Preparation method and application of zein-based Pickering emulsion controlled-release pesticide

Abstract

The invention discloses a preparation method and application of a zein-based Pickering emulsion controlled-release pesticide, firstly zein is modified, deamidation treatment is performed, then covalent coupling with sodium lignin sulfonate is performed to obtain a stabilizer, plant essential oil is further selected as a solvent of the pesticide and a dispersion phase of the emulsion, and the zein-based Pickering emulsion controlled-release pesticide is prepared. The sterilization effect is enhanced, meanwhile, a pesticide delivery system is constructed, complex and harsh preparation conditions are not needed, and toxic and harmful organic solvents are also prevented from being used; the preparation technology is simple and stable, the pesticide delivery system is low in cost and environmentally friendly, the delivery system shows pH response release performance, the release efficiency of effective components of the applied pesticide can be controlled by adjusting the pH value of the delivery system itself or the pH value of the application environment, and the delivery system is applied to the field of preparing antibacterial agents to kill fungi or used for preventing and controlling soil-borne diseases and has broad application prospects. The harm to the environment and non-target organisms can be reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticide formulations. More specifically, it relates to a preparation method and application of zein-based Pickering emulsion controlled-release pesticides for constructing an efficient and stable controlled-release pesticide delivery system. Background Art

[0002] Chemical pesticides play a crucial role in modern agriculture. However, traditional pesticide application methods often have the problem of low efficiency, which not only leads to waste of resources but also causes serious environmental pollution and threatens the stability of the ecosystem. To solve the problems of low efficiency of pesticide utilization and the resulting environmental issues, the development of a pesticide delivery system with low cost, stability, and high transmission and controlled release characteristics has become a current research hotspot. Summary of the Invention

[0003] The present invention aims to solve one of the above-mentioned technical problems existing in the prior art. To overcome the deficiencies of the prior art, the present invention provides a preparation method and application of zein-based Pickering emulsion controlled-release pesticides, which have high environmental friendliness and biocompatibility at low cost, can effectively protect and control the release of pesticides, and can effectively improve the utilization efficiency of pesticides and reduce environmental pollution.

[0004] The technical solution adopted for one of the purposes of the present invention is:

[0005] Provide a preparation method of zein-based Pickering emulsion controlled-release pesticides, comprising the following steps:

[0006] S1. Dissolve zein in a strong base solution for deamidation treatment, stir magnetically until evenly mixed, dialyze thoroughly using a dialysis bag, and then perform freeze-drying treatment to obtain deamidated zein;

[0007] S2. Dissolve sodium lignosulfonate in deionized water, stir until evenly dissolved, and adjust the pH to 8 - 9;

[0008] S3. Drop epichlorohydrin (abbreviated as ECH) into the solution obtained in step S2, and stir magnetically until evenly mixed;

[0009] S4. Dissolve the deamidated zein obtained in S1 in deionized water, then pour it into the solution obtained in step S3, adjust the pH to 11 - 12, react fully, dialyze thoroughly using a dialysis bag, and then perform freeze-drying treatment to obtain a covalent conjugate of sodium lignosulfonate and zein;

[0010] S5. Stir and disperse the covalent conjugate obtained in step S4 evenly in the aqueous phase to obtain an aqueous dispersion solution;

[0011] S6. Ultrasonically dissolve the pesticide molecules in the plant essential oil to obtain an oily dispersion solution. Mix the aqueous dispersion solution obtained in step S5 with the oily dispersion solution and then homogenize them using a homogenizer to obtain a zein-based Pickering emulsion controlled-release pesticide.

[0012] The technical principle of the above technical solution is as follows:

[0013] Unmodified zein (abbreviated as Zein) faces challenges in terms of interfacial adsorption, hydrophobic-hydrophilic balance, acid-base resistance, and thermal stability. Therefore, it is necessary to modify zein (deamidated zein, abbreviated as ZD) through step S1 to improve its stability and functionality. Further, by covalently interacting with other plant-derived particles, Zein can create a composite system, significantly enhancing the stability of the zein-based Pickering emulsion controlled-release pesticide of the present invention. Lignin is the main component of lignocellulosic biomass and has a unique aromatic structure and functional groups (such as phenolic hydroxyl groups, aliphatic hydroxyl groups, and aldehydes), which contribute to amphiphilicity, anti-ultraviolet, antioxidant, and antibacterial properties. Sodium lignosulfonate (abbreviated as SLS) with amphiphilicity is covalently bonded to Zein and the coupling ratio and conditions are optimized, which can improve the interfacial adsorption ability of the stabilizer during the preparation of the controlled-release pesticide of the present invention, thereby enhancing the oil-water interface stability. At the same time, the combination of SLS and Zein as natural functional materials has the advantages of low cost, strong availability, easy processing, and modification.

[0014] The present invention realizes the efficient delivery and controlled release of pesticides by constructing a stable Pickering emulsion (abbreviated as Pickering emulsion) using zein (abbreviated as Zein) and sodium lignosulfonate (abbreviated as SLS). Zein is deamidated and covalently coupled with sodium lignosulfonate to form a covalent conjugate (abbreviated as S-O-Z) complex of sodium lignosulfonate and zein, which improves its hydrophobicity and interfacial stability, enables it to be stably dispersed at the emulsion interface, forms a physical barrier to prevent oil droplet coalescence, and enhances the emulsion stability. As a natural surfactant, SLS further improves the solubility and emulsifying ability of zein and regulates the release behavior of pesticides, extending the effective period of pesticides.

[0015] Preferably, the plant essential oil is tea tree oil. Using tea tree oil (abbreviated as TTO) as the dispersed phase can not only dissolve the bactericide, but also its inherent bactericidal effect can synergistically enhance the bactericidal effect and reduce environmental toxicity. In other embodiments, tea tree oil can also be replaced with other plant essential oils.

[0016] Preferably, the dialysis time in dialysis bag in step S1 is 24 - 36 h; in step S1 and / or S3, the magnetic stirring time is 4 - 8 h, and the magnetic stirring is carried out under heating conditions.

