CN112514918A - Embedding body composite insecticidal probiotic agent and preparation method and application thereof - Google Patents
Embedding body composite insecticidal probiotic agent and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
- A01N63/23—B. thuringiensis
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/26—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests in coated particulate form
- A01N25/28—Microcapsules or nanocapsules
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/32—Yeast
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- 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
- C12N1/16—Yeasts; Culture media therefor
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- 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
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses an embedding body composite insecticidal probiotic and a preparation method and application thereof. In the embedding process, an inner layer embedding wall structure and an outer layer embedding wall structure are adopted, thalli are adsorbed and fixed on the inner layer wall, strength and a protective layer are provided on the outer layer wall, the structural strength and the internal thalli activity of an embedding body are fully guaranteed, the slow release performance is also provided, the long-acting property of the microbial inoculum effect is guaranteed, low-temperature freeze-drying and the addition of a protective agent are adopted in the microbial inoculum preparation process, the maintenance of the microbial inoculum activity is guaranteed, the increase of the microbial inoculum biomass after use is facilitated, and the functional effect of the microbial inoculum is more obvious. When high-rise courtyards are built, the high-rise courtyards are inoculated and mixed into soil for courtyard gardens according to a certain proportion, so that the degradation of courtyard garbage can be promoted, insects can be resisted, and the activities of pests such as mosquitoes, flies and the like can be inhibited.
Description
Technical Field
The invention relates to an embedding body composite insecticidal probiotic preparation and a preparation method and application thereof, belonging to the technical field of soil conditioner preparation
Technical Field
The high-rise building courtyard (urban forest garden) saves land resources, improves urban greening rate, has excellent living environment and has good environmental protection, social and economic values. However, the growing of garden plants in open high-rise yards creates a series of problems: a) compared with the traditional courtyard cleaning, the cleaning of the courtyard garbage (leaves, branches and the like) is more difficult, and the generated rotten gas is not cleaned for a long time, so that the courtyard landscape and the sensory experience are damaged; b) insects and flies are easy to breed in the garden garbage of the high-rise courtyard, so that the living human health is threatened; c) because the bearing capacity of the high-rise building courtyard is limited, the thickness of planting soil is thinner than that of the traditional courtyard, and the water and fertilizer retention is poorer, so that the difficulty is brought to the growth of plants in the courtyard.
The yard waste is treated by the traditional treatment modes of landfill and incineration, few parts are recycled, and special treatment and disposal places are needed. And the problems of land occupation, smoke dust and the like can be caused by direct landfill and simple incineration. Valuable organic matter of the yard waste is not effectively utilized.
On the other hand, the measures for maintaining the soil fertility of the high-rise courtyard building depend on base fertilizers and additional fertilizers before soil planting. After the yard is built, the soil of the elevation yard is difficult to topdressing, and large labor and cost are required.
At present, the method for solving the problem of yard garbage in high-rise yards comprises regular cleaning and ex-situ centralized treatment, and more manpower and material resources are consumed for the collection and transportation process of yard garbage. The nutrient components in the soil are taken away along with the cleaning of the yard rubbish, and are often supplemented by periodically supplementing fertilizers, and the costs of fertilizer purchasing, transporting, fertilizing and the like are also consumed.
On the other hand, if the high-rise courtyard is not cleaned in time, organic matters are rotten and deteriorated and are easy to breed mosquitoes, and the traditional treatment method is to regularly spray insecticide, so that the physical health of resident personnel is not facilitated.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an embedding body composite insecticidal probiotic preparation with a spherical microcapsule shape and a slow release effect, a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention relates to a preparation method of an embedding body composite insecticidal probiotic, which comprises the following steps:
step one, rejuvenation of microbial inoculum
Inoculating EM microbial inoculum and BT mixed bacteria in a sterilization bottle, then adding honey and water to obtain a mixed solution, then sealing and storing the mixed solution, fermenting under magnetic stirring for more than or equal to 7 days, and testing the pH value to be less than or equal to 3.5 to obtain a rejuvenation bacteria solution; centrifuging the rejuvenation bacterial liquid, and diluting the obtained solid phase to 8-12g/L by using physiological saline to obtain concentrated bacterial liquid;
step two microbial inoculum embedding
Adding a protective agent into the concentrated bacterial liquid to obtain a microbial inoculum containing the protective agent, mixing the microbial inoculum containing the protective agent with an inner wall material to obtain microbial inoculum microspheres, mixing the microbial inoculum microspheres with an outer wall material to obtain pasty slurry, and carrying out vacuum freeze-drying on the pasty slurry to obtain the embedded body composite insecticidal probiotic; the inner wall material is porous starch; the outer wall material is a mixture containing sodium alginate, dextrin and gelatin.
