CN111328803B - Bacillus thuringiensis thallus microcapsule and preparation method and application thereof - Google Patents
Bacillus thuringiensis thallus microcapsule and preparation method and application thereof Download PDFInfo
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- CN111328803B CN111328803B CN201811560015.8A CN201811560015A CN111328803B CN 111328803 B CN111328803 B CN 111328803B CN 201811560015 A CN201811560015 A CN 201811560015A CN 111328803 B CN111328803 B CN 111328803B
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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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Agronomy & Crop Science (AREA)
- Environmental Sciences (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a bacillus thuringiensis thallus microcapsule, which is prepared by a layer-by-layer self-assembly method, takes bacillus thuringiensis thallus as a core and selects two polyelectrolyte materials with opposite charges under preparation conditions, namely chitosan and sodium alginate. The invention also provides a preparation method of the bacillus thuringiensis thallus microcapsule, which loads the bacillus thuringiensis thallus under a neutral condition to obtain a microcapsule preparation capable of controlling the release under an alkaline condition. The microcapsule preparation provided by the invention maintains the insecticidal activity of thalli, protects the active insecticidal substance-protein through double defenses of cell walls and coating (capsule walls) outside the cell walls, and helps the active insecticidal substance-protein to resist the influence of some environmental factors.
Description
Technical Field
The invention belongs to the technical field of agricultural biological preparations, and relates to a bacillus thuringiensis thallus microcapsule as well as a preparation method and application thereof.
Background
Bacillus thuringiensis (Bt) is a gram-positive bacterium with specific insecticidal activity, and can generate a parasporal crystal with specific insecticidal activity in the process of forming spores. Each type of parasporal crystal is only specifically directed against a narrow spectrum of insects, mainly against a variety of insects such as isoptera, homoptera, orthoptera, hymenoptera, lepidoptera, coleoptera, and the like, as well as nematodes, protozoa, and the like. Meanwhile, the bacillus thuringiensis is harmless to vertebrates and plants when playing a role in killing insects, is an environment-friendly biological insecticide, accounts for about 2 percent in the pesticide market, accounts for about 95 percent in the microbial pesticide market, and is very widely applied in the field of plant protection.
Bt produces parasporal crystals during the stationary phase of growth, which is the greatest difference between Bt and other bacilli. Parasporal crystals consist of one or more cytotoxic (cyt) and crystal (cry) toxin proteins, collectively referred to as delta-endotoxins. The parasporal crystals account for about 25% of the dry weight of the cells, and Bt cells need to produce 10 per parasporal crystal synthesized in the stationary growth phase6-2×106The delta-endotoxin molecule of (1). Specific binding of insect midgut cell surface receptor proteins to Cry toxinsIs the determining factor of the insecticidal specificity. Currently, studies on specific binding of insect larvae midgut cell surface receptor proteins to Cry toxins have focused primarily on Cry1 class proteins and lepidopteran insect larvae.
The insecticidal mechanism of the bacillus thuringiensis toxin protein has two forms: respectively, an active toxin form and a protoxin form. For active toxins, the higher ALP or APN receptors in the cell membrane bind to the active toxin first with lower binding strength, while transporting the toxin to the midgut brush border membrane. Cadherin then binds to the complex to a high degree. For protoxins, after ingestion by an insect, the parasporal crystals are digested by the digestive juices of the insect midgut, thereby releasing the protoxin. Cadherin can then bind to protoxins, allowing further cleavage of the protoxin by midgut trypsin or juice protease to form a monomeric toxin, which can aggregate to form oligomers. The GPI-anchored APN has a high affinity for ALP and oligomerized toxin proteins, promoting the insertion of oligomers into membrane lipid rafts, which then form tunnels in the midgut epithelial cells. Eventually, the osmotic pressure of the target insect midgut cells is destroyed, and the target insect is dehydrated and killed.
As is known in the art, protein drugs have the characteristics of easy degradation and inactivation, and how to maintain the activity of proteins to effectively exert the effects becomes a great problem in the application of protein drugs. As a class of widely applied biological pesticides, Bt plays a virulence role in exactly killing crystallin or protein aggregates (parasporal crystals) thereof, but the Bt also has some problems in application, such as slow killing speed, short residual period, easy influence of environmental factors and the like. Thus, the defects of protein drugs also become a key factor restricting the further development thereof.
Therefore, the problem at present is that research and development of a bacillus thuringiensis insecticide with stronger insecticidal efficacy, longer efficacy and better stability are urgently needed.
Disclosure of Invention
The invention aims to solve the technical problem of providing a bacillus thuringiensis thallus microcapsule aiming at the defects of the prior art, wherein the microcapsule is prepared by a layer-by-layer self-assembly method, takes bacillus thuringiensis thallus as a core and selects two polyelectrolyte materials with opposite charges under preparation conditions. The invention also provides a preparation method of the bacillus thuringiensis thallus microcapsule, which loads the bacillus thuringiensis thallus under a neutral condition to obtain a microcapsule preparation capable of controlling the release under an alkaline condition. The microcapsule preparation provided by the invention maintains the insecticidal activity of thalli, protects the active insecticidal substance-protein through double defenses of cell walls and coating (capsule walls) outside the cell walls, and helps the active insecticidal substance-protein to resist the influence of some environmental factors.
