A complex microcapsule suspension targeting the bee and a preparation method thereof

Abstract

The present application relates to the technical field of pesticide suspending agent, and discloses a compound microcapsule suspending agent for targeting needle bees and a preparation method thereof.The suspending agent comprises propargite, spinetoram, cyromazine, methyl eugenol, methyl oleate, feruloylated O-carboxymethyl chitosan, yeast autolysate protein-peptide component and a preparation aid.During preparation, the oil phase is emulsified in the water phase containing feruloylated O-carboxymethyl chitosan, and then the yeast autolysate protein-peptide component is added for second-stage oxidative crosslinking after first-stage oxidative crosslinking by laccase, so that a microcapsule dispersion liquid is obtained; and then the microcapsule dispersion liquid is compounded with a cyromazine suspension liquid prepared by wet grinding.The prepared agent has good suspending stability, storage stability and persistence in killing needle bees.

Description

Technical Field

[0001] This invention relates to the field of pesticide suspension technology, and in particular to a compound microcapsule suspension for targeting wasps and its preparation method. Background Technology

[0002] Fruit flies are a common pest in the production of fruit trees, melons, and other fruit and vegetable crops, and are commonly known as "needle flies" in some regions. For example, the citrus fruit fly (Bactrocera dorsalis) is recorded as a "needle fly" in relevant local monitoring technical regulations. Fruit flies are characterized by a wide host range and strong adult activity. Their occurrence can affect the normal growth and commercial quality of fruit and vegetable crops. Therefore, effective monitoring and control of fruit flies is a crucial aspect of fruit and vegetable production.

[0003] Currently, the control of nematode wasps mainly employs methods such as trapping, baiting, and chemical control. Among these, combining attractant substances with insecticidal active ingredients is a commonly used technique that can simultaneously attract and kill pests. Existing nematode wasp baiting formulations typically use direct mixing or conventional emulsification and dispersion methods to prepare the attractant components and insecticidal active ingredients. However, due to differences in volatility, solubility, and dispersibility between attractant components and different insecticidal active ingredients, problems such as rapid loss of attractant components, inconsistent release behaviors of different active ingredients, and insufficient dispersion stability in aqueous systems can easily arise. Furthermore, when using a single insecticidal active ingredient, its mode of action is relatively singular, making it difficult to address the pest control needs at different stages of action.

[0004] Therefore, it is necessary to provide a compound microcapsule suspension targeting bees and its preparation method to solve the above problems. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a compound microcapsule suspension for targeting wasps and its preparation method. By rationally compounding insecticidal active ingredients, attractant components, microcapsule wall materials, and formulation adjuvants, and combining staged oxidation crosslinking and wet grinding processes, different functional components are stably dispersed in the same aqueous system, so as to balance the attraction and control of wasps, the effective effect of active ingredients, and the stability of the formulation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A compound microcapsule suspension for targeting bees, comprising the following components by weight percentage: propoxur 3.0-7.0%, ethyl spinosad 0.8-2.5%, cyromazine 3.0-6.0%, methyl eugenol 4.0-9.0%, methyl oleate 4.0-10.0%, ferulic acid-acylated O-carboxymethyl chitosan 0.8-2.5%, yeast autolysate protein-peptide component 0.30-1.20%, emulsifier 0.2-0.8%, dispersant 0.4-1.2%, antifreeze 2.0-5.0%, thickener 0.10-0.40%, preservative 0.05-0.20%, pH adjuster 0.05-0.20%, and the balance being water.

[0008] Furthermore, the targeted bee compound microcapsule suspension comprises the following components by weight percentage: propoxur 5.0%, ethyl spinosad 1.5%, cyromazine 4.5%, methyl eugenol 6.0%, methyl oleate 6.0%, ferulic acid-oxidized O-carboxymethyl chitosan 1.6%, yeast autolysate protein-peptide component 0.60%, emulsifier 0.5%, dispersant 0.8%, antifreeze 3.0%, thickener 0.25%, preservative 0.10%, pH adjuster 0.10%, and the balance being water.

[0009] Furthermore, the mass ratio of propoxur to spinosad is (2.5-4.0):1. By combining propoxur and spinosad in the above ratio, two insecticidal active ingredients with different characteristics can work together to control adult spinosads.

[0010] Furthermore, the mass ratio of methyl eugenol to methyl oleate is 1:(0.8-1.5). Methyl oleate serves as an oil phase medium, on the one hand supporting methyl eugenol, propoxur, and spinosad, and on the other hand facilitating the formation of a stable oil-in-water emulsion, providing an oil phase basis for subsequent microencapsulation.

[0011] Furthermore, the mass ratio of the yeast autolysate protein-peptide component to ferulic acylated O-carboxymethyl chitosan is (0.30-0.60):1. By controlling the amount of yeast autolysate protein-peptide component relative to ferulic acylated O-carboxymethyl chitosan, the protein and peptide components can participate in the subsequent second-stage oxidative cross-linking process, while avoiding insufficient participation due to too low a amount of protein-peptide component or adverse effects on the emulsion dispersion state due to too high a amount.

