Nanometer pesticide with photolysis resistance based on enzymatic hydrolysis lignin and preparation method

Abstract

The invention relates to a nano-pesticide with photolysis resistance based on enzymatic hydrolysis lignin and a preparation method, and belongs to the technical field of nano-pesticides. The nano-pesticide is a black sphere, the effective particle size is 160-200 nm, the drug loading rate is 35.72%, the nano-pesticide has good photolysis resistance, and the photolysis half-life period is prolonged by 1.56 times compared with that of prothioconazole original drug; the composition is used for rice blast diseases. According to the nano pesticide, enzymatic hydrolysis lignin is used as a carrier, a prothioconazole bactericide is used as a loading drug, and tannic acid and copper ion coatings are used for coating; functional groups such as phenol and ketone in the enzymatic hydrolysis lignin can effectively absorb a large range of ultraviolet rays and reduce ultraviolet radiation, so that the enzymatic hydrolysis lignin has excellent oxidation resistance and ultraviolet resistance; meanwhile, the enzymatic hydrolysis lignin has renewability, environmental protection property and degradability, and the coating of tannic acid and copper ions can further improve the antifungal ability of the nano pesticide, improve the thermal stability of the nano pesticide, increase the release rate of the pesticide and have the ability to reduce the loss of the pesticide.

Description

Technical Field

[0001] The invention belongs to the technical field of nano pesticides and discloses a preparation method and application of a nano pesticide based on enzymatically degraded lignin with anti-photolysis performance. Background Art

[0002] Rice blast is a serious disease that affects rice yield and quality. Chemical control using fungicides is an important method for preventing and controlling diseases. Prothioconazole has a novel and unique structure and has a broader spectrum of antifungal activity than other triazole fungicides. However, prothioconazole is easily photolyzed in aqueous solution, and photolysis is an important non-biological degradation pathway that has a significant impact on pesticide residues, drug efficacy maintenance, toxicity, and safety to the ecosystem. The main metabolite of prothioconazole is desulfurized prothioconazole, which affects the female reproductive system and is teratogenic. Therefore, it is extremely important to reduce the metabolism of prothioconazole and increase the fungicidal activity. However, conventional preparations do not have the ability to protect and control the sustained release of active ingredients. Therefore, it is necessary to develop controlled-release preparations of prothioconazole to reduce toxicity risks, extend the shelf life, and reduce environmental pollution.

[0003] Functional groups such as phenol and ketone in enzymatic lignin can effectively absorb a wide range of ultraviolet rays, reduce ultraviolet radiation, and make enzymatic lignin have excellent antioxidant capacity and anti-ultraviolet properties. Nanoenzymatic lignin can replace some inorganic nanoparticles in some fields due to its renewability, environmental protection and degradability. When mixed with different polymers, enzymatic lignin nanoparticles can be evenly distributed in the polymer matrix. Enzymatic lignin as a nanocarrier can not only avoid the photolysis of active ingredients of pesticides and extend the shelf life, but also reduce toxicity and environmental stress to non-target organisms. Therefore, the development of a nanopesticide based on enzymatic lignin with anti-photolysis properties, whose pH value, enzyme response release and photostability are of great significance to reducing the amount of pesticide application and improving the utilization rate of pesticides. Summary of the invention

[0004] In order to reduce the metabolism of prothioconazole, control the sustained release of active ingredients and improve the fungicidal activity, the present invention provides a method for preparing a nano pesticide with anti-photolysis performance based on enzymatic lignin.

[0005] A nanopesticide based on enzymatic lignin with anti-photolysis performance uses enzymatic lignin as a carrier, prothioconazole fungicide as a loaded drug, and is coated with tannic acid and copper ion coating; The nanopesticide is a black sphere with an effective particle size of 160-200 nm and a drug loading rate of 35.72%.