Therefore, the invention provides a bacillus thuringiensis thallus microcapsule which is composed of bacillus thuringiensis thallus serving as a core and a capsule wall coated outside the core, wherein the core is a single complete bacillus thuringiensis thallus.
In the invention, the capsule wall is formed by alternately adsorbing polyelectrolyte macromolecules with positive charges and polyelectrolyte macromolecules with negative charges as wall materials on the outer surface of a core layer by layer; wherein the positively charged polyelectrolyte macromolecules comprise positively charged chitosan and/or polylysine, preferably positively charged chitosan; the macromolecule with negative charges comprises one or more of sodium alginate with negative charges, sodium carboxymethylcellulose, chondroitin sulfate and sodium hyaluronate, and is preferably sodium alginate with negative charges.
In some embodiments of the present invention, the number N of wall material layers of the capsule wall is an integer greater than or equal to 5, preferably the number N of wall material layers of the capsule wall is an integer from 6 to 20, and more preferably the number N of wall material layers of the capsule wall is an integer from 6 to 8.
In some particularly preferred embodiments of the present invention, the microcapsules have a particle size of (1-1.5 μm) × (1.8-2.7 μm).
In a second aspect, the present invention provides a process for preparing a bacillus thuringiensis bacterial microcapsule according to the first aspect of the invention, which comprises:
step A, uniformly mixing polyelectrolyte macromolecules with positive charges or negative charges as a wall material and precipitates of bacillus thuringiensis thalli as a first core material in a salt solution, oscillating, forming a first layer of wall material on the outer surface of the bacillus thuringiensis thalli, further separating, treating and washing to obtain the bacillus thuringiensis thalli microcapsule with the wall material layer number of 1;
step B, uniformly mixing polyelectrolyte macromolecules which are used as wall materials and have charges opposite to those of the wall materials in the previous step with precipitates of the bacillus thuringiensis thallus microcapsules with the wall material layer number of 1, which are used as second core materials, in a salt solution, oscillating, forming a second layer of wall materials on the outer surface of the bacillus thuringiensis thallus microcapsules with the wall material layer number of 1, and further separating, processing and washing to obtain the bacillus thuringiensis thallus microcapsules with the wall material layer number of 2;
c, repeating the step B for N-2 times, and adding a layer of wall material every time, thereby obtaining the bacillus thuringiensis thallus microcapsule with the N layers of wall materials;
wherein the pH value of the salt solution is 6.8-7.2.
In some preferred embodiments of the invention, the salt solution has a pH of 7.0 to 7.2.
In some preferred embodiments of the invention, the salt solution has a molarity of 0.5M.
In some particularly preferred embodiments of the invention, the salt is sodium chloride.
According to the method of the invention, the concentration of the wall material in the salt solution is 0.1 g/L.
In some embodiments of the present invention, the mass ratio of the wall material to the core material is greater than or equal to 1: 1, and preferably, the mass ratio of the wall material to the core material is 1: (1-2).
According to the method, the oscillation time is more than or equal to 20 minutes.
In the invention, the precipitate of the bacillus thuringiensis thallus is obtained by separating and washing the suspension of the bacillus thuringiensis thallus.
In some embodiments of the present invention, the bacillus thuringiensis suspension is obtained by inoculating activated bacillus thuringiensis seed liquid into LB liquid culture medium to culture for 20-24 hours after logarithmic growth phase, and then transferring to LP culture medium to culture.
In some embodiments of the invention, the separation is a centrifugation during the precipitation of the Bacillus thuringiensis thallus, preferably at a speed of 4500-.
In some embodiments of the invention, the separating is a precipitation separation during precipitation of the bacillus thuringiensis thallus, which comprises adding CaCl to a suspension of the bacillus thuringiensis thallus2Stirring the hydrate at a high speed until the hydrate is completely dissolved, stirring the hydrate at a low speed until floccule is formed, and standing the floccule for precipitation; preferably, the rotation speed of the high-speed stirring is 75-100rpm, preferably 85-90rpm, and the time of the high-speed stirring is 3-10 minutes, preferably 5-7 minutes; the rotation speed of the low-speed stirring is 25-50rpm, preferably 35-40rpm, and the time of the low-speed stirring is 8-20 minutes, preferably 10-15 minutes.
According to some embodiments of the present invention, in steps A-C, the separation is a centrifugation at 3500rpm, preferably 2500 rpm and 3000rpm, for 15-60 minutes, preferably 15-30 minutes.
According to some embodiments of the invention, in steps a-C, the separation is a precipitation separation comprising adding CaCl to the shaken mixture2Stirring the hydrate at a high speed until the hydrate is completely dissolved, stirring the hydrate at a low speed until floccule is formed, and standing the floccule for precipitation; preferably, the rotation speed of the high-speed stirring is 75-100rpm, preferably 85-90rpm, and the time of the high-speed stirring is 3-10 minutes, preferably 5-7 minutes; the rotation speed of the low-speed stirring is 25-50rpm, preferably 35-40rpm, and the time of the low-speed stirring is 8-20 minutes, preferably 10-15 minutes.
In a third aspect, the present invention provides a bacillus thuringiensis microcapsule according to the first aspect of the present invention or a bacillus thuringiensis microcapsule prepared by the method according to the second aspect of the present invention for use in agricultural pest control.