In the invention, the EM microbial inoculum is a commercialized microbial inoculum which contains five types of various probiotic microorganisms, namely various microorganisms of photosynthetic bacteria, lactic acid bacteria, saccharomycetes, gram-positive actinomycetes and filamentous bacteria of a fermentation system.
The EM microbial inoculum used in the invention is purchased from EM strain stock EM microbial inoculum of Guangyu biotechnology limited company of Anhui, is in liquid state, and has the effective component content of 107～108CFU/mL。
The BT mixed bacteria are commercial bacteria agents, and are mainly mixed with three Bacillus strains of Bacillus thuringiensis subsp.
The BT mixed bacteria are purchased from Infinite ecological technology company of Hubei agricultural science and technology, are concentrated emulsions, and are tested by a plate colony counting method, and the spore content of the BT mixed bacteria is more than or equal to 1010CFU/ml。
The inventor finds that the effect of rejuvenating the two microbial inoculum together is better than the effect of rejuvenating and mixing the two microbial inoculum respectively.
Preferably, in the first step, in the mixed solution, the volume ratio of the EM microbial inoculum: BT mixed bacteria: and (3) honey: water-1.5-2.5: 0.5-1.5:2.5-3.5: 90-100.
Preferably, in the first step, the temperature for sealed storage is 25-30 ℃.
In the actual operation process, the mixture is stored in a sealed way, slowly fermented under magnetic stirring, and after 7 days, the pH value is tested to be less than or equal to 3.5; the success of the strain rejuvenation is shown along with the sour and sweet smell, and the biomass reaches 10 by adopting a plate counting method for detection8CFU/mL or more.
Preferably, in the step one, the rotation speed of the centrifugation of the rejuvenation bacteria liquid is 2500-.
Preferably, in the second step, the protective agent comprises: according to the mass ratio: whey powder, glycerin and cane sugar in a ratio of 4-6:0.5-1.5: 4-6. The protective agent of the inventor can ensure the activity and storage resistance of the microbial inoculum.
Preferably, in the second step, the solid-liquid mass-volume ratio of the protective agent to the concentrated bacterial liquid is 0.15-0.3 g:1 mL.
In the preferred scheme, in the second step, the process of mixing the microbial inoculum containing the protective agent and the inner wall material is as follows: and (3) adding the inner wall material into a microbial inoculum containing a protective agent, and stirring for 30-60 min at a speed of 50-100 r/min to obtain microbial inoculum microspheres. In the above operation, the porous starch can sufficiently adsorb the microbial inoculum.
In the embedding process, the porous starch forms the inner wall of the composite pesticidal probiotic and adsorbs and fixes thalli.
Preferably, in the second step, the liquid-solid volume mass ratio of the concentrated bacterial liquid to the inner wall material is 1 mL: 0.6 to 1.2 g.
In the preferable scheme, in the second step, the mass ratio of sodium alginate, gelatin and dextrin is 1.5-2.5: 3.5-4.5: 4-6.
Preferably, in the second step, the solid-liquid mass-volume ratio of the outer wall material to the concentrated bacterial liquid is 0.03-0.06 g:1 ml.
In the embedding process, the outer wall material forms the outer wall of the composite insecticidal probiotic, and the outer wall provides strength and a protective layer, so that the structural strength and the internal thallus activity of an embedding body are fully guaranteed, the slow release performance is also provided, and the long-acting property of the action of the microbial inoculum is guaranteed.
Preferably, in the second step, the paste slurry is subjected to vacuum freeze-drying at the temperature of below 10 ℃ until the water content is less than or equal to 5 percent, and the embedded body composite insecticidal probiotic is obtained
The invention also provides the embedding body composite insecticidal probiotic prepared by the preparation method.
The invention also provides application of the embedding body composite insecticidal probiotic prepared by the preparation method, and the embedding body composite insecticidal probiotic is applied to planting soil of an ecological courtyard.
In the application process, the embedding body composite insecticidal probiotic is doped into the planting matrix for the courtyard, and the beneficial effects are that
The composite microbial agent provided by the invention encapsulates the microbial agent in a selective permeation membrane through an embedding process to form spherical microcapsules, has the effects of slow release, improvement of adaptability and long-acting property, and can promote the degradation of yard garbage, resist insects and inhibit the activities of pests such as mosquitoes and flies when being inoculated and mixed into soil for yard gardens according to a certain proportion during the construction of high-rise yards.
