Long-acting mosquito repellent emulsion containing insecticidal protein and preparation method thereof

Abstract

The application discloses a long-acting mosquito repellent emulsion containing insecticidal protein and a preparation method thereof, and belongs to the mosquito repellent field. First, the application obtains a compounded essential oil with better mosquito repellent effect by compounding citronella oil, peppermint oil and mugwort leaf oil, and uses patchouli oil and cypress oil as a fixative to improve the volatility uniformity and durability of the essential oil; then, the application uses Bacillus thuringiensis crystal protein to prepare nano-particle emulsion to embed the essential oil component, so as to slow down the volatilization rate of the essential oil, and finally, the long-acting mosquito repellent emulsion is prepared. The mosquito repellent emulsion provided by the application does not contain chemical components such as diethyltoluamide and mosquito repellent ester, has obvious mosquito repellent effect and long duration, has a high knockdown rate on mosquitoes, and will not cause resistance of mosquitoes.

Description

Technical Field

[0001] This invention belongs to the field of mosquito repellents, specifically relating to a long-lasting mosquito repellent emulsion containing insecticidal proteins and its preparation method. Background Technology

[0002] Mosquitoes, belonging to the family Culicidae in the order Diptera, are diverse in species and reproduce rapidly, causing considerable annoyance to people's lives every summer. Mosquitoes not only bite and harass humans but also carry various pathogenic microorganisms, leading to a range of diseases. Effectively preventing mosquito bites and diseases is a global public health challenge. Currently, humans mostly use insecticides to control mosquitoes. However, the extensive use of insecticides can easily lead to increased mosquito resistance and exacerbate environmental pollution. Even more serious consequences include the reabsorption of chemical components from insecticides through the skin, harming human health. With increasing awareness of ecological protection and health, the development of highly effective, environmentally friendly, and safe mosquito repellents is urgently needed.

[0003] my country has a long tradition of using natural plants to repel mosquitoes, such as mosquito-repelling grass, lavender, eucalyptus leaves, citronella, and mugwort. However, these natural plants have limited mosquito-repelling efficiency and can only be used as supplementary methods. With the development of the chemical industry, extracting essential oils from these mosquito-repelling plants and using their special aromas to repel mosquitoes has become more convenient and feasible. Mosquito-repelling essential oils are popular because they are derived from natural raw materials and have advantages such as safety, non-toxicity, low risk of developing resistance, and easy degradation. However, single plant essential oils generally suffer from low mosquito-repelling efficiency and short effective protection time. Therefore, researchers have designed combinations of multiple plant essential oils to improve their mosquito-repelling efficiency and effective protection time. However, mosquito-repelling essential oils obtained using this method still have the problem of short effective protection time. Therefore, developing mosquito-repelling products with good mosquito-repelling effect and long effective protection time remains one of the key goals in the field of mosquito repellent technology and a common aspiration of the general public.

[0004] Pickering emulsions are stable emulsions formed by the irreversible adsorption of solid / colloidal particles at the oil-water interface. They possess advantages such as high safety, physical stability, and good loading / release characteristics. Preparing essential oils into Pickering emulsions stabilized by solid particles can effectively solve problems such as high volatility and poor chemical stability of essential oils. Currently, the solid particles used to stabilize Pickering emulsions are generally divided into two categories: inorganic particles and organic particles. Inorganic particles mainly include modified silica, titanium dioxide, and clay particles, while organic particles mainly include proteins, polysaccharides, nanocellulose, and bioactive small molecules such as flavonoids and phytosterols. Using these solid particles, stable mosquito-repellent essential oil Pickering emulsions can be prepared, slowing down the evaporation rate of the essential oils and thus increasing their effective mosquito-repellent protection time. However, these solid particles only play an emulsifying role in the emulsion and do not enhance the mosquito-repellent efficiency. Summary of the Invention

[0005] The purpose of this invention is to provide a long-lasting mosquito repellent emulsion containing insecticidal proteins and its preparation method. By emulsifying and encapsulating mosquito repellent essential oils with Bacillus thuringiensis crystalline protein nanoparticles, the prepared mosquito repellent product does not produce toxic side effects on the human body, has a significant and long-lasting mosquito repellent effect, a high mosquito knockdown rate, and does not induce mosquito resistance, thus meeting people's needs for mosquito repellent in summer.

