Slow-release capsule capable of killing mosquitoes by adjusting intestinal flora of mosquitoes

Abstract

The invention discloses a sustained-release capsule capable of killing mosquitoes by adjusting intestinal flora of mosquitoes, and belongs to the technical field of functional microcapsules. The thymol and D-limonene mixed microcapsule emulsion for killing mosquitoes by adjusting intestinal flora of mosquitoes provided by the invention has an optimal insecticidal effect, LC50 is 48.98 mg / L and is lower than that of a single-component microcapsule emulsion, and the insecticidal effect is better. A sustained-release stability test shows that the microcapsule emulsion mixed according to the ratio of 1: 1 can still keep the insecticidal rate of 50% or above after 120 h, and the sustained-release stability of thymol is superior to that of D-limonene. Intestinal flora detection shows that different natural plant extracts have different effects on mosquito intestinal flora, and the mixed microcapsule emulsion can act on various flora, so that the insecticidal efficiency is improved. The natural plant extract microcapsule emulsion which is relatively high in stability and good in insecticidal and slow-release effects is successfully prepared, and a new direction is provided for application in the fields related to mosquito control and food.

Description

Technical Field

[0001] The invention relates to a slow-release capsule capable of killing mosquitoes by regulating mosquito intestinal flora, and belongs to the technical field of functional microcapsules. Background Art

[0002] Traditional chemical mosquito killers have limitations, such as the development of mosquito resistance, environmental pollution, and toxicity to non-target organisms caused by organophosphates and pyrethroids. Pyrethroids are a broad-spectrum insecticide that can control a wide range of pests, with insecticidal potency 10 to 100 times greater than older-generation insecticides such as organochlorines, organophosphates, and carbamates. However, long-term use of pyrethroids can easily lead to mosquito resistance, and some pyrethroid insecticides have a certain odor and irritation.

[0003] Natural plant extracts are widely available in nature, and most are low-toxic, playing different roles in various fields. Essential oils, as a common natural plant extract, play an important role in food preservation and antiseptic treatment.

[0004] Plant essential oils can be used as preservatives during postharvest storage of fruits and vegetables. For example, Abedi et al. found that a rosemary essential oil-whey protein coating significantly reduced the total microbial count and coliform count in fresh spinach while maintaining chlorophyll content. Tabassum et al. used a sodium alginate coating containing thyme essential oil on fresh-cut papaya, significantly reducing the papaya's weight loss rate and organic acid consumption, effectively delaying its aging process. Furthermore, many natural plant extracts exhibit mosquito repellent activity. Essential oils, alkaloids, and aromatic compounds from various plants are commonly used in plant-based mosquito repellents. When applied to human skin or used as indoor sprays, these compounds have been shown to interfere with mosquito host-seeking behavior. This demonstrates the feasibility of using natural plant extracts to kill mosquitoes and their potential to bring significant social value.

[0005] Studies have shown that using natural plant extracts to kill mosquitoes is very effective. However, the effects of different natural plant extracts and their ability to kill different mosquitoes vary. In addition, natural plant extracts are generally more active and need to be used in a specific environment. They are difficult to maintain long-term activity after extraction, and their stability, sustained release, and applicability are also areas that need to be considered. Nanoemulsion, microcapsule and other encapsulation technologies are an effective way to protect active substances. In the field of mosquito control, studies have found that the toxicity of perilla essential oil nanoemulsion is stronger than that of perilla essential oil to mosquito larvae of all ages, indicating that the emulsion encapsulation system will enhance the killing effect of perilla essential oil on mosquito larvae.

[0006] How to screen suitable natural plant extracts to kill mosquitoes, the improvement of embedding technology, the selection of emulsifiers and the mechanism of action of natural plant extracts on mosquito larvae are not yet clear. These are the limitations of using natural plant extracts to kill mosquitoes. Summary of the Invention

[0007] This study aims to select natural plant secretions and crude extracts to investigate their mosquito-killing efficacy. Effective natural plant extracts will be microencapsulated and emulsified to enhance their stability and sustained release. The stability and entrapment properties of the resulting microcapsule emulsions will be characterized, while their effectiveness against mosquito larvae will also be measured. Furthermore, the mosquito-killing or repellent mechanisms of these natural substances will be analyzed in an effort to uncover their underlying mechanisms. Based on these findings, we hope to develop a natural, non-toxic, highly effective, stable-release insecticidal microcapsule emulsion with a long shelf life and broad application, providing a new solution for mosquito control.

[0008] (1) Screening natural products with mosquito-killing activity and optimizing their embedding process;

[0009] Preliminary experiments were conducted on the selected natural plant extracts to identify the two most effective mosquito killers. Based on the chemical and structural properties of the two substances, along with references to literature and patents, the appropriate encapsulation technology was selected. While varying some details and parameters, such as encapsulation time, temperature, packaging material, emulsifier, and emulsification method, the optimal encapsulation effect was achieved.

[0010] (2) Characterization of the structure, stability and sustained-release performance of the encapsulation system

[0011] For the prepared microcapsule emulsion, we observed the structure of the microcapsules by fluorescent staining, measured its turbidity, Zeta potential and particle size to evaluate its stability, and studied its sustained-release performance by releasing mosquito larvae into the environment for different drug release times and observing their death.

