Nanoscale sustained-release microbial pesticide degrading agent and preparation method thereof

Abstract

The application provides a nano-slow-release microbial pesticide degrading agent and a preparation method thereof, and belongs to the technical field of pesticide degradation and slow release. The self-assembled peptide nanotube is used as a carrier, then a complex microbial group composed of pseudomonas and bacillus is loaded on the self-assembled peptide nanotube, and a nano-composite system which can be slowly released, degradable and environment-friendly is constructed. The self-assembled peptide nanotube can be cut into free amino acids by proteases secreted by bacillus or soil fungi, so that the amino acids can be used by microorganisms, the activity of the microorganisms is maintained, and a high degradation rate is achieved.

Description

Technical Field

[0001] This invention relates to the field of pesticide degradation slow-release technology, and in particular to a nano-slow-release microbial pesticide degrading agent and its preparation method. Background Technology

[0002] With the continuous development of agriculture, the application of pesticides in the prevention and control of pests and diseases inevitably leads to the increasingly prominent problem of pesticide residues in soil and water. Due to the low actual usage rate of pesticides, most pesticides are lost into the environment, resulting in pesticide residues in the soil. In the treatment of pesticide residues, there are three main treatment approaches: physical methods, chemical methods and biological methods. Although traditional chemical degradation agents (such as hydrogen peroxide and ozone) can quickly oxidize pesticides, they are prone to producing toxic byproducts, posing a risk of secondary pollution, and are difficult to effectively treat deep soil residues.

[0003] Microbial degradative agents (such as Pseudomonas and Bacillus) have become a research hotspot due to their green and sustainable characteristics, but they face three major bottlenecks: poor sustained-release effect, difficulty in maintaining activity, and low degradation efficiency. In particular, their short action period (only 5-7 days) cannot cover the pesticide half-life. To extend the action time of microbial degradative agents, nanocarrier technology (such as mesoporous silica and chitosan) has been introduced. However, existing nanocarriers have limitations such as poor biocompatibility, lack of synergistic nutritional effects, and high cost. These carrier materials often cannot provide the necessary nutritional support for microorganisms and have limited adaptability in complex soil environments.

[0004] Therefore, this invention is proposed to solve the above-mentioned technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a nano-controlled-release microbial pesticide degrading agent, comprising the following steps:

[0006] S1. Mix the peptide RADA16-I powder with ultrapure water, sonicate for 30 minutes to completely dissolve the peptide powder, then adjust the pH of the solution to 7 to form a 0.5~2 mg / mL RADA16-I solution.

[0007] S2. A composite bacterial solution is prepared by mixing Pseudomonas bacterial solution and Bacillus bacterial solution.

[0008] S3. Mix the RADA16-I solution and composite bacterial solution prepared in steps S1 and S2 at a volume ratio of (8~10):1, incubate at a constant temperature of 37℃ for 12h, and then collect the product by centrifugation to obtain wet self-assembled peptide nanotubes.

[0009] S4. The wet self-assembled peptide nanotubes were placed in a freeze dryer and dried for 24 hours to obtain a white powder solid. The white powder solid was pressed and sieved to obtain a nano-slow-release microbial pesticide degrading agent with a particle size of 1~2 mm.

[0010] Preferably, in step S1, the ultrasonic treatment temperature is room temperature and the frequency is 40kHz.

[0011] Preferably, in step S2, the Pseudomonas aeruginosa is Pseudomonas aeruginosa with a viable count of 1.0 × 10⁻⁶. 9 CFU / mL, the Bacillus species being Bacillus subtilis, with a viable count of 6.0 × 10⁻⁶. 8 CFU / mL.

[0012] Preferably, in step S2, the Pseudomonas and Bacillus are mixed in a mass ratio of (1~3):1.

[0013] Preferably, in step S3, the centrifugation speed is 5000 rpm and the centrifugation time is 15 min.

[0014] Preferably, in step S4, the freeze dryer is set to a temperature of -50°C and a pressure of 0.1 mbar.

[0015] The present invention also provides a nano-slow-release microbial pesticide degrader prepared by the preparation method described above.

[0016] The beneficial effects of this invention are:

[0017] Compared with traditional nanocarrier technology, this invention can adsorb and enrich pesticide molecules in soil through self-assembled peptide nanotubes. The loaded complex microbial community can not only directly degrade and enrich pesticides, but also use the degradation products of the self-assembled peptide carrier, amino acids, as nitrogen and carbon sources to provide necessary nutritional support. Furthermore, because the microbial complex community is loaded inside the peptide nanotubes, a slow-release effect can be achieved. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1

[0020] The preparation method of a nano-controlled-release microbial pesticide degrading agent according to this embodiment includes the following steps:

[0021] S1. Mix 500mg of peptide RADA16-I powder with 1000mL of ultrapure water, sonicate for 30min, set the sonication temperature to room temperature and the frequency to 40kHz, and adjust the pH of the solution to 7 after the peptide powder is completely dissolved to form a 0.5mg / mL RADA16-I solution.