[0017] Preferably, the sufficient reaction in step S4 is carried out at room temperature for 4 - 6 h, and the dialysis time with a dialysis bag is 3 - 5 days.

[0018] Preferably, in step S6, the homogenizer first performs high - speed homogenization for 6 - 10 min, pauses for 5 - 10 min, and then performs high - speed homogenization again for 6 - 10 min.

[0019] Preferably, in step S1, the strong base solution is NaOH or KOH.

[0020] Preferably, in step S1 and / or step S4, the cut - off molecular weight of the dialysis bag is 800 - 1200 daltons.

[0021] Preferably, in step S5, the mass fraction of the aqueous dispersion solution is 1.5 - 3.5%.

[0022] Preferably, in step S6, the volume fraction of the oily dispersion solution is 30 - 50%.

[0023] Preferably, the pesticide molecule is tebuconazole (abbreviated as Teb) or triadimefon (abbreviated as Tri); or the pesticide molecule is other medicinal molecules of non - agricultural pesticides applied in the fields of food preservation, medicine, and cosmetics. In other embodiments, the present invention can also be used to load other pesticide molecules or non - pesticide molecules. For example, in the fields of food preservation, medicine, and cosmetics, non - pesticide molecules can be loaded through the delivery system of the present invention and then applied in the corresponding fields. For example, in the food preservation field, antibacterial agents or preservatives (preferably antibacterial agents and preservatives harmless to the human body) can be loaded; in the medicine field, medicinal molecules required by the human skin can be loaded and applied to the human skin; in the cosmetics field, medicinal molecules beneficial to the human skin can be loaded and combined with plant essential oils to produce beneficial effects, etc.

[0024] The second object of the present invention is to provide the application of the zein-based Pickering emulsion controlled-release pesticide of the present invention, wherein the pesticide molecule is tebuconazole or the pesticide molecule is triadimefon. The pH value of the zein-based Pickering emulsion controlled-release pesticide is adjusted to acidic or neutral, or it is applied to an acidic or neutral environment. The controlled-release pesticide of the present invention can increase the release amount of the pesticide molecule under the same time by adjusting the pH of the application environment to acidic or neutral, or applying it to an acidic or neutral environment, which is more than the release amount under alkaline conditions in the same time. Under alkaline conditions, the hydroxyl group, imino group, carboxyl group and sulfonic acid group in S-O-Z are deprotonated, and the negative charge generated on tebuconazole or triadimefon causes electrostatic repulsion. This repulsive effect inhibits the diffusion of the pesticide molecule from tea tree oil to the release medium, thereby reducing the release amount. That is, the present invention can regulate the release amount of the pesticide by adjusting the pH value, improving the effective utilization rate and reducing the environmental residue. The present invention realizes the controlled release of the pesticide at a specific pH, significantly improves the utilization efficiency of the pesticide, and provides a low-cost, stable and sustainable pesticide delivery solution. In the aforementioned steps 2 and 4 of the present invention, there are process steps of adjusting to alkaline, and it is also disclosed that it can be adjusted to alkaline by sodium hydroxide and potassium hydroxide. Here, the alkaline is for the deamidation treatment of zein, which is based on the treatment of the preparation materials. And the technical solution in the second object of the present invention needs to be adjusted to acidic to create the most suitable application conditions, and dilute hydrochloric acid, dilute acetic acid or dilute sulfuric acid can be used to adjust the acidity without affecting the effect of the original solution.

[0025] The third object of the present invention is to provide the application of the zein-based Pickering emulsion controlled-release pesticide of the present invention for preparing an antibacterial agent to kill fungi; or for preventing and controlling soil-borne diseases. The zein-based Pickering emulsion controlled-release pesticide of the present invention can not only effectively protect the active ingredients of the pesticide by controlled release and stable delivery, improve its utilization efficiency, but also promote crop health and reduce the occurrence of diseases under environmentally friendly conditions. Therefore, it can be used for delivering an antibacterial agent to kill fungi and for preventing and controlling soil-borne diseases such as southern blight of peanut.

[0026] The technical solution provided by the present invention has the following beneficial effects:

[0027] The present invention provides a preparation method and application of a zein-based Pickering emulsion controlled-release pesticide. This controlled-release pesticide has high environmental friendliness and biocompatibility. By the selection of natural materials (such as zein and tea tree oil), the dependence on chemically synthesized surfactants is reduced, thereby reducing environmental pollution and toxic effects on non-target organisms. In addition, the functionalized zein is stably dispersed in the emulsion, improving its hydrophobicity and interfacial stability, enabling long-term stable dispersion and effective encapsulation of pesticides, extending their shelf life, reducing the frequency of use, and improving the utilization efficiency of pesticides. This controlled-release pesticide system can regulate the release amount of pesticides by adjusting the pH value under different environmental conditions, increasing the release amount of pesticides within the required time, enhancing the effective utilization rate and reducing environmental residues.

[0028] Meanwhile, as a pesticide solvent and emulsion dispersed phase, tea tree oil not only has a natural bactericidal effect but also synergistically enhances the bactericidal effect with fungicides, reducing the amount of pesticides used, and further reducing the environmental toxicity risk. The deamidation treatment of Zein and its covalent coupling with SLS during the preparation process significantly improve the interfacial stability of the emulsion, preventing stratification or demulsification phenomena and ensuring long-term dispersion stability. The raw materials selected for this system are widely available and inexpensive, with low cost and sustainability, providing an efficient, environmentally friendly and cost-effective solution for pesticide delivery, and showing significant application value in pesticide protection, controlled release and environmental toxicity control. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention can be further understood from the following description in conjunction with the drawings.

[0030] Figure 1 It is a schematic diagram of the steps for preparing the zein-based Pickering emulsion controlled-release pesticide of the present invention.

[0031] Figure 2 It is the FTIR spectra of SLS, ZD and stabilizer S-O-Z.

[0032] Figure 3 It is the Raman spectra of SLS, ZD and stabilizer S-O-Z.

[0033] Figure 4 It is the DTG curves of SLS, ZD and stabilizer S-O-Z.

