 25%, by 15 pieces
The above-mentioned cloth handled through steam high-temperature sterilization or sponge block are put into 100mL mixed liquor, and every 5S stirs the cloth in mixed once liquid
Block or sponge block after 60S, after cloth or sponge block absorb saturation, take out to obtain the cloth for being soaked with mixed liquor or the leaching
There is the sponge block of mixed liquor,
2.4 cultures: the cloth for being soaked with mixed liquor or the sponge block loading for being soaked with mixed liquor are had been subjected into surface
The bottom of disinfection have in through-hole and stackable plastics fermenting case (outside dimension: 480 × 355 × 170mm, inside dimension: 445 ×
320 × 165mm), it is transferred to fermenting cellar culture immediately, is placed in the fermenting cellar that temperature is 25-28 DEG C and carries out fermentation 6 days.
3 cultivation results
Surface to the cloth for being soaked with mixed liquor or the sponge block for being soaked with mixed liquor covers with the mitogenetic spore of green muscardine fungus
Son, by tunning be placed at shady and cool ventilation naturally dry dust base (or 12-24h is dried in 28-30 DEG C of baking oven, it is aqueous
Rate≤10%), it is then sieved, is closed the discharge port of vibrating screen when screening, on culture medium using 200 mesh vibrating screens
Conidia powder opens discharge port after having sieved, and the high cryptogam of green muscardine fungus is made；The high cryptogam agent 5g of green muscardine fungus obtained is weighed at random, is used
0.03% Tween-80 solution is diluted, and blood counting chamber counts, and the average spore content of the high cryptogam of green muscardine fungus is 4.5 ×
1010Spore/g.
Embodiment three
The preparation method of the high cryptogam of the green muscardine fungus of the present embodiment, includes the following steps:
The culture of 1 green muscardine fungus hypha fermentation liquid
1.1 strains: green muscardine fungus (MaFZ-01) bacterial strain, forest conservation research institute, Fujian Academy of Forestry save, and are
Known bacterial strain.
1.2 slant strains: in advance by green muscardine fungus strain test tube slant under the conditions of 25 ± 1 DEG C secondary culture, activation, when
Chamfered surface can be used when covering with spore.Slant medium is potato dextrose agar general in microculture
(PDA) culture medium.
1.3 fluid nutrient mediums: 20 parts of corn flour by weight, 20 parts of rice flour and 40 parts of white granulated sugar are added after mixing
1000 parts of water, fluid nutrient medium is obtained, prepares fluid nutrient medium 3L, loading 200mL culture medium in each 500mL triangular flask, 121
Steam high-temperature sterilization 40min at DEG C, it is spare after cooling.
1.4 inoculations: in aseptic operating platform, a small amount of spore of picking and lawn access aforesaid liquid culture from strain inclined plane
In base.
1.5 shaking table cultures: shaken cultivation 66h in 25 ± 1 DEG C of constant-temperature shaking incubators, after growing up to sticky mycelium as
Green muscardine fungus hypha fermentation liquid.
The inoculated and cultured of 2 fungus blocks
The preparation method of 2.1 nutrient solutions: it by 300g wheat bran is added in every 1000g water, boils 10min and filters to take filtrate, gained
Filtrate adds water to mend to 1000g to obtain the wheat bran juice, 1000 parts of the wheat bran juice, 20 parts of rice flour and 40 parts of white granulated sugar are mixed to obtain institute
Nutrient solution is stated, the triangular flask of each 500mL is packed into nutrient solution described in 200mL, and the steam high-temperature sterilization 40min at 121 DEG C is cooling
It is spare afterwards.