[0012] Furthermore, the mass ratio of the total amount of cyromazine to propoxur and spinosad is 1:(1.2-2.2), ensuring that the dosage of cyromazine, propoxur, and spinosad in the compound system is reasonably matched, so as to achieve compound control by utilizing the characteristics of different insecticidal active ingredients.

[0013] Furthermore, the feruloyl substitution amount of the feruloyl-substituted O-carboxymethyl chitosan is 0.06-0.18 mmol / g. By introducing a feruloyl group that can participate in oxidation reactions into the water solubility and film-forming properties of O-carboxymethyl chitosan, the feruloyl-substituted O-carboxymethyl chitosan can undergo oxidative coupling in the presence of laccase and oxygen, thereby forming a microcapsule crosslinking wall material. By controlling the amount of feruloyl substitution, it is beneficial to balance the dispersion performance and oxidative crosslinking ability of the feruloyl-substituted O-carboxymethyl chitosan in the aqueous phase.

[0014] Further, the yeast autolysate protein-peptide component is obtained by treating the yeast autolysate with an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, collecting the retentate, and then drying it. Preferably, based on the normalized peak area of ​​size exclusion chromatography, the proteins and peptides with an apparent molecular weight of 5–100 kDa account for 60–90% of the obtained protein-peptide component, and tyrosine residues account for 0.8–4.0% of the total molar amount of amino acid residues in the proteins and peptides. This invention, through ultrafiltration fractionation of yeast autolysate, ensures that the obtained component has a suitable protein and peptide composition, and enables the proteins and peptides therein to participate in the construction of the microcapsule wall system during the second-stage oxidative cross-linking process. In this document, the total mass of the retentate refers to the mass of the dried product obtained after drying the ultrafiltration retentate.

[0015] Furthermore, the emulsifier is a sucrose fatty acid ester. Sucrose fatty acid esters are used to promote the dispersion and emulsification of the oil phase in the aqueous phase and are beneficial for maintaining the stability of the emulsion droplets during subsequent crosslinking processes.

[0016] Furthermore, the dispersant is composed of a polycarboxylate dispersant and a polyoxyethylene-polyoxypropylene block copolymer at a mass ratio of 1:(0.3-1.0). The polycarboxylate dispersant and the polyoxyethylene-polyoxypropylene block copolymer are used in combination to improve the wetting, dispersion, and suspension stability of the cyromazine fine particles in the aqueous phase.

[0017] Furthermore, the antifreeze is propylene glycol, the thickener is composed of xanthan gum and magnesium aluminum silicate in a mass ratio of 1:(2-4), the preservative is potassium sorbate, and the pH adjuster is one or both of citric acid and sodium citrate. Xanthan gum and magnesium aluminum silicate combine to form an aqueous rheology adjustment system to improve the suspension stability of the microcapsule particles and cyromazine suspended particles in the formulation.

[0018] This invention also provides a method for preparing the above-mentioned targeted bee compound microcapsule suspension, comprising the following steps:

[0019] S1. Mix ferulic acid-modified O-carboxymethyl chitosan, emulsifier, pH adjuster and water, adjust pH to 4.8-5.8 to obtain aqueous phase; mix propoxur, spinosad, methyl eugenol and methyl oleate to obtain oil phase.

[0020] S2. The oil phase obtained in step S1 is added to the aqueous phase for emulsification, dispersing the oil phase in droplet form in the continuous aqueous phase to obtain an oil-in-water emulsion. The system temperature is controlled at 25-35℃ and the pH at 4.8-5.8. Oxygen is supplied to the system and the dissolved oxygen concentration is maintained at 3.5-7.0 mg / L. Laccase is added to perform the first-stage oxidative crosslinking for 20-50 min to obtain a first-stage crosslinked emulsion. The first-stage oxidative crosslinking is used to induce preliminary oxidative coupling of ferulic acylated O-carboxymethyl chitosan in the emulsion system, gradually increasing the degree of crosslinking at the droplet interface, while reserving reaction space for subsequent protein-peptide components to participate in further crosslinking.

[0021] S3. Stop oxygen supply, add yeast autolysate protein-peptide components to the first-stage cross-linked emulsion obtained in step S2, and stir for 10-30 min to ensure sufficient contact between the yeast autolysate protein-peptide components and the first-stage cross-linked emulsion; then supply oxygen again to achieve a dissolved oxygen concentration of 2.0-5.0 mg / L, and perform the second-stage oxidative cross-linking for 20-60 min to obtain a microcapsule emulsion. By performing the first-stage oxidative cross-linking first and then introducing the yeast autolysate protein-peptide components for the second-stage oxidative cross-linking, it is beneficial to avoid interference with the emulsification process and the initial interface assembly of ferulic acylated O-carboxymethyl chitosan caused by adding the protein-peptide components at the initial emulsification stage, and it is also beneficial to form a composite cross-linked microcapsule wall system in which ferulic acylated O-carboxymethyl chitosan and protein-peptide components participate together.

[0022] S4. The microcapsule emulsion obtained in step S3 is subjected to tangential flow microfiltration to remove free laccase and water-soluble small molecule components that can permeate the microfiltration membrane, thereby obtaining a microcapsule dispersion. Washing after oxidative cross-linking helps reduce excessive cross-linking caused by continued action of free laccase and improves the stability of the microcapsule dispersion system during subsequent compounding processes.