[0006] The objective of this invention is achieved through the following technical solution: First, a compound essential oil with good mosquito-repellent effect is obtained by compounding three plant essential oils: citronella oil, peppermint oil, and mugwort oil. Then, patchouli oil and cypress oil are added to the compound essential oil as fixatives in a specific ratio to prolong its mosquito-repellent effect. Finally, nanoparticles are prepared using Bacillus thuringiensis crystal protein to emulsify and encapsulate the essential oil components, further slowing down the evaporation rate of the essential oil and improving its knockdown rate against mosquitoes.

[0007] Specifically, a method for preparing a long-lasting mosquito repellent emulsion containing insecticidal proteins includes the following steps:

[0008] S1. Plant essential oil extraction: Lemongrass, peppermint leaves, artemisia leaves, patchouli leaves, and cypress wood chips were freeze-dried and pulverized, passed through a 30-mesh sieve, and 800g of each plant powder was weighed and placed in a 5L extraction vessel. The vessel was loaded into a supercritical CO2 extraction device and extracted under the following conditions: temperature, pressure, time, and CO2 flow rate of 38-42℃, 24-26Mpa, 80-100min, and 23-27L / h, respectively. The mixture was then refrigerated at 4℃ for 12-15h and filtered to obtain lemongrass oil, peppermint oil, artemisia oil, patchouli oil, and cypress wood oil, respectively.

[0009] S2. Preparation of mosquito repellent essential oil: Citronella oil, peppermint oil, and artemisia oil are combined and blended in a volume ratio of 1–3:1–3:1, and stirred at 120–150 r / min for 2–2.5 h to obtain a compound essential oil with good mosquito repellent effect; patchouli oil and cypress oil are combined and blended in a volume ratio of 1:1–3, and stirred at 120–150 r / min for 2–2.5 h to obtain a compound fixative; the compound fixative is added to the compound essential oil at a mass content of 10%–30%, and stirred at 120–150 r / min for 2–2.5 h to obtain a mosquito repellent essential oil with good mosquito repellent duration.

[0010] S3. Extraction of Bacillus thuringiensis crystal protein: Bacillus thuringiensis was inoculated into NB medium and cultured in a shaker at 28–30℃ and 1180–200 r / min for 72–80 h. 1 L of fermentation broth was collected by centrifugation. After removing most of the spores by foam separation, the product was washed with sterile pre-cooled 0.5 mol / L NaCl solution and deionized water. The washed solid product was dissolved in 150–160 mL of lysis buffer (50 mmol / L EDTA, 50 mmol / L Na2CO3, 3% mercaptoethanol) and lysed at 3–4℃ for 6–7 h. The supernatant was collected by centrifugation and neutralized with 4 mol / L sodium acetate-acetic acid solution to pH 5.0 ± 0.1. The supernatant was precipitated at 3–4℃ for 4–4.5 h. The precipitate was collected by centrifugation, washed with sterile pre-cooled deionized water, dialyzed to desalt, and then freeze-dried under vacuum to obtain Bacillus thuringiensis crystal protein.

[0011] S4. Preparation of Bacillus thuringiensis crystal protein nanoparticles: Bacillus thuringiensis crystal protein was added to water with a pH of 9-11 at a mass content of 1-5% to prepare a Bacillus thuringiensis crystal protein solution. Anhydrous ethanol was added to the Bacillus thuringiensis crystal protein solution at a volume ratio of 1-4:1. When the solution showed a blue opalescence, genipin, a cross-linking agent, was added to make the mass concentration of genipin in the mixed solution 0.5-1%. Cross-linking was carried out at 37-38℃ for 12-36 hours. Ethanol was removed by rotary evaporation to obtain a colloidal solution. The colloidal solution was centrifuged and the precipitate was washed with pure water. The precipitate was then freeze-dried to obtain Bacillus thuringiensis crystal protein nanoparticles.

[0012] S5. Preparation of long-lasting mosquito repellent and mosquito-killing emulsion: Mix equal amounts of mosquito repellent essential oil and water to obtain an oil-water medium. Disperse Bacillus thuringiensis crystal protein nanoparticles in the oil-water medium at a ratio of 1g:5-10mL. Stir at 250-300r / min for 2-2.5h, and then homogenize at 10000-14000r / min for 1-3min to obtain the long-lasting mosquito repellent and mosquito-killing emulsion.

