Nanodispersion for preventing and treating swine respiratory disease and method for preparing the same

Abstract

The application provides a nanoemulsion for preventing and treating pig respiratory diseases and a preparation method thereof, and belongs to the field of nanoemulsion preparation. The nanoemulsion comprises an external water phase and first, second, third and fourth water-in-oil emulsion cores dispersed in the external water phase; wherein the oil phase of the first and second water-in-oil emulsion cores comprises eucalyptus globulus oil and peppermint oil; the D90 particle size of the internal water phase of the first water-in-oil emulsion core is between 100 nm and 150 nm; the D90 particle size of the internal water phase of the second water-in-oil emulsion core is between 250 nm and 300 nm; the oil phase of the third and fourth water-in-oil emulsion cores comprises carvacrol and thymol; the D90 particle size of the internal water phase of the third water-in-oil emulsion core is 200-300 nm; and the D90 particle size of the internal water phase of the fourth water-in-oil emulsion core is 500-700 nm. The nanoemulsion has long drug efficacy and long-term stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanoemulsion preparation, in particular, to a nanoemulsion for preventing and treating pig respiratory diseases and a preparation method thereof. BACKGROUND

[0002] The large-scale breeding of pigs has become a trend in the contemporary pig industry. Under the large-scale breeding mode, the pig population density is large, and the stress factors are increased, which can easily cause pigs to be sick or even die. Pig respiratory diseases have become one of the common and serious diseases in pig farms. Antibiotics are an important means to cope with pig respiratory diseases, but the widespread use of antibiotics can easily lead to antibiotic residues and drug resistance in pigs.

[0003] In some antibiotic-free pig farms, attempts have been made to use natural substances such as plant essential oils to prevent or treat pig respiratory diseases and reduce the amount of antibiotics used. In pig houses, the main way to use plant essential oils at present is to temporarily extract plant essential oils from plants by means of fumigation or steaming and directly apply them. This application mode has many constraints. In some other attempts, plant essential oils can be prepared into emulsions and applied by spraying or adding to drinking water. However, the embedding rate and stability of these plant essential oil emulsions are poor, and they can only meet the needs of temporary / short-term use. Therefore, there is an urgent need for a long-acting and stable preparation for the prevention and treatment of respiratory diseases in pig breeding.

[0004] It should be noted that the information disclosed in the above background section is only used to strengthen the understanding of the background of the present disclosure, and therefore can include information that does not constitute prior art known to those of ordinary skill in the art. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art. The present application provides a nanoemulsion for preventing and treating pig respiratory diseases and a preparation method thereof, which improves the long-acting property and long-term stability of the nanoemulsion.

[0006] According to a first aspect of the present application, a nanoemulsion for preventing and treating pig respiratory diseases is provided, comprising an external water phase and first, second, third and fourth water-in-oil emulsion cores dispersed in the external water phase.

[0007] The oil phase of the first and second water-in-oil emulsion cores is a mixture of bluegum oil and peppermint oil, and Span 80, and the internal water phase of the first and second water-in-oil emulsion cores is water; the D90 particle size of the internal water phase of the first water-in-oil emulsion core is between 100 nm and 150 nm; and the D90 particle size of the internal water phase of the second water-in-oil emulsion core is between 250 nm and 300 nm.

[0008] The oil phase of the third water-in-oil emulsion core is a mixture of carvacrol, thymol, Span 60, vitamin E and MCT oil, and the inner water phase of the third water-in-oil emulsion core is a sodium alginate aqueous solution; the D90 particle size of the inner water phase of the third water-in-oil emulsion core is 200-300 nm;

[0009] The oil phase of the fourth water-in-oil emulsion core is a mixture of carvacrol, thymol, Span 60, vitamin E, ascorbic acid palmitate and MCT oil, and the inner water phase of the fourth water-in-oil emulsion core is a sodium alginate and hydroxypropyl-β-cyclodextrin aqueous solution; the D90 particle size of the inner water phase of the fourth water-in-oil emulsion core is 500-700 nm;

[0010] The outer water phase is a Tween 80, xanthan gum, sodium caseinate and sodium benzoate aqueous solution.