[0006] The preparation steps of a nanopesticide based on enzymatic lignin with anti-photolysis performance are as follows: (1) Preparation of self-assembled enzymatic lignin nanocarrier particles Solution A was prepared by using enzymatic lignin and dimethyl sulfoxide at a mass volume ratio of 1 g:10 mL of enzymatic lignin and dimethyl sulfoxide; solution B was prepared by using cetyl trimethyl ammonium bromide, ethanol and water at a mass volume ratio of 1:2 of enzymatic lignin and cetyl trimethyl ammonium bromide; solution A and solution B were mixed, water was added dropwise, allowed to stand, centrifuged and freeze-dried to obtain purified self-assembled enzymatic lignin nanocarrier particles; (2) Preparation of prothioconazole-loaded nanoparticles The self-assembled enzymatic lignin nanocarrier particles and prothioconazole are dissolved in dimethyl sulfoxide at a mass ratio of 2:1 to obtain an organic phase; water is added dropwise, ultrasonic dispersion is performed to obtain a suspension of drug-loaded microspheres in which prothioconazole is embedded in the enzymatic lignin carrier, centrifugation washing is performed, and freeze drying is performed to obtain prothioconazole-loaded nanocarrier particles; The volume ratio of the organic phase to the added water is 1:4; (3) Preparation of enzymatic lignin nanopesticides The prothioconazole-loaded nanoparticles were dispersed in water at a mass volume ratio of 3 mg:1 mL, and copper chloride was added; tannic acid was then added, and the mass ratio of the prothioconazole-loaded nanoparticles to copper chloride was 10:1; the mass ratio of the prothioconazole-loaded nanoparticles to tannic acid was 3:1; ultrasonic treatment was performed, washing was performed with water, and excess copper chloride and tannic acid were removed by centrifugation; freeze-drying was performed, and the nanopesticide with anti-photolysis properties was collected.

[0007] The further preparation technology scheme is as follows: In step (1), 1 g of enzymatic lignin is dissolved in 10 mL of dimethyl sulfoxide, the volume is fixed to 100 mL with ultrapure water, and the mixture is stirred at 700 rpm for 24 hours at room temperature to obtain solution A; 2 g of hexadecyltrimethylammonium bromide is weighed and dissolved in a mixed solvent of 6 mL of ethanol and 4 mL of ultrapure water, the volume is fixed to 100 mL with ultrapure water, and the mixture is stirred at 600 rpm for 24 hours at room temperature to obtain solution B; solution A and solution B are mixed, stirred at 500 rpm, and 800 mL of ultrapure water is added dropwise at a speed of 4 mL / min; the mixture is allowed to stand for 24 hours, the precipitate is centrifuged at a speed of 10,000 rpm, the centrifuged precipitate is washed 3 times with ultrapure water, and freeze-dried at -50°C to obtain purified self-assembled enzymatic lignin nanocarrier particles; In step (2), 0.1 g of self-assembled enzymatic lignin nanocarrier particles and 0.05 g of prothioconazole original drug are dissolved in 10 mL of dimethyl sulfoxide to obtain an organic phase; the organic phase is ultrasonically dispersed for 5 min, stirred at a speed of 200 rpm, 40 mL of ultrapure water is added dropwise, and ultrasonic dispersion is continued for 3 min to obtain a suspension of drug-loaded microspheres in which the enzymatic lignin carrier encapsulates prothioconazole; the suspension is centrifuged at a speed of 10,000 rpm, the centrifugal precipitate is washed 3 times with ultrapure water, and freeze-dried to obtain prothioconazole-loaded nanocarrier particles; In step (3), 300 mg of the prothioconazole-loaded nanoparticles are dispersed in 100 mL of ultrapure water and stirred continuously at room temperature, and 30 mg of copper chloride is added; 100 mg of tannic acid is then added, ultrasonically treated for 10 min, washed with water, and centrifuged to remove excess copper chloride and tannic acid; freeze-dried, and the enzymatically hydrolyzed lignin nanopesticide is collected.