[0013] The Bacillus thuringiensis was purchased from the China Industrial Microbial Culture Collection Center, with the number CICC20559.

[0014] This invention employs the aforementioned technical solution. First, it combines and blends three plant essential oils: citronella oil, peppermint oil, and mugwort oil. Utilizing the synergistic effect between these different essential oils, a compound essential oil with high mosquito-repellent efficiency can be obtained. Then, patchouli oil and cypress oil are added as fixatives to the compound essential oil to obtain a mosquito-repellent essential oil. Through the adsorption effect between the polymer structures, the uniformity and persistence of the mosquito-repellent essential oil's volatility are improved, helping to prolong its mosquito-repellent effect. Finally, nanoparticles are prepared using Bacillus thuringiensis crystal protein to emulsify and encapsulate the essential oil components, further slowing down the essential oil's evaporation rate. Simultaneously, the introduction of Bacillus thuringiensis crystal protein also enhances the knockdown rate of mosquitoes, thus obtaining a long-lasting mosquito-repellent emulsion.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) This invention combines three kinds of plant essential oils with good mosquito repellent effect, which further improves the mosquito repellent efficiency and effective protection time of the essential oils, and the aroma of the essential oils is effectively improved.

[0017] (2) The present invention uses patchouli oil and cypress oil as fixatives, which improves the uniformity and persistence of the essential oil's volatility and effectively prolongs the mosquito-repelling effect of the essential oil components.

[0018] (3) The present invention uses Bacillus thuringiensis crystal protein nanoparticles to emulsify and encapsulate mosquito repellent essential oil, thereby controlling the evaporation rate of essential oil and achieving a stable and continuous mosquito repellent effect. At the same time, Bacillus thuringiensis crystal protein has a good insecticidal effect, which makes the mosquito repellent emulsion have a high mosquito knockdown rate.

[0019] (4) The mosquito repellent product prepared by the above technical solution of the present invention contains effective components such as citronella oil, peppermint oil, artemisia oil, patchouli oil, cypress oil, and Bacillus thuringiensis crystal protein, and does not contain chemical components such as DEET and IR3535, and has no toxic side effects on the human body. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the embodiments.

[0021] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0022] Example 1

[0023] A method for preparing a long-lasting mosquito repellent emulsion containing insecticidal proteins includes the following steps:

[0024] S1. Plant essential oil extraction: Lemongrass, peppermint leaves, mugwort leaves, patchouli leaves, and cypress wood chips were freeze-dried, pulverized, and passed through a 30-mesh sieve. 800g of each plant powder was weighed and placed in a 5L extraction vessel, loaded into a supercritical CO2 extraction device, and extracted under the following conditions: temperature, pressure, time, and CO2 flow rate of 40℃, 25MPa, 90min, and 25L / h, respectively. After refrigeration at 4℃ for 12h, the mixture was filtered to obtain lemongrass oil, peppermint oil, mugwort oil, patchouli oil, and cypress wood oil, respectively.

[0025] S2. Preparation of mosquito repellent essential oil: Citronella oil, peppermint oil, and artemisia oil were combined and blended in a volume ratio of 2:2:1, and stirred at 120 rpm for 2 hours to obtain a compound essential oil with good mosquito repellent effect. Patchouli oil and cypress oil were combined and blended in a mass-volume ratio of 1:2, and stirred at 120 rpm for 2 hours to obtain a compound fixative. The compound fixative was added to the compound essential oil at a mass content of 20%, and stirred at 120 rpm for 2 hours to obtain a mosquito repellent essential oil with good long-lasting mosquito repellent effect.

[0026] S3. Extraction of Bacillus thuringiensis crystal protein: Bacillus thuringiensis (CICC 20559) was inoculated into NB medium and cultured at 30℃ and 200 r / min for 72 h on a shaker. 1 L of fermentation broth was collected by centrifugation. After removing most of the spores through foam separation, the cells were washed with sterile, pre-cooled 0.5 mol / L NaCl solution and deionized water. The washed solid product was dissolved in 150 mL of lysis buffer (50 mmol / L EDTA, 50 mmol / L Na2CO3, 3% mercaptoethanol) and lysed at 4℃ for 6 h. The supernatant was collected by centrifugation. The supernatant was neutralized to pH 5.0 with 4 mol / L sodium acetate-acetic acid solution and precipitated at 4℃ for 4 h. The precipitate was collected by centrifugation, washed with sterile, pre-cooled deionized water, dialyzed to desalt, and then freeze-dried under vacuum to obtain Bacillus thuringiensis crystal protein.

