Biocontrol bacillus microcapsule and preparation method thereof

Abstract

The application provides a biocontrol bacillus microcapsule and a preparation method thereof, and belongs to the technical field of biological bacterial agents. The application provides a bacillus velezensis microcapsule and a preparation method thereof, prolongs the action time of the strain through microcapsule technology, improves the adhesion of the drug, and realizes more effective biological control and green control. The microcapsule technology can protect the strain from the influence of the external environment, improve the colonization ability of the strain in the soil, and prolong the control effect. Through the method, the application efficiency and range of the bacillus velezensis can be significantly improved, and an environment-friendly and efficient biological control means is provided for agricultural production.

Description

Technical Field

[0001] This invention relates to the field of biological agents, and in particular to a biocontrol Bacillus microcapsule and its preparation method. Background Technology

[0002] Biological control is a method that utilizes the interactions between biological species to reduce the population density of harmful organisms. Compared to abiotic control methods such as chemical pesticides, biological control offers advantages such as long-lasting effects, high safety, environmental friendliness, and cost savings. In biological control, using biocontrol bacteria to control pests is an important approach. It involves using biocontrol bacteria to suppress pests and harmful pathogens while simultaneously improving soil quality and population structure, providing safety and nutritional support for plant growth. However, the application of biocontrol bacteria is limited by various ecological factors, such as soil environment, making it difficult for them to establish themselves in different soil types, thus affecting their actual effectiveness. Furthermore, a single biocontrol bacteria typically can only control a limited number of pest and disease species.

[0003] Bacillus velezensis, a novel biocontrol bacterium, exhibits broad-spectrum inhibitory effects against various plant pathogenic fungi and bacteria. It is widely distributed in nature and is harmless to humans and animals, causing no environmental pollution. Studies have shown that Bacillus velezensis can produce a variety of antimicrobial substances, such as polypeptides, antimicrobial proteins, lipopeptide antibiotics, and polyketides, demonstrating good inhibitory effects against plant pathogenic fungi and bacteria. Furthermore, Bacillus velezensis can induce plant resistance and secrete substances that promote plant growth. Nevertheless, in practical applications, Bacillus velezensis still faces challenges such as a short shelf life and a limited duration of its disease-resistant effects. Summary of the Invention

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

[0005] The Bacillus belye ZC-214 used in this invention was deposited on December 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32921.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

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

[0008] Sodium alginate solution was mixed with an oxidizing agent and subjected to oxidation treatment to obtain the oxidation product;

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

[0010] Sodium alginate and sodium polyacrylate are dissolved to obtain a sodium alginate and sodium polyacrylate solution.

[0011] Sodium alginate and sodium polyacrylate solution were mixed with biocontrol bacteria solution to obtain a mixture;

[0012] The mixture was sprayed into a calcium chloride solution to obtain a precipitate;

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

[0014] Preferably, the oxidant 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 to 0.03 g.

[0016] Preferably, the oxidation treatment time is 100-140 min.

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

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

[0019] Preferably, the heat treatment reaction temperature is 70–90°C, and it is carried out under nitrogen protection;

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

[0021] The dialysis time is 2–4 days;

[0022] The freeze-drying process takes 12–72 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 being sprayed into the calcium chloride solution, the sedimentation time is 20-40 minutes.

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

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

[0028] This invention also provides the application of the above-mentioned biocontrol microcapsules in the preparation of biological control products.

[0029] The beneficial effects of this invention are:

[0030] This invention proposes a microcapsule for Bacillus berleis and its preparation method. Utilizing specific microcapsule technology, the effect of the strain is prolonged, and the adhesion of the drug is improved, achieving more effective biological and environmentally friendly control. This microcapsule technology protects the strain from external environmental influences, enhances its colonization ability in soil, and extends its control effect. This method can significantly improve the application efficiency and scope of Bacillus berleis, providing an environmentally friendly and efficient biological control method for agricultural production. Attached Figure Description

[0031] Figure 1 Figure 1 shows the effect of sodium alginate concentration on the encapsulation efficiency of microcapsules after microcapsule formation.

[0032] Figure 2 The graph shows the effect of calcium ion concentration on the encapsulation efficiency of microcapsules after microcapsule formation.

[0033] Figure 3 This is a comparison of the survival rates of Bacillus belyss microcapsules and unencapsulated bacterial solutions under ultraviolet light irradiation. Detailed Implementation

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection 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 belyssus* ZC-214 was added to 100 mL of LB medium and incubated at 37 °C for 16 h. The OD600 value of the strain was measured to be 4.82 using a UV spectrophotometer. The bacterial suspension was centrifuged at 5000 rpm and 4 °C for 10 min, resuspended in PBS, and centrifuged three times. The precipitate was collected, washed with sterile physiological saline, and diluted to a colony count of approximately 10. 8 CFU / mL bacterial culture, used for coating process or viability detection.

[0038] Weigh 1.0g of sodium alginate powder and dissolve it in 200mL of deionized water. Under a nitrogen atmosphere, stir at a constant temperature of 30℃ and 200rpm until completely dissolved to form a homogeneous solution. Add sodium periodate for oxidation, then heat to 60℃. Neutralize 0.72g of redistilled acrylic monomer with sodium hydroxide solution under ice-water bath conditions. Slowly add the neutralized monomer dropwise to the sodium alginate solution, heat to 80℃, and react under nitrogen protection for 3h. Dialyze to water for 3d, and freeze-dry for 24h to obtain sodium alginate polyacrylate (SA-PAAs) material.