[0034] Figure 5 It is the DSC curves of SLS, ZD and stabilizer S-O-Z.

[0035] Figure 6 It is the scanning electron microscope (SEM) image of S-O-Z.

[0036] Figure 7Are the particle size distribution, average particle size and Zeta potential of S-O-Z.

[0037] Figure 8 Are digital photos of five Pickering emulsions when freshly prepared (0 d).

[0038] Figure 9 Are digital photos of five Pickering emulsions after storage for 21 d.

[0039] Figure 10 Are digital photos of five Pickering emulsions after being shaken well again after standing for 21 d.

[0040] Figure 11 Are the emulsified phase volume fractions of five Pickering emulsions after standing for different days.

[0041] Figure 12 Are optical microscope pictures of five Pickering emulsions when freshly prepared (0 d).

[0042] Figure 13 Are optical microscope pictures of five Pickering emulsions after storage for 21 d.

[0043] Figure 14 Are the particle size distributions of five Pickering emulsions when freshly prepared (0 d).

[0044] Figure 15 Are the average particle sizes of five Pickering emulsions when freshly prepared (0 d).

[0045] Figure 16 Are the particle size distributions of five Pickering emulsions after storage for 21 d.

[0046] Figure 17 Are the average particle sizes of five Pickering emulsions after storage for 21 d.

[0047] Figure 18 Are the surface tensions of water, ZD, SLS, S-O-Z and Pickering emulsions.

[0048] Figure 19 Are the contact angles of water and Pickering emulsions on cucumber leaves.

[0049] Figure 20 Are the remaining curves of TTO in Pickering emulsions and TTO+Teb under ultraviolet light irradiation.

[0050] Figure 21 Are the Teb release curves of Pickering emulsions at different pH values in 60% ethanol / water solution.

[0051] Figure 22 Are the TTO release curves of Pickering emulsions at different pH values in 60% ethanol / water solution.

[0052] Figure 23 It is the antibacterial activity diagram of Pickering emulsion against Sclerotium rolfsii at different concentrations.

[0053] Figure 24 It is the germination situation of peanut seeds treated with water, S-O-Z, Pickering emulsion, 1% ethanol / water solution, TTO+Teb, TTO, and Teb on the 2nd day and the 7th day.

[0054] Among them (the same English words are not translated repeatedly):

[0055] Figure 3 In it, Raman shift is the Raman shift; Figure 4 In it, Derivative Weight is the derivative weight; Temperature is the temperature; Figure 5 In it, Heat Flow is the heat flow; Figure 14 In it, Droplet size is the droplet size; Figure 21 In it, Cumulative Release is the cumulative release.

[0056] The meanings of these words are as follows:

[0057] Raman shift: In Raman spectroscopy, it refers to the degree of change in the wavelength of light after the interaction between light and matter. Raman shift reflects the changes in molecular vibration, rotation, or other internal energy levels.

[0058] Derivative weight: Usually in thermogravimetric analysis (TGA), it represents the rate of change of the mass of a substance with temperature during heating or cooling. It is usually the derivative of the mass-versus-temperature curve.

[0059] Temperature: It represents the degree of hotness or coldness of a substance, and usually refers to the temperature at the measurement point in an experiment.

[0060] Heat flow: It represents the rate of heat transfer with time or the temperature difference between objects. In a differential scanning calorimeter (DSC), heat flow is used to describe the heat absorption or release of materials.

[0061] Droplet size: Usually used to describe the size of liquid droplets, which may refer to the diameter or volume of the droplets.

[0062] Cumulative release: Usually used to describe the total amount of drug released gradually over time. It represents the total amount released over a period of time and may be used in drug release experiments or similar studies. Specific implementation methods

[0063] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. For those skilled in the art, after referring to the following detailed description, other systems, methods, and / or features of this embodiment will become obvious. It is intended that all such additional systems, methods, features, and advantages be included within this specification, be included within the scope of the present invention, and be protected by the appended claims.

[0064] In the experimental methods in the following embodiments, unless otherwise specified, they are all conventional methods. The test materials used in the following embodiments, unless otherwise specified, can all be obtained through conventional commercial channels.

[0065] A preparation method of a zein-based Pickering emulsion controlled-release pesticide includes the following steps:

[0066] S1. Dissolve zein in a strong alkali solution for deamidation treatment, stir magnetically until evenly mixed, dialyze thoroughly using a dialysis bag, and then perform freeze-drying treatment to obtain deamidated zein;

[0067] S2. Dissolve sodium lignosulfonate in deionized water, stir until evenly dissolved, and adjust the pH to 8-9;

[0068] S3. Drop epichlorohydrin (abbreviated as ECH) into the solution obtained in step S2, and stir magnetically until evenly mixed;

[0069] S4. Dissolve the deamidated zein obtained in S1 in deionized water, then pour it into the solution obtained in step S3, adjust the pH to 11-12, react fully, dialyze thoroughly using a dialysis bag, and then perform freeze-drying treatment to obtain a covalent conjugate of sodium lignosulfonate and zein;

[0070] S5. Stir and disperse the covalent conjugate obtained in step S4 in the aqueous phase to obtain an aqueous dispersion solution;

[0071] S6. Ultrasonically dissolve the pesticide molecules in tea tree oil to obtain an oily dispersion solution, mix the aqueous dispersion solution obtained in step S5 with the oily dispersion solution, and homogenize using a homogenizer to obtain a zein-based Pickering emulsion controlled-release pesticide.

[0072] The specific experiments and steps are as follows:

[0073] 1. Materials and Methods

[0074] 1.1. Test Materials

[0075] Experimental reagents: Zein (purity 91%), sodium hydroxide (NaOH, 97%), Macklin Biochemical Technology Co., Ltd. Medicinal grade tea tree oil (TTO), epichlorohydrin, potassium bromide, xanthan gum, Aladdin Biochemical Technology Co., Ltd. Tebuconazole, triadimefon, Shenzhen Noposion Agrochemicals Co., Ltd. LB medium, nutrient agar, Solarbio Science & Technology Co., Ltd. Absolute ethanol, Damao Chemical Reagent Co., Ltd. Peanut seeds (Lushou 2), Shandong Shouhe Seed Industry Co., Ltd.