[0012] (3) Evaluate the killing effect and mechanism of natural products on mosquitoes before and after encapsulation.

[0013] Preliminary experiments determined the appropriate gradient concentration and prepared a series of microcapsule emulsion concentration gradients to meet the mosquito larvae Lc50 test standard. The insecticidal efficacy of three microcapsule emulsions (two containing 100% screened natural extracts and one containing two natural extracts in a 1:1 ratio) was measured, using Lc50 values ​​as the indicator. The intestinal flora of dead larvae was analyzed to understand the drug's mechanism of action and to elucidate the underlying mechanism of larval mortality.

[0014] The present invention provides a method for preparing a microcapsule emulsion, which comprises the following steps:

[0015] (1) Add 1.0-2.0 g of β-cyclodextrin to 98.5-99 g of distilled water, heat and stir in a water bath at 40-60° C. (speed: 250-300 rpm), and mix until the liquid becomes clear and transparent to obtain a cyclodextrin solution;

[0016] (2) adding 18-20 g of natural plant extract to the cyclodextrin solution obtained in step (1), stirring at 45-55° C. and 250-300 rpm for 10-15 min to obtain a mixed solution;

[0017] (3) 2.5-30 g of an emulsifier, Tween-20 or Tween-80, and 2.0-3.0 g of an emulsifier, ethanol, are stirred at 50-55° C. to form an emulsifier solution; the emulsifier solution and 65-70 g of distilled water are added to the mixed solution of step (2), and the mixture is stirred at 50-55° C. and 250-300 rpm for 20-25 minutes to emulsify and obtain a microcapsule emulsion;

[0018] The natural plant extract is obtained by mixing thymol and D-limonenal in a mass ratio of 1:1.

[0019] In one embodiment of the present invention, step (1) is: adding 1.5 g of β-cyclodextrin to 98.5 g of distilled water, heating and stirring in a 50° C. water bath (rotation speed: 300 rpm), and mixing until the liquid becomes clear and transparent to obtain a cyclodextrin solution with a concentration of 1.5%.

[0020] In one embodiment of the present invention, step (2) is: adding 20 g of natural plant extract to the cyclodextrin solution obtained in step (1), stirring at 50° C. and 300 rpm for 10 min to obtain a mixed solution.

[0021] In one embodiment of the present invention, step (3) comprises: stirring 10 g of an emulsifier, Tween-20, and 2.5 g of an emulsifier, ethanol, at 50° C. to form an emulsifier solution; adding the emulsifier solution and 67.5 g of distilled water to the mixed solution of step (2), stirring at 50° C. and 300 rpm for 20 minutes for emulsification, and obtaining a microcapsule emulsion of the corresponding natural extract.

[0022] The present invention also provides a natural mosquito-killing microcapsule emulsion, which is prepared according to the following method:

[0023] (1) Add 1.0-2.0 g of β-cyclodextrin to 98.5-99 g of distilled water, heat and stir in a water bath at 40-60° C. (speed: 250-300 rpm), and mix until the liquid becomes clear and transparent to obtain a cyclodextrin solution;

[0024] (2) adding 18-20 g of natural plant extract to the cyclodextrin solution obtained in step (1), stirring at 45-55° C. and 250-300 rpm for 10-15 min to obtain a mixed solution;

[0025] (3) 2.5-30 g of emulsifier Tween-20 or Tween 80 and 2.0-3.0 g of co-emulsifier ethanol are stirred at 50-55° C. to form an emulsifier solution; the emulsifier solution and 65-70 g of distilled water are added to the mixed solution of step (2), and the mixture is stirred at 50-55° C. and 250-300 rpm for 20-25 minutes to emulsify and obtain a microcapsule emulsion;

[0026] The natural plant extract is obtained by mixing thymol and D-limonenal in a mass ratio of 1:1.

[0027] In one embodiment of the present invention, step (1) is: adding 1.5 g of β-cyclodextrin to 98.5 g of distilled water, heating and stirring in a 50° C. water bath (rotation speed: 300 rpm), and mixing until the liquid becomes clear and transparent to obtain a cyclodextrin solution with a concentration of 1.5%.

[0028] In one embodiment of the present invention, step (2) is: adding 20 g of natural plant extract to the cyclodextrin solution obtained in step (1), stirring at 50° C. and 300 rpm for 10 min to obtain a mixed solution.

[0029] In one embodiment of the present invention, step (3) comprises: stirring 10 g of an emulsifier, Tween-20, and 2.5 g of an emulsifier, ethanol, at 50° C. to form an emulsifier solution; adding the emulsifier solution and 67.5 g of distilled water to the mixed solution of step (2), stirring at 50° C. and 300 rpm for 20 minutes for emulsification, and obtaining a microcapsule emulsion of the corresponding natural extract.

[0030] The present invention also provides the use of the above method or the above microcapsule emulsion in killing mosquitoes or in preparing mosquito killing products.

[0031] In one embodiment of the present invention, the products include but are not limited to disc-type mosquito coils, mosquito coil sheets, mosquito repellent liquids, mosquito repellent bracelets, mosquito repellent patches, and mosquito repellent medicine bags.