[0023] S5. The cyromazine, dispersant, and water are wet-milled to obtain a cyromazine suspension with a particle size D50 of 150–350 nm. This cyromazine suspension is then added to the microcapsule dispersion obtained in step S4, followed by the addition of an antifreeze, thickener, and preservative. The mixture is stirred until homogeneous, yielding a compound microcapsule suspension targeting cyromazine bees. By first wet-milling cyromazine independently and then compounding it with the already formed microcapsule dispersion, the participation of cyromazine in the aforementioned emulsification and laccase oxidation cross-linking processes can be avoided. This also facilitates the acquisition of a composite aqueous dispersion system containing both microcapsule particles and fine cyromazine particles.

[0024] Furthermore, in step S2, pre-emulsification is first performed at 1000-3000 r / min for 3-8 min, followed by shear emulsification at 7000-11000 r / min for 8-18 min. By combining pre-emulsification and high-speed shear emulsification, the oil phase first forms a relatively uniform coarse emulsion, which further reduces the droplet size and improves the uniformity of droplet distribution.

[0025] Further, the amount of laccase added is 20-100 U / g, based on the mass of ferulic acylated O-carboxymethyl chitosan. U represents laccase activity units, and the specific activity determination conditions are determined according to the methods described in the specific embodiments of the instruction manual.

[0026] Further, the preparation method of the ferulic acid-oxygenated O-carboxymethyl chitosan includes: dispersing ferulic acid in an aqueous medium, adjusting the pH to 4.8-5.5, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and activating for 15-60 min; then adding O-carboxymethyl chitosan, adjusting the pH to 6.0-7.0, reacting at 20-35℃ for 4-16 h, and removing unreacted substances by dialysis or ultrafiltration to obtain ferulic acid-oxygenated O-carboxymethyl chitosan. The molar ratio of ferulic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:(1.0-1.5):(0.5-1.0). The carboxyl group of ferulic acid was activated by the EDC / NHS system, enabling ferulic acid to couple with O-carboxymethyl chitosan, thereby introducing feruloyl groups onto O-carboxymethyl chitosan.

[0027] Furthermore, in step S5, the wet grinding uses zirconium oxide grinding media with a diameter of 0.1-0.3 mm, and the grinding temperature is not higher than 30°C, so as to reduce the adverse effects of temperature rise during the grinding process on the performance of the formulation while reducing the particle size of cyromazine.

[0028] Compared with the prior art, the present invention provides a compound microcapsule suspension targeting bees and its preparation method, which has the following beneficial effects:

[0029] 1. This invention combines propoxur, spinosad, and cyromazine, and introduces methyl eugenol as an attractant component. By controlling the content and ratio of each active component and methyl eugenol, the attractant effect and the effects of different insecticidal active ingredients are synergistic, which is beneficial to improving the overall control effect of serpentine wasps.

[0030] 2. This invention uses ferulic acidified O-carboxymethyl chitosan as a microcapsule crosslinking material. Under the presence of laccase and oxygen, a first-stage oxidative crosslinking is carried out, followed by the addition of yeast autolysate protein-peptide components for a second-stage oxidative crosslinking. This allows the formation of microcapsules and the participation of protein-peptide components in crosslinking to proceed in stages, which is beneficial for maintaining the sustained effects of methyleugenol and oil-phase active ingredients and improving the sustained inducing and killing properties of the formulation.

[0031] 3. In this invention, cyromazine is wet-milled separately and then compounded with microcapsule dispersion, so that the fine particles of cyromazine and the microcapsule particles coexist in the aqueous system in different dispersion forms, which is beneficial to improving the coexistence stability of the cyromazine suspension particles and the microcapsule system. At the same time, the combination of dispersants, thickeners, antifreeze and other adjuvants is beneficial to improving the dispersibility, suspension stability and storage stability of the compound preparation. Detailed Implementation

[0032] 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.

[0033] In this embodiment, the emulsifier is sucrose fatty acid ester; the dispersant is a combination of polycarboxylate dispersant and polyoxyethylene-polyoxypropylene block copolymer; the antifreeze is propylene glycol; the thickener is a combination of xanthan gum and magnesium aluminum silicate; the preservative is potassium sorbate; and the pH adjuster is citric acid and / or sodium citrate.

[0034] In this embodiment, laccase activity was determined using the ABTS method. A sodium acetate buffer solution at pH 4.5 was used as the reaction medium to achieve a final ABTS concentration of 1.0 mmol / L. The absorbance change from ABTS oxidation was continuously measured at 420 nm at 25°C, and the reaction rate was calculated based on the molar extinction coefficient of the ABTS oxidation product at 420 nm. The amount of laccase required to catalyze the oxidation of 1 μmol of ABTS per minute under the above conditions was defined as 1 U. The amount of laccase added in each embodiment was calculated according to the above activity determination method.

[0035] I. Preparation of Ferulaylated O-Carboxymethyl Chitosan

[0036] The ferulic acidified O-carboxymethyl chitosan used in Example 1 was prepared according to the following method:

[0037] 0.40 g of ferulic acid was added to 100 mL of deionized water and stirred to disperse. The pH of the system was adjusted to 5.2 using sodium hydroxide solution. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added in a molar ratio of 1:1.25:0.75. The mixture was stirred and activated at room temperature for 35 min to obtain the activated solution.

