CN106350457B - A kind of preparation method of the high cryptogam of muscardine - Google Patents
A kind of preparation method of the high cryptogam of muscardine Download PDFInfo
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Abstract
The present invention relates to a kind of preparation methods of the high cryptogam of muscardine, including: muscardine hypha fermentation liquid mixes to obtain culture solution 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 culture solution or the sponge block for being soaked with culture solution, 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 water, 20 parts of corn flour, and 20 parts of rice flour and 40 parts of white granulated sugar.The preparation method of the high cryptogam of muscardine 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 muscardine of this method preparation is 8.0 × 1010~9.5 × 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 muscardine.
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.
Muscardine (Beauveria spp) is one of the disinsection fungal for being widely used in biological control of insect pests both at home and abroad, is recognized
For a kind of insect pathogenic fungus for being most potentiality to be exploited, can infect 15 Ge Mu149Ge sections 700 various insects and a variety of mites
Class has exceeded a century using the research and practice of muscardine pest control, strong with pathogenicity, free from environmental pollution, the lasting period is long
The features such as, be a kind of elegant formulations of sustainable pest control, and the current main fungal insecticide for biological control it
One, there is important application value in biological control.Conidium is the main component of muscardine dosage form, and the high spore of muscardine
Powder (female powder) refers to the conidia of beauveria bassiana high by the spore content for extracting or sieving, and the high cryptogam of muscardine can be used as
How largely, quickly a variety of dosage form raw materials such as wettable powder, finish, microcapsule formulations, purposes is very extensive, therefore, prepare white
Stiff bacterium spore is the basis of quick, a large amount of high cryptogams of preparation muscardine.
Currently, the preparation of conidia of beauveria bassiana is mainly in such a way that liquid consolidates biphasic fermentation 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 muscardine
The production application and control efficiency of high cryptogam.
As an improvement, the Chinese invention patent of 1212773 C of Publication No. CN discloses beauveria bassiana non-woven fabric fungal band
Manufacturing technique method, this method include five production processes, the i.e. acquirement of strain, primary inclined plane Spawn incubation, liquid seeds training
It supports, the inoculated and cultured of liquid fermentation tank production and bacterium item.Using sterile cloth as the immobilization carrier of muscardine, finally by muscardine
Non-woven fabric fungal band is directly used in control of insect, although avoiding the problem of spore is separated with matrix difficulty to a certain extent, its
Liquid fermentation uses three grade fermemtation, and fermentation time needs 8-10 days, and fermentation period is longer, affects production efficiency, and it is made
Most product can only use as bacterium item, limit its use form, wettable cannot be can be used as the high cryptogam of separation
The raw material of pulvis, finish, microcapsule formulations etc., meanwhile, bacterium item is larger, and surface area is smaller to also limit inoculum concentration to a certain degree,
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 muscardine.
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 muscardine
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 muscardine, comprising: muscardine hypha fermentation liquid with through steam height
Temperature sterilizes and the nutrient solution of cooling room temperature mixes to obtain culture solution in the ratio of 1:1-3, by the cloth for being soaked with culture solution or is soaked with training
The sponge block of nutrient solution, 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 water, 20 parts of corn flour, and 20 parts of rice flour and white
40 parts of granulated sugar.
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 culture solution as muscardine 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 muscardine；(2) due to muscardine point
Spore is given birth to as the raw spore of gas, in incubation, since round middle layer has through-hole, keeps the ventilatory effect of middle layer good,
In addition matrix height≤3cm is conducive to spore generation, and entire round is closed, and the moisture in cloth or sponge block is hardly
It can scatter and disappear, so also can guarantee high humility needed for muscardine mycelia growth course without Regulate Environment humidity, simplify life
Production. art；(3) immobilization carrier of spore is produced and produced as muscardine using cloth or sponge block, it is small in size, facilitate collection spore
When operate；(4) above-mentioned nutrient solution is added in muscardine hypha fermentation liquid, it is ensured that the Regeneration of Mycelium being inoculated on cloth or sponge block
Nutrition that is long and producing spore, while increasing breeding coefficient, increase the yield of cloth or the conidia of beauveria bassiana on sponge block.
