Biocontrol bacillus microcapsule and preparation method thereof

Abstract

The invention provides a biocontrol bacillus microcapsule and a preparation method thereof, and belongs to the technical field of biological agents. According to the bacillus velezensis microcapsule and the preparation method thereof, the acting time of a strain is prolonged through a microcapsule technology, the adhesiveness of a medicine is improved, and more effective biological control and green control are achieved. The microcapsule technology can protect the strains from being influenced by the external environment, improve the planting capacity of the strains in soil and prolong the control effect of the strains. Through the method, the application efficiency and range of bacillus velezensis can be remarkably improved, and an environment-friendly and efficient biological control means is provided for agricultural production.

Description

Technical Field

[0001] The invention relates to the technical field of biological bacterial agents, and in particular to a biocontrol bacillus microcapsule and a preparation method thereof. Background Art

[0002] Biological control is a method that uses interactions between biological species to reduce the density of pest populations. Compared with non-biological control methods such as chemical pesticides, biological control has the advantages of lasting effects, high safety, environmental protection and cost savings. In biological control, using bacteria to control insects is an important method. It uses biocontrol bacteria to inhibit pests and harmful pathogens, while improving soil quality and population structure, providing safety and nutritional guarantees for plant growth. However, the application of biocontrol bacteria is limited by a variety of ecological factors, such as soil environment, which makes it difficult for them to colonize in different soils, affecting their actual effects. In addition, a type of biocontrol bacteria can usually only control a limited number of pests and diseases.

[0003] Bacillus velezensis is a new type of biocontrol bacteria that has a wide range of inhibitory effects on a variety of plant pathogenic fungi and bacteria. It is widely distributed in nature, harmless to humans and animals, and does not pollute the environment. Studies have shown that Bacillus velezensis can produce a variety of antibacterial substances, such as peptides, antimicrobial proteins, lipopeptide antibiotics, and polyketide compounds, which exhibit good inhibitory effects on plant pathogenic fungi and bacteria. In addition, Bacillus velezensis can also induce plant resistance and secrete substances that promote plant growth. Despite this, Bacillus velezensis still faces the problems of short storage period and limited duration of anti-disease effect in practical applications. Summary of the invention

[0004] The purpose of the present invention is to provide a biocontrol Bacillus microcapsule and a preparation method thereof. By embedding the strain in a specific microcapsule, not only can the action time of the microcapsule be prolonged, but also the adhesion of the drug can be enhanced, thereby achieving the goals of biological control and green control.

[0005] The Bacillus Velezii ZC-214 used in the present invention has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on December 5, 2024, with the deposit number CGMCC No.32921.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing biocontrol bacteria microcapsules, comprising the following steps:

[0008] The sodium alginate solution is mixed with an oxidant to perform oxidation treatment to obtain an oxidation product;

[0009] The oxidation product is mixed with a sodium hydroxide solution and a redistilled acrylic acid monomer, and then subjected to heating treatment, dialysis, and freeze drying treatment in sequence to obtain sodium alginate sodium polyacrylate;

[0010] Dissolving sodium alginate sodium polyacrylate to obtain a sodium alginate sodium polyacrylate solution;

[0011] Mixing the sodium alginate sodium polyacrylate solution with the biocontrol bacteria solution to obtain a mixed solution;

[0012] The mixed solution is sprayed into a calcium chloride solution to obtain a precipitate;

[0013] The precipitate is freeze-dried to obtain biocontrol bacteria microcapsules.

[0014] Preferably, the oxidizing agent is sodium periodate.

[0015] Preferably, the volume ratio of the sodium alginate solution to the weight ratio of the oxidant is 200 mL: 0.01-0.03 g.

[0016] Preferably, the oxidation treatment time is 100 to 140 minutes.

[0017] Preferably, the concentration of the calcium chloride solution is 0.1-0.3 mol / L.

[0018] Preferably, the temperature of the oxidation treatment is 50-70°C.

[0019] Preferably, the reaction temperature of the heating treatment is 70-90°C and is carried out under nitrogen protection;

[0020] The heating treatment time is 2 to 4 hours;

[0021] The dialysis time is 2 to 4 days;

[0022] The freeze-drying treatment time is 12 to 72 hours;

[0023] The concentration of the sodium alginate sodium polyacrylate solution is 1-3%;

[0024] The concentration of the biocontrol bacteria solution is 10 8 ~10 11 CFU / mL;

[0025] After the spraying to the calcium chloride solution, the precipitation is allowed to stand for 20 to 40 minutes.

[0026] Preferably, the biocontrol bacteria are selected from one or more of Bacillus Velezii, Bacillus subtilis, Pseudomonas fluorescens, Trichoderma, Streptomyces, Pseudomonas, Chaetomium, Penicillium or Verticillium chlamydospores.