[0032] Beneficial effects

[0033] Combining the encapsulation technology of food science, a new natural product controlled-release system is developed to fill the application gap in the field of mosquito control and provide new ideas for the research and development of green mosquito killers.

[0034] (1) Ten natural plant extracts were screened and it was found that thymol and D-limonene had significant killing effects on mosquito larvae. At a concentration of 100 mg / L, thymol had a 100% killing rate against mosquito larvae, while D-limonene had a 90% killing rate. The remaining eight extracts had weaker insecticidal activity, with larval killing rates below 50%. This indicates that thymol and D-limonene have high development potential in the field of mosquito control.

[0035] (2) Thymol and D-limonene were prepared into microcapsule emulsions. Characterization showed that the three microcapsule emulsions (thymol microcapsule emulsion THY, D-limonene microcapsule emulsion LIM, and thymol and D-limonene 1:1 mixed microcapsule emulsion TL) all had a uniform, milky white, opaque liquid appearance. No obvious phase separation or precipitation occurred after standing, and the physical stability was good. Among them, THY had the best suspension stability, LIM particles were larger than THY, and TL particles were the largest and less stable. This provides a stable formulation basis for subsequent bioactivity testing and practical applications.

[0036] (3) The insecticidal effects of the three microcapsule emulsions were dose-dependent. As the concentration increased, the mortality rate of the third-instar larvae of Aedes albopictus increased. The LC50 of TL was 48.98 mg / L, which was lower than that of THY (54.22 mg / L) and LIM (55.50 mg / L). This indicated that the microcapsule emulsion after mixing the two extracts had a better insecticidal effect and a synergistic effect. At the same time, all three microcapsule emulsions had good sustained-release stability. TL could still maintain an insecticidal rate of more than 50% after 120 hours. THY had a better sustained-release stability than LIM, and had the potential to continuously release active ingredients and prolong the efficacy in actual mosquito control.

[0037] (4) Different microcapsule emulsions have different effects on the intestinal flora of mosquito larvae. Compared with the control group, the Alpha diversity of the intestinal flora of larvae treated with microcapsule emulsions increased, and the increase was the highest in the TL group. At the genus level, the three microcapsule emulsions significantly increased the abundance of bacterial genera such as Acinetobacter, Clostridium and Paraclostridium, and inhibited beneficial bacteria such as Limnohabitans, Rahnella and Serratia, which may cause damage to the intestinal barrier and abnormal function of mosquito larvae, affecting their normal body functions and even leading to death. This reveals a new mechanism for the killing of mosquitoes by natural plant extract microcapsule emulsions, that is, to achieve insecticidal effects by affecting the intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Pictures of three finished products of microcapsule emulsions.

[0039] Figure 2 : Fluorescence staining images.

[0040] Figure 3 : Graph showing the sedimentation effects of three microcapsule emulsions at different times.

[0041] Figure 4 : Changes of absorbance of three microcapsule emulsions over time.

[0042] Figure 5 : Insecticidal effect curves of three microcapsule emulsions.

[0043] Figure 6 : Data chart related to intestinal flora; Note: Control in the picture represents the blank group, D-Limonene represents the D-limonene group, Thymol represents the thymol group, and TL represents the mixed group.

[0044] Figure 7 :Intestinal flora related data diagram; Note: Control in the picture represents the blank group, D-Limonene represents the D-limonene group, Thymol represents the thymol group, and TL represents the mixed group

[0045] Figure 8 :Data chart related to intestinal flora. DETAILED DESCRIPTION

[0046] Thymol, cinnamaldehyde, geranium oil, lemon eucalyptus essential oil, peppermint oil, capsaicin, neem oil, D-limonene, citronellal, trans-caryophyllene, β-cyclodextrin, Tween-20, anhydrous ethanol, and Nile red dye involved in the following examples are all commercially available reagents;

[0047] The third instar larvae of Aedes albopictus were provided by Jiangsu Institute of Schistosomiasis Control and Prevention.

[0048] The equipment information involved in the following examples is as follows:

[0049] BHC-1300ⅡA / B2 biological cleanroom safety cabinet, Shanghai Hujing Medical Instrument Co., Ltd.; UPW-30UV ultrapure water machine, Nanjing Aipure Environmental Protection Technology Co., Ltd.; DF-101S heat-collecting magnetic stirrer, Xicheng Xinrui Instrument Factory, Jintan District; KS-5200DE liquid ultrasonic cleaner, Kunshan Jielimei Ultrasonic Instrument Co., Ltd.; HDPN series electric heating constant temperature incubator, Shanghai Yuejin Medical Instrument Co., Ltd.; D-7pc UV-visible spectrophotometer, Nanjing Feile Instrument Co., Ltd.; SOPTOPCX 40 laboratory biological microscope, Ningbo Sunny Optical Instrument Co., Ltd.; NanoBrook Omni multi-angle nanoparticle size and zeta potential analyzer, Brookhaven Instrument Company, USA

[0050] The detection methods involved in the following embodiments are as follows:

[0051] Characterization of the stability and encapsulation effect of microcapsule emulsions

[0052] (1) Observe the embedding of microcapsules:

[0053] Weigh 5 mg of Nile Red and add 5 mL of acetone. Stir thoroughly with a blender to dissolve the Nile Red powder completely. Once dissolved, dilute the solution to the desired volume with acetone to prepare a Nile Red dye solution of a desired concentration.