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 muscardine fluid nutrient medium and nutrient solution will be soaked with
The immobilization carrier of spore, cloth or sponge block are produced and produced as muscardine for nutrient matrixes such as conventional rice, wheat bran, corn flour
Surface area it is big, muscardine is in cloth or sponge block fast-growth and produces spore, and beauveria bassiana spore is then sieved into muscardine
High cryptogam.
The present invention provides a kind of preparation method of high cryptogam of muscardine, comprising: muscardine hypha fermentation liquid with through steam height
Temperature sterilizes and the nutrient solution of cooling room temperature mixes to obtain culture solution in the ratio of 1:1-3, by the cloth for being soaked with culture solution or is soaked with training
The sponge block of nutrient solution, 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
Fermentation 5-6 days is carried out, the nutrient solution includes that following raw material is prepared: 1000 parts of water, 20 parts of corn flour, rice flour 20
Part and 40 parts of white granulated sugar.
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 culture solution
The immobilization carrier that spore is produced and produced as muscardine, 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, shortens incubation time.Therefore, the production efficiency and stable product quality of the high cryptogam of muscardine are substantially increased；Due to white
Stiff bacterium conidium is the raw spore of gas, in incubation, since round middle layer has through-hole, the ventilation of middle layer is kept to imitate
Fruit is good, in addition matrix height≤3cm, is conducive to spore generation, entire round is closed, and the moisture in cloth or sponge block is several
It will not scatter and disappear, so also can guarantee high humility needed for muscardine mycelia growth course without Regulate Environment humidity, simplify
Production technology；The immobilization carrier of spore is produced and produced as muscardine using cloth or sponge block, it is small in size, facilitate collection spore
When operate；Above-mentioned nutrient solution is added in muscardine hypha fermentation liquid, it is ensured that the Regeneration of Mycelium being inoculated on cloth or sponge block is long
Nutrition with spore is produced, while increasing the yield of the conidia of beauveria bassiana on cloth or sponge block.
Further, the culture of the muscardine hypha fermentation liquid: by the examination for filling slant medium of muscardine 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 25 ± 1 DEG C of constant temperature incubation 60-72h of constant temperature oscillation case, wait grow up to sticky bacterium
Up to the muscardine hypha fermentation liquid after filament.
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 culture solution or the sponge block for being soaked with culture solution the preparation method comprises the following steps: will
Cloth described in 10-15 block or the sponge block are put into the culture solution described in 100mL, take out after 20-60S and are soaked with culture up to described
The cloth of liquid or the sponge block for being soaked with culture solution.
Seen from the above description, the beneficial effects of the present invention are: the cloth or sponge block are directly immersed in culture solution,
Quick and easy completion inoculation, and nutrient solution has been adsorbed on cloth or sponge block, muscardine 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 muscardine mycelia and production spore
Regulation.
A specific embodiment of the invention has:
A kind of preparation method of the high cryptogam of muscardine, includes the following steps:
The culture of 1 muscardine hypha fermentation liquid
1.1 strains: the good muscardine of spore is produced.Generally with inclined-plane cryo-conservation.
1.2 slant strains: in advance by muscardine 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, 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 muscardine hypha fermentation liquid.
The inoculated and cultured of 2 fungus blocks
The preparation method of 2.1 nutrient solutions: 20 parts of corn flour by weight, 20 parts of rice flour and 40 parts of white granulated sugar are uniformly mixed
Afterwards, 1000 parts of water are added, the nutrient solution is obtained, the triangular flask of each 500mL is packed into nutrient solution described in 200mL, steams at 121 DEG C
Vapour high-temperature sterilization 30min, it is spare after cooling.
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
Work fabric panel or high density water-absorbing sponge it is packaged enter polypropylene plastics pocket (being resistant to moist heat sterilization), the steam under the conditions of 121 DEG C
High-temperature sterilization 30min.