The embedding body composite insecticidal probiotic prepared by the invention has the advantages that compared with the prior art:
1. the embedding body material is a natural compound, is safe and nontoxic, and is suitable for the environment of the house courtyard, which is closely contacted with the human body.
2. The bactericide has a long use history of killing insects and probiotics, and is proved to be a strain safe to a human body, so that the bactericide is suitable for the environment of a residential yard, and the like, which is in close contact with the human body.
3. The structure of the inner layer and the outer layer of embedding wall is adopted, thalli are adsorbed and fixed on the inner layer wall, and the outer layer wall provides strength and a protective layer, so that the structural strength and the internal thalli activity of an embedding body are fully guaranteed, the slow release performance is provided, and the long-acting property of the microbial inoculum effect is guaranteed.
4. The low-temperature freeze-drying and the addition of the protective agent are adopted in the preparation process of the microbial inoculum, so that the activity of the microbial inoculum is maintained, the biomass of the microbial inoculum is improved after the microbial inoculum is used, and the functional effect of the microbial inoculum is more obvious.
Detailed Description
Example 1
1. Selecting rejuvenation strains: purchasing commodity BT microbial inoculum and EM microbial inoculum, and packaging the standard effective spore (bacterial) number as follows: not less than 1010CFU/ml and 108 CFU/ml;
2. rejuvenation of strains: 6mL of EM bacterial liquid and 3mL of mixed bacterium agent are inoculated into a 500mL sterilized triangular flask in a laboratory, 9mL of honey is added, water is added to 300mL, and 5 flasks are inoculated in the same way, wherein the total volume is 1500 mL.
In a 28 ℃ incubator, the mixture is slowly fermented for 50r/min in a shaking table, after 7 days, the pH value is tested to be 3, the success of strain rejuvenation is shown along with sweet and sour smell, and the biomass is detected to reach 3 multiplied by 10 by adopting a plate counting method10CFU/mL。
3. Preparation of a microbial inoculum centrifugal liquid: and centrifuging the prepared rejuvenation bacterium liquid at the low temperature of 3000r/min for 20min, skimming a supernatant, and diluting the precipitate (thallus) of the centrifuge to 10g/L by using normal saline to obtain 500ml of concentrated bacterium liquid.
4. Preparing and manufacturing an embedding body material:
pre-adding a protective agent: adding 45g of whey powder, 10g of glycerol and 45g of sucrose into 500mL of concentrated bacterial liquid;
preparing slow-release microbial agent microspheres:
(2) preparing slow-release microbial agent microspheres: adding 400g of porous starch, stirring at the speed of 60r/min for 60min, and making into microbial inoculum microspheres after full adsorption;
(3) manufacturing an outer wall material: adding 5g of sodium alginate, 10g of gelatin and 11g of dextrin, and uniformly stirring to form paste.
5. Preparing an embedding body: and (3) putting the paste into a stainless steel plate, putting the paste into a vacuum low-temperature freeze dryer, and drying at the temperature of below 10 ℃ until the water content is less than or equal to 5%, thus completing the preparation of the embedding body microbial inoculum to obtain about 1.2kg of the embedding body microbial inoculum.
6. Adding the mixture into planting soil according to the mixing proportion of 2 percent (w/w) for use.
7. Adding planting soil, culturing at 20 deg.C for 1 week, sampling soil with thickness of less than or equal to 30cm around the embedding body, soaking in a triangular flask at a ratio of 1kg soil to 1L water to obtain suspension, and performing drop test on the supernatant with LD50 of about 500 μ g/head.
8. After the embedding body microbial inoculum is mixed into planting soil, the effect of the embedding body microbial inoculum on degrading the courtyard garbage is tested, 3% (w/w) of materials mainly comprising the courtyard garbage (leaves) are mixed into the soil, a control group is a sample which is not mixed with the embedding body microbial inoculum and is mixed with the same proportion of the courtyard garbage, the mixture is cultured for 3 weeks at the temperature of about 20 ℃, the concentration of the microbial communities in the courtyard garbage in the soil is detected, the ratio of the mixed embedding body microbial inoculum to the control group is 100 times higher, and the magnitude of an experimental group is 105CFU/mg, 10 for the control group3CFU/mg。
9. In a plot experiment, the insecticidal effect and the strain activity described in 7 and 8 strips are still maintained after 6 months, which shows that the slow release effect is effective in practical application, and 10 corn plants are planted, compared with a control group, the amount of the planted corn plants is 7-10% more than that of the control group on average in biomass (weight), and the plant height and the plant diameter are 3-5% higher and 3-5% thicker than that of the control group.