2.2 cloths or sponge block sterilization treatment: it is 5cm, width 3cm by length, is highly the geotextile cloth of 0.4cm
Block or high density water-absorbing sponge it is packaged enter polypropylene plastics pocket (being resistant to moist heat sterilization), the steam high-temperature sterilization under the conditions of 121 DEG C
40min。
2.3 inoculations: in the closed room for carrying out space sterilizing in advance, by the green muscardine fungus hypha fermentation liquid and through steam
High-temperature sterilization and the nutrient solution of cooling room temperature are mixed in the ratio of 1:2, stir to obtain mixed liquor, i.e. inoculum concentration is 33.3%, by 12
The above-mentioned cloth handled through steam high-temperature sterilization of block or sponge block are put into 100mL mixed liquor, and every 5S is stirred in mixed once liquid
Cloth or sponge block after 40S, after cloth or sponge block absorb saturation, take out to obtain the cloth or described for being soaked with mixed liquor
It is soaked with the sponge block of mixed liquor,
2.4 cultures: the cloth for being soaked with mixed liquor or the sponge block loading for being soaked with mixed liquor are had been subjected into surface
The bottom of disinfection have in through-hole and stackable plastics fermenting case (outside dimension: 480 × 355 × 170mm, inside dimension: 445 ×
320 × 165mm), it is transferred to fermenting cellar culture immediately, is placed in the fermenting cellar that temperature is 25-28 DEG C and carries out fermentation 5.5 days.
3 cultivation results
Surface to the cloth for being soaked with mixed liquor or the sponge block for being soaked with mixed liquor covers with the mitogenetic spore of green muscardine fungus
Son, by tunning be placed at shady and cool ventilation naturally dry dust base (or 12-24h is dried in 30-35 DEG C of baking oven, it is aqueous
Rate≤10%), it is then sieved, is closed the discharge port of vibrating screen when screening, on culture medium using 200 mesh vibrating screens
Conidia powder opens discharge port after having sieved, and the high cryptogam of green muscardine fungus is made；The high cryptogam agent 5g of green muscardine fungus obtained is weighed at random, is used
0.03% Tween-80 solution is diluted, and blood counting chamber counts, and the average spore content of the high cryptogam of green muscardine fungus is 4.8 ×
1010Spore/g.
In conclusion the preparation method of the high cryptogam of green muscardine fungus provided by the invention, by using the cloth for being soaked with mixed liquor
Or sponge block produces and produces the immobilization carrier of spore as green muscardine fungus, compared with the nutrient matrixes such as traditional rice, wheat bran, corn flour,
Rate of drying is fast, spore is easily separated, and small in size due to cloth or sponge block, and surface area makes greatly its inoculum concentration, and big (it connects
Kind amount is 2.5-5 times of the isometric culture medium inoculated amount of tradition), the average fermentation period is 5-6 days, compared with conventional solid matrix culture
7-10 days, substantially reduce incubation time, therefore, substantially increase the production efficiency of the high cryptogam of green muscardine fungus and product quality is steady
It is fixed；Since green muscardine fungus conidium is the raw spore of gas, in incubation, since round middle layer has through-hole, keep intermediate
The ventilatory effect of layer is good, in addition matrix height≤3cm, is conducive to spore generation, and entire round is closed in incubation, cloth
Moisture in block or sponge block hardly scatters and disappears, so also can guarantee green muscardine fungus mycelia growth course without Regulate Environment humidity
Needed for high humility, simplify production technology；The immobilization carrier of spore is produced and produced as green muscardine fungus using cloth or sponge block,
Facilitate operation when collecting spore；Above-mentioned nutrient solution is added in green muscardine fungus hypha fermentation liquid, it is ensured that be inoculated on cloth or sponge block
Regeneration of Mycelium it is long and produce the nutrition of spore, while increasing the conidial yield of green muscardine fungus on cloth or sponge block.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalents made by bright description are applied directly or indirectly in relevant technical field, are similarly included in this hair
In bright scope of patent protection.
Claims (5)
1. a kind of preparation method of the high cryptogam of green muscardine fungus characterized by comprising green muscardine fungus hypha fermentation liquid with through steam high temperature
It sterilizes and the nutrient solution for cooling down room temperature mixes to obtain mixed liquor in the ratio of 1:1-3, by the cloth for being soaked with mixed liquor or be soaked with mixing
The sponge block of liquid is fitted into bottom with through-hole and in stackable round, be placed in the fermenting cellar that temperature is 25-28 DEG C into
Row fermentation 5-6 days；
The nutrient solution includes that following raw material is prepared: 1000 parts of wheat bran juice, 20 parts of rice flour and 40 parts of white granulated sugar；
The wheat bran juice adds 300g wheat bran the preparation method comprises the following steps: pressing in every 1000g water, boil 10min and filter to take filtrate, gained filter
Liquid adds water to mend to 1000g to obtain the wheat bran juice；
The cloth is geotextile cloth, and specification is 700~1000g/m2, the sponge block is PVA high density water suction sea
It is continuous；
The length of the cloth or sponge block is 4-6cm, width 2-4cm, is highly 0.3-0.5cm.