The invention uses single complete bacillus thuringiensis as core by layer-by-layer self-assembly method, and selects two polyelectrolyte materials with opposite charges under preparation condition, namely chitosan and sodium alginate, to prepare the microcapsule. The method can load the bacillus thuringiensis thalli under the neutral condition to obtain the microcapsule preparation which can be controlled and released under the alkaline condition that the pH value is more than or equal to 9. The preparation maintains the insecticidal activity of thallus to the maximum extent, and can help the thallus to resist the influence of environmental factors such as ultraviolet radiation, high temperature, rain wash and the like.
Drawings
The invention is described in further detail below with reference to the attached drawing figures:
FIG. 1 shows the formation of parasporal crystals in Bt cells.
FIG. 2 shows the form of Bt bacterial microcapsules under a scanning electron microscope; wherein, 2A is HBF-1 thallus microcapsule (single thallus visual field); 2B is HBF-1 thallus (single thallus visual field); 2C is HBF-1 thallus microcapsule (multiple thallus visual fields); 2D HBF-1 cells (multiple cell fields).
FIG. 3 is a graph showing the comparison of Bt bacterial microcapsules and uncoated Bt bacterial plaques after 5 days of treatment at 50 ℃; wherein, 3A is HBF-1 microcapsule suspension stored at 4 ℃; 3B is HBF-1 microcapsule processed at 50 deg.C for 5 days; 3C is HBF-1 cell treated at 50 ℃ for 5 days.
FIG. 4 shows a Bt microbial cell microcapsule prepared by the method of the present invention; wherein, 4A is HBF-1 thallus microcapsule prepared by flocculation technology; 4B is HBF-1 thallus microcapsule prepared by centrifugal technology; 4C is HBF-1 thallus.
Detailed Description
In order that the invention may be readily understood, a more particular description of the invention briefly described below will be rendered by reference to specific embodiments that are illustrated in the appended drawings. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
I. Term(s) for
The term "larvae" as used herein refers to developmental stages of an insect that are morphologically and physiologically distinct and embryonic in development.
The term "active ingredient" as used herein means an ingredient having a killing or inhibitory effect on insects or their larvae.
The term "water" as used herein means deionized water, distilled water or ultrapure water unless otherwise specified or limited.
The terms "number of layers of capsule wall" and "number of layers of wall material" are used interchangeably in the present invention.
Embodiments of
As mentioned above, Bt is just its insecticidal crystal protein or its protein aggregate (parasporal crystal) which exerts its virulence effect as a class of widely used biopesticides, but it has problems such as slow insecticidal speed, short residual life, susceptibility to environmental factors, etc. in its application. In view of this, the present inventors have conducted extensive studies on the preparation method, drug effect, efficacy and stability of bacillus thuringiensis insecticides.
Microcapsules are considered to be one of the best controlled release (controlled released) carrier tools at present, and the technology is a substance which forms the space isolation between the inside and the outside of the capsule by using various wall materials and enables partial space structure to be generated in the capsule, and a plurality of functional substances can be loaded in the space structure in the capsule.
There are many methods for preparing microcapsules. Microcapsules prepared by layer self-assembly (LbL) technology can accurately control the size, shape, composition, thickness, structural form and the like of wall materials, and thus the microcapsules are concerned. The self-assembly technology of the microcapsule is a technology based on the interaction between positive and negative charges carried by polyelectrolyte anions and cations. The technology is mainly characterized in that polyelectrolyte anions and cations with opposite charges are alternately adsorbed on the surface of a substrate with charged surface through electrostatic interaction. The microcapsules obtained by this method exhibit unique structural and versatile properties compared to those of the traditional microcapsules: the preparation process is simple; the preparation condition is mild, the preparation can be carried out in a normal-temperature aqueous solution, and the natural conformation of the biological molecules with the bioactivity can be ensured; in addition, the method is applicable to various materials, and can be realized on devices and materials with complex body-type structures. Therefore, the technology is widely applied to the research in the field of biomedical materials.
The inventor successfully utilizes a layer-by-layer self-assembly technology to adsorb polyelectrolyte macromolecules with positive and negative charges, such as chitosan and sodium alginate, on the outer layer of the parasporal crystal of Bt layer by layer alternately to form a capsule wall, and obtains the microcapsule containing the parasporal crystal of Bt. Different Bt strains can produce different types of parasporal crystals, such as rhombus, spherical, biconical, cuboid, ellipsoid, irregular and the like, wherein rhombus is common. The inventor discovers that if the surface of a protein aggregate (parasporal crystal) is smooth without edges and corners in the assembling process, the deposition of the wall material on the surface of the core material is uniform after the assembly; if the surface of the protein aggregates (parasporal crystals) is angular, the sharp corners may affect the assembly of the microcapsules to some extent, i.e. the encapsulation is not uniform enough. This affects the efficacy, duration and stability of Bt containing microcapsules.
As previously described, Bt produces parasporal crystals during the stationary phase of growth. The parasporal crystal formation process is shown in fig. 1, and as can be seen from fig. 1, the parasporal crystal and spore formation in the Bt thallus requires seven steps: stage 0 and stage I are axonal formation in the cell; stage II is propagule septal formation and cell asymmetric division; stage III is phagocytic collapse, preporocyte formation and parasporal crystal synthesis; stage IV to stage VI are to form spore cortex, bud exocarp and outer wall components, and meanwhile, companion cell crystals are continuously synthesized; stage VII is spore maturation, blast cell lysis releasing spore and crystal.