2.3 inoculations: in the closed room for carrying out space sterilizing in advance, by the muscardine 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 culture solution, 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 culture solution, every 5S stirs primary culture
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 culture solution
Block or the sponge block for being soaked with culture solution,
2.4 cultures: the cloth for being soaked with culture solution or the sponge block loading for being soaked with culture solution 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 1000g water；
Nutrient solution described in step 2.3, specific formula are as follows: 1000 parts of water by weight, 20 parts of corn flour, 20 parts of rice flour
And 40 parts of white granulated sugar；
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 muscardine of the present embodiment, includes the following steps:
The culture of 1 muscardine hypha fermentation liquid
1.1 strains: muscardine (BbMA-01) bacterial strain, forest conservation research institute, Fujian Academy of Forestry save, and are
Known bacterial strain.
1.2 slant strains: in advance by muscardine 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
Muscardine hypha fermentation liquid.
The inoculated and cultured of 2 fungus blocks
The preparation method of 2.1 nutrient solutions: 20 parts of corn flour by weight, 20 parts of rice flour and 40 parts of white granulated sugar are uniformly mixed
Afterwards, 1000 parts of water are added, the nutrient solution is obtained, the triangular flask of each 500mL is packed into nutrient solution described in 200mL, steams at 121 DEG C
Vapour high-temperature sterilization 30min, it is spare after cooling.
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 muscardine 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 culture solution, 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 culture solution, and every 5S stirs the cloth in a culture solution
Block or sponge block after 20S, after cloth or sponge block absorb saturation, take out to obtain the cloth for being soaked with culture solution or the leaching
There is the sponge block of culture solution,
2.4 cultures: the cloth for being soaked with culture solution or the sponge block loading for being soaked with culture solution 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 culture solution or the sponge block for being soaked with culture solution covers with the mitogenetic spore of muscardine
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 muscardine is made；The high cryptogam agent 5g of muscardine 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 muscardine is 8.5 ×
1010Spore/g.
Embodiment two
The preparation method of the high cryptogam of the muscardine of the present embodiment, includes the following steps:
The culture of 1 muscardine hypha fermentation liquid
1.1 strains: muscardine (BbSMYXTR-01) bacterial strain, forest conservation research institute, Fujian Academy of Forestry are protected
It deposits, is known bacterial strain.
1.2 slant strains: in advance by muscardine 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
Muscardine hypha fermentation liquid.
The inoculated and cultured of 2 fungus blocks
The preparation method of 2.1 nutrient solutions: 20 parts of corn flour by weight, 20 parts of rice flour and 40 parts of white granulated sugar are uniformly mixed
Afterwards, 1000 parts of water are added, the nutrient solution is obtained, the triangular flask of each 500mL is packed into nutrient solution described in 200mL, steams at 121 DEG C
Vapour high-temperature sterilization 50min, it is spare after cooling.
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 muscardine 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 culture solution, 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 culture solution, and every 5S stirs the cloth in a culture solution
Block or sponge block after 60S, after cloth or sponge block absorb saturation, take out to obtain the cloth for being soaked with culture solution or the leaching
There is the sponge block of culture solution,
2.4 cultures: the cloth for being soaked with culture solution or the sponge block loading for being soaked with culture solution 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 culture solution or the sponge block for being soaked with culture solution covers with the mitogenetic spore of muscardine
Son, by tunning be placed at shady and cool ventilation naturally dry dust base (or 12-24h is dried in 20-30 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 muscardine is made；The high cryptogam agent 5g of muscardine 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 muscardine is 9.2 ×
1010Spore/g.
Embodiment three
The preparation method of the high cryptogam of the muscardine of the present embodiment, includes the following steps:
The culture of 1 muscardine hypha fermentation liquid
1.1 strains: muscardine (BbTK-01) bacterial strain, forest conservation research institute, Fujian Academy of Forestry save, and are
Known bacterial strain.
1.2 slant strains: in advance by muscardine 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
Muscardine hypha fermentation liquid.
The inoculated and cultured of 2 fungus blocks
The preparation method of 2.1 nutrient solutions: 20 parts of corn flour by weight, 20 parts of rice flour and 40 parts of white granulated sugar are uniformly mixed
Afterwards, 1000 parts of water are added, the nutrient solution is obtained, the triangular flask of each 500mL is packed into nutrient solution described in 200mL, steams at 121 DEG C
Vapour high-temperature sterilization 40min, it is spare after cooling.