[0054] Add Nile red staining solution to the centrifuge tube containing the extract to thoroughly mix the dye and extract. The volume ratio of dye to extract is generally 1:20. Gently shake or vortex the centrifuge tube, then let it stand at room temperature in the dark for 20 minutes to stain. Take an appropriate amount of the mixed extract and drop the prepared microcapsule emulsion onto a glass slide. Cover with a coverslip, minimizing the formation of bubbles. Place the prepared slide under an inverted fluorescence microscope, adjust the light source emitter to the green emission band, use green light excitation, and observe in the corresponding emission channel. The lipid components in the emulsion will be stained red by Nile red. The intensity and distribution of the fluorescence can be used to analyze the lipid content and state in the emulsion.

[0055] (2) Determination of particle size and zeta potential of microcapsule emulsion

[0056] Using a nanoparticle size and zeta potential analyzer, the particle size and zeta potential of the three microcapsule emulsions were measured according to the instrument's instructions. All three microcapsule emulsions were diluted to a concentration between 0.01 and 1 mg / mL in advance to ensure that the light beam passed through the solution without deflection or refraction and could pass completely through the diluted microcapsule emulsion. The three diluted microcapsule emulsions were placed in a cuvette. Zeta potential measurements require the insertion of electrodes, but particle size measurements do not require electrodes. The cuvette was placed in the instrument, and the system software adjusted parameters such as the number of cycles, equilibration time, particle size, and solvent before measurement.

[0057] (3) Suspension stability and sedimentation effect

[0058] Equal volumes of the prepared microcapsule emulsion were placed in a cuvette. Using a UV spectrophotometer set at a wavelength of 900 nm, the absorbance changes of the emulsions were monitored over 120 minutes. The relative absorbance was used to reflect the suspension stability of the three microcapsule emulsions: relative absorbance (%) at a given time = (absorbance at a given time / initial absorbance) × relative absorbance at 100%. Plotting relative absorbance against time yielded a curve of relative absorbance change over time, reflecting the suspension stability of the different microcapsule emulsions.

[0059] The prepared microcapsule emulsions were respectively placed in 2 mL glass bottles and allowed to stand under the same conditions. The bottles were photographed with a camera at regular intervals to observe the apparent stability of the microcapsule emulsions and reflect the sedimentation of the microcapsule emulsions.

[0060] Mosquito gut flora detection

[0061] Under the same growth conditions and at the same time, normally growing larvae and dead larvae exposed to a 100 mg / L concentration were selected. After surface disinfection with 75% alcohol, the larvae were quickly frozen in liquid nitrogen and stored at -80°C. PE300 microbial community diversity sequencing and bioinformatics analysis were performed to obtain data related to their intestinal flora and analyze the effects of natural plant extract microcapsules on the microbiome of mosquito larvae.

[0062] LC 50 Determination of value:

[0063] Two microcapsule emulsions were used to kill mosquito larvae at 8 gradient concentrations. According to the preliminary experimental results, the concentration gradient of the microcapsule emulsion was set to 0.5, 1, 10, 25, 50, 100, 120, and 150 mg / L, with three parallel experiments for each concentration. Twenty Aedes albopictus larvae were placed in each 360 mL round lunch box. The growth environment was a mixed solution of 200 mL pure water and natural extracts. The boxes were placed in an electric constant temperature incubator at a temperature of 27.5°C and a humidity of 60%. The boxes were cultured at a constant temperature and humidity for 24 hours. The death of the mosquito larvae was observed, the data was recorded, and the data was summarized using GraphPad Prism software to generate a curve graph to obtain the LC50 value and measure the drug effect.

[0064] Determination of sustained-release stability:

[0065] Select the microcapsule emulsion drug concentration that results in the highest number of mosquito larvae deaths. Under this concentration, add two microcapsule emulsions to 200 mL of pure water and place them at a constant temperature and humidity for 0, 24, 48, 72, 96, and 120 hours. Then, add 20 mosquito larvae. Three parallel groups of each microcapsule emulsion are placed in an electric constant temperature incubator at 27.5°C and 60% humidity for 24 hours. Observe the death of mosquito larvae to measure the release stability of the microcapsule emulsion.

[0066] Example 1: Preliminary screening of different natural plant extracts

[0067] Aedes albopictus larvae were grouped into 20 mL round plastic cups, each containing 10 individuals. The larvae were maintained in chlorine-free water. Natural plant extracts were diluted with acetone to 100 mg / L and added to the round plastic cups, with triplicates for each concentration. Each cup was filled to 10 mL with the aqueous solution and placed in an electrically controlled incubator at 27.5°C and 60% humidity for 24 hours. The number of mosquito larvae that died was then observed. The results of the preliminary screening of the effectiveness of different natural plant extracts against Aedes albopictus larvae are shown in Table 1.