The inventor further researches and discovers that by controlling the culture process of Bt to produce parasporal crystals, after Bt enters into the growth stationary phase, spore cortex, spore coat and outer wall components are formed, and a large amount of parasporal crystals are synthesized, before spores are mature and mother cells are cracked, namely, the culture is stopped at stages V to VI before stage VII, the obtained bacterial suspension agent is subjected to separation treatment, can obtain complete bacillus thuringiensis thalli containing parasporal crystals and spores, polyelectrolyte macromolecules with positive and negative charges such as chitosan, sodium alginate and the like are alternately adsorbed on the outer surface of the cell wall of the complete bacillus thuringiensis thalli layer by utilizing a layer-by-layer self-assembly technology to form a capsule wall, the prepared bacillus thuringiensis thallus microcapsule has the advantages of uniform package, good drug effect, long efficacy, good stability and particularly excellent stress resistance. The present invention has been made based on the above findings.
Therefore, the bacillus thuringiensis thallus microcapsule according to the first aspect of the invention is composed of a single complete bacillus thuringiensis thallus serving as a core and a capsule wall covering the core, wherein the capsule wall is formed by alternately adsorbing polyelectrolyte macromolecules with positive charges and polyelectrolyte macromolecules with negative charges on the outer surface of the core layer by layer as a wall material. That is, each microcapsule has one complete Bacillus thuringiensis cell embedded inside the wall and the cell contains parasporal crystal and spore. This is understood to mean that each bacillus thuringiensis thallus microcapsule comprises: parasporal crystals and spores as active ingredients, and a cell wall outside the active ingredients and a cyst wall outside the cell wall formed by polyelectrolyte macromolecules with positive and negative charges as wall materials alternately layer by layer.
Obviously, the wall material is another limiting factor limiting the application of the microcapsule, and particularly in the case of using the microcapsule preparation as an insecticide, a substance which is cheap and/or has biocompatibility is required to be used as the wall material, so that the microcapsule has higher use value. Sodium alginate is a linear anionic natural polysaccharide compound, and is mainly extracted from marine plants such as giant kelp, gulfweed, kelp and the like. Sodium alginate has a pKa of 3.38-3.65, and a large number of hydroxyl and carboxyl groups are carried on a molecular chain, so that the formed microcapsule has low permeability, but the pH is easy to influence the solubility. At present, the most common way is to deposit a new layer of cationic polymer wall material, such as chitosan, on the surface of sodium alginate. Chitosan is the only basic weak cationic polysaccharide which is known in nature and exists in large quantity at present, the pKa of the chitosan is about 6.3, the chitosan can be opposite to the charge of the negatively charged polyelectrolyte due to the charge, and therefore the chitosan and the negatively charged polyelectrolyte interact with each other due to electrostatic force to form a polyelectrolyte multilayer composite microcapsule which can play a role in loading living cells, proteins and the like.
In the present invention, the positively charged polyelectrolyte macromolecules include positively charged chitosan and/or polylysine, preferably positively charged chitosan; the macromolecule with negative charges comprises one or more of sodium alginate with negative charges, sodium carboxymethylcellulose, chondroitin sulfate and sodium hyaluronate, and is preferably sodium alginate with negative charges.
As known in the art, chitosan is a natural high molecular polysaccharide prepared by removing more than 50% of acetyl groups from chitin (chitin). The primary amino groups in the chitosan molecular chain have different numbers due to different deacetylation degrees. The Degree of Deacetylation (DD) is an important parameter of chitosan materials, which can affect their physicochemical properties (solubility, crystallinity, swelling properties, mechanical properties) and biological characteristics (protein absorption capacity). The higher the degree of deacetylation, the higher the tensile strength and elasticity of the chitosan, but the higher its brittleness. The chitosan used in the present invention comprises 90% DD chitosan and/or 95% DD chitosan.
It will be appreciated by those skilled in the art that, with a smaller number of wall layers, the coating thickness will be thinner, thus reducing the protein protection and resulting in poor controlled release. If the number of the layers of the capsule wall is too large, not only the drug effect is affected, but also unnecessary workload is increased, and the cost is increased. And under the proper capsule wall layer number, the active ingredients in the capsule can be effectively protected, and can effectively act on a target under the synergistic controlled release action of the cell wall and the capsule wall. Therefore, in some embodiments of the present invention, the number N of wall material layers of the bag wall is an integer greater than or equal to 5, preferably the number N of wall material layers of the bag wall is an integer from 6 to 20, more preferably the number N of wall material layers of the bag wall is an integer from 6 to 8, and even more preferably the number N of wall material layers of the bag wall is an integer from 6.
In some particularly preferred embodiments of the present invention, the microcapsules have a particle size of (1-1.5 μm) × (1.8-2.7 μm). The particle size of the microcapsule is basically the particle size of bacillus thuringiensis somatic cells, namely, the wall material of the bacillus thuringiensis somatic microcapsule has little influence on the particle size of the microcapsule. Compared with the microcapsules prepared by a plurality of template etching methods, the microcapsules have smaller particle size and are more suitable to be used as insecticides for feeding insect larvae and spraying in fields.