[0027] The invention also provides biocontrol bacteria microcapsules prepared by the preparation method.

[0028] The present invention also provides the use of the biocontrol bacteria microcapsule in the preparation of biocontrol products.

[0029] Beneficial effects of the present invention:

[0030] The present invention proposes a Bacillus Velez microcapsule and a preparation method thereof, which prolongs the duration of action of the strain through a specific microcapsule technology, improves the adhesion of the drug, and achieves more effective biological control and green control. This microcapsule technology can protect the strain from the influence of the external environment, improve its colonization ability in the soil, and prolong its control effect. Through this method, the application efficiency and scope of Bacillus Velez can be significantly improved, providing an environmentally friendly and efficient biological control method for agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the result diagram of the effect of sodium alginate concentration on the embedding rate of microcapsules after molding;

[0032] Figure 2 This is the result diagram of the effect of calcium ion concentration on the embedding rate of microcapsules after molding;

[0033] Figure 3 This is a comparison chart of the survival rate of Bacillus Velez microcapsules and unencapsulated bacterial solution under ultraviolet light irradiation. DETAILED DESCRIPTION

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Example 1

[0036] Components: sodium alginate (4 mg / mL, 100 mL); calcium chloride solution (0.2 mol / L, 500 mL); acrylic acid (0.8 g); sodium periodate (0.02 g).

[0037] 100 μL of Bacillus Velezii ZC-214 was added to 100 mL of LB medium and then cultured in a 37°C incubator for 16 h. The OD600 value of the strain was 4.82 determined by UV spectrophotometer. The bacterial solution was centrifuged at 5000 rpm and 4°C for 10 min, resuspended in PBS, and centrifuged three times. The centrifugal precipitate was collected and washed with sterile saline and diluted to a colony count of about 10 8 CFU / mL bacterial solution, used for coating process or vitality detection.

[0038] 1.0 g of sodium alginate powder was weighed and dissolved in 200 mL of deionized water. Under a nitrogen atmosphere, the mixture was stirred at a constant temperature of 30 ° C and 200 rpm until it was completely dissolved to form a uniform solution. Sodium periodate was added for oxidation, and then the mixture was heated to 60 ° C. 0.72 g of redistilled acrylic acid monomer was neutralized with sodium hydroxide solution under ice-water bath conditions. The neutralized monomer was slowly added dropwise to the sodium alginate solution, and the temperature was raised to 80 ° C. The reaction was protected by nitrogen for 3 h, and the mixture was dialyzed against water for 3 d. The sodium alginate polyacrylate (SA-PAAs) material was obtained by freeze-drying for 24 h.

[0039] The obtained SA-PAAs material was sterilized and dissolved in sterile water to obtain a 2% SA-PAAs solution. 10 mL of 10% SA-PAAs solution was added. 8 CUF / mL Bacillus liquid was stirred at 500 rpm for 5 min to mix evenly, then the mixture was added to the sprayer, and the mixture was evenly sprayed in the calcium chloride solution, allowed to settle for 30 min, and the precipitate was collected and freeze-dried to obtain microcapsules.

[0040] Comparative Example 1

[0041] The number of colonies of Bacillus velez ZC-214 obtained by the method in Example 1 was about 10 8 CFU / mL of bacterial solution.

[0042] 0.5 g of the microcapsules prepared with the best ratio selected in Example 1 and 0.5 mL of the bacterial solution prepared in this comparative example were added to sterile culture dishes, 10 mL of sterile water was added, and the dishes were sealed and placed in parallel in three groups. The survival rates were measured at 1 h, 2 h, and 3 h under ultraviolet light. The results are as follows: Figure 3 As shown. Figure 3 It can be seen that under ultraviolet light irradiation, the survival rate of the microcapsule-embedded strain reached 62.31% under a 20W ultraviolet light at 30CM for three hours, which was 29.08% higher than the survival rate of the unencapsulated strain of 33.23%. And the survival rate of Example 1 (microcapsules prepared by the optimal ratio) was always higher than that of Comparative Example 1 (unencapsulated bacterial solution), indicating that the microencapsulation technology provided by the present invention significantly improves the survival rate of Bacillus Velezii under ultraviolet light irradiation and enhances the stress resistance of the strain.

[0043] Experimental example

[0044] Based on the method in Example 1, the following experimental settings were performed:

[0045] ①Except for the oxidation of sodium alginate by increasing the amount of oxidant from 0.01g to 0.1g in equal steps of 0.01g, the oxidation time and the amount of other drugs added remained unchanged. Finally, the embedding rate after microcapsule formation was determined to select the optimal amount of oxidant.