[0076] 1.2 Experimental methods

[0077] Combined Figure 1 As shown, 4 g of Zein was dissolved in 100 mL of 0.1 mol / L NaOH solution, magnetically stirred at 80 °C for 4 h, dialyzed with a 1000 Da dialysis bag for 24 h, and freeze-dried. 1 g of SLS was dissolved in 10 mL of deionized water, stirred and dissolved at room temperature, and the pH was adjusted to 9. 0.34 g of ECH was slowly added dropwise to the SLS solution, and stirred at 80 °C for 6 h. 2 g of the above-treated Zein was dissolved in 20 mL of deionized water, then poured into the above SLS solution, the pH was adjusted to 12, and reacted at room temperature for 5 h. Dialyzed with a 1000 Da dialysis bag for 4 d and freeze-dried to obtain a covalent conjugate of SLS and Zein (S-O-Z).

[0078] According to the ratio in Table 1, different masses of S-O-Z were weighed and fully stirred and dispersed in the aqueous phase to obtain S-O-Z dispersions with different mass fractions. Teb was ultrasonically dissolved in TTO to obtain an oil phase (where 1 mL of TTO dissolves 100 mg of Teb). Different volumes of the oil phase were measured according to the ratio and added to the corresponding S-O-Z solution. The homogenizer was used to homogenize at high speed for 8 min first, paused for 10 min, and then homogenized at high speed again for 8 min with the homogenizer to obtain Pickering emulsions stabilized by S-O-Z with different ratios.

[0079] Table 1 Preparation parameters of Pickering emulsions

[0080]

[0081]

[0082] 1.3 Structural characterization

[0083] The microscopic morphology of S-O-Z was analyzed by scanning electron microscopy (SEM) (TESCAN MIRALMS, Czech Republic) and transmission electron microscopy (TEM) (JEOL JEM-F200, Japan). The liquid morphology of the Pickering emulsion was observed by optical microscopy (OM) (Olympus CX41, Japan), and the sample was diluted 50 times before observation. The Fourier transform infrared spectroscopy (FT-IR) of the sample was performed using a Spectrum-100 infrared spectrometer (PerkinElmer Inc., USA). The Raman spectrum of the sample was obtained by a DXR2 microscopic Raman spectrometer equipped with an Olympus BX 41 optical microscope, operating in the automatic exposure mode. The thermogravimetric analysis (TG) and derivative thermogravimetric analysis (DTG) of the sample were completed by a thermogravimetric analyzer (Mettler Toledo, Switzerland). The differential scanning calorimetry (DSC) analysis of the sample was carried out using a DSC-Q2000 calorimeter (USA). The droplet size and potential of the emulsion were measured by a laser particle size analyzer (Brookhaven90Plus, USA).

[0084] 1.4. Storage stability:

[0085] 4 mL of emulsions with different ratios were sealed in transparent glass bottles and placed in an environment at 25 °C for different days. The changes in the morphology and size of the emulsion before and after standing for 21 d were observed by optical microscopy and laser particle size analyzer. In addition, the emulsified phase volume fraction (EVF, %) of the emulsion was calculated according to Equation (1).

[0086]

[0087] where He is the height of the emulsified phase after storage for a certain time, and Ht is the total height of the freshly prepared emulsion.

[0088] 1.5. Evaluation of wetting performance

[0089] Before the test, solid samples of SLS, ZD, and S-O-Z were dispersed in deionized water at a concentration of 0.35 g / L. The liquid Pickering emulsion was diluted with deionized water to a volume fraction of 1% for testing. The contact angle and surface tension of the samples were measured by a contact angle measuring instrument (Theta, Sweden) by the sessile drop method and the pendant drop method, respectively. Freshly picked cucumber leaves were trimmed into slender strips, avoiding the main veins, and fixed flat on a glass slide with double-sided tape. The test solution was then applied to the leaf surface with a microsyringe, and the contact angle measuring instrument recorded the contact angle, droplet image, and surface tension value.

[0090] 1.6. UV resistance:

[0091] Take 2 mL of the newly prepared emulsion and place it in a 50 mL brown volumetric flask. Dilute it to the mark with deionized water. Transfer the above-diluted sample solution into different transparent glass test tubes and place them in a photoreactor. Each sample is placed 5 cm away from a mercury lamp (300 W, Emax = 365 nm). After ultraviolet irradiation at specific time intervals, take out 1 mL of the emulsion solution and place it in a 10 mL brown volumetric flask. Dilute it to the mark with 90% acetonitrile. Take out 1 mL of the emulsion and place it in a 10 mL brown volumetric flask. Dilute it to the mark with 90% ethanol. Use a high-performance liquid chromatograph to determine the content of TTO. Calculate the residual rate (RR) of TTO in the sample after irradiation for different times through formula (2).

[0092]

[0093] Where C t represents the concentration of TTO in the sample after irradiation for a specific time, and C 0 represents the concentration of TTO in the sample before irradiation.

[0094] 1.7. Controlled release performance:

[0095] The dialysis bag method was used to test the controlled release behavior of Pickering emulsion in a controlled release medium of 60% ethanol solution with different pH values. Take 2 mL of the newly prepared emulsion and place it in a dialysis bag. Then put it into a brown conical flask containing 50 mL of a 60% ethanol-water controlled release medium with different pH values (3, 5, 7, 9). Place it in a shaker at 30 °C and shake. At intervals, take out 1 mL of the controlled release medium and replenish 1 mL of the buffer medium with the corresponding pH value into the conical flask. Use a high-performance liquid chromatograph to determine the contents of Teb and TTO in the controlled release medium. Calculate the cumulative release rate (R i ) through formula (3).

[0096]

[0097] Where C i is the concentration of Teb or TTO in the controlled release medium at different times (mg / L), and m Teb or TTO is the total mass of Teb or TTO added to the dialysis bag.