[0011] According to another aspect of the present application, a preparation method of a nanoemulsion for preventing and treating porcine respiratory disease is provided, comprising:

[0012] Tween 80, xanthan gum, sodium caseinate and sodium benzoate are dissolved in water to form a first aqueous solution;

[0013] A mixture of bluegum oil, peppermint oil and Span 80 is prepared into a first coarse emulsion with water, and then a first water-in-oil emulsion is prepared by high-pressure microfluidic nanoemulsification technology under a pressure of 150-200 MPa; the first water-in-oil emulsion is mixed with part of the first aqueous solution and stirred at a speed of 4000-6000 rpm for 1-3 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsion core, and the D90 particle size of the inner water phase of the first water-in-oil emulsion core is 100-150 nm;

[0014] A mixture of bluegum oil, peppermint oil and Span 80 is prepared into a second coarse emulsion with water, and then a second water-in-oil emulsion is prepared by high-pressure microfluidic nanoemulsification technology under a pressure of 100-130 MPa; the second water-in-oil emulsion is mixed with part of the first aqueous solution and stirred at a speed of 4000-6000 rpm for 1-3 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 250-300 nm;

[0015] Sodium alginate is dissolved in water to form a second aqueous solution;

[0016] Sodium alginate and hydroxypropyl-β-cyclodextrin are dissolved in water to form a third aqueous solution;

[0017] After a mixture of carvacol, thymol, Span 60, vitamin E, ascorbyl palmitate, MCT oil is prepared into a fourth coarse emulsion with the third aqueous solution, a fourth water-in-oil emulsion is prepared by high-pressure microfluidic nano-emulsification technology under a pressure of 50MPa-70MPa; after the fourth water-in-oil emulsion is mixed with part of the first aqueous solution, stirring is carried out at a speed of 4000rpm-6000rpm for 1-3 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsification core, and the D90 particle size of the inner aqueous phase of the fourth water-in-oil emulsification core is between 500nm-700nm;

[0018] After a mixture of carvacol, thymol, Span 60, vitamin E, ascorbyl palmitate, MCT oil is prepared into a fourth coarse emulsion with the third aqueous solution, a fourth water-in-oil emulsion is prepared by high-pressure microfluidic nano-emulsification technology under a pressure of 50MPa-70MPa; after the fourth water-in-oil emulsion is mixed with part of the first aqueous solution, stirring is carried out at a speed of 4000rpm-6000rpm for 1-3 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsification core, and the D90 particle size of the inner aqueous phase of the fourth water-in-oil emulsification core is between 500nm-700nm;

[0019] After the first water-in-oil-in-water emulsion, the second water-in-oil-in-water emulsion, the third water-in-oil-in-water emulsion, and the fourth water-in-oil-in-water emulsion are all cooled to 10℃-15℃, they are mixed and stirred at a speed of 2000rpm-3000rpm for 1-3 minutes to form the nanoemulsion.

[0020] The present application disperses eucalyptus oil and mint oil in one oil phase, and disperses carvacol and thymol in another oil phase. In this way, the prepared emulsion has high stability. The particle size of the eucalyptus oil / mint oil emulsified core is divided into two levels, the particle size of the first water-in-oil emulsified core is smaller and the volatilization speed is faster, and the particle size of the second water-in-oil emulsified core is larger and the volatilization speed is slower. When spraying, the first water-in-oil emulsified core volatilizes quickly to quickly increase the concentration of eucalyptus oil / mint oil in the pig house, achieving the effect of quickly repelling mosquitoes. The second water-in-oil emulsified core has a certain slow-release effect, so that the concentration of eucalyptus oil / mint oil in the pig house can be maintained for a relatively long time, achieving the purpose of prolonging the effective time of insect repelling, which is beneficial to reducing the spraying frequency. At the same time, the total particle size of the first water-in-oil emulsified core and the second water-in-oil emulsified core is nanoscale, and the overall volatilization speed is relatively fast, which is easy to form a local high concentration to kill in the dead angle of spraying, and improve the effectiveness and comprehensiveness of bacteriostatic killing. The particle size of the carvacol / thymol emulsified core is slightly larger, the release is slightly later and slower, which can achieve relay in insect repelling effect, and further achieve the purpose of long-acting insect repelling; at the same time, the release speed is slightly slower, which can achieve long-acting killing effect. The particle size of the carvacol / thymol emulsified core (i.e. the third water-in-oil emulsified core and the fourth water-in-oil emulsified core) is divided into two levels, achieving the effect of controlled release, and further improving the effect of long-acting killing and long-acting insect repelling. The particle size of the eucalyptus oil / mint oil emulsified core is divided into two levels, and the particle size of the carvacol / thymol emulsified core is divided into two levels, which not only realizes the slow release of components to prolong the effect, but also improves the stability of the nanoemulsion.