[0027] S4. Nanoparticle Preparation: Bacillus thuringiensis crystal protein was added to water at pH 10 at a mass concentration of 3% to prepare a Bacillus thuringiensis crystal protein solution. Anhydrous ethanol was added to the Bacillus thuringiensis crystal protein solution at a volume ratio of 2:1. Once the solution exhibited a blue opalescent appearance, genipin, a cross-linking agent, was added to bring the genipin concentration in the mixture to 0.75%. Cross-linking was carried out at 37°C for 24 hours. Ethanol was removed by rotary evaporation to obtain a colloidal solution. The colloidal solution was centrifuged, and the precipitate was washed with pure water and freeze-dried to obtain Bacillus thuringiensis crystal protein nanoparticles.

[0028] S5. Preparation of long-lasting mosquito repellent emulsion: Mix equal amounts of mosquito repellent essential oil and water to obtain an oil-water medium. Disperse Bacillus thuringiensis crystal protein nanoparticles in the oil-water medium at a ratio of 1g:7.5mL. Stir at 250r / min for 2h and then homogenize at 12000r / min for 2min to obtain the long-lasting mosquito repellent emulsion.

[0029] Example 2

[0030] A method for preparing a long-lasting mosquito repellent emulsion containing insecticidal proteins includes the following steps:

[0031] S1. Plant essential oil extraction: Lemongrass, peppermint leaves, mugwort leaves, patchouli leaves, and cypress wood chips were freeze-dried and pulverized, then passed through a 30-mesh sieve. 800g of each plant powder was weighed and placed in a 5L extraction vessel, which was then loaded into a supercritical CO2 extraction device. Extraction was carried out at 40℃, 25MPa, 90min, and 25L / h CO2 flow rates, respectively. The mixture was then refrigerated at 4℃ for 12h and filtered to obtain lemongrass oil, peppermint oil, mugwort oil, patchouli oil, and cypress wood oil, respectively.

[0032] S2. Preparation of mosquito repellent essential oil: Citronella oil, peppermint oil, and artemisia oil were combined and blended in a 1:1:1 volume ratio, and stirred at 120 rpm for 2 hours to obtain a compound essential oil with good mosquito repellent effect. Patchouli oil and cypress oil were combined and blended in a 1:1 volume ratio, and stirred at 120 rpm for 2 hours to obtain a compound fixative. The compound fixative was added to the compound essential oil at a mass content of 10%, and stirred at 120 rpm for 2 hours to obtain a mosquito repellent essential oil with good long-lasting mosquito repellent effect.

[0033] S3. Extraction of Bacillus thuringiensis crystal protein: Bacillus thuringiensis (CICC 20559) was inoculated into NB medium and cultured at 30℃ and 200 r / min for 72 h on a shaker. 1 L of fermentation broth was collected by centrifugation. After removing most of the spores through foam separation, the cells were washed with sterile, pre-cooled 0.5 mol / L NaCl solution and deionized water. The washed solid product was dissolved in 150 mL of lysis buffer (50 mmol / L EDTA, 50 mmol / L Na2CO3, 3% mercaptoethanol) and lysed at 4℃ for 6 h. The supernatant was collected by centrifugation. The supernatant was neutralized to pH 5.0 with 4 mol / L sodium acetate-acetic acid solution and precipitated at 4℃ for 4 h. The precipitate was collected by centrifugation, washed with sterile, pre-cooled deionized water, dialyzed to desalt, and then freeze-dried under vacuum to obtain Bacillus thuringiensis crystal protein.

[0034] S4. Nanoparticle Preparation: Bacillus thuringiensis crystal protein was added to water at pH 9 at a mass concentration of 1% to prepare a Bacillus thuringiensis crystal protein solution. Anhydrous ethanol was added to the Bacillus thuringiensis crystal protein solution at a volume ratio of 1:1. Once the solution exhibited a blue opalescent appearance, genipin, a cross-linking agent, was added to bring the genipin concentration in the mixture to 0.5%. Cross-linking was carried out at 37°C for 12 hours. Ethanol was removed by rotary evaporation to obtain a colloidal solution. The colloidal solution was centrifuged, and the precipitate was washed with pure water and freeze-dried to obtain Bacillus thuringiensis crystal protein nanoparticles.