[0098] 1.8. Antibacterial activity:

[0099] The mycelial growth method was used to test the antibacterial effect of Pickering emulsion against *Sclerotium rolfsii* at different concentrations (0.03, 0.06, 0.13, 0.25, 0.33, 0.50 mg / L). 100 μL of Pickering emulsion was added to 20 mL of PDA agar that had been sterilized and cooled to 50 °C, shaken well and then poured into plates. After the agar solidified, an *Sclerotium rolfsii* disc with a diameter of 8 mm was placed upside down in the center of the agar plate. The edges of the petri dish were sealed with parafilm and incubated in an incubator for 3 - 4 d, and then the mycelial growth diameter was measured. The antibacterial rate of Pickering emulsion against *Sclerotium rolfsii* was calculated by formula (4).

[0100] Antibacterial rate (%) = (average colony diameter of the control group - average colony diameter of the experimental group) / (average colony diameter of the control group - disc diameter) × 100%

[0101] 1.9. Phytotoxicity:

[0102] The toxicity of the sample to plants was tested by a seed germination experiment. 4 mL of Pickering emulsion was diluted with 96 mL of water. 1.6 mL of the oil phase (TTO + Teb) dissolved with Teb was first dissolved in 1 mL of absolute ethanol and then dispersed in 97.4 mL of deionized water. 1.6 mL of pure TTO was first dissolved in 1 mL of absolute ethanol and then dispersed in 97.4 mL of deionized water. 160 mg of tebuconazole was first dissolved in 1 mL of absolute ethanol and then dispersed in 99 mL of deionized water. 140 mg of S - O - Z was dispersed in 100 mL of deionized water. 60 peanut seeds of similar size and intact were selected, surface - sterilized with 1% sodium hypochlorite solution for 10 min, then rinsed with water 5 - 6 times and air - dried at room temperature. The air - dried peanut seeds were respectively soaked in the above different diluted sample solutions, then sealed and shaken in a shaker at 25 °C for 5 h. They were taken out and air - dried, and then placed in a germination box lined with two layers of germination paper (20 seeds in each box), and then 5 mL of deionized water was added to each germination box. They were cultured for 7 d under the conditions of 25 °C, 12 h of light and 12 h of darkness alternating. Equal amounts of deionized water and 1% ethanol solution were used as controls, and the operations were the same as above.

[0103] 2. Results and Analysis

[0104] 2.1. Preparation and Characterization

[0105] Figure 2In it, in the SLS spectrogram, the characteristic absorption peaks of the lignin benzene ring skeleton vibration are at 1423 cm-1 and 1601 cm-1; the symmetric vibration of S=O in the sulfonate group is at 1043 cm-1. In the S-O-Z spectrogram, it can be clearly seen that the characteristic absorption peaks attributed to the amide I band and amide II band of Zein are at 1658 cm-1 and 1537 cm-1. Compared with ZD, new characteristic peaks belonging to the aromatic ring skeleton vibration and the symmetric vibration of S=O in the sulfonate group appear at 1423 cm-1 and 1043 cm-1 in S-O-Z respectively. In addition, the C-N stretching vibration peak originally attributed to the protein amino group at 1249 cm-1 undergoes a red shift (1240 cm-1), and the relative intensity is significantly enhanced. This may be due to the nucleophilic addition reaction of the free amino group in Zein with the epoxy group grafted on SLS under alkaline conditions, that is, the nitrogen atom in the amino group attacks the carbon atom on the epoxy ring, opening the epoxy ring and forming a new carbon-nitrogen bond. From the figure, we can also observe that in the SLS spectrogram, the peak at 1116 cm-1 is caused by the vibration of the C-O bond between the aromatic ring carbon-carbon bond and the adjacent hydroxyl group in the benzene ring. Compared with the ZD spectrogram, a new characteristic peak attributed to C-O-C also appears at 1121 cm-1 in the S-O-Z spectrogram. From this, we can speculate that the hydroxyl group on SLS undergoes a nucleophilic substitution reaction with ECH to generate an SLS derivative containing an epoxy group, and then further undergoes a nucleophilic addition reaction with the amino group on Zein through the epoxy group to form an SLS-Zein covalent conjugate (S-O-Z).

[0106] In Figure 3 it, it can be clearly observed that in the S-O-Z spectrogram, characteristic peaks related to SLS appear at approximately 1095 cm-1, 792 cm-1 and 560 cm-1, indicating the successful binding of Zein and SLS.

[0107] In Figure 4Among them, the DTG curve of SLS shows a relatively wide thermal decomposition temperature range, and its thermal decomposition is divided into three stages. The first stage is around 219 °C, attributed to the pyrolysis of small molecules. The second stage is at 270 °C, attributed to the cleavage of ether bonds and the cleavage of aliphatic side chains. The third stage is around 325 °C, attributed to the elimination and depolymerization reactions of polyhydroxyl groups. The thermal decomposition temperature of S-O-Z shows an increase compared to that of ZD. This is because the addition of SLS increases the aromatic ring content in the Zein structure, and aromatic rings are not easily decomposed, so the thermal decomposition temperature is increased. In addition, ZD is obtained by appropriately treating Zein under high temperature and alkaline conditions. During this process, some of the amide groups on the side chain of Zein will be removed and transformed into carboxyl groups, and its spatial structure will also change, thus increasing the molecular flexibility and reducing the thermal stability compared to untreated Zein. After covalently coupling with SLS, due to the increase in active groups in the conjugate structure, the hydrogen bond interaction inside the molecule is also enhanced, which also improves the thermal stability of S-O-Z to a certain extent.

[0108] In Figure 5 Among them, there is no obvious change in the Tg of S-O-Z compared to the Tg of ZD. Generally speaking, when rigid groups such as phenyl groups and conjugated double bonds are introduced into the main chain of the molecule, the rigidity of the chain increases and the Tg increases; while when flexible groups such as ether bonds are introduced into the main chain, the chain becomes flexible and the Tg decreases. When ZD is coupled with SLS, although aromatic ring rigid groups are introduced into the molecular chain, it is worth noting that flexible groups such as ether bonds and amino alcohols are also introduced at the same time. This may be one of the reasons for the lack of obvious difference between the Tg of S-O-Z and the Tg of ZD. Combining the DTG and DSC diagrams, it can be inferred that the introduction of SLS can enhance the heat resistance of ZD without changing its flexibility.