[0008] The beneficial technical effects of the present invention are embodied in the following aspects: The nano pesticide with anti-photolysis performance of the present invention selects enzymatic lignin with excellent anti-ultraviolet and enzyme response release performance, and forms spherical nano carrier particles with hexadecyltrimethylammonium bromide through electrostatic self-assembly, and the polyphenol compound tannic acid from plant sources forms a complex with copper ions. Tannic acid / copper ions are used for coating on the surface of nano drug-carrying particles, and the complex is easily degraded in an acidic medium. In addition, the coating can further improve the anti-photolysis performance, loading rate, antifungal and anti-pesticide leakage capabilities. Its photolysis half-life is extended by 1.56 times compared with prothioconazole original medicine and 1.31 times compared with 30% prothioconazole suspension. The release of prothioconazole original drug reached equilibrium after 10 hours, and the release rate was only about 40%. At pH = 7, 5, and 3, the release rates of enzymatic lignin nanopesticides were 46.53%, 58.64%, and 65.29% after 10 hours, respectively; at pH = 3 and 72 hours, the release rate of enzymatic lignin nanopesticides could reach more than 70%. In the presence of pectinase, at pH = 7, the cumulative release rate could also reach 73.22% after 36 hours, realizing pH-responsive and enzyme-responsive drug release, improving the effectiveness of pesticides, improving the stability of active ingredients, extending their shelf life, and reducing the biological toxicity and environmental burden of pesticides.

[0009] 2. The functional groups such as phenol and ketone in the enzymatic lignin used in the present invention can effectively absorb a wide range of ultraviolet rays, reduce ultraviolet radiation, and make the enzymatic lignin have excellent antioxidant capacity and anti-ultraviolet performance. Nano enzymatic lignin can replace some inorganic nanoparticles in certain fields due to its renewability, environmental protection and degradability. When mixed with different polymers, the enzymatic lignin nanoparticles can be evenly distributed in the polymer matrix. Enzymatic lignin as a nanocarrier can not only avoid the photolysis of pesticide active ingredients and extend the shelf life, but also reduce toxicity and environmental stress to non-target organisms. Therefore, the development of a nanopesticide based on enzymatic lignin with anti-photolysis performance, whose pH value, enzyme response release and photostability are of great significance to reducing the amount of pesticide application and improving the utilization rate of pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The transmission electron micrographs of the enzymatically hydrolyzed lignin self-assembled carrier obtained in Example 1, the prothioconazole-loaded nanoparticles, and the enzymatically hydrolyzed lignin nanopesticide particles.

[0011] Figure 2 The average particle size and Zeta potential diagram of the enzymatically hydrolyzed lignin self-assembled carrier obtained in Example 1, the nanoparticles loaded with prothioconazole, and the enzymatically hydrolyzed lignin nanopesticide particles.

[0012] Figure 3 The physicochemical properties of the enzymatically hydrolyzed lignin self-assembled carrier obtained in Example 1, the nanoparticles loaded with prothioconazole, and the enzymatically hydrolyzed lignin nanopesticide.

[0013] Figure 4 This is a surface element analysis diagram of the enzymatically hydrolyzed lignin self-assembled carrier obtained in Example 1, the nanoparticles loaded with prothioconazole, and the enzymatically hydrolyzed lignin nanopesticide.

[0014] Figure 5 This is a diagram showing the drug loading of the prothioconazole-loaded nanoparticles and the enzymatically hydrolyzed lignin nanopesticide obtained in Example 1.

[0015] Figure 6 The release curves of the enzymatically hydrolyzed lignin nanopesticide obtained in Example 1 under different conditions.

[0016] Figure 7 This is a graph showing the anti-photolysis performance of the enzymatically hydrolyzed lignin nanopesticide obtained in Example 1.

[0017] Figure 8 The graphs are mycelium growth experiments of the fungicidal activity of prothioconazole-loaded nanoparticles, enzymatic lignin nanopesticides, and prothioconazole original drug against rice blast pathogens at different concentrations obtained in Example 1.

[0018] Fig. 9This is a graph showing the mycelium inhibition rate of the fungicidal activity of prothioconazole-loaded nanoparticles, enzymatic lignin nanopesticides, and prothioconazole original drug against rice blast pathogens at different concentrations obtained in Example 1. DETAILED DESCRIPTION

[0019] The present invention will be further described below by way of embodiments in conjunction with the accompanying drawings.