[0021] In addition, vitamin E and ascorbyl palmitate can be added to the oil phase of the large particle size carvacol / thymol emulsified core to improve the antioxidant property, and the vitamin E and ascorbyl palmitate in the oil phase have a certain synergistic effect on the antioxidant effect.

[0022] It should be understood that the foregoing general description and the following detailed description are only exemplary and explanatory, and cannot limit the present disclosure. DETAILED DESCRIPTION

[0023] The present application provides a nanoemulsion for preventing and treating pig respiratory diseases, comprising an external water phase and a first water-in-oil emulsified core, a second water-in-oil emulsified core, a third water-in-oil emulsified core and a fourth water-in-oil emulsified core dispersed in the external water phase.

[0024] The oil phase of the first water-in-oil emulsion core and the second water-in-oil emulsion core is a mixture of eucalyptus oil and peppermint oil, and Span 80, and the internal water phase of the first water-in-oil emulsion core and the second water-in-oil emulsion core is water; the D90 particle size (the particle size value corresponding to 90% in the cumulative particle size distribution curve) of the internal water phase of the first water-in-oil emulsion core is between 100 nm and 150 nm; and the D90 particle size of the internal water phase of the second water-in-oil emulsion core is between 250 nm and 300 nm.

[0025] The oil phase of the third water-in-oil emulsion core is a mixture of carvacrol, thymol, Span 60, vitamin E and MCT oil, and the internal water phase of the third water-in-oil emulsion core is a sodium alginate aqueous solution; and the D90 particle size of the internal water phase of the third water-in-oil emulsion core is 200-300 nm.

[0026] The oil phase of the fourth water-in-oil emulsion core is a mixture of carvacrol, thymol, Span 60, vitamin E, ascorbyl palmitate and MCT oil, and the internal water phase of the fourth water-in-oil emulsion core is a sodium alginate and hydroxypropyl-β-cyclodextrin aqueous solution; and the D90 particle size of the internal water phase of the fourth water-in-oil emulsion core is 500-700 nm.

[0027] The external water phase is a water solution of Tween 80, xanthan gum, sodium caseinate and sodium benzoate.

[0028] The nanoemulsion provided by the present application contains four different plant essential oils, namely eucalyptus oil, peppermint oil, carvacrol and thymol. The eucalyptus oil is a volatile essential oil extracted from the leaves of eucalyptus trees, which not only has the effect of repelling insects (such as mosquitoes, ants, etc.), but also has a certain inhibitory effect on some viruses and bacteria. Furthermore, the eucalyptus oil has a certain relieving effect on respiratory symptoms (such as nasal congestion, cough, etc.). The nanoemulsion of the present application can be administered by spraying, and the volatiles of eucalyptus oil / peppermint oil can effectively repel insects in the pig house, reducing the risk of disease transmission by insects. At the same time, when the eucalyptus oil / peppermint oil adheres to the facilities of the pig house or the skin of the pig, it can produce a certain inhibitory and killing effect on the bacteria and viruses on the pig house or the skin. In this way, the nanoemulsion helps to prevent the occurrence of respiratory diseases in pigs, and alleviates the respiratory symptoms when respiratory diseases occur in pigs. When the pig respiratory disease breaks out or the symptoms are more serious, the nanoemulsion can also be directly added to the drinking water. Carvacrol and thymol are both relatively natural monoterpenoid phenolic antibacterial compounds, and their antibacterial spectrum has a certain complementarity. Furthermore, both of them can also alleviate the inflammation caused by infection. The nanoemulsion of the present application can provide more powerful and more sustained bacteriostatic and bactericidal effects by adding carvacrol and thymol, and can more effectively prevent and treat pig respiratory diseases.