In a second aspect of the present invention, there is provided a process for producing a Bacillus thuringiensis bacterial microcapsule according to the first aspect of the present invention, comprising:
step A, uniformly mixing polyelectrolyte macromolecules (such as chitosan) with positive charges or polyelectrolyte macromolecules (such as sodium alginate) with negative charges, which are used as wall materials, with precipitates of Bacillus thuringiensis thalli, which are used as first core materials, in a salt solution, oscillating, forming a first layer of wall material on the outer surface of the Bacillus thuringiensis thalli, and further performing separation treatment and water washing to obtain the Bacillus thuringiensis thalli microcapsule with the wall material layer number of 1;
step B, uniformly mixing polyelectrolyte macromolecules (such as sodium alginate or chitosan) which are used as wall materials and have charges opposite to those of the wall materials in the previous step with precipitates of the bacillus thuringiensis thallus microcapsules with the wall material layer number of 1, which are used as second core materials, in a salt solution, oscillating, forming a second layer of wall materials on the outer surface of the bacillus thuringiensis thallus microcapsules with the wall material layer number of 1, and further performing separation treatment and water washing to obtain the bacillus thuringiensis thallus microcapsules with the wall material layer number of 2;
and C, repeating the step B for N-2 times, and adding a layer of wall material every time, thereby obtaining the bacillus thuringiensis thallus microcapsule with the N layers of wall materials.
In the present invention, the layer-by-layer alternate coating of Bacillus thuringiensis bacteria with positively or negatively charged polyelectrolyte macromolecules can be carried out under neutral conditions, for example, in some cases, the pH of the salt solution is 6.8 to 7.2, preferably 7.0 to 7.2.
In some particularly preferred embodiments of the invention, the salt solution is a 0.5M molar aqueous sodium chloride solution.
In some particularly preferred embodiments of the invention, the concentration of the wall material in the salt solution is 0.1 g/L.
In some embodiments of the present invention, the mass ratio of the wall material to the core material is greater than or equal to 1: 1, and preferably, the mass ratio of the wall material to the core material is 1: (1-2).
According to the method, in the step A and the step B, the oscillation time is more than or equal to 20 minutes.
In the invention, the precipitate of the bacillus thuringiensis thallus is obtained by separating and washing the bacillus thuringiensis thallus suspension; wherein the bacillus thuringiensis thallus in the bacillus thuringiensis thallus suspension is a complete bacillus thuringiensis thallus cell containing parasporal crystals and spores.
In the invention, the complete bacillus thuringiensis somatic cells containing parasporal crystals and spores are obtained by controlling the culture process of Bt, namely, after Bt enters a growth stationary phase to form a spore cortex, a spore coat and an outer wall assembly and synthesize a large amount of parasporal crystals, the culture is stopped before spores are mature and mother cells are cracked, namely, in a stage V to a stage VI before a stage VII, and the obtained bacterial suspension agent is separated, so that the complete bacillus thuringiensis somatic cells containing parasporal crystals and spores can be obtained. For example, in some embodiments of the present invention, the bacillus thuringiensis suspension is obtained by inoculating the activated wild-type HBF-1 bacillus thuringiensis seed liquid into LB liquid medium, culturing the seed liquid after logarithmic growth phase, and transferring the seed liquid to LP medium for culturing for 20-24 hours, preferably 20-22 hours, and more preferably 22 hours.
The preparation method of the LB liquid culture medium comprises the following steps: 1.0% Tryptone (Tryptone), 0.5% yeast extract, 1% NaCl, deionized water, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min.
The preparation method of the LP beef extract peptone medium comprises the following steps: 3 per mill of beef extract, 5 per mill of soybean peptone and deionized water, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min.
In the present invention, the method for separating and treating the bacterial cells of Bacillus thuringiensis from the Bacillus thuringiensis bacterial suspension is not particularly limited, and a separation method which is conventional in the art can be used. For example, in some embodiments of the present invention, the cells of the Bacillus thuringiensis are separated from the suspension of Bacillus thuringiensis cells by centrifugation at 4500-6500rpm, preferably 5500-6000rpm, and more preferably 6000rpm for 10-60 minutes, preferably 20-30 minutes, and more preferably 20 minutes.
The flocculation precipitation method is a method of adding a flocculating agent into a fermentation liquid by using a flocculation technology to change the dispersibility of colloidal particles such as thalli or proteins and the like, so that the colloidal particles are aggregated to form floccules and finally precipitate. The method is simple to operate and does not need special equipment. CaCl2·2H2O is used as a common environment-friendly flocculating agent, is non-toxic to human bodies and has an ideal flocculating effect. The supernatant after flocculation is centrifuged to find that the residual bacteria amount in the supernatant is less, and the supernatant can be used as the flocculant in the invention.
For example, in some embodiments, flocculation precipitation is used to separate the bacteria from the suspension of Bacillus thuringiensis bacteria by adding CaCl to the shaken mixture2Stirring the hydrate at a high speed until the hydrate is completely dissolved, stirring the hydrate at a low speed until floccule is formed, and standing the floccule for precipitation; preferably, the rotation speed of the high-speed stirring is 75-100rpm, preferably 85-90rpm, further preferably 90rpm, and the time of the high-speed stirring is 3-10 minutes, preferably 5-7 minutes, further preferably 5 minutes; the rotation speed of the low-speed stirring is 25-50rpm, preferably 35-40rpm, and more preferably 40rpm, and the time of the low-speed stirring is 8-20 minutes, preferably 10-15 minutes, and more preferably 10 minutes.