[0020] Unless otherwise defined, all technical and scientific terms used herein belong to the technical field of the present invention. Example 1

[0021] The preparation steps of a nanopesticide based on enzymatic lignin with anti-photolysis performance are as follows: Preparation of self-assembled enzymatic lignin nanocarrier particles 1 g of enzymatically hydrolyzed lignin was dissolved in 10 mL of dimethyl sulfoxide, and the volume was made up to 100 mL with ultrapure water. The mixture was stirred at 700 rpm for 24 hours at room temperature to obtain solution A. Weigh 2 g of hexadecyltrimethylammonium bromide in a mixed solvent of 6 mL of ethanol and 4 mL of ultrapure water, make up to 100 mL with ultrapure water, and stir at 600 rpm for 24 hours at room temperature to obtain solution B; Solution A and solution B were mixed, stirred at 500 rpm, and 800 mL of ultrapure water was added dropwise at a rate of 4 mL / min; the mixture was allowed to stand for 24 hours, the precipitate was centrifuged at 10,000 rpm, the centrifuged precipitate was washed three times with ultrapure water, and freeze-dried at -50°C to obtain purified self-assembled enzymatic lignin nanocarrier particles.

[0022] Preparation of prothioconazole-loaded nanoparticles 0.1 g of self-assembled enzymatic lignin nanocarrier particles and 0.05 g of prothioconazole original drug were dissolved in 10 mL of dimethyl sulfoxide to obtain an organic phase; the organic phase was ultrasonically dispersed for 5 min, stirred at 200 rpm, 40 mL of ultrapure water was added dropwise, and ultrasonic dispersion was continued for 3 min to obtain a suspension of prothioconazole-loaded microspheres encapsulated in the enzymatic lignin carrier; the mixture was centrifuged at 10,000 rpm, the centrifugal precipitate was washed 3 times with ultrapure water, and freeze-dried to obtain prothioconazole-loaded nanoparticles.

[0023] Preparation of enzymatically hydrolyzed lignin nanopesticide particles 300 mg of the prothioconazole-loaded nanoparticles were dispersed in 100 mL of ultrapure water and stirred continuously at room temperature, and 30 mg of copper chloride was added; 100 mg of tannic acid was then added, and ultrasonic treatment was performed for 10 min, and the mixture was washed with water and centrifuged to remove excess copper chloride and tannic acid; the mixture was freeze-dried and the enzymatic lignin nanopesticide was collected.

[0024] The self-assembled enzymatically hydrolyzed lignin nano-carrier particles, prothioconazole-loaded nano-drug-carrying particles and enzymatically hydrolyzed lignin nano-pesticide particles prepared in Example 1 were analyzed by transmission electron microscopy.

[0025] See also Figure 1 , Figure 1 A in the figure is a nanocarrier particle formed by self-assembly of enzymatically hydrolyzed lignin and hexadecyltrimethylammonium bromide. Figure 1 B in the figure is a nanoparticle loaded with prothioconazole. Figure 1 C in the figure is the enzymatically degraded lignin nanopesticide particles after tannic acid / copper ion coating. Figure 1 A in the figure shows that the self-assembled carrier is a sphere with smooth and regular surface. Figure 1 B in the figure is a black sphere with a regular shape. Figure 1 A in the figure indicates that most of the prothioconazole was condensed on the microspheres. After the tannic acid / copper ion coating, the surface of the nanoparticles became no longer smooth. Tannic acid / copper ions formed microparticles that adhered to the surface of the spheres. Figure 1 C in.

[0026] See also Figure 2 , Figure 2 A in the figure is the average particle size. Figure 2 B in the figure is the Zeta potential diagram. The average particle size of enzymatic lignin, self-assembled carrier, prothioconazole-loaded nanoparticles and enzymatic lignin nanopesticides are all less than 200 nm. Enzymatic lignin is an anionic surfactant, and hexadecyltrimethylammonium bromide is a cationic surfactant. When enzymatic lignin and hexadecyltrimethylammonium bromide meet at the isoelectric point, they will attract each other through electrostatic interaction and self-assemble into regular nanospheres. Therefore, the Zeta potential shows that enzymatic lignin (-31.57 mV) and hexadecyltrimethylammonium bromide have a higher potential after forming a self-assembled carrier (-11.96 mV) through electrostatic interaction, while the potential of the prothioconazole-loaded nanoparticles is reduced (-37.43 mV), indicating that the self-assembled carrier can load drugs through electrostatic interaction. With the coating of tannic acid / copper ions, the potential of the final nanopesticide is -19.60 mV, indicating that tannic acid / copper ions are successfully deposited on the surface of the prothioconazole-loaded nanoparticles.