[0038] Separately, 10.0 g of O-carboxymethyl chitosan was added to 400 mL of deionized water and stirred until homogeneous. The resulting O-carboxymethyl chitosan solution was then added to the above activation solution. The pH of the system was adjusted to 6.5 using sodium hydroxide solution, and the reaction was carried out at 28 °C with stirring for 10 h. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with deionized water for 48 h, with the deionized water being replaced every 6 h. After dialysis, the retentate was concentrated and freeze-dried to obtain ferulic acylated O-carboxymethyl chitosan. The ferulic acylation amount was determined by the free amino difference method, and the ferulic acylation amount of the obtained ferulic acylated O-carboxymethyl chitosan was 0.12 mmol / g.

[0039] The feruloylated O-carboxymethyl chitosan used in Examples 2-4 was prepared using the same coupling reaction and purification method as in Example 1. Specifically, in Example 2, the reaction was carried out at 25°C for 6 h, resulting in feruloylated O-carboxymethyl chitosan with a feruloyl substitution amount of 0.06 mmol / g; in Example 3, the reaction was carried out at 32°C for 16 h, resulting in feruloylated O-carboxymethyl chitosan with a feruloyl substitution amount of 0.18 mmol / g; and in Example 4, the reaction was carried out at 30°C for 12 h, resulting in feruloylated O-carboxymethyl chitosan with a feruloyl substitution amount of 0.14 mmol / g.

[0040] II. Preparation of yeast autolysate protein-peptide components

[0041] The yeast autolysate protein-peptide components used in the examples were prepared according to the following method:

[0042] Take 100 g of yeast autolysate (dry matter equivalent) and add deionized water to a total system mass of 1000 g. Stir at 25°C for 30 min to ensure thorough dispersion. Perform tangential flow ultrafiltration on a polyethersulfone (PES) membrane with a molecular weight cutoff of 5 kDa, controlling the material temperature at 28°C and the transmembrane pressure at 0.10 MPa. After concentrating the retentate to approximately one-third of its initial volume, continuously add deionized water for constant-volume washing while maintaining a relatively constant retentate volume. The cumulative volume of deionized water added is twice the volume of the concentrated retentate. After washing, continue concentrating the retentate and freeze-dry the resulting concentrate to obtain the yeast autolysate protein-peptide fraction.

[0043] The molecular weight distribution of the obtained proteins and peptides was determined by size exclusion chromatography. Based on the normalized peak area, proteins and peptides with an apparent molecular weight of 5-100 kDa accounted for 78.0% of the obtained protein-peptide components. After hydrolysis of the obtained protein-peptide components, the amino acid composition was determined by an amino acid analyzer. Tyrosine residues accounted for 2.4% of the total molar amount of amino acid residues in the proteins and peptides.

[0044] Example 1

[0045] The components are shown in the table below, based on the preparation of 1000 g of compound microcapsule suspension.

[0046] Components Dosage / g mass percentage / % massacre 50.0 5.0 Ethyl spinosad 15.0 1.5 Flymethrin 45.0 4.5 Methyleugenol 60.0 6.0 Methyl oleate 60.0 6.0 Ferula-acylated O-carboxymethyl chitosan 16.0 1.6 Yeast autolysate protein-peptide components 6.0 0.60 Sucrose fatty acid esters 5.0 0.50 dispersant 8.0 0.80 Propylene glycol 30.0 3.0 Thickener 2.5 0.25 Potassium sorbate 1.0 0.10 pH adjuster 1.0 0.10 water 700.5 70.05

[0047] The dispersant consists of polycarboxylate dispersant and polyoxyethylene-polyoxypropylene block copolymer in a mass ratio of 1:0.6; the thickener consists of xanthan gum and magnesium aluminum silicate in a mass ratio of 1:3; and the pH adjuster consists of citric acid and sodium citrate.

[0048] The preparation process is as follows:

[0049] S1. Take about 500 g of formulated water, add 16.0 g of ferulic acid-modified O-carboxymethyl chitosan, 5.0 g of sucrose fatty acid ester and 1.0 g of pH adjuster, and stir evenly. The pH adjuster is composed of citric acid and sodium citrate. The pH of the resulting aqueous phase is adjusted to 5.3 by adjusting the ratio of the two. Separately, mix 50.0 g of propoxur, 15.0 g of ethyl spinosad, 60.0 g of methyl eugenol and 60.0 g of methyl oleate evenly to obtain the oil phase.

[0050] S2. Under stirring, the oil phase is slowly added to the aqueous phase. Pre-emulsification is performed at 2000 r / min for 5 min, followed by shear emulsification at 9000 r / min for 13 min to obtain an oil-in-water emulsion. The system temperature is controlled at 30℃, the pH at 5.3, and air or oxygen is introduced to maintain the dissolved oxygen concentration at approximately 5.0 mg / L. Based on the mass of ferulic acid-oxidized O-carboxymethyl chitosan, 60 U / g of laccase (approximately 960 U of total activity) is added to initiate the first-stage oxidative crosslinking for 35 min, yielding the first-stage crosslinked emulsion.