[0109] Figure 6 shows the scanning electron microscope (SEM) image of S-O-Z, indicating that the size of the nanoparticles is less than 500 nm. Figure 7 The particle size distribution of S-O-Z was measured and found to be uneven. Its average particle size is 379.24 ± 1.40 nm, and the average potential is -19.72 ± 1.10 mV. The absolute value of the Zeta potential of S-O-Z is less than 20 mV, which may cause S-O-Z to have a tendency to agglomerate and settle.

[0110] 2.2. Stability analysis

[0111] The stability of Pickering emulsions is crucial for their encapsulation and delivery of pesticides. Therefore, during the preparation process, the physical and chemical properties of the emulsions must be examined. This analysis will help determine the optimal preparation parameters required to achieve stable Pickering emulsions.

[0112] According to Table 1, we first prepared five groups of emulsions, namely A, B, C, D, and E. FromFigure 8 It can be seen that the five groups of freshly prepared Pickering emulsions are all emulsions with uniform dispersion. Figure 9 It shows that obvious stratification and partial emulsifier sedimentation occurred in all five groups of emulsions after 21 days of storage. Among them, oil leakage occurred in the emulsion layer of group C. After the stratified emulsions stored for 21 days were shaken well, obvious oil droplets could be observed on the bottle wall and in the emulsions of group C, indicating the irreversible deterioration of the emulsions ( Figure 10 ). However, the other four groups of stratified emulsions could still present a uniform dispersion state after being shaken well, and no oil droplets were observed. Figure 11 It is the volume fraction of the emulsified phase of the static emulsions at different time periods (since oil leakage occurred in the emulsions of group C on the 7th day, the volume fractions of the emulsified phase of the emulsions of group C at 7 days, 14 days and 21 days are no longer presented). It can be seen from groups A, B and C that when the solid particle content is kept constant, as the volume fraction of the oil phase increases, the volume fraction of the emulsified phase in the system also increases. And it can be seen from groups B, D and E that when the volume fraction of the oil phase is kept constant, increasing the solid particle content of the system will also increase the volume fraction of the emulsified phase in the system. It can be obtained from the figure that, without oil leakage, the order of the volume fractions of the emulsified phase of the emulsions at different time periods is E > D > B > A.

[0113] Figure 12 It is the optical microscope image of the five groups of emulsions when they were freshly prepared (0 d). It can be seen that at the same dilution, the droplets of the five groups of emulsions were in a uniformly dispersed state at 0 d, but the droplets of groups C, D and E were denser, while those of groups A and B were sparser.

[0114] Figure 13 It is the optical microscope image of the five groups of emulsions when they were stored for 21 d. After the stratified emulsions stored for 21 days were shaken well and diluted at the same dilution, it can be seen under the optical microscope that the droplet size of the emulsions in group C increased significantly and the size distribution was uneven. Similar situations also occurred in the other four groups of emulsions. However, relatively speaking, the droplet density, size and distribution of the emulsions in group E were better.

[0115] Figure 14 It is the particle size distribution of emulsions A, B, C, D and E when they were freshly prepared (0 d).

[0116] Figure 15 It is the average particle size of emulsions A, B, C, D and E when they were freshly prepared (0 d). The order of the average particle sizes of the emulsions in groups A, B and C is C < B < A, but the particle size distribution width is C > A > B. The order of the average particle sizes of the emulsions in groups B, D and E is E < D < B, but the particle size distribution width of B is the widest, and those of D and E are similar.

[0117] Figure 16 It is the particle size distribution of emulsions A, B, C, D and E after being stored for 21 d.

[0118] Figure 17 It is the average particle size of emulsions A, B, C, D, and E after 21 days of storage. After 21 days of placement, the order of the average particle sizes of the three groups of emulsions A, B, and C is A < B < C, and the particle size distribution width is C > B > A. The order of the average particle sizes of the three groups of emulsions B, D, and E is E < D < B, and the particle size distribution width is B > D > E. Judging from the figure, the particle sizes of the five groups of emulsions all increase after being placed at room temperature for 21 days. Among them, the particle size of group C changes the most, and the particle size of group E changes the least. The size distribution of emulsion droplets and the change of particle size during storage can reflect the stability of the emulsion. A wider particle size distribution indicates a larger size difference between droplets, a larger difference in sedimentation velocity between droplets, and a greater tendency to stratify and become unstable. Therefore, the formulation of group E is selected for the preparation and performance testing of subsequent emulsions.

[0119] 2.4, Wetting performance

[0120] Teb has systemic properties and can be transported to all parts of the plant after being absorbed by the plant. And Teb is a broad-spectrum fungicide. In addition to controlling some soil-borne diseases, it can also control some foliar diseases. Therefore, it is necessary to test the contact angle of the sample solution on plant leaves. In actual applications, pesticide emulsions often need to be diluted to a certain extent before application because the relative content of the active ingredient in undiluted emulsions is relatively high, and direct application may cause phytotoxicity to plants. The same is true for the Pickering emulsion system for delivering pesticides, which needs to be appropriately diluted during application. Therefore, the emulsion is diluted 100 times here before testing the surface tension and leaf surface contact angle (the concentrations of S-O-Z, ZD, and SLS are all prepared at 0.35 mg / L for testing the surface tension).

[0121] From Figure 18 it can be seen that under the same low concentration conditions, both ZD and S-O-Z show significantly lower surface tensions than water, with average values of 51.43 ± 0.07 mN / m and 51.34 ± 0.09 mN / m respectively, while SLS shows a surface tension closer to that of water at this concentration, with a value of 69.72 ± 0.08 mN / m. The emulsion after being diluted 100 times also shows a significantly lower surface tension than water, with an average value of 43.54 ± 0.06 mN / m. The low surface tension of the Pickering emulsion is mainly attributed to the good emulsifying property of S-O-Z and the low surface tension of TTO.