[0027] See also Figure 3 , Figure 3 A and Figure 3 B in the figure is the Fourier infrared visible spectrum. Figure 3 C in is the UV-visible absorption spectrum. Figure 3 D in the figure is the thermogravimetric analysis curve. Figure 3 A and Figure 3 B in the figure shows that the infrared spectrum of prothioconazole original drug is 1482 cm -1 The stretching vibration peak of the CH bond is shown at 750 cm -1 There is a stretching vibration peak caused by the C-Cl bond on the benzene ring. The nanoparticles loaded with prothioconazole and the nanopesticide coated with tannic acid / copper ions are both at 1480 cm -1 and 750 cm -1 The infrared characteristic peak was observed at , proving that prothioconazole has been successfully loaded. Figure 3 C in the figure, from the results of the UV-visible absorption spectrum, it can be seen that both the enzymatic lignin and the self-assembled carrier have absorption peaks at 280 nm, which is the characteristic peak of enzymatic lignin. The characteristic peak of prothioconazole is around 250 nm. It can be seen from the figure that the nanoparticles loaded with prothioconazole and the nanopesticide after the final coating have absorption peaks at 250 nm, which further indicates that prothioconazole is successfully loaded on the self-assembled carrier. Figure 3 D in Figure 1, thermogravimetric analysis was performed under nitrogen flow to determine the decomposition pattern and thermal stability of prothioconazole, self-assembled carrier, nanoparticles loaded with prothioconazole, and nanopesticide coated with coating. Prothioconazole had its first weight loss before 100°C, which was attributed to the evaporation of water; the weight fraction of prothioconazole began to drop sharply after 305°C, and the total weight loss of prothioconazole was about 70.29% when the sample was heated to 600°C. When the temperature increased from 240°C to 600°C, the weight loss of the self-assembled carrier was about 50.38%, which may be caused by the decomposition of the nanoparticles themselves or the organic functional groups therein. After prothioconazole was loaded on the self-assembled carrier, it began to lose weight from about 240°C, and the total weight loss was only 7.20%. The nanopesticide after tannic acid / copper ion coating also began to lose weight from about 240°C, and the total weight loss was less, only 4.77%. This further proves that the nanoparticles successfully loaded prothioconazole, and there is a strong interaction between prothioconazole and the self-assembled carrier, and the tannic acid / copper ion coating can further improve the thermal stability of the nanodrug-loaded particles.

[0028] See also Figure 4 , the elemental composition and valence state of the self-assembled carrier, prothioconazole-loaded nanoparticles and nanopesticides were analyzed using X-ray photoelectron spectroscopy. Figure 4In A, we can see that carbon, nitrogen and oxygen are detected on the surface of the self-assembled support, and the binding energies at 284.8 eV, 400.6 eV and 532.8 eV correspond to C1s, N1s and O1s, respectively. Figure 4 B in), nitrogen ( Figure 4 C in), oxygen ( Figure 4 D) element in the nanoparticles loaded with prothioconazole ( Figure 4 E in the final coating, and the copper element of the nanopesticide ( Figure 4 F) was fitted by peak fitting, and the binding energy peak of Cl2p appeared at 200.4 eV, which proved that prothioconazole was successfully loaded onto the self-assembled carrier, and the binding energy peaks of Cu2p appeared at 932.4 eV and 952.1 eV, which proved that tannic acid / Cu 2+ Successfully coated on nano drug-loaded particles.

[0029] Nanopesticide loading rate: See also Figure 5 , Figure 5 A in the figure is the standard curve of prothioconazole determined by ultra-high performance liquid chromatography. Figure 5 B is the drug loading rate of nanoparticles loaded with prothioconazole and nanopesticide. The results show that the drug loading rate of nanoparticles loaded with prothioconazole is 31.75%, and the drug loading rate of nanopesticide coated with tannic acid / copper ion coating is 35.72%, indicating that tannic acid / copper ion can effectively reduce the loss of pesticides and increase the pesticide loading rate.