[0068] Table 1: Results of the initial screening test

[0069]

[0070] The results show:

[0071] Among the 10 natural plant extracts that showed a mosquito larval killing effect, thymol achieved a 100% kill rate, while D-limonene achieved a 90% kill rate. Meanwhile, acetone and dechlorinated tap water, used as control groups, showed no effect on mosquito larvae. The other eight natural plant extracts all achieved kill rates of less than 50%. Therefore, thymol and D-limonene were selected for subsequent experiments.

[0072] Example 2: Preparation of microcapsule emulsion

[0073] The natural extract is prepared into microcapsule emulsion as follows:

[0074] (1) Add 1.5 g of β-cyclodextrin to 98.5 g of distilled water, heat and stir in a 50°C water bath (speed: 300 rpm), and mix until the liquid becomes clear and transparent to obtain a cyclodextrin solution with a concentration of 1.5%;

[0075] (2) adding 20 g of natural plant extract to the cyclodextrin solution obtained in step (1), stirring at 50° C. and 300 rpm for 10 min to obtain a mixed solution;

[0076] (3) 10 g of emulsifier Tween-20 and 2.5 g of co-emulsifier ethanol were stirred at 50° C. to form an emulsifier solution; the emulsifier solution and 67.5 g of distilled water were added to the mixed solution of step (2), and the mixture was stirred at 50° C. and 300 rpm for 20 min for emulsification to obtain a microcapsule emulsion of the corresponding natural extract.

[0077] The natural extract is thymol, D-limonene, and is obtained by adding thymol and D-limonene in a mass ratio of 1:1.

[0078] Finally, thymol:D-limonenal (1:1) microcapsule emulsion, thymol microcapsule emulsion and D-limonene microcapsule emulsion were obtained.

[0079] Example 3: Characterization of the stability and embedding effect of microcapsule emulsion

[0080] (1) Appearance of microcapsule emulsion

[0081] Mosquito larvae usually live in aquatic environments. The emulsion can effectively release the mosquito-killing substance into the water, meeting the needs of subsequent experiments and practical applications. Observation Example 2 yielded three microcapsule emulsions: thymol microcapsule emulsion, D-limonene microcapsule emulsion, and thymol: D-limonene aldehyde microcapsule emulsion. The results are shown in Figure 2. Figure 1 shown.

[0082] It can be found that all three emulsions have a milky white and opaque appearance, which is consistent with the characteristics of emulsions, and there is no obvious stratification, stable properties, and no precipitation occurs in the short term.

[0083] (2) Embedding effect

[0084] The three microcapsule emulsions were fluorescently stained, and the results of fluorescence microscopy were as follows: Figure 2 As shown, both thymol and D-limonene are encapsulated in β-cyclodextrin. The extract can also be seen encapsulated in β-cyclodextrin in the same 1:1 mixture. Furthermore, the size of the microcapsule particles can be determined based on the area of ​​fluorescence. The particles in the D-limonene microcapsule emulsion are significantly larger than those in the thymol microcapsule emulsion, while those in the mixed microcapsule emulsion are somewhere in between. Particle size is related to its stability, and the difference in stability between different microcapsule emulsions is an important indicator of microcapsule emulsion product stability. Generally, larger particles make the emulsion less stable.

[0085] (2) Stability of microcapsule emulsion

[0086] Suspension stability test results are as follows Figure 3 As shown in the figure, the relative absorbance of the three microcapsule emulsions gradually decreased within the first hour and then remained relatively stable. This is likely because the relatively large microcapsule particles sank to the bottom of the bottle during the first hour, leaving the relatively smaller microcapsule particles suspended in the solution as a colloid. Furthermore, the mixed microcapsule emulsion showed greater sedimentation within the first hour, likely due to the reduced microcapsule uniformity caused by the simultaneous addition of the two extracts.

[0087] from Figure 4 It can be observed from the photos that after standing for 48 hours, the three emulsions are still turbid, among which the two microcapsule emulsions such as thymol and mixed have no obvious separation compared with the D-limonene group after 48 hours.

[0088] Smaller average particle size and lower PDI indicate that the microcapsule emulsion system has higher stability. The higher the absolute value of Zeta potential, the greater the electrostatic repulsion between particles and the more stable the dispersion system. Zeta potential and particle size data (Table 2) show that D-limonene microcapsule particles are negatively charged and have the smallest absolute value of Zeta potential, while the average particle size and PDI values ​​are the highest, indicating that they have the worst stability. The other two microcapsule particles are positively charged, have larger absolute values ​​of Zeta potential, and have smaller average particle sizes and PDI values, indicating higher stability. This is consistent with Figure 4 The results were consistent with the obvious stratification.