[0051] S3. Stop oxygen supply, add 6.0 g of yeast autolysate protein-peptide component, and react under stirring conditions for 20 min; then resume oxygen supply to maintain the dissolved oxygen concentration of the system at about 3.5 mg / L, and carry out the second stage of oxidative cross-linking for 40 min to obtain microencapsulated emulsion.

[0052] S4. Transfer the microcapsule emulsion obtained in step S3 to a tangential flow microfiltration device and perform washing filtration using a polyethersulfone microfiltration membrane with a pore size of 0.10 μm, controlling the material temperature at 28℃ and the transmembrane pressure at 0.08 MPa. During the retentate circulation process, deionized water is continuously added while maintaining the retentate volume at approximately constant, and a total of four times the initial microcapsule emulsion volume of deionized water is added for constant-volume washing filtration. After washing filtration, tangential flow microfiltration is continued to concentrate the retentate to the volume before washing filtration, obtaining a microcapsule dispersion.

[0053] S5. Mix 45.0 g of cyromazine, 8.0 g of dispersant, and approximately 150 g of formulated water. Add zirconium oxide grinding media with a diameter of approximately 0.2 mm and perform wet grinding, controlling the material temperature below 30℃, until the cyromazine particle D50 is approximately 250 nm. Add the obtained cyromazine suspension to the microcapsule dispersion obtained in step S4, then add 30.0 g of propylene glycol, 2.5 g of thickener, and 1.0 g of potassium sorbate sequentially, add the remaining formulated water, and stir evenly to obtain a compound microcapsule suspension for targeted beekeeping.

[0054] Example 2

[0055] The formula for preparing 1000 g of product is as follows: 30.0 g of propoxur, 8.0 g of ethyl spinosad, 30.0 g of cyromazine, 40.0 g of methyl eugenol, 40.0 g of methyl oleate, 8.0 g of ferulic acid-oxidized O-carboxymethyl chitosan, 3.0 g of yeast autolysate protein-peptide component, 2.0 g of emulsifier, 4.0 g of dispersant, 20.0 g of propylene glycol, 1.0 g of thickener, 0.5 g of potassium sorbate, 0.5 g of pH adjuster, and 813.0 g of water.

[0056] The mass ratio of propoxur to spinosad is 3.75:1, the mass ratio of methyl eugenol to methyl oleate is 1:1, the mass ratio of yeast autolysate protein-peptide component to ferulic acid O-carboxymethyl chitosan is 0.375:1, and the mass ratio of cyromazine to the total mass of propoxur and spinosad is 1:1.27.

[0057] Example 2 was prepared according to the method of Example 1, with the following differences: S1 The pH of the aqueous phase was adjusted to 4.8; S2 Pre-emulsification was carried out at 1000 r / min for 3 min, followed by shear emulsification at 7000 r / min for 8 min, with the temperature controlled at 25℃, the dissolved oxygen concentration at 3.5 mg / L, and the amount of laccase added at 20 U / g, and the first stage of oxidation cross-linking was carried out for 20 min; S3 After the oxygen supply was stopped, the protein-peptide component was added, stirred for 10 min, and then oxygen was supplied again to make the dissolved oxygen concentration 2.0 mg / L, and the second stage of oxidation cross-linking was carried out for 20 min; S5 Zirconia grinding media with a diameter of 0.1 mm was used to control the D50 of the cyromazine particles to about 150 nm.

[0058] Example 3

[0059] The formula for preparing 1000 g of product is as follows: 70.0 g of propoxur, 25.0 g of ethyl spinosad, 60.0 g of cyromazine, 90.0 g of methyl eugenol, 100.0 g of methyl oleate, 25.0 g of ferulic acid-oxidized O-carboxymethyl chitosan, 12.0 g of yeast autolysate protein-peptide component, 8.0 g of emulsifier, 12.0 g of dispersant, 50.0 g of propylene glycol, 4.0 g of thickener, 2.0 g of potassium sorbate, 2.0 g of pH adjuster, and 540.0 g of water.

[0060] The mass ratio of propoxur to spinosad is 2.8:1, the mass ratio of methyl eugenol to methyl oleate is 1:1.11, the mass ratio of yeast autolysate protein-peptide component to ferulic acid O-carboxymethyl chitosan is 0.48:1, and the mass ratio of cyromazine to the total mass of propoxur and spinosad is 1:1.58.

[0061] Example 3 was prepared according to the method of Example 1, with the following differences: S1 The pH of the aqueous phase was adjusted to 5.8; S2 Pre-emulsification was carried out at 3000 r / min for 8 min, followed by shear emulsification at 11000 r / min for 18 min, with the system temperature controlled at 35℃, the dissolved oxygen concentration at 7.0 mg / L, and the amount of laccase added at 100 U / g, and the first stage of oxidative cross-linking was carried out for 50 min; S3 After stopping the oxygen supply, the protein-peptide component was added and stirred for 30 min, and then oxygen was supplied again to make the dissolved oxygen concentration 5.0 mg / L, and the second stage of oxidative cross-linking was carried out for 60 min; S5 Zirconia grinding media with a diameter of 0.3 mm was used, and the grinding temperature was controlled not to exceed 30℃, and the D50 of the cyromazine particles was controlled at about 350 nm.