[0022] S2. A composite bacterial solution was prepared by mixing *Pseudomonas aeruginosa* bacterial solution and *Bacillus subtilis* bacterial solution at a mass ratio of 1:1, wherein the viable count of *Pseudomonas aeruginosa* was 1.0 × 10⁻⁶. 9 CFU / mL, Bacillus subtilis viable count was 6.0 × 10⁻⁶. 8 CFU / mL;

[0023] S3. The RADA16-I solution and composite bacterial solution prepared in steps S1 and S2 are mixed evenly at a volume ratio of 8:1. After static incubation at 37℃ for 12h, the product is collected by centrifugation at 5000rpm for 15min to obtain wet self-assembled peptide nanotubes.

[0024] S4. The wet self-assembled peptide nanotubes were placed in a freeze dryer and dried at a temperature of -50℃ and a pressure of 0.1mbar for 24 hours to obtain a white powder solid. The white powder solid was pressed and sieved to obtain a nano-slow-release microbial pesticide degrading agent with a particle size of 1~2mm.

[0025] Example 2

[0026] The preparation method of a nano-controlled-release microbial pesticide degrading agent according to this embodiment includes the following steps:

[0027] S1. Mix 100mg of peptide RADA16-I powder with 1000mL of ultrapure water, sonicate for 30min, set the sonication temperature to room temperature and the frequency to 40kHz, and adjust the pH of the solution to 7 after the peptide powder is completely dissolved to form a 1mg / mL RADA16-I solution.

[0028] S2. A composite bacterial solution was prepared by mixing *Pseudomonas aeruginosa* bacterial solution and *Bacillus subtilis* bacterial solution at a mass ratio of 2:1, wherein the viable count of *Pseudomonas aeruginosa* was 1.0 × 10⁻⁶. 9 CFU / mL, Bacillus subtilis viable count was 6.0 × 10⁻⁶. 8 CFU / mL;

[0029] S3. The RADA16-I solution and composite bacterial solution prepared in steps S1 and S2 are mixed evenly at a volume ratio of 9:1. After static incubation at 37℃ for 12h, the product is collected by centrifugation at 5000rpm for 15min to obtain wet self-assembled peptide nanotubes.

[0030] S4. The wet self-assembled peptide nanotubes were placed in a freeze dryer and dried at a temperature of -50℃ and a pressure of 0.1mbar for 24 hours to obtain a white powder solid. The white powder solid was pressed and sieved to obtain a nano-slow-release microbial pesticide degrading agent with a particle size of 1~2mm.

[0031] Example 3

[0032] The preparation method of a nano-controlled-release microbial pesticide degrading agent according to this embodiment includes the following steps:

[0033] S1. Mix 200mg of peptide RADA16-I powder with 1000mL of ultrapure water, sonicate for 30min, set the sonication temperature to room temperature and the frequency to 40kHz, and adjust the pH of the solution to 7 after the peptide powder is completely dissolved to form a 2mg / mL RADA16-I solution.

[0034] S2. A composite bacterial solution was prepared by mixing *Pseudomonas aeruginosa* bacterial solution and *Bacillus subtilis* bacterial solution at a mass ratio of 3:1, wherein the viable count of *Pseudomonas aeruginosa* was 1.0 × 10⁻⁶. 9 CFU / mL, Bacillus subtilis viable count was 6.0 × 10⁻⁶. 8 CFU / mL;

[0035] S3. Mix the RADA16-I solution and the composite bacterial solution prepared in steps S1 and S2 at a volume ratio of 10:1. After static incubation at 37℃ for 12h, collect the product by centrifugation at 5000rpm for 15min to obtain wet self-assembled peptide nanotubes.

[0036] S4. The wet self-assembled peptide nanotubes were placed in a freeze dryer and dried at a temperature of -50℃ and a pressure of 0.1mbar for 24 hours to obtain a white powder solid. The white powder solid was pressed and sieved to obtain a nano-slow-release microbial pesticide degrading agent with a particle size of 1~2mm.

[0037] Comparative Example 1

[0038] The difference between this comparative example and Example 2 is that the carrier used in step S1 of the preparation process of this comparative example is mesoporous silica.

[0039] Comparative Example 2

[0040] The difference between this comparative example and Example 2 is that no carrier is used; the composite bacterial solution is freeze-dried into powder and used directly.