[0030] The drug release experiment of prothioconazole was carried out by ultra-high performance liquid chromatography to determine the release amount of prothioconazole concentration detected at different time intervals. The release behavior of nanopesticides was investigated by dialysis bag method, and 30% ethanol phosphate buffer solution was used as the release medium. 0.1 g of nanopesticide and prothioconazole original drug were weighed and suspended in 10 mL of release medium with different pH values ​​(3, 5, 7), and placed in the dialysis bag. The dialysis bag was sealed and placed in a 250 mL triangular flask, and 190 mL of release medium was added to the triangular flask. The triangular flask was placed in a thermostatic table shaker with a speed of 150 rpm. 3 mL of release medium was taken with a pipette at intervals (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72 h). 3 mL of fresh release medium was added to the triangular flask after each sample was taken. The released medium was injected into the injection bottle through a 0.22 μm needle filter using a disposable syringe, labeled and stored at 4°C for testing. The concentration of the active ingredient in the release medium was determined by ultra-high performance liquid chromatography.

[0031] To further explore the mechanism of pesticide release, the first-order, Higuchi and Ritger-Peppas models were used to analyze the release kinetics of nanopesticides. The relevant equations are as follows: First-order model:

[0032] Higuchi Model:

[0033] Ritger-Peppas Model:

[0034] in Prothioconazole t Cumulative release ratio at time (%), a is the initial concentration, k is the kinetic constant, n is the diffusion index.

[0035] See also Figure 6 , Figure 6 A in the figure is the release curve of nanopesticide and prothioconazole original drug at pH values ​​of 3, 5 and 7; Figure 6 B in the figure is the release curve of nanopesticide and prothioconazole original drug at pH 7 in the presence of pectinase. The correlation coefficient (R 2 ) to judge the fitting accuracy of the model. The fitting results are shown in Table 1. The first-order kinetic release model is better than the Higuchi model and the Ritger-Peppas model in fitting the release process of prothioconazole original medicine and nanopesticide. It can be seen from the release kinetic curve that the release rate of prothioconazole in the nanopesticide is higher than that of the original medicine. The release of prothioconazole original medicine reaches equilibrium after 10 hours, and the release rate is only about 40%, while the nanopesticide release rates are 46.53%, 58.64% and 65.29% at pH = 7, 5, and 3, respectively, after 10 hours. At 72 hours, the release rate of nanopesticide at pH = 3 reached more than 70%. This pH-dependent release under acidic conditions may be due to the release of tannic acid / copper ions in acidic media, which increases the release rate of pesticides. In addition, in the presence of pectinase, at pH = 7, the cumulative release rate of nanopesticides reached 73.22% after 36 hours. In the absence of the enzyme, the release rate of the nanopesticide was slow, with a cumulative release rate of 50.67%, while the cumulative release rate of prothioconazole was only 42.32%. During the same period, the cumulative release rates increased by 22.55% and 30.90%, respectively. In the presence of pectinase, a large amount of prothioconazole was released rapidly, so pectinase was the main reason for the accelerated release.

[0036] Table 1 Fitting results of release curves of nanopesticides under different conditions

[0037] Anti-photolysis performance of nanopesticides: See also Figure 7 , Figure 7 The curve of the relationship between the retention rate of prothioconazole and time under the same experimental conditions, where the slope represents the rate of photodegradation. Lignin has a good anti-ultraviolet effect due to its rich phenolic hydroxyl and quinone structure. Figure 7 As shown in Table 2, the photodegradation rates of prothioconazole are as follows: nanopesticide (0.61%) < nanoparticles loaded with prothioconazole (0.70%) < prothioconazole preparation (1.17%) < prothioconazole original drug (1.18%). After 60 h, the retention rate of prothioconazole in nanopesticide (65.51%) > nanoparticles loaded with prothioconazole (58.37%) > prothioconazole preparation (29.03%) > prothioconazole original drug (21.33%). It was calculated that the half-life of nanoparticles loaded with prothioconazole was 1.11 times longer than that of prothioconazole original drug and 0.9 times longer than that of prothioconazole preparation. The half-life of the nanopesticide is 1.56 times longer than that of the original prothioconazole and 1.31 times longer than that of the prothioconazole preparation, which indicates that the enzymatic lignin self-assembled carrier can effectively reduce the photolysis rate of prothioconazole, and the tannic acid / copper ion coating further improves the anti-photolysis performance.