[0029] In the preliminary exploration of the present application, eucalyptus oil, peppermint oil, carvacol and thymol were mixed to prepare an emulsion. However, no matter how the emulsifier and the water phase were adjusted, the emulsion prepared by these preliminary explorations had serious stability problems, it was difficult to achieve full emulsification, and oil and water separation easily occurred. Moreover, the prepared emulsion also had the problem of rapid discoloration, and it was possible that carvacol and thymol and other substances were rapidly oxidized.

[0030] To solve this problem, in further exploration, the eucalyptus oil and the peppermint oil were dispersed in one oil phase, and the carvacol and the thymol were dispersed in another oil phase. In this way, the stability of the prepared emulsion was improved. However, in further tests, it was found that the effective time of mosquito repellent and the duration of antibacterial effect needed to be improved, otherwise the problem of high frequency of application would occur.

[0031] In further exploration, the technical solution proposed in the present application was formed. In the present application, the particle size of the eucalyptus oil / peppermint oil emulsion core (the first water-in-oil emulsion core and the second water-in-oil emulsion core) is divided into two levels, the particle size of the first water-in-oil emulsion core is smaller and the volatilization speed is faster, and the particle size of the second water-in-oil emulsion core is larger and the volatilization speed is slower. When spraying, the first water-in-oil emulsion core volatilizes quickly to quickly increase the concentration of eucalyptus oil / peppermint oil in the pig house, achieving the effect of quickly repelling mosquitoes. The second water-in-oil emulsion core has a certain slow-release effect, so that the concentration of eucalyptus oil / peppermint oil in the pig house can be maintained for a relatively long time, achieving the purpose of prolonging the effective time of mosquito repellent, which is beneficial to reducing the spraying frequency. At the same time, the overall particle size of the first water-in-oil emulsion core and the second water-in-oil emulsion core is nanoscale, the overall volatilization speed is relatively fast, it is easy to form a local high concentration to kill in the dead angle of spraying, and improve the onset time and comprehensiveness of bacteriostatic and killing. In the present application, the particle size of the carvacol / thymol emulsion core is slightly larger, the release is slightly later and slower, which can achieve relay in mosquito repellent effect, and thus achieve the purpose of long-acting mosquito repellent; at the same time, the release speed is slightly slower, which can achieve the effect of long-acting killing. The particle size of the carvacol / thymol emulsion core (i.e. the third water-in-oil emulsion core and the fourth water-in-oil emulsion core) is divided into two levels, achieving the effect of controlled release, and further improving the effects of long-acting killing and long-acting mosquito repellent. It is more unexpected that the particle size of the eucalyptus oil / peppermint oil emulsion core is divided into two levels, and the particle size of the carvacol / thymol emulsion core is divided into two levels, which not only achieves the slow release of components to prolong the effect, but also improves the stability of the nano-emulsion. This may be because the small-particle-size emulsion core is embedded in the gap between the large-particle-size emulsion cores, reducing the migration of each emulsion core and thus reducing the ripening process.

[0032] In addition, the present application found in the study that the large particle size carvacol / thymol emulsion core oil phase can improve the antioxidant property by adding vitamin E and ascorbic acid palmitate, and simply increasing the amount of vitamin E without adding ascorbic acid palmitate can cause limited improvement in the antioxidant effect, and simply using ascorbic acid palmitate also has a general antioxidant effect. Therefore, it is speculated that vitamin E and ascorbic acid palmitate in the large particle size carvacol / thymol emulsion core oil phase have a certain synergistic effect on the antioxidant effect.

[0033] In addition, the present application found in the study that the large particle size carvacol / thymol emulsion core oil phase can improve the antioxidant property by adding vitamin E and ascorbic acid palmitate, and simply increasing the amount of vitamin E without adding ascorbic acid palmitate can cause limited improvement in the antioxidant effect, and simply using ascorbic acid palmitate also has a general antioxidant effect. Therefore, it is speculated that vitamin E and ascorbic acid palmitate in the large particle size carvacol / thymol emulsion core oil phase have a certain synergistic effect on the antioxidant effect.