[0041] Experiment Example 1: Microbial Survival Rate Experiment

[0042] Take 10g of the finished product from Examples 1-3 and Comparative Example 1 and add it to 50g of sterilized soil (autoclave at 121℃ for 30min). Adjust the soil moisture to 60% and maintain the temperature at around 25℃. Take samples from 0 to 7 days, a total of seven times, 1g each time. Add 9mL of PBS buffer (0.01M), vortex for 30min, take the eluent, and serially dilute it with sterile water. Then spread the diluted solution on LB solid medium and incubate at 37℃ for 24h. Count the number of colonies on the medium.

[0043] Wherein, the microbial survival rate (%) = (number of viable bacteria at a certain time point / initial number of viable bacteria) × 100%;

[0044] The initial viable count was the viable count at 0h, and the viable count at a certain time point was the viable count after elution at the corresponding time points from 1 to 7 days. The experiment was repeated 3 times, and the data is the average of the 3 times. The experimental data are shown in Table 1 below.

[0045] Table 1

[0046]

[0047] Experiment Example 2: Degradation of Organochlorine Pesticides in Soil

[0048] The degradation rates of organochlorine pesticides in Examples 1-3 and Comparative Examples 1-2 were determined using pot experiments. Dichlorophenyltrichloroethane (DDT), 2,4-dichlorophenol, or fenthion were uniformly mixed into the test soil at a concentration of 150 mg / kg. The prepared products from Examples 1-3 and Comparative Examples 1-2 were also mixed into the soil at a concentration of 150 mg / kg and stirred thoroughly. Each pot contained 5 kg of soil, with a pH of 7.0, and was placed in an incubator at room temperature. The concentrations of DDT, 2,4-dichlorophenol, and fenthion in the soil were measured 2, 4, 6, and 8 days after application. A blank control was not applied without any degradation agent. The experiment was repeated three times, and the data are the average of the three trials. The data are shown in Table 2 below.

[0049] The degradation rate is calculated as follows: (pesticide content in the soil before degradation treatment - pesticide content in the soil after degradation treatment) / pesticide content in the soil before degradation treatment × 100%.

[0050] Table 2

[0051]

[0052] As can be seen from the table, compared with Comparative Example 1, the self-assembled peptide nanotube carrier used in the examples can significantly improve the survival rate of microorganisms compared with traditional mesoporous silica carriers. This is because, compared with traditional mesoporous silica carriers, the self-assembled peptide nanotubes of the present invention may decompose into free amino acids during degradation, which can be used as carbon and nitrogen sources for further utilization by the complex bacterial community, maintaining the activity of microorganisms. Therefore, compared with Comparative Examples 1-2, in the degradation rate experiment of organochlorine pesticides, the pesticide degradation agent prepared by the present invention still achieved a degradation rate of over 90% for organochlorine pesticides after 8 days of use.

[0053] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a nano-controlled-release microbial pesticide degrading agent, characterized in that, Includes the following steps: S1. Mix the peptide RADA16-I powder with ultrapure water, sonicate for 30 minutes to completely dissolve the peptide powder, then adjust the pH of the solution to 7 to form a 0.5~2 mg / mL RADA16-I solution. S2. A composite bacterial solution is prepared by mixing Pseudomonas bacterial solution and Bacillus bacterial solution. S3. Mix the RADA16-I solution and composite bacterial solution prepared in steps S1 and S2 at a volume ratio of (8~10):1, incubate at a constant temperature of 37℃ for 12h, and then collect the product by centrifugation to obtain wet self-assembled peptide nanotubes. S4. The wet self-assembled peptide nanotubes were placed in a freeze dryer and dried for 24 hours to obtain a white powder solid. The white powder solid was pressed and sieved to obtain a nano-slow-release microbial pesticide degrading agent with a particle size of 1~2 mm.

2. The preparation method of the nano-controlled-release microbial pesticide degrading agent according to claim 1, characterized in that, In step S1, the ultrasonic treatment temperature is room temperature and the frequency is 40kHz.

3. The preparation method of the nano-controlled-release microbial pesticide degrading agent according to claim 1, characterized in that, In step S2, the Pseudomonas aeruginosa is *Pseudomonas aeruginosa*, with a viable count of 1.0 × 10⁻⁶. 9 CFU / mL, the Bacillus species being Bacillus subtilis, with a viable count of 6.0 × 10⁻⁶. 8 CFU / mL.

4. The preparation method of the nano-controlled-release microbial pesticide degrading agent according to claim 1, characterized in that, In step S2, the Pseudomonas and Bacillus are mixed at a mass ratio of (1~3):

1.

5. The preparation method of the nano-controlled-release microbial pesticide degrading agent according to claim 1, characterized in that, In step S3, the centrifugation speed is 5000 rpm and the centrifugation time is 15 min.

6. The preparation method of the nano-controlled-release microbial pesticide degrading agent according to claim 1, characterized in that, In step S4, the freeze dryer is set to a temperature of -50°C and a pressure of 0.1 mbar.

7. The nano-slow-release microbial pesticide degrader prepared by the preparation method according to any one of claims 1 to 6.

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