Comparative example 1-rejuvenation separately (insecticidal Performance reduced)
1. Selecting rejuvenation strains: purchasing commodity BT microbial inoculum and EM microbial inoculum, and packaging the standard effective spore (bacterial) number as follows: not less than 1010CFU/ml and ≥ 108CFU/ml；
2. Rejuvenation of strains: respectively inoculating 6mL of EM bacterial liquid and 3mL of mixed bacterial agent of LBT into two 500mL sterilized triangular flasks in a laboratory, adding 9mL of honey, adding water to 300mL, and inoculating 5 flasks by the same method to obtain 1500mL in total;
placing the two kinds of bacteria in 28 deg.C incubator, fermenting slowly in shaker for 50r/min, testing pH to 3 after 7 days, and detecting biomass of EM bacteria to 10 by plate counting method10CFU/mL, BT bacterial biomass up to 108CFU/mL。
3. Preparation of a microbial inoculum centrifugal liquid: and (2) respectively centrifuging the prepared rejuvenation EM and BT bacterial liquids at the low temperature of 4000r/min for 20min, skimming supernate, diluting the precipitate (thallus) of the centrifuge to 10g/L by using normal saline to obtain 500ml of concentrated bacterial liquid, and mixing the two concentrated bacterial liquids according to the proportion of 1:1(w/w) to prepare mixed bacterial liquid.
4. Preparing and manufacturing an embedding body material:
pre-adding a protective agent: adding 45g of whey powder, 10g of glycerol and 45g of sucrose into 500mL of mixed concentrated bacterial liquid;
preparing slow-release microbial agent microspheres:
(2) preparing slow-release microbial agent microspheres: adding 400g of porous starch, stirring at the speed of 60r/min for 60min, and making into microbial inoculum microspheres after full adsorption.
(3) Manufacturing an outer wall material: adding 5g of sodium alginate, 10g of gelatin and 11g of dextrin, and uniformly stirring to form paste.
5. Preparing an embedding body: and (3) putting the paste into a stainless steel plate, putting the paste into a vacuum low-temperature freeze dryer, and drying at the temperature of below 10 ℃ until the water content is less than or equal to 5%, thus completing the preparation of the embedding body microbial inoculum to obtain about 1.2kg of the embedding body microbial inoculum.
6. Adding the mixture into planting soil according to the mixing proportion of 2 percent (w/w) for use.
7. Adding planting soil, culturing at 20 deg.C for 1 week, sampling soil with thickness of less than or equal to 30cm around the embedding body, soaking in a triangular flask at a ratio of 1kg soil to 1L water to obtain suspension, and performing drop test on the supernatant with LD50 of about 1000 μ g/head.
8. In a plot experiment, 10 corn plants are planted, compared with a control group, the biomass (weight) of the corn plants is 5-7% more than that of the control group on average, and the plant height and the plant diameter are 2-3% higher and 2-3% thicker than those of the control group.
Comparative example 2-Change of protectant and dehydration Process (reduction of Biomass)
1. Selecting rejuvenation strains: purchasing commodity BT microbial inoculum and EM microbial inoculum, and packaging the standard effective spore (bacterial) number as follows: not less than 1010CFU/ml and 108 CFU/ml;
2. rejuvenation of strains: 6mL of EM bacterial liquid and 3mL of mixed bacterium agent are inoculated into a 500mL sterilized triangular flask in a laboratory, 9mL of honey is added, water is added to 300mL, and 5 flasks are inoculated in the same way, wherein the total volume is 1500 mL.
In a 28 ℃ incubator, the mixture is slowly fermented for 50r/min in a shaking table, after 7 days, the pH value is tested to be 3, the success of strain rejuvenation is shown along with sweet and sour smell, and the biomass is detected to reach 3 multiplied by 10 by adopting a plate counting method10CFU/mL。
3. Preparation of a microbial inoculum centrifugal liquid: and centrifuging the prepared rejuvenation bacterium liquid at the low temperature of 3000r/min for 20min, skimming a supernatant, and diluting the precipitate (thallus) of the centrifuge to 10g/L by using normal saline to obtain 500ml of concentrated bacterium liquid.