[0062] Example 4

[0063] Based on the preparation of 1000 g of compound microcapsule suspension, the components are as follows: 45.0 g of propoxur, 14.0 g of ethyl spinosad, 40.0 g of cyromazine, 70.0 g of methyl eugenol, 75.0 g of methyl oleate, 18.0 g of ferulic acid-oxygenated O-carboxymethyl chitosan, 8.0 g of yeast autolysate protein-peptide component, 4.0 g of emulsifier, 7.0 g of dispersant, 35.0 g of propylene glycol, 2.0 g of thickener, 1.2 g of potassium sorbate, 0.8 g of pH adjuster, and 680.0 g of water.

[0064] The mass ratio of propoxur to spinosad is 3.21:1, the mass ratio of methyl eugenol to methyl oleate is 1:1.07, the mass ratio of yeast autolysate protein-peptide component to ferulic acid O-carboxymethyl chitosan is 0.44:1, and the mass ratio of cyromazine to the total mass of propoxur and spinosad is 1:1.48.

[0065] Example 4 was prepared according to the method of Example 1, with the following differences: In S1, the pH of the aqueous phase was adjusted to 5.5; in S2, the mixture was first pre-emulsified at 2500 r / min for 6 min, then sheared and emulsified at 10000 r / min for 15 min, with the system temperature controlled at 32℃, the dissolved oxygen concentration at 5.8 mg / L, and the amount of laccase added at 70 U / g, and the first stage of oxidative cross-linking was carried out for 40 min; in S3, after the oxygen supply was stopped, the yeast autolysate protein-peptide component was added, stirred for 25 min, and then oxygen was supplied again to make the dissolved oxygen concentration 4.0 mg / L, and the second stage of oxidative cross-linking was carried out for 50 min; in S5, wet grinding was performed using a zirconia grinding medium with a diameter of 0.2 mm, and the D50 of the obtained cyromazine suspension particles was approximately 280 nm.

[0066] Comparative Example 1

[0067] The formulation is the same as in Example 1. The difference is that the yeast autolysate protein-peptide component and laccase are added immediately after emulsification. Instead of stopping oxygen supply and protein-peptide pre-adsorption steps, oxidative cross-linking is carried out continuously under oxygen supply conditions, with the total oxidative cross-linking time maintained at 75 min. The remaining steps are the same as in Example 1.

[0068] Comparative Example 2

[0069] The formulation and preparation conditions are the same as in Example 1, except that an equal mass of O-carboxymethyl chitosan is used instead of ferulic acylated O-carboxymethyl chitosan.

[0070] Comparative Example 3

[0071] The formulation and preparation conditions are the same as in Example 1, except that: ordinary yeast autolysate of equal dry weight is used instead of the yeast autolysate protein-peptide component in Example 1. The ordinary yeast autolysate is from the same source as the yeast autolysate used in Example 1, but it is not subjected to ultrafiltration fractionation with a molecular weight cutoff of 5 kDa. Instead, it is directly concentrated and dried after autolysis.

[0072] Comparative Example 4

[0073] The formulation and other preparation conditions are the same as in Example 1. The only difference is that in step S5, wet grinding with zirconium oxide grinding media is not used. Instead, the cyromazine, dispersant and water are directly added to the microcapsule dispersion obtained in step S4 after high-speed dispersion. All other steps are the same as in Example 1.

[0074] Comparative Example 5

[0075] Keeping the total mass percentage of propoxur and spinosad unchanged at 6.5%, the mass ratio of the two was adjusted to 5:1, that is, propoxur about 5.417% and spinosad about 1.083%. The remaining components and preparation conditions were the same as in Example 1.

[0076] Comparative Example 6

[0077] Except for the absence of methyl eugenol, the preparation conditions were the same as in Example 1, and the deleted methyl eugenol was replaced with an equal mass of methyl oleate.

[0078] Performance testing

[0079] 1. The suspension rate was determined according to the method specified in GB / T 14825-2023.

[0080] 2. The stability of the thermal storage shall be evaluated according to the method specified in GB / T 19136-2021. Before and after the thermal storage treatment, observe whether the samples show obvious water separation, clumping, stratification or irreversible sedimentation, and measure the suspension rate after thermal storage.

[0081] 3. Low-temperature stability shall be evaluated according to the method specified in GB / T 19137-2003, and the sample shall be observed to see if irreversible crystallization, stratification or sedimentation occurs after treatment.

[0082] 4. The D50 of the cyromazine suspended particles was determined by laser diffraction particle size analysis, and the measurement method was carried out in accordance with the principles of GB / T19077-2024.

[0083] 5. Evaluation of Indoor Trapping Efficacy: The trapping performance of each formulation against the oriental fruit fly was evaluated using the indoor cage method. Male adults 13–16 days after emergence, without pretreatment with methyleugenol, were selected as test insects. The experimental conditions were: temperature (25±1)℃, relative humidity (70±5)%, and photoperiod of 12 h:12 h (L:D). Each treatment was set up in 6 replicates, with 50 male adults per replicate. The experimental cage size was 50 cm × 50 cm × 50 cm.