[0035] S5. Preparation of long-lasting mosquito repellent and mosquito-killing emulsion: Mix mosquito repellent essential oil and water in equal amounts, disperse Bacillus thuringiensis crystal protein nanoparticles in the oil-water medium at a ratio of 1g:5mL, stir at 250r / min for 2h, and then homogenize at 10000r / min for 1min to obtain the long-lasting mosquito repellent and mosquito-killing emulsion.

[0036] Example 3

[0037] A method for preparing a long-lasting mosquito repellent emulsion containing insecticidal proteins includes the following steps:

[0038] S1. Plant essential oil extraction: Lemongrass, peppermint leaves, mugwort leaves, patchouli leaves, and cypress wood chips were freeze-dried and pulverized, then passed through a 30-mesh sieve. 800g of each plant powder was weighed and placed in a 5L extraction vessel, which was then loaded into a supercritical CO2 extraction device. Extraction was carried out at 40℃, 25MPa, 90min, and 25L / h CO2 flow rates, respectively. The mixture was then refrigerated at 4℃ for 12h and filtered to obtain lemongrass oil, peppermint oil, mugwort oil, patchouli oil, and cypress wood oil, respectively.

[0039] S2. Preparation of mosquito repellent essential oil: Citronella oil, peppermint oil, and artemisia oil were combined and blended in a volume ratio of 3:3:1, and stirred at 120 rpm for 2 hours to obtain a compound essential oil with good mosquito repellent effect. Patchouli oil and cypress oil were combined and blended in a volume ratio of 1:3, and stirred at 120 rpm for 2 hours to obtain a compound fixative. The compound fixative was added to the compound essential oil at a mass content of 30%, and stirred at 120 rpm for 2 hours to obtain a mosquito repellent essential oil with good long-lasting mosquito repellent effect.

[0040] S3. Extraction of Bacillus thuringiensis crystal protein: Bacillus thuringiensis (CICC 20559) was inoculated into NB medium and cultured at 30℃ and 200 r / min for 72 h on a shaker. 1 L of fermentation broth was collected by centrifugation. After removing most of the spores through foam separation, the cells were washed with sterile, pre-cooled 0.5 mol / L NaCl solution and deionized water. The washed solid product was dissolved in 150 mL of lysis buffer (50 mmol / L EDTA, 50 mmol / L Na2CO3, 3% mercaptoethanol) and lysed at 4℃ for 6 h. The supernatant was collected by centrifugation. The supernatant was neutralized to pH 5.0 with 4 mol / L sodium acetate-acetic acid solution and precipitated at 4℃ for 4 h. The precipitate was collected by centrifugation, washed with sterile, pre-cooled deionized water, dialyzed to desalt, and then freeze-dried under vacuum to obtain Bacillus thuringiensis crystal protein.

[0041] S4. Nanoparticle Preparation: Bacillus thuringiensis crystal protein was added to water at pH 11 at a mass concentration of 5% to prepare a Bacillus thuringiensis crystal protein solution. Anhydrous ethanol was added to the Bacillus thuringiensis crystal protein solution at a volume ratio of 4:1. Once the solution exhibited a blue opalescent appearance, genipin, a cross-linking agent, was added to bring the genipin concentration in the mixture to 1%. Cross-linking was carried out at 37°C for 36 hours. Ethanol was removed by rotary evaporation to obtain a colloidal solution. The colloidal solution was centrifuged, and the precipitate was washed with pure water and freeze-dried to obtain Bacillus thuringiensis crystal protein nanoparticles.

[0042] S5. Preparation of long-lasting mosquito repellent and mosquito-killing emulsion: Mix mosquito repellent essential oil and water in equal amounts to obtain an oil-water medium. Disperse Bacillus thuringiensis crystal protein nanoparticles in the oil-water medium at a ratio of 1g:10mL. Stir at 250r / min for 2h and then homogenize at 14000r / min for 3min to obtain the long-lasting mosquito repellent and mosquito-killing emulsion.

[0043] Comparative Example 1

[0044] A mosquito repellent and insecticidal emulsion containing insecticidal proteins differs from Example 1 in that it does not contain peppermint oil and artemisia oil.