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 muscardine 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 culture solution, 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 culture solution, and every 5S is stirred in a culture solution
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 culture solution
It is soaked with the sponge block of culture solution,
2.4 cultures: the cloth for being soaked with culture solution or the sponge block loading for being soaked with culture solution 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 culture solution or the sponge block for being soaked with culture solution covers with the mitogenetic spore of muscardine
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 muscardine is made；The high cryptogam agent 5g of muscardine 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 muscardine is 9.5 ×
1010Spore/g.
In conclusion the preparation method of the high cryptogam of muscardine provided by the invention, by using the cloth for being soaked with culture solution
Or sponge block produces and produces the immobilization carrier of spore as muscardine, 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 muscardine and product quality is steady
It is fixed；Since conidia of beauveria bassiana 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, entire round is closed, cloth or sponge block
Interior moisture hardly scatters and disappears, so also can guarantee height needed for muscardine mycelia growth course without Regulate Environment humidity
Humidity simplifies production technology；The immobilization carrier of spore, small in size, side are produced and produced as muscardine using cloth or sponge block
It is operated when just collecting spore；Above-mentioned nutrient solution is added in muscardine hypha fermentation liquid, it is ensured that be inoculated on cloth or sponge block
Regeneration of Mycelium is long and produces the nutrition of spore, while increasing breeding coefficient, increases cloth or the conidia of beauveria bassiana on sponge block
Yield.
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 muscardine characterized by comprising muscardine hypha fermentation liquid with through steam high temperature
It sterilizes and the nutrient solution for cooling down room temperature mixes to obtain culture solution in the ratio of 1:1-3, by the cloth for being soaked with culture solution or be soaked with culture
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 water, 20 parts of corn flour, and 20 parts of rice flour and white granulated sugar
40 parts；
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 muscardine according to claim 1, which is characterized in that the muscardine mycelia
The culture of fermentation liquid: by muscardine 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 muscardine hypha fermentation liquid after growing up to sticky mycelium.
3. a kind of preparation method of the high cryptogam of muscardine 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 muscardine according to claim 3, which is characterized in that described to be soaked with culture solution
Cloth or the sponge block for being soaked with culture solution the preparation method comprises the following steps: cloth described in 10-15 block or the sponge block are put into
The culture solution described in 100mL takes out after 20-60S up to the cloth for being soaked with culture solution or the sponge for being soaked with culture solution
Block.
5. a kind of preparation method of the high cryptogam of muscardine according to claim 1, which comprises the steps of:
Muscardine 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 in perseverance
Temperature oscillation 25 ± 1 DEG C of constant temperature incubation 60-72h of case, up to the muscardine 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, 20 parts of agar and 1000 parts of water；The liquid
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 muscardine 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 culture solution, will be put into the culture solution 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 culture solution that be soaked with culture solution, by the culture solution that is soaked with
Cloth or the sponge block for being soaked with culture solution 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 water, 20 parts of corn flour, and 20 parts of rice flour and white granulated sugar
40 parts；The length of the cloth or sponge block is 4-6cm, width 2-4cm, is highly 0.3-0.5cm.
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| CN114621908B (en) * | 2022-03-16 | 2024-03-22 | 福建省林业科学研究院 | Fermentation method of beauveria bassiana serosa |
Citations (2)
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|---|---|---|---|---|
| CN1419824A (en) * | 2002-11-22 | 2003-05-28 | 安徽林苑虫草研究所 | Process for producing Beauveria globisporus non-woven fibric ribbon |
| 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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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1419824A (en) * | 2002-11-22 | 2003-05-28 | 安徽林苑虫草研究所 | Process for producing Beauveria globisporus non-woven fibric ribbon |
| 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 |
Non-Patent Citations (1)
| Title |
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| Conidia production of Beauveria sp. by solid-state fermentation for biocontrol of llex paraguariensis caterpillars;Dalla Santa HS等;《Folia Microbiol(Praha).》;20041231;第49卷(第4期);全文 |
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