[0084] 0.20 g of the test formulation was uniformly loaded onto an adsorbent carrier of the same specification and placed in a test cage. After 24 h, the number of adult insects attracted was recorded, and the attraction rate was calculated according to "attraction rate (%) = number of attracted insects / total number of test insects × 100%". Insecticidal performance was evaluated under the same conditions. After the test insects were in contact with the trapping carrier for 4 h, the carrier was removed, and the insects were continued to be reared normally for 24 h. The number of dead insects was recorded. A corresponding substrate without methyl eugenol and the insecticidal active ingredient was used as a blank control. When natural mortality occurred in the blank control, the corrected mortality rate was calculated.

[0085] To evaluate the sustained efficacy of the formulation, the traps for each treatment were placed at a temperature of (25±1)℃ and a relative humidity of (60±5)% under conditions of darkness and ventilation. The trapping rate and 24-hour corrected mortality rate were measured at 0, 14 and 28 days, respectively, according to the above method.

[0086] Performance test results

[0087] The formulations obtained in the examples and comparative examples were subjected to performance tests according to the above test methods. The test items included suspension rate, thermal storage stability, low temperature stability, particle size of cyromazine, and its attraction and insecticidal performance on needle wasps.

[0088] Table 1. Formulation performance test results of the examples and comparative examples.

[0089] Example 1 248 98.1 95.6 No layering, no crystallization, and no hard precipitate. Example 2 158 96.4 92.1 No layering, no crystallization, and no hard precipitate. Example 3 342 95.2 90.3 No layering, no crystallization, and no hard precipitate. Example 4 282 97.2 94.0 No layering, no crystallization, and no hard precipitate. Comparative Example 1 253 91.8 82.4 Slight delamination, recoverable after shaking. Comparative Example 2 261 86.7 72.8 Obvious stratification and settlement were observed. Comparative Example 3 255 93.4 85.6 Slight delamination, recoverable after shaking. Comparative Example 4 1280 81.3 69.2 Significant settlement and stratification were observed.

[0090] As shown in Table 1, the formulations obtained in Examples 1-4 all exhibited good suspension and storage stability, with Example 1 showing the best overall performance. Compared to Example 1, the suspension rate and thermal storage stability of the formulations obtained in Comparative Example 1 (using a single-stage oxidative crosslinking), Comparative Example 2 (using unferuloylated O-carboxymethyl chitosan), and Comparative Example 3 (using unfiltered, unfractionated ordinary yeast autolysate) all decreased to varying degrees. In Comparative Example 4, the particle size of the cyromazine increased significantly after unrefined grinding, resulting in a significant decrease in suspension stability. This indicates that the wall material system, specific protein-peptide components, staged oxidative crosslinking, and refined dispersion method of the cyromazine in this invention are beneficial for improving the stability of the compound formulations.

[0091] Table 2. Results of the trapping performance of the exemplified and comparative examples on the bee-killing effect.

[0092] sample Initial trapping rate / % Initial 24-hour corrected mortality rate / % 14d trapping rate / % 14-day corrected mortality rate / % 28d trapping rate / % 28-day corrected mortality rate / % Example 1 93.4 96.2 86.8 91.5 76.9 84.7 Example 2 89.8 92.7 79.6 85.4 67.2 76.8 Example 3 88.6 91.3 77.1 83.6 64.9 74.1 Example 4 91.8 94.3 82.5 88.0 71.9 80.2 Comparative Example 1 87.3 89.1 64.8 72.6 46.9 57.8 Comparative Example 2 84.9 86.8 56.1 64.5 39.6 50.7 Comparative Example 3 86.7 88.5 62.3 70.1 44.8 55.9 Comparative Example 5 — 82.6 — 73.8 — 62.9 Comparative Example 6 17.2 — 14.9 — 12.7 —

[0093] Note: Comparative Example 5 only examined the insecticidal component ratio, and the attractant component was the same as in Example 1, so the attraction rate was not listed; Comparative Example 6 did not contain methyl eugenol, and the number of insects attracted was extremely small, so the corrected mortality rate was not statistically significant, and therefore it was not listed.

[0094] As shown in Table 2, Examples 1-4 all exhibited good attracting, insecticidal, and sustained-effect properties for nematodes, with Example 1 showing the best overall effect. Comparative Examples 1-3 showed significant decreases in attraction rate and corrected mortality rate after 14 and 28 days of exposure, indicating that the synergistic effect of feruloylated O-carboxymethyl chitosan, ultrafiltration-fractionated yeast autolysate protein-peptide components, and two-stage oxidative cross-linking helps maintain the sustained-effect attraction and killing properties of the formulation. Comparative Example 5 showed a decrease in insecticidal effect after changing the mass ratio of propoxur to spinosad, and Comparative Example 6 showed a significant decrease in attraction rate when methyl eugenol was not present, further demonstrating that the main functional components of this invention and their synergistic relationship can jointly improve the overall control effect against nematodes.