In some embodiments, the bacillus thuringiensis mycelia are sequentially subjected to separation treatment and water washing at least 2 times.
Similarly, the method for separating and treating the Bacillus thuringiensis bacterial microcapsules in the steps A to C is not particularly limited in the present invention, and any separation method conventionally used in the art can be used. For example, in the steps A-C, the Bacillus thuringiensis bacterial microcapsules are separated by a centrifugal separation method, wherein the centrifugal rotation speed is 1500-3500rpm, preferably 2500-3000rpm, more preferably 3000rpm, and the centrifugal time is 15-60 minutes, preferably 15-30 minutes, more preferably 15 minutes.
For another example, in some embodiments, the separation of the bacillus thuringiensis microcapsules by flocculation precipitation comprises adding CaCl to the shaken mixture of the microcapsules containing bacillus thuringiensis2Stirring the hydrate at a high speed until the hydrate is completely dissolved, stirring the hydrate at a low speed until floccule is formed, and standing the floccule for precipitation; preferably, the rotation speed of the high-speed stirring is 75-100rpm, preferably 85-90rpm, further preferably 90rpm, and the time of the high-speed stirring is 3-10 minutes, preferably 5-7 minutes, further preferably 5 minutes; the rotation speed of the low-speed stirring is 25-50rpm, preferably 35-40rpm, and more preferably 40rpm, and the time of the low-speed stirring is 8-20 minutes, preferably 10-15 minutes, and more preferably 10 minutes.
In some embodiments, the bacillus thuringiensis microcapsules in the mixed liquid containing the bacillus thuringiensis microcapsules in the steps A-C are sequentially subjected to separation treatment and water washing for at least 2 times.
In a third aspect, the present invention provides a bacillus thuringiensis microcapsule according to the first aspect of the present invention or a bacillus thuringiensis microcapsule prepared by the method according to the second aspect of the present invention for use in agricultural pest control. The agricultural pests include all insects, in particular larvae of these insects, for which bacillus thuringiensis is effective in inhibiting or killing.
In some embodiments of the invention, the insect comprises a larva of one or more of an insect of the order isoptera, an insect of the order homoptera, an insect of the order orthoptera, an insect of the order hymenoptera, an insect of the order lepidoptera, an insect of the order coleoptera, a nematode, and a protozoan, preferably a larva of the order coleoptera, and more preferably a larva of a chafer aeruginosa.
III, examples
The present invention will be specifically described below with reference to specific examples. The experimental methods described below are, unless otherwise specified, all routine laboratory procedures. The experimental materials described below, unless otherwise specified, are commercially available.
The size and the shape of the microcapsule are observed by a scanning electron microscope: taking Bt thallus microcapsule samples, diluting with deionized water to a proper concentration, then respectively taking 20 mu L of the diluted Bt thallus microcapsule samples and dripping the diluted Bt thallus microcapsule samples on clean aluminum foil paper, spraying gold on the samples by using a sputtering coating machine after natural drying, and observing the size and the shape of the microcapsules by using a scanning electron microscope (JSM-6700F cold field emission scanning electron microscope, Japan electron).
Example 1: HBF-1 thallus microcapsule prepared by taking HBF-1 thallus as core
(1) Preparation of HBF-1 bacterial core
The wild type HBF-1 (provided by plant protection research institute of academy of agriculture and forestry, North river) cryopreserved bacteria are transferred to LB liquid medium according to the proportion of 1 percent and cultured for about 10 hours at 30 ℃ and 180 rpm. Transferring the activated bacteria liquid into another LB liquid culture medium according to the proportion of 1 percent, and culturing for 4-5h at 30 ℃ and 180rpm to ensure that the bacteria reach the logarithmic phase. Then transferring the bacterial liquid into an LP culture medium according to the proportion of 1%, culturing at 30 ℃ and 220rpm for about 22h, and collecting. Centrifuging at 6000rpm for 20min, collecting precipitate, washing the precipitate with water, centrifuging at 6000rpm for 20min, and discarding the supernatant. Adding appropriate amount of deionized water into the precipitate, and mixing to obtain thallus extract (shown as 2B and 2D in FIG. 2).
(2) Preparing wall material solution
0.5M sodium chloride solution: weighing 58.5g of sodium chloride, adding deionized water, and fixing the volume to 2L;
0.1g/L chitosan (degree of deacetylation is more than or equal to 95%): dissolving 1.0g of chitosan (Cs) in 1L of 0.5M sodium chloride solution, and stirring for 5 hours by using a magnetic stirrer;
0.1g/L sodium alginate: 1.0g sodium alginate (Alg) was dissolved in 1L0.5M sodium chloride solution and stirred for 15h with a magnetic stirrer.