4. Preparing and manufacturing an embedding body material:
preparing slow-release microbial agent microspheres:
(2) preparing slow-release microbial agent microspheres: adding 400g of porous starch, stirring at the speed of 60r/min for 60min, and making into microbial inoculum microspheres after full adsorption;
(3) manufacturing an outer wall material: adding 5g of sodium alginate, 10g of gelatin and 11g of dextrin, and uniformly stirring to form paste.
5. Preparing an embedding body: and (3) putting the paste into a stainless steel plate, putting the paste into a normal-temperature dryer, and drying the paste at the temperature of below 25 ℃ until the water content is less than or equal to 6%, thus completing the preparation of the embedding body microbial inoculum to obtain about 1.5kg of the embedding body microbial inoculum.
6. Adding the mixture into planting soil according to the mixing proportion of 2 percent (w/w) for use.
7. Adding planting soil, culturing at 20 deg.C for 1 week, sampling soil with thickness of less than or equal to 30cm around the embedding body, soaking in a triangular flask at a ratio of 1kg soil to 1L water to obtain suspension, and performing drop test on the supernatant with LD50 of about 1000 μ g/head.
9. In a plot experiment, 10 corn plants are planted, compared with a control group, the biomass (weight) of the corn plants is increased by 6-8% on average, and the plant height and the plant diameter are 3-5% higher and 3-5% thicker than those of the control group.
Comparative example 3-changing the embedding agent ratio (the combination property is reduced)
1. Selecting rejuvenation strains: purchasing commodity BT microbial inoculum and EM microbial inoculum, and packaging the standard effective spore (bacterial) number as follows: not less than 1010CFU/ml and ≥ 108CFU/ml；
2. Rejuvenation of strains: 6mL of EM bacterial liquid and 3mL of mixed bacterium agent are inoculated into a 500mL sterilized triangular flask in a laboratory, 9mL of honey is added, water is added to 300mL, and 5 flasks are inoculated in the same way, wherein the total volume is 1500 mL.
In a 28 ℃ incubator, the mixture is slowly fermented for 50r/min in a shaking table, after 7 days, the pH value is tested to be 3, the success of strain rejuvenation is shown along with sweet and sour smell, and the biomass is detected to reach 3 multiplied by 10 by adopting a plate counting method10CFU/mL。
3. Preparation of a microbial inoculum centrifugal liquid: and centrifuging the prepared rejuvenation bacterium liquid at the low temperature of 3000r/min for 20min, skimming a supernatant, and diluting the precipitate (thallus) of the centrifuge to 10g/L by using normal saline to obtain 500ml of concentrated bacterium liquid.
4. Preparing and manufacturing an embedding body material:
(1) pre-adding a protective agent: adding 45g of whey powder, 10g of glycerol and 45g of sucrose into 500mL of concentrated bacterial liquid;
preparing slow-release microbial agent microspheres:
(2) preparing slow-release microbial agent microspheres: adding 400g of porous starch, stirring at the speed of 60r/min for 60min, and making into microbial inoculum microspheres after full adsorption;
(3) manufacturing an outer wall material: adding 15g of sodium alginate, 30g of gelatin and 30g of dextrin, and uniformly stirring to form paste.
5. Preparing an embedding body: and (3) putting the paste into a stainless steel plate, putting the paste into a vacuum low-temperature freeze dryer, and drying at the temperature of below 10 ℃ until the water content is less than or equal to 5%, thus completing the preparation of the embedding body microbial inoculum to obtain about 1.2kg of the embedding body microbial inoculum.
6. Adding the mixture into planting soil according to the mixing proportion of 2 percent (w/w) for use.
7. Adding planting soil, culturing at 20 deg.C for 1 week, sampling soil with thickness of less than or equal to 30cm around the embedding body, soaking in a triangular flask at a ratio of 1kg soil to 1L water to obtain suspension, and performing drop test on the supernatant with LD50 of about 1200 μ g/head.
8. After the embedding body microbial inoculum is mixed into planting soil, the effect of the embedding body microbial inoculum on degrading the courtyard garbage is tested, 3% (w/w) of materials mainly comprising the courtyard garbage (leaves) are mixed into the soil, a control group is a sample which is not mixed with the embedding body microbial inoculum and is mixed with the same proportion of the courtyard garbage, the mixture is cultured for 3 weeks at the temperature of about 20 ℃, the concentration of the microbial communities in the courtyard garbage in the soil is detected, the concentration of the microbial communities in the embedding body is 10 times higher than that of the control group, and the magnitude of the experimental4CFU/mg, 10 for the control group3CFU/mg。
9. In a plot experiment, the insecticidal effect and the strain activity described in 7 and 8 strips are still maintained after 6 months, which shows that the slow release effect is effective in practical application, and 10 corn plants are planted, compared with a control group, the number of the corn plants is more than that of the control group by 3% on the biomass (weight), the plant height and the plant diameter are both more than that of the control group by 3%, the plant height and the plant diameter are within 3%, and the difference is not obvious.