[0089] Table 2: Particle size and Zeta potential results

[0090]

[0091] Note: Data marked with different lowercase letters in the same group have significant differences (P < 0.05)

[0092] Example 4: Verification of insecticidal ability of microcapsule emulsion

[0093] A series of concentration gradients of the natural plant extract were prepared: 250, 200, 150, 120, 100, 80, 50, 10, 1, and 0.5 mg / L. Preliminary experiments determined the concentration range of the microcapsule series, with larval mortality less than 20% at the lowest concentration and greater than 80% at the highest concentration. Each 360 mL round lunch box was filled with 149 mL of dechlorinated tap water, and then the corresponding amount of microcapsule emulsion was added dropwise (pre-calculated how much microcapsule emulsion would need to be added to 200 mL of water to achieve the desired concentration. Because both encapsulation and emulsification occur simultaneously, the effective concentration of the natural plant extract in the microcapsule emulsion can be assumed to be the concentration used to prepare the microcapsule emulsion). The mixture was stirred thoroughly with a glass rod, and then 20 preselected larvae (from another cup) were added to the lunch box along with 50 mL of water. The entire experiment was maintained in a greenhouse at 27°C ± 1°C and a relative humidity of 60% to 80%. After 24 hours in an electrically controlled temperature and humidity incubator, mortality was checked. Each concentration was repeated three times. Dechlorinated tap water was used as a control. Table 3 shows the larvicidal effects of the three microcapsule emulsions on the third instar larvae of Aedes albicans.

[0094] Table 3: Larval killing results of three microcapsule emulsions

[0095]

[0096] The results show:

[0097] The mortality rate of larvae was dose-dependent and increased with the increase of insecticide concentration. The three microcapsule emulsions had a mortality rate of 100% against the third instar larvae of Aedes albopictus at lower concentrations, but the LC 50 Slightly different. The LC of thymol 50 The LC50 of D-limonene was 54.22 mg / L, and the LC50 of the 1:1 mixture of the two extracts was 55.50 mg / L. 50 The insecticidal effect of the microcapsule emulsion prepared by mixing the two extracts is better than that of a single microcapsule emulsion, and the combination of different insecticides has a synergistic effect on its insecticidal effect.

[0098] Example 5: Sustained-release stability of microcapsule emulsion

[0099] The sustained-release stability of the three microcapsule emulsions is quite good, as shown in Table 4.

[0100] Table 4: Sustained release results of three microcapsule emulsions

[0101]

[0102] The results show:

[0103] All three microcapsule emulsions have good sustained-release stability. During the 120-hour release cycle in the water environment, each emulsion system continued to maintain an effective mosquito-killing concentration. A systematic comparison found that the sustained-release performance of the 1:1 compound microcapsule emulsion was particularly outstanding, and it could still maintain a larval killing rate of more than 50% after 120 hours, showing a significant long-term controlled-release advantage. At the same time, among the single-component microcapsule emulsions, thymol showed better sustained-release properties than D-limonene. Microcapsule emulsions with good sustained-release stability have greater application potential in actual mosquito control scenarios for continuously releasing active ingredients and prolonging the duration of drug efficacy, and can be used as efficient, long-lasting, green mosquito-killing preparations.

[0104] Example 6: Effect of the Dosage of β-Cyclodextrin on the Stability and Dilution Performance of Microcapsule Emulsion

[0105] The specific method is the same as Example 2, except that the amount of β-cyclodextrin is adjusted. Thymol: D-limonene aldehyde microcapsule emulsion (1:1) is prepared according to the method of Example 2 to explore the effect of the amount of β-cyclodextrin on stability and dilution performance.

[0106] The stability and dilution performance of the system were tested under different dextrin dosages;

[0107] The results show:

[0108] When the amount of dextrin was 1.5 g (Example 2), 1 g, and 2 g, the system was uniform and stable at 25°C, 45°C, and 65°C, and could be diluted (with water) in any proportion during use, and the emulsion system was uniform and stable.

[0109] When the dosage is 0g and 0.5g, the stratification is unstable at all temperatures, and the emulsion system is stratified when diluted with water; when the dosage is 2.5g, the precipitation is unstable at all temperatures, and the emulsion system is precipitated when diluted.

[0110] Example 7: Effect of different emulsifiers on stability and dilution performance

[0111] The specific details are the same as Example 2, except that the emulsifiers are adjusted to Tween 80, Tween 20, polyoxyethylene octylphenol ether-10, sodium lauroyl sarcosinate (LS-30N), and PEG-40 hydrogenated castor oil, respectively. Thymol: D-limonenal microcapsule emulsion (1:1) is prepared according to the method of Example 2, and the effects of various emulsifiers on the stability and dilution performance of the system are explored.

[0112] The results show:

[0113] Tween 80 and Tween 20 are uniformly stable at all temperatures, but can only be diluted within the range of 1-10 times during use. At other dilutions, the emulsion system will separate.

[0114] Polyoxyethylene octylphenol ether-10, sodium lauroyl sarcosinate (LS-30N), and PEG-40 hydrogenated castor oil were unstable at all temperatures and the emulsion systems separated upon dilution;

[0115] Example 8: Effect of emulsifier dosage on stability and dilution performance

[0116] The specific details are the same as Example 2, except that the amount of emulsifier is adjusted, and a thymol: D-limonenal microcapsule emulsion (1:1) is prepared according to the method of Example 2 to explore the effects of different emulsifier amounts on stability and dilution performance.

[0117] The results show:

[0118] When the emulsifier dosage is 10g (Example 2), 2.5g, 5g, 20g, 30g, and 40g, the system is uniform and stable at 25°C, 45°C, and 65°C, and can be diluted (with water) in any proportion during use, and the emulsion system is uniform and stable;

[0119] When the dosage is 40g, the system is uniform and stable but viscous; when the dosage is 0g, 0.5g, and 1g, the emulsion system is unstable and stratified at all temperatures, and stratifies when diluted.