[0034] In an embodiment of the present application, the mass ratio of the first water-in-oil emulsion core to the second water-in-oil emulsion core is in the range of 7:3 to 3:7; and the mass ratio of the third water-in-oil emulsion core to the fourth water-in-oil emulsion core is in the range of 7:3 to 3:7.

[0035] In an example, the mass ratio of the first water-in-oil emulsion core to the second water-in-oil emulsion core is 2:1; and the mass ratio of the third water-in-oil emulsion core to the fourth water-in-oil emulsion core is 6:5. In this way, a balance can be achieved between long-acting mosquito repellent, long-acting antibacterial, and ensuring long-term stability of the emulsion.

[0036] In an example, the mass of the eucalyptus oil in the first water-in-oil emulsion core is 2.8% to 4.5% of the total mass of the nanoemulsion. The mass of the eucalyptus oil in the second water-in-oil emulsion core is 2.5% to 4.1% of the total mass of the nanoemulsion. The mass of the peppermint oil in the first water-in-oil emulsion core is 1.2% to 2.1% of the total mass of the nanoemulsion. The mass of the peppermint oil in the second water-in-oil emulsion core is 0.9% to 1.8% of the total mass of the nanoemulsion.

[0037] In an example, the mass of the carvacol in the third water-in-oil emulsion core is 1.5% to 2.1% of the total mass of the nanoemulsion. The mass of the thymol in the third water-in-oil emulsion core is 1.5% to 2.1% of the total mass of the nanoemulsion. The mass of the carvacol in the fourth water-in-oil emulsion core is 0.9% to 1.8% of the total mass of the nanoemulsion. The mass of the thymol in the fourth water-in-oil emulsion core is 0.9% to 1.8% of the total mass of the nanoemulsion.

[0038] In an example, the D90 particle size of the inner water phase of the first water-in-oil emulsion core is between 115 nm and 125 nm; the D90 particle size of the inner water phase of the second water-in-oil emulsion core is between 280 nm and 290 nm; the D90 particle size of the inner water phase of the third water-in-oil emulsion core is between 250 nm and 260 nm; and the D90 particle size of the inner water phase of the fourth water-in-oil emulsion core is between 500 nm and 550 nm.

[0039] In an example, in the third water-in-oil emulsion core, the mass of vitamin E is 3% to 7% of the total mass of carvacol and thymol; and in the fourth water-in-oil emulsion core, the mass of vitamin E is 3% to 7% of the total mass of carvacol and thymol, and the mass of ascorbyl palmitate is 3% to 5% of the total mass of carvacol and thymol. In this way, the oxidation of carvacol and thymol can be reduced, and the bacteriostatic duration can be verified.

[0040] In an example, in the fourth water-in-oil emulsion core, the mass content of hydroxypropyl-β-cyclodextrin in the inner water phase is 0.1% to 0.3%. This helps to improve the bacteriostatic duration of the crosslinked emulsion.

[0041] In an embodiment of the present application, in the nanoemulsion, the mass content of the bluegum oil is 6.3% to 7.5%, the mass content of the peppermint oil is 2.7% to 3.5%, the mass content of the carvacol is 2.5% to 3.6%, and the mass content of the thymol is 2.5% to 3.6%.

[0042] In an example, the mass of the bluegum oil in the first water-in-oil emulsion core is 4.5% of the total mass of the nanoemulsion. The mass of the bluegum oil in the second water-in-oil emulsion core is 2.5% of the total mass of the nanoemulsion. The mass of the peppermint oil in the first water-in-oil emulsion core is 2.0% of the total mass of the nanoemulsion. The mass of the peppermint oil in the second water-in-oil emulsion core is 1.0% of the total mass of the nanoemulsion. The mass of the carvacol in the third water-in-oil emulsion core is 1.8% of the total mass of the nanoemulsion. The mass of the thymol in the third water-in-oil emulsion core is 1.8% of the total mass of the nanoemulsion. The mass of the carvacol in the fourth water-in-oil emulsion core is 1.2% of the total mass of the nanoemulsion. The mass of the thymol in the fourth water-in-oil emulsion core is 1.2% of the total mass of the nanoemulsion. In an example, the D90 particle size of the inner water phase of the first water-in-oil emulsion core is 120 nm; the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm; the D90 particle size of the inner water phase of the third water-in-oil emulsion core is 255 nm; and the D90 particle size of the inner water phase of the fourth water-in-oil emulsion core is 520 nm.