[0039] The obtained SA-PAAs material was sterilized and then dissolved in sterile water to obtain a 2% SA-PAAs solution. 10 mL of 10% SA-PAAs solution was then added to 100 mL of this solution. 8 Bacillus cereus culture with a concentration of CUF / mL was stirred at 500 rpm for 5 minutes until homogeneous. The mixture was then added to a sprayer and sprayed evenly onto a calcium chloride solution. The mixture was allowed to stand and precipitate for 30 minutes. The precipitate was collected and freeze-dried to obtain microcapsules.

[0040] Comparative Example 1

[0041] The number of colonies of Bacillus belyssus ZC-214 obtained according to the method in Example 1 was approximately 10. 8 CFU / mL bacterial culture.

[0042] Take 0.5g of the microcapsules prepared with the optimal ratio selected in Example 1 and 0.5mL of the bacterial suspension prepared in this comparative example, add them to sterile culture dishes, add 10mL of sterile water, seal and let stand. Three parallel groups were formed, and the survival rate was measured under ultraviolet light at 1h, 2h and 3h. The results are as follows. Figure 3 As shown. From Figure 3 As can be seen, under ultraviolet light irradiation, the survival rate of the microcapsule-encapsulated strains irradiated at 30 cm under a 20W ultraviolet lamp for three hours reached 62.31%, which is 29.08% higher than the survival rate of the unencapsulated strains (33.23%). Furthermore, the survival rate of Example 1 (microcapsules prepared with the optimal ratio) was consistently higher than that of Comparative Example 1 (unencapsulated bacterial solution), indicating that the microencapsulation technology provided by this invention significantly improves the survival rate of Bacillus belyssus under ultraviolet light irradiation and enhances the strain's resistance.

[0043] Experimental Example

[0044] Based on the method in Example 1, the following experimental setup was performed:

[0045] ① Except for the amount of oxidant added in increments of 0.01g to 0.1g for the oxidation of sodium alginate, the oxidation time and the amount of other drugs added remained unchanged. Finally, the encapsulation rate after microcapsule formation was measured to select the optimal amount of oxidant.

[0046] ② After determining the amount of oxidant, keep all other conditions unchanged and only change the oxidation time. The reaction time was 30 min, 60 min, 90 min, 120 min, 150 min and 180 min for six different times. Finally, the encapsulation rate of the microcapsules was measured to select the optimal oxidation time.

[0047] ③ Under the optimal oxidant dosage and oxidation time conditions determined in the previous step, calcium chloride solution was used to prepare microcapsules with different calcium ion concentrations, ranging from 0.1 mol / L to 1 mol / L. The encapsulation efficiency of the microcapsules was then measured to select the optimal calcium ion concentration. The results are as follows: Figure 2 As shown.

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

[0049]

[0050] Experimental results: The optimal solution selected in the final experiment was a sodium periodate oxidant content of 0.02 g in 200 mL of sodium alginate solution, an oxidation time of 120 min, and a calcium chloride solution concentration of 0.2 mol / L. Microcapsules with an encapsulation efficiency of 85.32% were obtained.

[0051] As can be seen from the above embodiments, the present invention provides a Bacillus microcapsule technology that significantly improves the application effect of Bacillus belyss 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 microcapsules made of materials such as sodium alginate. Microencapsulation not only improves the adhesion of the strain, enabling it to adhere more effectively to plant surfaces and soil particles, but also extends the product's shelf life, achieving a slower and more precise release of the strain, thereby improving the accuracy and durability of biological control.

[0052] The Bacillus microcapsules provided by this invention can reduce the use of chemical pesticides, meeting the requirements of green agriculture and sustainable development, and helping to protect the environment and improve the safety of agricultural products. They can also increase crop yield and quality, improve soil microbial community structure, 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 this invention offers an environmentally friendly, efficient, and durable new approach to biological control in agricultural production, contributing to the green transformation and sustainable development goals of agricultural production.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing biocontrol microcapsules, characterized in that, Includes the following steps: Sodium alginate solution was mixed with an oxidizing agent and subjected to oxidation treatment to obtain the oxidation product; The oxidation product was mixed with sodium hydroxide solution and redistilled acrylic acid monomer, and then subjected to heat treatment, dialysis and freeze drying in sequence to obtain sodium alginate polyacrylate. Sodium alginate and sodium polyacrylate are dissolved to obtain a sodium alginate and sodium polyacrylate solution. Sodium alginate and sodium polyacrylate solution were mixed with biocontrol bacteria solution to obtain a mixture; The mixture was sprayed into a calcium chloride solution to obtain a precipitate; The precipitate was freeze-dried to obtain biocontrol microcapsules; The biocontrol bacteria is Bacillus belyssus ZC-214, with the accession number CGMCC No. 32921.

2. The preparation method according to claim 1, characterized in that, The oxidant 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 100~140 min.

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 oxidation treatment temperature is 50~70℃.

7. The preparation method according to claim 1, characterized in that, The heat treatment reaction temperature is 70~90℃, and it is carried out under nitrogen protection; The heat treatment time is 2-4 hours; The dialysis time is 2-4 days; The freeze-drying process takes 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 being sprayed into the calcium chloride solution, the sedimentation time is 20-40 minutes.

8. Biocontrol microcapsules prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the biocontrol microcapsules according to claim 8 in the preparation of biological control products.

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