[0120] Example 9: Detection of mosquito intestinal flora

[0121] Normal larvae and dead larvae at a concentration of 100 mg / L obtained in Example 4 were selected, and after surface disinfection with 75% alcohol, they were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator. The intestinal flora of the larvae was sequenced with PE300 microbial community diversity and bioinformatics analysis to obtain relevant data on its intestinal flora and analyze the effects of natural plant extract microcapsules on the microorganisms in the mosquito larvae. The intestinal flora of the dead mosquito larvae in different groups obtained from the experimental results were sequenced with 16s rRNA amplicon to evaluate the effects of different microcapsule emulsions on the composition of the intestinal flora of the dead mosquito larvae. The results are as follows: Figures 6-8 Control represents the blank group, D-Limonene represents the D-limonene group, Thymol represents the thymol group, and TL represents the mixed group.

[0122] The results show that:

[0123] (1) Compared with the group of mosquito larvae that died by normal liquid nitrogen quick freezing, i.e., the control group (Control group / CON group), the mosquito larvae killed by microcapsule emulsion increased the Alpha diversity of the microbiome (Ace, Shannon, Simpson and Chao indexes), and the increase in the TL group was the highest.

[0124] (2) Diversity results showed that the intestinal microbiota of the THY, LIM, and TL groups may exhibit different microbial community characteristics from the normal liquid nitrogen quick-frozen mosquito larvae group (Con group). Cluster forest analysis showed that there were significant differences between the THY, LIM, and TL groups compared with the Con group.

[0125] (3) The Adonis test was used to evaluate the diversity of bacterial community structure, and the experimental results in the principal component analysis (PCA) score diagram revealed that there were very significant differences in the OTU levels between the Con group and the THY, LIM, and TL groups.

[0126] (4) This study also conducted enterotype analysis at the genus level of the dominant microbial community to facilitate grouping different samples with similar dominant microbial community structures according to the relative abundance of microbial groups.

[0127] (5) At the genus level, the three microcapsule emulsions significantly increased the abundance of Acinetobacter, Clostridium, and Paraclostridium, while inhibiting beneficial bacteria such as Limnohabitans, Rahnella, and Serratia. In addition, it can be seen that the THY group significantly increased the abundance of Acinetobacter, the LIM group significantly increased the abundance of Aeromonas and Chromobacterium, and the TL group significantly increased the abundance of Clostridium and Enterococcus. Some bacteria in the Acinetobacter and Aeromonas genera are opportunistic pathogens that can cause intestinal infections; Chromobacterium is not a normal flora and is relatively rare in the intestine; Clostridium may produce toxins; and Enterococcus is an important pathogen. This shows that microcapsule emulsions may damage the intestinal barrier of mosquito larvae. The increase in the abundance of these genera can lead to abnormal intestinal function, thereby affecting normal body functions and even causing death.

[0128] (6) Through the above cluster analysis, the microbial groups with significant differences between the Con group and the THY, LIM, and TL groups were identified as Aeromonas, Clostridium, and Paraclostridium intestinal microbial groups. From the results, it can be seen that the intestinal flora structure of the dead mosquito larvae caused by the microcapsule emulsion is different from that of the control group. The harm of the first two genera was mentioned in the results (5). In addition, Paraclostridium, as part of the normal intestinal flora, can maintain the stability and balance of the intestinal microecological system under normal circumstances. However, once the intestinal flora is out of balance, it can also cause intestinal infection, thereby affecting normal body functions.

[0129] (7) Linear discriminant effect size (LEfSe) analysis was performed on the Con, THY, LIM, and TL groups from the phylum to the genus level; the role of key microbial groups was further studied and their relationships within the community were explored. The linear discriminant histogram showed that the main characteristic microbial group branch of the CON group was Serratia (LDA value = 8.98). The main characteristic microbial group branch of the THY group was Pseudomonadaceae (LDA value = 7.48) and Anaerolineaceae (LDA = 7.27). The main characteristic microbial group branch of the LIM group was Luteitalea (LDA value = 7.10). The main characteristic microbial group branch of the TL group was Cryomorphaceae (LDA value = 7.21) and Owenweeksia (LDA = 7.14). The genus Serratia can interact with the intestinal immune system, enhance immune defense, and inhibit the growth of harmful bacteria. Pseudomonadaceae and Anaerolineaceae bacteria have been shown to protect against other harmful microorganisms. Exposure of mosquito larvae to microencapsulated emulsion disrupts the intestinal microbiome, leading to an increase in harmful microorganisms, highlighting the importance of these two bacterial groups in protecting against other microorganisms. Luteitalea, Cryomorphaceae, and Owenweeksia bacteria play a crucial role in mosquito metabolism, metabolizing excess substances. Exposure to microencapsulated emulsion may disrupt normal metabolic pathways, thus increasing the nutritional support provided by these bacteria and boosting their numbers.