[0043] The present application also provides a method for preventing and treating respiratory diseases of pigs by using the nanoemulsion. The nanoemulsion can be diluted with water and sprayed, or added to the drinking water of pigs. For example, when prevention is performed in the high incidence season, the nanoemulsion can be diluted 200 times with water and sprayed in the environment, or added to the drinking water (diluted 3000-4000 times). When the pigs have early symptoms, the nanoemulsion can be added to the drinking water (diluted 3000-4000 times). Of course, if the pigs have more symptoms, the nanoemulsion can also be used as an auxiliary treatment, for example, combined with antibiotics (for example, added to the drinking water, diluted 3000 times).

[0044] The present application also provides a method for preparing the nanoemulsion, comprising:

[0045] Dissolving Tween 80, xanthan gum, sodium caseinate, and sodium benzoate in water to form a first aqueous solution;

[0046] Preparing a first coarse emulsion by mixing the mixture of Eucalyptus globulus oil, peppermint oil, and Span 80 with water, and then preparing a first water-in-oil emulsion by high-pressure microfluidic nanomixing technology under a pressure of 150-200 MPa; mixing the first water-in-oil emulsion with part of the first aqueous solution and stirring at a speed of 4000-6000 rpm for 1-3 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsion core, and the D90 particle size of the inner water phase of the first water-in-oil emulsion core is between 100 nm and 150 nm;

[0047] Preparing a second coarse emulsion by mixing the mixture of Eucalyptus globulus oil, peppermint oil, and Span 80 with water, and then preparing a second water-in-oil emulsion by high-pressure microfluidic nanomixing technology under a pressure of 100-130 MPa; mixing the second water-in-oil emulsion with part of the first aqueous solution and stirring at a speed of 4000-6000 rpm for 1-3 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is between 250 nm and 300 nm;

[0048] Dissolving sodium alginate in water to form a second aqueous solution;

[0049] Dissolving sodium alginate and hydroxypropyl-β-cyclodextrin in water to form a third aqueous solution;

[0050] The mixture of carvacol, thymol, Span 60, vitamin E, MCT oil is prepared into a third coarse emulsion with the second aqueous solution, and then a third water-in-oil emulsion is prepared by high-pressure microfluidic nano-emulsification technology under a pressure of 90 MPa-130 MPa; the third water-in-oil emulsion is mixed with part of the first aqueous solution and stirred at a speed of 4000 rpm-6000 rpm for 1-3 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsification core, and the D90 particle size of the inner water phase of the third water-in-oil emulsification core is between 200 nm and 300 nm;

[0051] The mixture of carvacol, thymol, Span 60, vitamin E, ascorbic acid palmitate, MCT oil is prepared into a fourth coarse emulsion with the third aqueous solution, and then a fourth water-in-oil emulsion is prepared by high-pressure microfluidic nano-emulsification technology under a pressure of 50 MPa-70 MPa; the fourth water-in-oil emulsion is mixed with part of the first aqueous solution and stirred at a speed of 4000 rpm-6000 rpm for 1-3 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsification core, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsification core is between 500 nm and 700 nm;

[0052] The first water-in-oil-in-water emulsion, the second water-in-oil-in-water emulsion, the third water-in-oil-in-water emulsion, and the fourth water-in-oil-in-water emulsion are all cooled to 10-15°C, mixed, and stirred at a speed of 2000 rpm-3000 rpm for 1-3 minutes to form the nanoemulsion.

[0053] The preparation method and effects of the nanoemulsion provided by the present application are introduced as follows, taking some examples / experimental examples in the research and development stage of the present application as examples.