[0095] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A compound microcapsule suspension targeting bees, characterized in that, The product comprises the following components by weight percentage: propoxur 3.0-7.0%, ethyl spinosad 0.8-2.5%, cyromazine 3.0-6.0%, methyl eugenol 4.0-9.0%, methyl oleate 4.0-10.0%, ferulic acid-acylated O-carboxymethyl chitosan 0.8-2.5%, yeast autolysate protein-peptide component 0.30-1.20%, emulsifier 0.2-0.8%, dispersant 0.4-1.2%, antifreeze 2.0-5.0%, thickener 0.10-0.40%, preservative 0.05-0.20%, pH adjuster 0.05-0.20%, and the balance being water.

2. The targeted bee-like compound microcapsule suspension according to claim 1, characterized in that, The product comprises the following components by weight percentage: propoxur 5.0%, ethyl spinosad 1.5%, cyromazine 4.5%, methyl eugenol 6.0%, methyl oleate 6.0%, ferulic acid-oxidized O-carboxymethyl chitosan 1.6%, yeast autolysate protein-peptide component 0.60%, emulsifier 0.5%, dispersant 0.8%, antifreeze 3.0%, thickener 0.25%, preservative 0.10%, pH adjuster 0.10%, and the balance being water.

3. The targeted bee microcapsule suspension according to claim 1, characterized in that, The mass ratio of propoxur to spinosad is (2.5-4.0):1, the mass ratio of methyl eugenol to methyl oleate is 1:(0.8-1.5), the mass ratio of yeast autolysate protein-peptide component to ferulic acid O-carboxymethyl chitosan is (0.30-0.60):1, and the mass ratio of cyromazine to the total amount of propoxur and spinosad is 1:(1.2-2.2).

4. The targeted bee microcapsule suspension according to claim 1, characterized in that, The feruloyl substitution amount of the feruloyl-substituted O-carboxymethyl chitosan is 0.06-0.18 mmol / g.

5. The targeted bee microcapsule suspension according to claim 1, characterized in that, The yeast autolysate protein-peptide components are obtained by treating yeast autolysate with an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, collecting the residue, and drying it.

6. The targeted bee microcapsule suspension according to claim 1, characterized in that, The emulsifier is a sucrose fatty acid ester, the dispersant is composed of a polycarboxylate dispersant and a polyoxyethylene-polyoxypropylene block copolymer in a mass ratio of 1:(0.3-1.0), the antifreeze is propylene glycol, the thickener is composed of xanthan gum and magnesium aluminum silicate in a mass ratio of 1:(2-4), the preservative is potassium sorbate, and the pH adjuster is one or both of citric acid and sodium citrate.

7. A method for preparing a compound microcapsule suspension of targeted bee venom as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Ferula-acylated O-carboxymethyl chitosan, emulsifier, pH adjuster and water are mixed, and the pH of the system is adjusted to 4.8-5.8 to obtain the aqueous phase; propoxur, spinosad, methyl eugenol and methyl oleate are mixed to obtain the oil phase; S2. The oil phase is added to the aqueous phase for emulsification to obtain an oil-in-water emulsion. The temperature is controlled at 25-35℃ and the pH is 4.8-5.

8. Oxygen is supplied to make the dissolved oxygen concentration 3.5-7.0 mg / L. Laccase is added to carry out the first stage of oxidative crosslinking for 20-50 min to obtain the first stage crosslinked emulsion. S3. Stop oxygen supply, add yeast autolysate protein-peptide component, stir for 10-30 min, then supply oxygen again to make the dissolved oxygen concentration 2.0-5.0 mg / L, and carry out the second stage of oxidative cross-linking for 20-60 min to obtain microcapsule emulsion. S4. The microcapsule emulsion obtained in step S3 is subjected to tangential flow microfiltration to remove free laccase and obtain a microcapsule dispersion. S5. The cyromazine, dispersant and water are wet-milled to obtain a cyromazine suspension with a particle size D50 of 150-350 nm. The cyromazine suspension is added to the microcapsule dispersion, and then antifreeze, thickener and preservative are added. The mixture is stirred evenly to obtain a compound microcapsule suspension for targeted bees.

8. The preparation method according to claim 7, characterized in that, In step S2, the mixture is first pre-emulsified at 1000-3000 r / min for 3-8 min, and then sheared and emulsified at 7000-11000 r / min for 8-18 min; the amount of laccase added is 20-100 U / g based on the mass of ferulic acylated O-carboxymethyl chitosan.

9. The preparation method according to claim 7, characterized in that, The preparation method of the ferulic acid-oxidized O-carboxymethyl chitosan includes the following steps: ferulic acid is dispersed in an aqueous medium, the pH is adjusted to 4.8-5.5, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added, activated for 15-60 min, then O-carboxymethyl chitosan is added, the pH is adjusted to 6.0-7.0, and the reaction is carried out at 20-35℃ for 4-16 h. Unreacted substances are removed by dialysis or ultrafiltration to obtain ferulic acid-oxidized O-carboxymethyl chitosan. The molar ratio of ferulic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:(1.0-1.5):(0.5-1.0).

10. The preparation method according to claim 7, characterized in that, In step S5, the wet grinding uses zirconium oxide grinding media with a diameter of 0.1-0.3 mm, and the grinding temperature is not higher than 30℃.