[0122] From Figure 19 it can be seen that after being diluted 100 times, the contact angle of the emulsion on cucumber leaves is still significantly smaller than that of pure water on the leaf surface, with an average value of 68.31 ± 0.52°. This indicates that the Pickering emulsion has good leaf surface affinity, which is related to its low surface tension value.

[0123] 2.4. Anti-ultraviolet performance

[0124] Figure 20 Show the remaining situation of pure oil phase (TTO + Teb) and TTO in the emulsion system under ultraviolet radiation for a certain period of time. It can be seen that TTO in the oil phase not coated with solid particle film drops below 30% at 40 min and is almost completely lost at 220 min, with a remaining rate of 0.48 ± 0.01%; while TTO in the Pickering emulsion system is still about 90% at 40 min and still retains 53.97 ± 0.28% at 220 min. This is because the solid particle film acts as a physical barrier to block most of the direct irradiation of ultraviolet rays on the active ingredient, and at the same time, the benzene ring structures in SLS and ZD structures in the solid particles can effectively absorb and filter out part of the ultraviolet light, so it plays a good protective role for TTO.

[0125] 2.5. Controlled release performance

[0126] From Figure 21 and Figure 22 it can be seen that the Pickering emulsion has obvious pH responsiveness, and the two active ingredients (Teb and TTO) have different drug release behaviors under the same conditions. The initial release rate of Teb is higher at pH = 3 and pH = 9 than at pH = 5 and pH = 7, and the release rate changes later, showing that the release rate is higher under slightly acidic conditions than under slightly alkaline conditions. In the TTO release curve, the total cumulative release amount of TTO is less than 25% under the condition of pH = 3, while the total cumulative release amount is higher than 60% at pH = 7 and pH = 9.

[0127] The above shows that acidic conditions are favorable for the release of Teb but unfavorable for the release of TTO, while alkaline conditions are favorable for the release of TTO but unfavorable for the release of Teb. When the controlled-release medium is acidic, the hydroxyl, imino, carboxyl, and sulfonic acid groups on the solid particles will all be protonated, enhancing the polarity and thus the hydrophilicity of the solid particles, accelerating the dissolution of the controlled-release medium in the emulsion solid particle film and the leakage of the internal oil phase. There can be hydrogen bonding interactions between the imidazole ring and hydroxyl groups in the Teb molecular structure and the complex components such as terpenes in TTO. Under acidic conditions, the hydrogen bonding interaction between Teb and TTO will be weakened. Additionally, Teb itself is negatively charged, while TTO is slightly acidic (around pH = 5.5). When the controlled-release medium has a pH of 3, there are a large number of proton hydrogens in the external environment, and due to electrostatic interactions, the effect of Teb dissolving out from TTO and diffusing into the external controlled-release medium increases. Under alkaline conditions, the hydroxyl, imino, carboxyl, and sulfonic acid groups on the solid particles will all be deprotonated. Since Teb is negatively charged, the electrostatic repulsion makes it not easy for Teb to dissolve out from TTO and diffuse into the controlled-release medium, thus showing a lower cumulative release amount. TTO also contains non-polar components such as alkanes and thus cannot be completely dissolved in the 60% ethanol controlled-release medium. When the controlled-release medium is acidic, the solubility of TTO will decrease, thus showing an even lower cumulative controlled-release amount; when the controlled-release medium is alkaline, the solubility of TTO increases, thus showing a higher cumulative controlled-release amount.

[0128] 2.6, Antibacterial Activity

[0129] Figure 23 The antibacterial activity of Pickering emulsions against Sclerotium rolfsii at different concentrations is shown, where the sample solution is the dilution after diluting 800 times according to the content of pesticide molecules. It can be seen that the Pickering emulsion at 0.03 mg / L has no antibacterial activity, indicating no inhibitory effect on Sclerotium rolfsii. As the concentration increases, the Pickering emulsion begins to show an inhibitory effect on Sclerotium rolfsii. Especially at a concentration of 0.5 mg / L, the inhibition rate of the Pickering emulsion against Sclerotium rolfsii reaches 90.72 - 93.74%.

[0130] 2.7, Phytotoxicity

[0131] Figure 24 This shows the germination of peanut seeds treated by soaking for 5 h with different sample solutions (the concentration of TTO in the system is 14.60 g / L, the concentration of Teb is 1.6 g / L, and the concentration of S - O - Z is 1.40 g / L) at the 2nd day and the 7th day. It can be clearly seen that the germination of peanut seeds in the water, S - O - Z, and Pickering emulsion treatment groups is good, while the germination of peanut seeds in the other groups is relatively poor.

[0132] In summary, the present invention provides a method for preparing zein-based Pickering emulsions and their application in pesticide delivery. This method functionalizes zein with a natural surfactant to improve its hydrophobicity and interfacial stability, enabling it to be stably dispersed in the Pickering emulsion system and preparing a pesticide delivery system with excellent dispersion stability and controlled-release characteristics. This system is formed by a covalent conjugate of zein and sodium lignosulfonate (SLS), which can regulate the release rate of pesticides under specific pH conditions and improve the utilization efficiency of pesticides. The zein-based Pickering emulsions prepared by the present invention not only have significant advantages in pesticide delivery but can also be applied in fields such as food preservation, medicine, and cosmetics to prepare emulsion systems with good stability and biocompatibility. Its unique structure can effectively control the release rate of active ingredients, thereby extending the shelf life, reducing the dosage, and at the same time reducing environmental pollution. This green and environmentally friendly emulsion preparation method meets the requirements of sustainable development and is expected to be widely applied in multiple industrial fields.