2. a kind of preparation method of the high cryptogam of green muscardine fungus according to claim 1, which is characterized in that the green muscardine fungus mycelia
The culture of fermentation liquid: by green muscardine fungus strain with filling the test tube of slant medium secondary culture, activation under the conditions of 24-26 DEG C,
When the chamfered surface of the slant medium covers with spore in aseptic operating platform, from the inclined-plane picking of the slant medium
A small amount of spore and lawn access are through in steam high-temperature sterilization and the fluid nutrient medium that is cooled to room temperature；Fluid nutrient medium after inoculation
In 25 ± 1 DEG C of constant temperature incubation 60-72h of constant temperature oscillation case, up to the green muscardine fungus hypha fermentation liquid after growing up to sticky mycelium.
3. a kind of preparation method of the high cryptogam of green muscardine fungus according to claim 2, which is characterized in that the fluid nutrient medium
It is prepared including following raw material: 1000 parts of water, 20 parts of corn flour, 20 parts of rice flour and 40 parts of white granulated sugar.
4. a kind of preparation method of the high cryptogam of green muscardine fungus according to claim 3, which is characterized in that described to be soaked with mixed liquor
Cloth or the sponge block for being soaked with mixed liquor the preparation method comprises the following steps: cloth described in 10-15 block or the sponge block are put into
The mixed liquor described in 100mL takes out after 20-60S up to the cloth for being soaked with mixed liquor or the sponge for being soaked with mixed liquor
Block.
5. a kind of preparation method of the high cryptogam of green muscardine fungus according to claim 1, which comprises the steps of:
Green muscardine fungus strain is used the test tube for filling slant medium secondary culture, activation under the conditions of 24-26 DEG C by step 1, works as institute
It states when the chamfered surface of slant medium covers with spore in aseptic operating platform, it is a small amount of from the inclined-plane picking of the slant medium
Spore and lawn access are through in steam high-temperature sterilization and the fluid nutrient medium that is cooled to room temperature；Fluid nutrient medium after inoculation is shaking
25 ± 1 DEG C of constant temperature incubation 60-72h of case are swung, up to the green muscardine fungus hypha fermentation liquid after growing up to sticky mycelium；
The slant medium is potato dextrose agar, and the potato dextrose agar is by weight
It is prepared including following raw material: 100 portions of potatos, 10 parts of glucose and 20 parts of agar and 1000 parts of water；The liquid
Body culture medium includes that following raw material is prepared: 1000 parts of water, 20 parts of corn flour, and 20 parts of rice flour and 40 parts of white granulated sugar；
Step 2, the ratio that the green muscardine fungus hypha fermentation liquid is pressed to 1:1-3 with the nutrient solution through steam high-temperature sterilization and cooling room temperature
Example mixes to obtain mixed liquor, will be put into the mixed liquor described in 100mL by the 10-15 block cloth or sponge block of steam high-temperature sterilization
In, after 20-60S the cloth or the sponge block for being soaked with mixed liquor that be soaked with mixed liquor, by the mixed liquor that is soaked with
Cloth or the sponge block for being soaked with mixed liquor are fitted into bottom in through-hole and stackable round, and being placed in temperature is
Fermentation 5-6 days is carried out in 25-28 DEG C of fermenting cellar；
The nutrient solution includes that following raw material is prepared: 1000 parts of wheat bran juice, 20 parts of rice flour and 40 parts of white granulated sugar, institute
State wheat bran juice by every 1000g water plus 300g wheat bran the preparation method comprises the following steps: boiling 10min and filtering to take filtrate, gained filtrate adds water
It mends to 1000g and obtains the wheat bran juice；The length of the cloth or sponge block is 4-6cm, width 2-4cm, is highly 0.3-
0.5cm。
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| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| 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 |
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