[0054] In the preparation process of the nanoemulsion in the embodiments of the present application, unless otherwise specified, the first aqueous solution, the second aqueous solution, and the third aqueous solution are prepared as follows. Tween 80 (4 g), xanthan gum (0.5 g), sodium caseinate (1 g), and sodium benzoate (0.2 g) are dissolved in water (200 g) to form the first aqueous solution. Sodium alginate (5 g) is dissolved in water (200 g) to form the second aqueous solution. Sodium alginate (5 g) and hydroxypropyl-β-cyclodextrin (0.5 g) are dissolved in water (200 g) to form the third aqueous solution.

[0055] In the preparation of the nanoemulsion of the embodiments of the present application, unless otherwise specified, the following method is used: four water-in-oil-in-water emulsions (first water-in-oil-in-water emulsion, second water-in-oil-in-water emulsion, third water-in-oil-in-water emulsion, and fourth water-in-oil-in-water emulsion) are prepared, and then the four water-in-oil-in-water emulsions are mixed to form the nanoemulsion. The mixing conditions are that the four emulsions are cooled to 10-15°C, mixed, and stirred at a speed of 2500 rpm for 2 minutes to form the nanoemulsion.

[0056] The following method is used to evaluate the mosquito repellent duration of the nanoemulsion of the present application.

[0057] A net cover is obtained, which has five side cloths (polyester mosquito net cloth, non-cotton cloth), each with a size of 50 cm x 50 cm. The opening of the net cover is used to be buckled on the test table. One of the side cloths is detachable, which is the side cloth for drug application. The side cloth for drug application is detached. The nanoemulsion is diluted 200 times with water, and then sprayed on the side cloth for drug application by spraying, with the spraying direction being directly towards the side cloth for drug application. The spraying head is 30 cm away from the cloth when spraying each time. After the drug application cloth is installed back to the net cover, the net cover is inverted and placed into 20 Culex quinquefasciatus, and the time is recorded as zero. Subsequently, every ten minutes, each non-drug application cloth of the net cover is shaken to make the mosquitoes fly. The mosquito staying on the drug application cloth is observed, and when the mosquito stays on the drug application cloth for more than ten seconds, it is recorded as one mosquito staying. The time when the fourth mosquito staying occurs is recorded as the mosquito repellent duration of the nanoemulsion.

[0058] The present application adopts the following method to detect the bacteriostatic / sterilization ability of nanoemulsion. The Actinobacillus pleuropneumoniae bacterial solution is mixed with nutrient solution (containing agar solution) uniformly, then diluted 3 times to form a low viscosity and nutrient bacterial solution. The diluted bacterial solution is uniformly sprayed on multiple culture dishes by spraying. Then the culture dishes are allowed to stand at room temperature for 15 minutes to volatilize the sprayed bacterial solution. At this time, multiple culture dishes are taken for bacterial culture, and the number of colonies is recorded after culture; the average number of colonies of the multiple culture dishes at this time is the bacterial number reference value N0. At this time, the remaining multiple culture dishes are sprayed with the nanoemulsion by spraying. The specific method of spraying is: the nanoemulsion is diluted 200 times with water, then sprayed at the back of the culture dish. The spraying direction is horizontal and towards the culture dish, the height of the spraying head is 40 cm from the height of the culture dish, and the distance between the spraying head and the culture dish in the horizontal direction is 30 cm. Spray twice. In this way, the process of the natural falling of the drug droplets to the surface of the pig house facility under the scene of spraying of the nanoemulsion is simulated. After 6 hours of spraying, part of the culture dishes are cultured for bacteria and the number of colonies N6 formed is recorded, and the 6-hour bacterial growth rate of the nanoemulsion is (N6-N0) / N0. After 20 hours of spraying, the remaining culture dishes are cultured for bacteria and the number of colonies N20 formed is recorded, and the 20-hour bacterial growth rate of the nanoemulsion is (N20-N0) / N0.