[0045] Comparative Example 2

[0046] A mosquito repellent and insecticidal emulsion containing insecticidal proteins differs from Example 1 in that it does not contain citronella oil or artemisia oil.

[0047] Comparative Example 3

[0048] A long-lasting mosquito repellent emulsion containing insecticidal proteins, which differs from Example 1 in that it does not contain citronella oil or peppermint oil.

[0049] Comparative Example 4

[0050] A long-lasting mosquito repellent emulsion containing insecticidal proteins differs from Example 1 in that it does not contain patchouli oil or cypress oil.

[0051] Comparative Example 5

[0052] A pure natural long-lasting mosquito repellent essential oil, which differs from Example 1 in that it does not contain Bacillus thuringiensis crystal protein nanoparticles or water.

[0053] Comparative Example 6

[0054] A pure natural mosquito repellent essential oil, which differs from Example 1 in that it does not contain patchouli oil, cypress oil, Bacillus thuringiensis crystal protein nanoparticles, or water.

[0055] Comparative Example 7

[0056] An effective mosquito repellent and insecticidal emulsion containing insecticidal proteins differs from Example 1 in that the Bacillus thuringiensis crystal protein in the emulsion is replaced with albumin.

[0057] The mosquito repellent emulsions prepared in Examples 1 to 3 and Comparative Examples 1 to 7 were used to test mosquito repellency rate, knockdown rate, and effective protection duration. The mosquito repellency efficiency test procedure was as follows: Two mosquito cages were connected by an plexiglass tube, with a movable baffle in the center of the passage. One hundred female Culex pipiens pallens were blown in from the left side, and an appropriate amount of the test sample was placed on the left side. After 60 minutes, the passage was cut off, and the number of mosquitoes and knockdowns in both chambers were observed. The repellency rate and knockdown rate were calculated from this. The effective protection duration test was conducted in accordance with GB / T 139179-2009 "Indoor Efficacy Tests and Evaluation of Pesticide Registration Sanitary Insecticides Part 9: Repellents". The results are shown in Table 1.

[0058] Table 1. Repellency rate, knockdown rate, and effective protection duration of mosquito repellent products.

[0059]

[0060] The data above show that the mosquito repellent products of Examples 1 to 3 all achieved 100% mosquito repellency, with effective protection times of 12.5h, 11h, and 11.5h, respectively, and mosquito knockdown rates of 69%, 58%, and 55%, respectively. The mosquito repellent products of Comparative Examples 1 to 3 showed lower mosquito repellency, knockdown rate, and effective protection time compared to Examples 1 to 3, indicating that the compound essential oils helped improve their mosquito repellency, knockdown rate, and effective protection duration. The effective protection time of the mosquito repellent product of Comparative Example 4 was shorter than that of Examples 1 to 3, indicating that patchouli oil and cypress oil, as fixatives, promoted the improvement of the effective mosquito repellency time. The mosquito repellent product of Comparative Example 5 showed significantly lower mosquito repellency, knockdown rate, and effective protection time than Examples 1 to 3, indicating that the emulsification and encapsulation of Bacillus thuringiensis crystal protein nanoparticles could improve the product's mosquito repellency, knockdown rate, and effective protection time. The results of Comparative Example 6 further confirmed the conclusions of Comparative Examples 4 and 5. The mosquito repellent lotion of Comparative Example 7 showed a significantly lower mosquito knockdown rate compared to Examples 1 to 3, indicating that Bacillus thuringiensis crystal protein helps to improve the mosquito knockdown rate of the mosquito repellent lotion.

[0061] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for preparing a long-lasting mosquito repellent emulsion containing insecticidal proteins, characterized in that, Includes the following steps: 1) Preparation of compound essential oil: Mix lemongrass oil, peppermint oil and artemisia oil evenly in a volume ratio of 1~3:1~3:1 to obtain compound essential oil; 2) Preparation of compound fixative: Patchouli oil and cypress oil are mixed evenly at a volume ratio of 1:1~3 to obtain compound fixative; 3) Preparation of mosquito repellent essential oil: Add the compound fixative to the compound essential oil at a mass content of 10%~30%, mix evenly, and obtain the mosquito repellent essential oil; 4) Extraction of Bacillus thuringiensis crystal protein: Bacillus thuringiensis was inoculated into NB medium and cultured on a shaker. 1 L of fermentation broth was collected by centrifugation. After removing the spores by foam separation, the cells were washed. The washed solid product was dissolved in 150-160 mL of lysis buffer and lysed at 3-4℃ for 6-7 h. The supernatant was collected by centrifugation and neutralized to pH 5.0±0.