[0130] (8) In order to confirm the evolutionary relationship of the intestinal flora, a phylogenetic tree and the relative abundance of species at the genus level for each sample were generated. The results showed that the three microcapsule emulsions changed the composition of the intestinal flora, causing it to form an intestinal flora composition different from that of the Con group. By establishing a network interaction diagram of three groups of microorganisms except the CON group, it was found that there was a strong correlation between Actinomycetota, Bacillota, Bacteroidota, and Pseudomonadota, indicating that after the microcapsule emulsion destroyed the intestinal flora of mosquito larvae, it may be that these types of bacteria have overlapping ecological niches in the mosquito larvae intestine and complementary functions. They may jointly participate in food digestion and nutrient absorption, maintain the stability of the intestinal microbial community, and protect the intestinal health of mosquito larvae.

[0131] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a microcapsule emulsion, characterized in that: The method comprises the following steps: (1) Add 1.0-2.0 g of β-cyclodextrin to 98.5-99 g of distilled water, heat and stir in a water bath at 40-60° C. at a speed of 250-300 rpm to obtain a cyclodextrin solution; (2) adding 18-20 g of natural plant extract to the cyclodextrin solution obtained in step (1), stirring at 45-55° C. and 250-300 rpm for 10-15 min to obtain a mixed solution; (3) 2.5-30 g of emulsifier Tween-20 or Tween 80 and 2.0-3.0 g of co-emulsifier ethanol are stirred at 50-55° C. to form an emulsifier solution; the emulsifier solution and 65-70 g of distilled water are added to the mixed solution of step (2), and the mixture is stirred at 50-55° C. and 250-300 rpm for 20-25 minutes to emulsify and obtain a microcapsule emulsion; The natural plant extract is obtained by mixing thymol and D-limonenal in a mass ratio of 1:

1.

2. The method according to claim 1, characterized in that Step (1) is: adding 1.5 g of β-cyclodextrin to 98.5 g of distilled water, heating and stirring in a 50° C. water bath at a rotation speed of 300 rpm, and mixing until the liquid becomes clear and transparent to obtain a cyclodextrin solution with a concentration of 1.5%.

3. The method according to claim 2, characterized in that Step (2) is: adding 20 g of natural plant extract to the cyclodextrin solution obtained in step (1), stirring at 50° C. and 300 rpm for 10 minutes to obtain a mixed solution.

4. The method according to claim 3, characterized in that Step (3) comprises: stirring 10 g of an emulsifier, Tween-20, and 2.5 g of an emulsifier, ethanol, at 50° C. to form an emulsifier solution; adding the emulsifier solution and 67.5 g of distilled water to the mixed solution of step (2), stirring at 50° C. and 300 rpm for 20 minutes for emulsification, and obtaining a microcapsule emulsion of the corresponding natural extract.

5. A natural mosquito-killing microcapsule emulsion, characterized in that: The microcapsule emulsion is prepared according to the following method: (1) Add 1.0-2.0 g of β-cyclodextrin to 98.5-99 g of distilled water, heat and stir in a water bath at 40-60° C., at a speed of 250-300 rpm, and mix until the liquid becomes clear and transparent to obtain a cyclodextrin solution; (2) adding 18-20 g of natural plant extract to the cyclodextrin solution obtained in step (1), stirring at 45-55° C. and 250-300 rpm for 10-15 min to obtain a mixed solution; (3) 2.5-30 g of emulsifier Tween-20 or Tween 80 and 2.0-3.0 g of co-emulsifier ethanol are stirred at 50-55° C. to form an emulsifier solution; the emulsifier solution and 65-70 g of distilled water are added to the mixed solution of step (2), and the mixture is stirred at 50-55° C. and 250-300 rpm for 20-25 minutes to emulsify and obtain a microcapsule emulsion; The natural plant extract is obtained by mixing thymol and D-limonenal in a mass ratio of 1:

1.

6. The microcapsule emulsion according to claim 5, characterized in that Step (1) is: adding 1.5 g of β-cyclodextrin to 98.5 g of distilled water, heating and stirring in a 50° C. water bath at a rotation speed of 300 rpm, and mixing until the liquid becomes clear and transparent to obtain a cyclodextrin solution with a concentration of 1.5%.

7. The microcapsule emulsion according to claim 6, characterized in that Step (2) is: adding 20 g of natural plant extract to the cyclodextrin solution obtained in step (1), stirring at 50° C. and 300 rpm for 10 minutes to obtain a mixed solution.

8. The microcapsule emulsion according to claim 7, characterized in that Step (3) comprises: stirring 10 g of an emulsifier, Tween-20, and 2.5 g of an emulsifier, ethanol, at 50° C. to form an emulsifier solution; adding the emulsifier solution and 67.5 g of distilled water to the mixed solution of step (2), stirring at 50° C. and 300 rpm for 20 minutes for emulsification, and obtaining a microcapsule emulsion of the corresponding natural extract.

9. Use of the method according to any one of claims 1 to 4 or the microcapsule emulsion according to any one of claims 5 to 8 in mosquito control or in the preparation of mosquito control products.

10. The use according to claim 9, characterized in that The products include but are not limited to disc-type mosquito coils, mosquito coil sheets, mosquito repellent liquid, mosquito repellent bracelets, mosquito repellent patches, and mosquito repellent medicine bags.

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