[0133] The present invention selects two natural products, SLS and zein, for covalent coupling to improve the hydrophobicity and interfacial stability of the initial zein, enabling it to be stably dispersed in the Pickering emulsion and regulating the release rate of pesticides under specific pH conditions. In addition, the present invention selects tebuconazole (Teb) and triadimefon (Tri) as fungicide model pesticides and uses tea tree oil (TTO), a plant essential oil, as the pesticide solvent and the dispersed phase of the emulsion. This natural solvent has a bactericidal effect and can synergistically enhance the effect of the fungicide, reducing the toxicity risk to the environment. The zein-based Pickering emulsion controlled-release pesticide prepared by the above method has excellent stability and controlled-release characteristics, low preparation cost, and wide raw material sources, significantly improving the delivery efficiency of pesticides. The present invention provides a low-cost, sustainable, and environmentally friendly pesticide delivery solution, which has important application value in pesticide protection and controlled release.

[0134] The present invention discloses a method for preparing and applying zein-based Pickering emulsion controlled-release pesticides. First, zein is modified by deamidation treatment and then covalently coupled with sodium lignosulfonate to obtain a stabilizer. Further, a plant essential oil is selected as the pesticide solvent and the dispersed phase of the emulsion, which not only enhances the bactericidal effect but also constructs a pesticide delivery system that does not require complex and harsh preparation conditions and avoids the use of toxic and harmful organic solvents; the preparation process is simple and stable, and it is a low-cost and environmentally friendly pesticide delivery system. This delivery system exhibits pH-responsive release properties, and the release efficiency of the applied pesticide can be controlled by adjusting the pH value of itself or the pH value of the application environment. It can be applied in the field of preparing antibacterial agents to kill fungi or for preventing and controlling soil-borne diseases, reducing the harm to the environment and non-target organisms.

[0135] The present invention selects a natural surfactant, sodium lignosulfonate (SLS), and obtains a stabilizer (S-O-Z) through covalent coupling with Zein. Using tebuconazole as a fungicide model pesticide, plant essential oil tea tree oil (TTO) is selected as the solvent of the pesticide and the dispersed phase of the Pickering emulsion. Through the above technical method, a pesticide delivery system for controlled-release pesticides based on zein Pickering emulsion is constructed. After ultraviolet light irradiation for 220 min, the pesticide residue rate in the delivery system is still 53.97 ± 0.28%. The results of the controlled-release test by the dialysis bag method show that the delivery system exhibits pH-responsive release. The seed germination experiment shows that the coating of the solid particle film of the Pickering emulsion can reduce the toxicity of the inner-phase active ingredient to peanut seeds to a certain extent. The zein-based Pickering emulsion pesticide delivery strategy constructed in the present invention does not require complex and harsh preparation conditions, and also avoids the use of toxic and harmful organic solvents. The preparation process is simple and stable, and it is a low-cost and environmentally friendly pesticide delivery system, which can reduce the harm to the environment and non-target organisms.

[0136] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions, and variations of these embodiments still fall within the protection scope of the present invention.

Claims

1. A method for preparing a zein-based Pickering emulsion controlled-release pesticide, characterized in that: The following steps are involved: S1, dissolving zein in a strong alkaline solution for deamidation treatment, stirring evenly with a magnetic stirrer, dialyzing sufficiently with a dialysis bag, and then freeze-drying to obtain deamidated zein; S2. Dissolve sodium lignin sulfonate in deionized water, stir and dissolve evenly, and adjust the pH to 8-9; S3, adding epichlorohydrin dropwise to the solution obtained in step S2, and stirring evenly with magnetic force; S4, dissolving the deamidated zein obtained in S1 in deionized water, and then pouring it into the solution obtained in step S3, adjusting the pH to 11-12, fully reacting, dialyzing using a dialysis bag, and then freeze-drying to obtain a covalent coupling body of sodium lignin sulfonate and zein; S5, fully stirring and dispersing the covalent coupling body obtained in step S4 in the aqueous phase to obtain an aqueous dispersion solution; S6. Ultrasonically dissolving the pesticide molecules in the plant essential oil to obtain an oily dispersion solution, mixing the aqueous dispersion solution obtained in step S5 with the oily dispersion solution, and homogenizing them using a homogenizer to obtain a zein-based Pickering emulsion controlled-release pesticide.

2. The preparation method according to claim 1, characterized in that: The plant essential oil is tea tree oil.

3. The preparation method according to claim 1, characterized in that: In step S1, the dialysis bag dialysis time is 24-36 hours; in step S1 and / or S3, the magnetic stirring time is 4-8 hours, and the magnetic stirring is carried out under heating conditions; the full reaction in step S4 is 4-6 hours at room temperature, and the dialysis bag dialysis time is 3 to 5 days.

4. The preparation method according to claim 1, characterized in that: In step S6, the homogenizer first homogenizes at high speed for 6 to 10 minutes, pauses for 5 to 10 minutes, and then homogenizes at high speed for another 6 to 10 minutes.

5. The preparation method according to claim 1, characterized in that: In step S1, the strong alkaline solution is NaOH or KOH.

6. The preparation method according to claim 1, characterized in that: In step S1 and / or step S4, the molecular weight cut-off of the dialysis bag is 800-1200 Daltons.

7. The preparation method according to claim 1, characterized in that: In step S5, the mass fraction of the aqueous dispersion solution is 1.5-3.5%; in step S6, the volume fraction of the oily dispersion solution is 30-50%.

8. The preparation method according to claim 1, characterized in that: The pesticide molecule is tebuconazole or triadimefon, or the pesticide molecule is other non-agricultural drug molecules used in the fields of food preservation, medicine and cosmetics.

9. Application of zein-based Pickering emulsion controlled release pesticide, characterized in that: The zein-based Pickering emulsion controlled-release pesticide prepared by any one of claims 1 to 8, wherein the pesticide molecule is tebuconazole or triadimefon, and the pH value of the zein-based Pickering emulsion controlled-release pesticide is adjusted to acidic or neutral, or applied in an acidic or neutral environment.

10. Application of zein-based Pickering emulsion controlled release pesticide, characterized in that: The zein-based Pickering emulsion controlled-release pesticide prepared by any one of claims 1 to 8 is used to prepare an antimicrobial agent to kill fungi; or to prevent and control soil-borne diseases.

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