[0038] Table 2 Anti-photolysis performance parameters of prothioconazole technical, prothioconazole preparation, prothioconazole-loaded nanoparticles and nanopesticides

[0039] Study on the biological activity of nanopesticides against plant pathogens: In order to determine the biological activity of the prepared prothioconazole-loaded nanoparticles and nanopesticides, rice blast was selected as the test strain, and the antibacterial activity of the nanopesticide was determined by the mycelial growth rate method. Prothioconazole original drug was used as a control. The prepared potato dextrose agar medium was sterilized at high temperature and high pressure, cooled to 50°C, and different concentrations (0.1, 0.5, 1, 5 and 10 mg / L) of prothioconazole original drug, prothioconazole-loaded nanoparticles and nanopesticide were added and mixed evenly, poured into a culture dish, and cooled and solidified to obtain a potato dextrose agar medium plate. The mycelial disk of the rice blast pathogen with a good growth and a diameter of 6 mm was inoculated on the potato dextrose agar medium plate. After culture at 26°C for 7 days, the colony diameters on days 3, 5 and 7 were measured and recorded by the cross method. The inhibition rate (%) of the fungus was calculated, and the relevant formula is as follows:

[0040] in, IR is the inhibition rate of fungi, dc and d t are the colony diameters of the control group and the treatment group, d 0 is the initial diameter of the mycelial disk (6 mm).

[0041] See also Figure 8 , which are experimental diagrams of the effects of different concentrations (0.1, 0.5, 1, 5 and 10 mg / L) of prothioconazole original drug, prothioconazole-loaded nanoparticles and nanopesticides on the mycelial growth of rice blast fungus. Figure 8 A in Figure 8 B and Figure 8 Figures C and B are the mycelial growth experiments on rice blast fungus on days 3, 5, and 7. Compared with prothioconazole and prothioconazole-loaded nanoparticles, the nanopesticide had a greater inhibitory effect on fungal growth after 7 days of cultivation (see the smaller colony size).

[0042] join Fig. 9 , the survival rate of fungi was calculated using the cross-over method. Fig. 9 A in Fig. 9 B and Fig. 9 C in the figure is the inhibition rate of prothioconazole technical, prothioconazole-loaded nanoparticles and nanopesticides at different concentrations (0.1, 0.5, 1, 5 and 10 mg / L) after 3, 5 and 7 days of cultivation. The results showed that prothioconazole technical, prothioconazole-loaded nanoparticles and nanopesticides all showed excellent biological activity, and the antibacterial activity against rice blast was dose- and time-dependent. Under the same conditions, the antibacterial activity of nanopesticide was significantly better than that of prothioconazole technical and prothioconazole-loaded nanoparticles. When the concentration of the active ingredient was 1 mg / L, on the 3rd, 5th and 7th days, the inhibition rate of nanopesticide against rice blast was 29.88%, 31.28% and 27.53% higher than that of prothioconazole technical, and 29.27%, 28.48% and 24.72% higher than that of nanoparticles loaded with prothioconazole. When the concentration of the active ingredient of prothioconazole reached 10 mg / L on the 7th day, the inhibition efficiency of the nanopesticide against rice blast fungus (96.25%) was still significantly higher than that of prothioconazole original drug (83.14%) and prothioconazole-loaded nanoparticles (88.38%).

[0043] The above results show that nanopesticides exhibit a greater inhibitory efficiency against rice blast. The tannic acid / copper ion modified nanopesticide carrier significantly improves the antifungal ability of the pesticide, which is attributed to the antifungal ability of copper ions and the ability of tannic acid to reduce pesticide losses.

Claims

1. A nanopesticide based on enzymatic lignin with anti-photolysis performance, characterized in that: The nano pesticide based on enzymatic lignin with anti-photolysis performance uses enzymatic lignin as a carrier, prothioconazole fungicide as a loaded drug, and is coated with tannic acid and copper ion coating; The nanopesticide is a black sphere with an effective particle size of 160-200 nm and a drug loading rate of 35.72%.