[0046] ② After determining the dosage of the oxidant, the other conditions remain unchanged, only the oxidation time is changed. The reaction time is 30min, 60min, 90min, 120min, 150min, and 180min for oxidation. Finally, the embedding rate after microcapsule formation is measured to select the optimal oxidation time.

[0047] ③ Under the conditions of the optimal oxidant dosage and oxidation time determined in the previous step, the calcium chloride solution was increased from 0.1mol / L to 1mol / L, with a concentration gradient of 0.1mol / L. This was used to prepare microcapsules with different calcium ion concentrations. Finally, the embedding rate after microcapsule formation was determined to select the optimal calcium ion concentration. The results are as follows: Figure 2 shown.

[0048] Table 1 Effect of oxidation time on microcapsule suspension rate, wetting time and bacterial count

[0049]

[0050] Test results: The best solution selected in the final experiment is that the amount of oxidant sodium periodate in 200mL sodium alginate solution is 0.02g, the oxidation time is 120min, and the concentration of calcium chloride solution is 0.2mol / L. The microcapsules with an embedding rate of 85.32% can be obtained.

[0051] As can be seen from the above examples, the present invention provides the Bacillus microcapsule technology provided by the present invention, which significantly improves the application effect of Bacillus Velez in agricultural biological control through an innovative method. This technology enhances the survival ability of the strain under adverse environmental conditions by encapsulating the strain in a microcapsule made of materials such as sodium alginate. Microencapsulation not only improves the adhesion of the strain, making it more effectively attached to the plant surface and soil particles, but also extends the storage period of the product, achieving a slower and more precise strain release, thereby improving the accuracy and durability of biological control.

[0052] The Bacillus microcapsules provided by the present invention can reduce the use of chemical pesticides, meet the requirements of green agriculture and sustainable development, help protect the environment and improve the safety of agricultural products; they can also increase crop yield and quality, improve the structure of soil microbial communities, enhance soil health, reduce chemical residues, provide farmers with more environmentally friendly planting options, and promote the sustainable development of agriculture. The Bacillus microcapsule technology provided by the present invention provides an environmentally friendly, efficient and long-lasting new biological control method for agricultural production, which helps to achieve the green transformation of agricultural production and sustainable development goals.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing biocontrol bacteria microcapsules, characterized in that: The following steps are involved: The sodium alginate solution is mixed with an oxidant to perform oxidation treatment to obtain an oxidation product; The oxidation product is mixed with a sodium hydroxide solution and a redistilled acrylic acid monomer, and then subjected to heating treatment, dialysis, and freeze drying treatment in sequence to obtain sodium alginate sodium polyacrylate; Dissolving sodium alginate sodium polyacrylate to obtain a sodium alginate sodium polyacrylate solution; Mixing the sodium alginate sodium polyacrylate solution with the biocontrol bacteria solution to obtain a mixed solution; The mixed solution is sprayed into a calcium chloride solution to obtain a precipitate; The precipitate is freeze-dried to obtain biocontrol bacteria microcapsules.

2. The preparation method according to claim 1, characterized in that: The oxidizing agent is sodium periodate.

3. The preparation method according to claim 2, characterized in that: The volume ratio of the sodium alginate solution to the weight ratio of the oxidant is 200 mL: 0.01-0.03 g.

4. The preparation method according to claim 3, characterized in that: The oxidation treatment time is 100 to 140 minutes.

5. The preparation method according to claim 1, characterized in that: The concentration of the calcium chloride solution is 0.1-0.3 mol / L.

6. The preparation method according to claim 4, characterized in that: The temperature of the oxidation treatment is 50-70°C.

7. The preparation method according to claim 1, characterized in that: The reaction temperature of the heating treatment is 70-90°C and is carried out under nitrogen protection; The heating treatment time is 2 to 4 hours; The dialysis time is 2 to 4 days; The freeze-drying treatment time is 12 to 72 hours; The concentration of the sodium alginate sodium polyacrylate solution is 1-3%; The concentration of the biocontrol bacteria solution is 10 8 ~10 11 CFU / mL; After the spraying to the calcium chloride solution, the precipitation is allowed to stand for 20 to 40 minutes.

8. The preparation method according to any one of claims 1 to 7, characterized in that: The biocontrol bacteria are selected from one or more of Bacillus Velez, Bacillus subtilis, Pseudomonas fluorescens, Trichoderma, Streptomyces, Pseudomonas, Chaetomium, Penicillium or Verticillium chlamydosporeans.

9. Biocontrol bacteria microcapsules obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the biocontrol bacteria microcapsule according to claim 9 in the preparation of biological control products.

Images