Claims (10)
1. A preparation method of an embedding body composite insecticidal probiotic is characterized by comprising the following steps: the method comprises the following steps:
step one, rejuvenation of microbial inoculum
Inoculating EM microbial inoculum and BT mixed bacteria in a sterilization bottle, then adding honey and water to obtain a mixed solution, then sealing and storing the mixed solution, fermenting under magnetic stirring for more than or equal to 7 days, and testing the pH value to be less than or equal to 3.5 to obtain a rejuvenation bacteria solution; centrifuging the rejuvenation bacterial liquid, and diluting the obtained solid phase to 8-12g/L by using physiological saline to obtain concentrated bacterial liquid;
step two microbial inoculum embedding
Adding a protective agent into the concentrated bacterial liquid to obtain a microbial inoculum containing the protective agent, mixing the microbial inoculum containing the protective agent with an inner wall material to obtain microbial inoculum microspheres, mixing the microbial inoculum microspheres with an outer wall material to obtain pasty slurry, and carrying out vacuum freeze-drying on the pasty slurry to obtain the embedded body composite insecticidal probiotic; the inner wall material is porous starch; the outer wall material is a mixture containing sodium alginate, dextrin and gelatin.
2. The preparation method of the inclusion complex pesticidal probiotic as claimed in claim 1, wherein the preparation method comprises the following steps: in the first step, in the mixed solution, according to the volume ratio, an EM microbial inoculum: BT mixed bacteria: and (3) honey: water-1.5-2.5: 0.5-1.5:2.5-3.5: 90-100.
3. The preparation method of the inclusion complex pesticidal probiotic as claimed in claim 1, wherein the preparation method comprises the following steps: in the first step, the temperature of sealed storage is 25-30 ℃.
4. The preparation method of the inclusion complex pesticidal probiotic as claimed in claim 1, wherein the preparation method comprises the following steps: in the first step, the rotation speed of the rejuvenation bacteria liquid centrifugation is 2500-.
5. The preparation method of the inclusion complex pesticidal probiotic as claimed in claim 1, wherein the preparation method comprises the following steps: in the second step, the protective agent comprises the following components: according to the mass ratio: whey powder, glycerin and cane sugar
＝4-6:0.5-1.5:4-6；
In the second step, the solid-liquid mass-volume ratio of the protective agent to the concentrated bacterial liquid is 0.15-0.3 g:1 mL.
6. The preparation method of the inclusion complex pesticidal probiotic as claimed in claim 1, wherein the preparation method comprises the following steps: in the second step, the process of mixing the microbial inoculum containing the protective agent and the inner wall material is as follows: adding the inner wall material into a microbial inoculum containing a protective agent, and stirring for 30-60 min at a speed of 50-100 r/min to obtain microbial inoculum microspheres;
in the second step, the liquid-solid volume mass ratio of the concentrated bacterial liquid to the inner wall material is 1 mL: 0.6 to 1.2 g.
7. The preparation method of the inclusion complex pesticidal probiotic as claimed in claim 1, wherein the preparation method comprises the following steps: in the second step, the mass ratio of sodium alginate to gelatin to dextrin is 1.5-2.5: 3.5-4.5: 4-6;
in the second step, the solid-liquid mass-volume ratio of the outer wall material to the concentrated bacterial liquid is 0.03-0.06 g:1 ml.
8. The preparation method of the inclusion complex pesticidal probiotic as claimed in claim 1, wherein the preparation method comprises the following steps: and in the second step, vacuum freeze-drying the pasty slurry at the temperature of below 10 ℃ until the water content is less than or equal to 5 percent, thus obtaining the embedded body composite insecticidal probiotic.
9. The inclusion complex pesticidal probiotic prepared by the preparation method according to any one of claims 1 to 8.
10. The application of the embedding body composite insecticidal probiotic prepared by the preparation method according to any one of claims 1 to 8 is characterized in that: the embedding body composite insecticidal probiotic is applied to planting soil of the ecological courtyard.
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