2. A method for preparing a nanopesticide with anti-photolysis performance based on enzymatic lignin, characterized in that: (1) Preparation of self-assembled enzymatic lignin nanocarrier particles Solution A was prepared by using enzymatic lignin and dimethyl sulfoxide at a mass volume ratio of 1 g:10 mL of enzymatic lignin and dimethyl sulfoxide; solution B was prepared by using cetyl trimethyl ammonium bromide, ethanol and water at a mass volume ratio of 1:2 of enzymatic lignin and cetyl trimethyl ammonium bromide; solution A and solution B were mixed, water was added dropwise, allowed to stand, centrifuged and freeze-dried to obtain purified self-assembled enzymatic lignin nanocarrier particles; (2) Preparation of prothioconazole-loaded nanoparticles The self-assembled enzymatic lignin nanocarrier particles and prothioconazole are dissolved in dimethyl sulfoxide at a mass ratio of 2:1 to obtain an organic phase; water is added dropwise, ultrasonic dispersion is performed to obtain a suspension of drug-loaded microspheres in which prothioconazole is embedded in the enzymatic lignin carrier, centrifugation washing is performed, and freeze drying is performed to obtain prothioconazole-loaded nanocarrier particles; The volume ratio of the organic phase to the added water is 1:4; (3) Preparation of enzymatic lignin nanopesticides The prothioconazole-loaded nanoparticles were dispersed in water at a mass volume ratio of 3 mg:1 mL, and copper chloride was added; tannic acid was then added, and the mass ratio of the prothioconazole-loaded nanoparticles to copper chloride was 10:1; the mass ratio of the prothioconazole-loaded nanoparticles to tannic acid was 3:1; ultrasonic treatment was performed, washing was performed with water, and excess copper chloride and tannic acid were removed by centrifugation; freeze-drying was performed, and the nanopesticide with anti-photolysis properties was collected.

3. The preparation method according to claim 2, characterized in that: In step (1), 1 g of enzymatic lignin is dissolved in 10 mL of dimethyl sulfoxide, the volume is fixed to 100 mL with ultrapure water, and the mixture is stirred at 700 rpm for 24 hours at room temperature to obtain solution A; 2 g of hexadecyltrimethylammonium bromide is weighed and dissolved in a mixed solvent of 6 mL of ethanol and 4 mL of ultrapure water, the volume is fixed to 100 mL with ultrapure water, and the mixture is stirred at 600 rpm for 24 hours at room temperature to obtain solution B; solution A and solution B are mixed, stirred at 500 rpm, and 800 mL of ultrapure water is added dropwise at a speed of 4 mL / min; the mixture is allowed to stand for 24 hours, the precipitate is centrifuged at a speed of 10,000 rpm, the centrifuged precipitate is washed 3 times with ultrapure water, and freeze-dried at -50°C to obtain purified self-assembled enzymatic lignin nanocarrier particles.

4. The preparation method according to claim 2, characterized in that: In step (2), 0.1 g of self-assembled enzymatic lignin nanocarrier particles and 0.05 g of prothioconazole original drug are dissolved in 10 mL of dimethyl sulfoxide to obtain an organic phase; the organic phase is ultrasonically dispersed for 5 min, stirred at a speed of 200 rpm, 40 mL of ultrapure water is added dropwise, and ultrasonic dispersion is continued for 3 min to obtain a suspension of drug-loaded microspheres in which the enzymatic lignin carrier encapsulates prothioconazole; the suspension is centrifuged at a speed of 10,000 rpm, and the centrifugal precipitate is washed with ultrapure water for 2 to 3 times, and freeze-dried to obtain enzymatic lignin nanocarrier drug-loaded particles loaded with prothioconazole.

5. The preparation method according to claim 2, characterized in that: In step (3), 300 mg of the prothioconazole-loaded nanoparticles are dispersed in 100 mL of ultrapure water and stirred continuously at room temperature, and 30 mg of copper chloride is added; 100 mg of tannic acid is then added, ultrasonically treated for 10 min, washed with water, and centrifuged to remove excess copper chloride and tannic acid; freeze-dried, and the enzymatically hydrolyzed lignin nanopesticide is collected.

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