1. The supernatant was precipitated at 3-4℃ for 4-4.5 h. The precipitate was collected by centrifugation, washed, dialyzed to desalt, and then freeze-dried under vacuum to obtain Bacillus thuringiensis crystal protein. The Bacillus thuringiensis was purchased from the China Industrial Microbial Culture Collection Center, with the number CICC20559. 5) Preparation of Bacillus thuringiensis crystal protein nanoparticles: Bacillus thuringiensis crystal protein was added to water with a pH of 9-11 at a mass content of 1-5% to prepare a Bacillus thuringiensis crystal protein solution. Anhydrous ethanol was added to the Bacillus thuringiensis crystal protein solution. When the solution showed a blue opalescence, the cross-linking agent genipin was added. The mixture was then cross-linked at 37-38℃ for 12-36 h. The ethanol was removed by rotary evaporation to obtain a colloidal solution. The colloidal solution was centrifuged and the precipitate was washed with water. The precipitate was then freeze-dried to obtain Bacillus thuringiensis crystal protein nanoparticles. The volume ratio of anhydrous ethanol to Bacillus thuringiensis crystal protein solution is 1-4:1, and the mass concentration of genipin in the mixture is 0.5-1%. 6) Preparation of long-lasting mosquito repellent and mosquito-killing emulsion: Mosquito repellent essential oil and water are mixed at a mass ratio of 1:1 to obtain an oil-water medium. Bacillus thuringiensis crystal protein nanoparticles are dispersed in the oil-water medium. The ratio of Bacillus thuringiensis crystal protein nanoparticles to oil-water medium is 1 g: 5~10 mL. The mixture is stirred evenly and then homogenized and emulsified to obtain a long-lasting mosquito repellent and mosquito-killing emulsion. The preparation methods of lemongrass oil, peppermint oil, artemisia oil, patchouli oil, and cypress oil are as follows: Lemongrass, peppermint leaves, artemisia leaves, patchouli leaves, and cypress wood chips are freeze-dried and pulverized respectively. Each plant powder is weighed and placed into an extraction vessel, which is then loaded into a supercritical CO2 extraction device. Extraction is carried out at temperatures of 38~42℃, pressures of 24~26 MPa, time of 80~100 min, and CO2 flow rates of 23~27 L / h, respectively. The mixture is then refrigerated at 4℃ for 12~15 h and filtered to obtain lemongrass oil, peppermint oil, artemisia oil, patchouli oil, and cypress oil, respectively.

2. The method for preparing a long-lasting mosquito repellent emulsion containing insecticidal proteins according to claim 1, characterized in that, In step 1), the mixing is carried out by stirring at a speed of 120~150 r / min for 2~2.5 h.

3. The method for preparing a long-acting mosquito repellent emulsion containing insecticidal proteins according to claim 1, characterized in that, In step 2), the mixing is carried out by stirring at a speed of 120~150 r / min for 2~2.5 h.

4. The method for preparing a long-lasting mosquito repellent emulsion containing insecticidal proteins according to claim 1, characterized in that, In step 3), the mixing is carried out by stirring at a speed of 120~150 r / min for 2~2.5 h.

5. A method for preparing a long-lasting mosquito repellent emulsion containing insecticidal proteins according to claim 1, characterized in that, In step 4), the culture conditions for Bacillus thuringiensis are: 28-30℃, 1180-200 r / min shaker culture for 72-80 h. The components and their final concentrations in the lysis buffer are as follows: 50 mmol / L EDTA, 50 mmol / L Na2CO3, 3% mercaptoethanol. The supernatant is neutralized with a 4 mol / L sodium acetate-acetic acid solution.

6. A method for preparing a long-lasting mosquito repellent emulsion containing insecticidal proteins according to claim 1, characterized in that, In step 6), the stirring is performed at a speed of 250-300 r / min for 2-2.5 h, and the homogenization and emulsification is performed at a speed of 10000-14000 r / min for 1-3 min.

7. A long-lasting mosquito repellent emulsion containing insecticidal proteins, prepared according to any one of claims 1 to 6.

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