(3) Microcapsule preparation by layer-by-layer self-assembly method
A first layer: 50mL of 0.1g/L Cs (95% DD) was mixed with the HBF-1 cell pellet, and the mixture was shaken for 20 min. Centrifuge at 3000rpm for 15min, and discard the supernatant. Washing the precipitate with water, centrifuging at 3000rpm for 15min, discarding the supernatant, and repeating for 3 times;
a second layer: 50mL of 0.1g/LAlg was mixed with the precipitate and shaken for 20 min. Centrifuge at 3000rpm for 15min, and discard the supernatant. Washing the precipitate with water, centrifuging at 3000rpm for 15min, discarding the supernatant, and repeating for 3 times;
repeating the steps, and sequentially assembling the 3 rd to 6 th layers of capsule walls, wherein the capsule walls are assembled from outside to inside in sequence: Alg-Cs-Alg-Cs-Alg-Cs. Finally, the (Cs (95% DD)/Alg) microcapsule with HBF-1 bacteria as the core is obtained. The wall of the microcapsule is 6 layers. The morphological structure thereof was observed by a scanning electron microscope to obtain microcapsules having a size of about 2.5X 1.2 μm as shown in FIGS. 2A and 2C.
Example 2: insecticidal activity assay of HBF-1 thallus microcapsule
The insecticidal activity of the newly prepared HBF-1 thallus microcapsules and HBF-1 thallus is respectively measured by a mode of feeding the larva of the Aerugo chafer (provided by the academy of agriculture and forestry in Hebei) with a mixed feed. The prepared bacterial microcapsules and bacterial extracts were diluted to 8 gradients (4.5. mu.g/g, 9.0. mu.g/g, 13.5. mu.g/g, 27.0. mu.g/g, 54.0. mu.g/g, 180.0. mu.g/g, 360.0. mu.g/g, 720.0. mu.g/g), respectively. Adding the microcapsule liquid and the bacterial liquid with different concentrations into 40g of potato strips, uniformly mixing, pouring the microcapsule liquid and the bacterial liquid into 200g of dry soil treated by ultraviolet rays (40W, 5h), and standing for 2-3 h. The potato shreds are uniformly distributed at the bottom of 20 sterilizing finger-shaped tubes, then the uniformly mixed soil is uniformly distributed and covered on the upper layer of the potato shreds, 1 head of active and individual and uniform larva of the Aerugo chafer with 15d age is connected into each tube, 20 larvae are repeated, and clean water and microcapsule wall materials are set as blank controls. The animals were kept in a climatic chamber at 27 + -1 deg.C, the number of live insects was investigated on day 7 and 14, respectively, and LC50 and 95% confidence intervals were calculated.
The bioassay results show that the bacterial microcapsules still maintain good insecticidal activity, as shown in table 1. The lethality of the microcapsule wall material and the clear water are both at a low level, belonging to normal mortality and not listed in the table.
TABLE 1 insecticidal Activity assay of HBF-1 thallus microcapsule and HBF-1 thallus on Aerugo chafer larva
Example 3: stability of microcapsules under high temperature conditions
(1) High temperature resistance of thallus and microcapsule
Respectively taking 5mL of HBF-1 thallus suspension and 5mL of HBF-1 thallus microcapsule suspension, uniformly coating the suspension in a culture dish, naturally airing, and storing in a constant temperature incubator at 50 ℃ for 5 days. The samples in the culture dishes are respectively suspended by 10mL of deionized water, evenly coated on an LB plate, the plate is placed in a constant temperature incubator at 30 ℃, and the colony number is observed after 15 h. The results are shown in FIG. 3 and Table 2. The result shows that compared with HBF-1 thallus (not embedded), the HBF-1 thallus microcapsule has higher bioactivity after high-temperature treatment, which indicates that the microcapsule has better high-temperature drying resistance.
TABLE 2 high temperature treatment of samples for plaque number statistics
(2) Lethality of thalli and microcapsules after high-temperature treatment
The lethality of HBF-1 cells and HBF-1 cell microcapsules was examined 15 days after treatment at 45 ℃ and 50 ℃ respectively, and the results are shown in Table 3. After high-temperature treatment, the HBF-1 thallus microcapsules and the HBF-1 thallus have certain insecticidal activity, but the toxicity of the HBF-1 thallus microcapsules is generally higher than that of the HBF-1 thallus (not embedded).
7-day lethality of HBF-1 thallus microcapsule and HBF-1 thallus treated at 345 ℃ and 50 ℃ respectively
Example 4: HBF-1 thallus microcapsule prepared by flocculation technology
(1) Preparation of HBF-1 bacterial core
Adding CaCl into HBF-1 thallus suspension2·2H2O, stirring at 90rpm for 5min to CaCl2·2H2And completely dissolving the O, stirring at 40rpm for 10min to form floccule, and standing for about 15min to completely precipitate the floccule. The supernatant was aspirated with a pipette until only floc and a small amount of liquid remained in the flask. Adding deionized water into the floccule, mixing, flocculating by the same method to obtain floccule precipitate, and repeating the water washing step for 3 times. FIG. 4C shows the HBF-1 cells thus obtained.
(2) Microcapsule preparation by layer-by-layer self-assembly method
A first layer: and (3) uniformly mixing 50mL of 1.0g/L chitosan with the HBF-1 floccule after washing, and oscillating for 20 min. Centrifuge at 3000rpm for 15min, and discard the supernatant. Washing the precipitate with water, centrifuging at 3000rpm for 15min, discarding the supernatant, and repeating for 3 times;
a second layer: 50mL of 1.0g/L sodium alginate is uniformly mixed with the precipitate, and the mixture is shaken for 20 min. Adding CaCl2·2H2O, stirring at 90rpm for 5min to CaCl2·2H2And completely dissolving the O, stirring at 40rpm for 10min to form floccule, and standing for about 15min after stirring until the floccule is completely precipitated. The supernatant was pipetted until only floc and a small amount of liquid remained in the flask. The mixture was washed 3 times with water in the same manner as described above.
Repeating the above steps, and sequentially assembling the 3 rd to 6 th layers. Finally, the (Cs (95% DD)/Alg) microcapsule with HBF-1 bacteria as the core is obtained. As shown in fig. 4A.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
Claims (10)
1. A bacillus thuringiensis thallus microcapsule is composed of a bacillus thuringiensis thallus serving as a core and a capsule wall covering the core, wherein the core is a single complete bacillus thuringiensis thallus;
the bacillus thuringiensis thallus is a complete bacillus thuringiensis thallus cell which contains parasporal crystals and spores inside;
the capsule wall is formed by alternately adsorbing polyelectrolyte macromolecules with positive charges and polyelectrolyte macromolecules with negative charges on the outer surface of the core layer by layer as wall materials; wherein the chitosan with positive charges has a deacetylation degree of more than or equal to 95 percent; the macromolecule with negative charges is sodium alginate with negative charges;
the number N of wall material layers of the capsule wall is 6;
the particle size of the microcapsules is (1-1.5 μm) × (1.8-2.7 μm).
2. A method of preparing a Bacillus thuringiensis bacterial microcapsule according to claim 1, comprising:
step A, uniformly mixing polyelectrolyte macromolecules with positive charges or negative charges as a wall material and precipitates of bacillus thuringiensis thalli as a first core material in a salt solution, oscillating, forming a first layer of wall material on the outer surface of the bacillus thuringiensis thalli, further separating, treating and washing to obtain the bacillus thuringiensis thalli microcapsule with the wall material layer number of 1;
step B, uniformly mixing polyelectrolyte macromolecules which are used as wall materials and have charges opposite to those of the wall materials in the previous step with precipitates of the bacillus thuringiensis thallus microcapsules with the wall material layer number of 1, which are used as second core materials, in a salt solution, oscillating, forming a second layer of wall materials on the outer surface of the bacillus thuringiensis thallus microcapsules with the wall material layer number of 1, and further separating, processing and washing to obtain the bacillus thuringiensis thallus microcapsules with the wall material layer number of 2;
c, repeating the step B for N-2 times, and adding a layer of wall material every time, thereby obtaining the bacillus thuringiensis thallus microcapsule with the N layers of wall materials;
wherein the pH value of the salt solution is 7.0-7.2;
the sediment of the bacillus thuringiensis thallus is obtained by separating and washing a bacillus thuringiensis thallus suspension, wherein the bacillus thuringiensis thallus in the bacillus thuringiensis thallus suspension is a complete bacillus thuringiensis thallus cell containing parasporal crystals and spores;
the complete bacillus thuringiensis somatic cell containing parasporal crystals and spores is obtained by forming a spore cortex, a spore coat and an outer wall component when Bt enters a growth stationary phase, stopping culturing in a stage V to VI before a stage VII after spores are mature and mother cells are cracked after a large number of parasporal crystals are synthesized, and separating the obtained bacterial suspension agent.
3. The method of claim 2, wherein the salt solution is at a molarity of 0.5M; the salt is sodium chloride; and/or the concentration of the wall material in the salt solution is 0.1 g/L; and/or the mass ratio of the wall material to the core material is 1 (1-2).
4. The method of claim 2, wherein the shaking time is 20 minutes or more.
5. The method as set forth in claim 2, wherein the Bacillus thuringiensis suspension is obtained by inoculating the activated Bacillus thuringiensis seed liquid into LB liquid medium, culturing until the logarithmic growth phase is reached, and then culturing for 20-24 hours by transferring to LP medium.
6. The method as claimed in claim 2, wherein the separation is a centrifugation at 5500-6000rpm for 20-30 minutes.
7. The method of claim 2, wherein the separating is a precipitation separation comprising adding CaCl to a suspension of bacillus thuringiensis cells2Stirring the hydrate at a high speed until the hydrate is completely dissolved, stirring the hydrate at a low speed until floccule is formed, and standing the floccule for precipitation; wherein the high-speed stirring speed is 85-90rpm, and the high-speed stirring time is 5-7 minutes; the rotating speed of the low-speed stirring is 35-40rpm, and the time of the low-speed stirring is 10-15 minutes.
8. The method as claimed in any one of claims 2 to 7, wherein in steps A to C, the separation is a centrifugation at 3000rpm and 2500 for 15 to 30 minutes.
9. The method according to any one of claims 2 to 7, wherein in steps A to C, the separation is a precipitation separation comprising adding CaCl to the shaken mixture2Stirring the hydrate at a high speed until the hydrate is completely dissolved, stirring the hydrate at a low speed until floccule is formed, and standing the floccule for precipitation; the high-speed stirring speed is 85-90rpm, and the high-speed stirring time is 5-7 minutes; the rotating speed of the low-speed stirring is 35-40rpm, and the time of the low-speed stirring is 10-15 minutes.
10. Use of a bacillus thuringiensis thallus microcapsule according to claim 1 or a bacillus thuringiensis thallus microcapsule prepared by a method according to any one of claims 2 to 9 for agricultural pest control.
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