[0059] The present application adopts the following method to test the stability of nanoemulsion. The nanoemulsion is divided into multiple test tubes and sealed, and stored at 45°C for constant temperature preservation. In the first ten days, observation is carried out once a day, and oil separation rating is carried out according to the observation results; after ten days, observation is carried out once every two days, and oil separation rating is carried out according to the observation results. When oil droplets appear but no oil film appears, it is defined as preliminary oil separation. When an oil film appears but the thickness of the oil film is not more than 0.3 mm, it is defined as moderate oil separation. When an oil film appears and the thickness of the oil film exceeds 0.3 mm, it is defined as severe oil separation. Record the preliminary oil separation time, moderate oil separation time and severe oil separation time. At most, monitor for fifty days.

[0060] Example 1

[0061] After a mixture of blue eucalyptus oil (7 g), peppermint oil (3 g) and Span 80 (1 g) is prepared into a first coarse emulsion with water (3 g), a first water-in-oil emulsion is prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 170 MPa; after the first water-in-oil emulsion is mixed with a first aqueous solution (36 g), stirring is carried out at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsification core, and the D90 particle size of the inner water phase of the first water-in-oil emulsification core is 120 nm.

[0062] A mixture of eucalyptus oil (7 g), peppermint oil (3 g), Span 80 (1 g) was prepared into a second coarse emulsion with water (3 g), and a second water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (36 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm.

[0063] A mixture of carvacrol (3 g), thymol (3 g), Span 60 (0.9 g), vitamin E (0.3 g), MCT oil (3 g) was prepared into a third coarse emulsion with the second aqueous solution (2.7 g), and a third water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 105 MPa; the third water-in-oil emulsion was mixed with the first aqueous solution (37.1 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsion core, and the D90 particle size of the inner water phase of the third water-in-oil emulsion core is 255 nm.

[0064] A mixture of carvacrol (3 g), thymol (3 g), Span 60 (0.9 g), vitamin E (0.3 g), ascorbyl palmitate (0.24 g), MCT oil (3 g) was prepared into a fourth coarse emulsion with the third aqueous solution (3.6 g), and a fourth water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (35.96 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsion core, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsion core is 520 nm.

[0065] The four water-in-oil-in-water emulsions were mixed to form a nanoemulsion 1.

[0066] The zeta potential of the nanoemulsion 1 was -38 V; the mosquito repellent duration was 6.2 h; the 6-hour bacterial colony growth rate was -75%; the 20-hour bacterial colony growth rate was 275%; the preliminary oil separation time was 12 days; the medium oil separation time was 36 days; no severe oil separation was observed.

[0067] Example 2

[0068] A mixture of eucalyptus oil (5.6 g), peppermint oil (2.4 g), Span 80 (0.8 g) and water (2.4 g) was prepared into a first coarse emulsion, and then a first water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with a first aqueous solution (28.8 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsion core, and the D90 particle size of the inner water phase of the first water-in-oil emulsion core is 120 nm.

[0069] A mixture of eucalyptus oil (8.4 g), peppermint oil (3.6 g), Span 80 (1.2 g) and water (3.6 g) was prepared into a second coarse emulsion, and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with a first aqueous solution (43.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm.

[0070] A mixture of carvacrol (3 g), thymol (3 g), Span 60 (0.9 g), vitamin E (0.3 g), MCT oil (3 g) and a second aqueous solution (2.7 g) was prepared into a third coarse emulsion, and then a third water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 105 MPa; the third water-in-oil emulsion was mixed with a first aqueous solution (37.1 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsion core, and the D90 particle size of the inner water phase of the third water-in-oil emulsion core is 255 nm.

[0071] A mixture of carvacrol (3 g), thymol (3 g), Span 60 (0.9 g), vitamin E (0.3 g), ascorbyl palmitate (0.24 g), MCT oil (3 g) and a third aqueous solution (3.6 g) was prepared into a fourth coarse emulsion, and then a fourth water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with a first aqueous solution (35.96 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsion core, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsion core is 520 nm.

[0072] The four water-in-oil-in-water emulsions were mixed at a low temperature to form a nanoemulsion 2.

[0073] The zeta potential of the nanoemulsion 2 was measured to be -39 V; the repellent duration was 5.1 h; the 6-hour colony growth rate was -73%; the 20-hour colony growth rate was 302%; the preliminary creaming time was 12 days; the moderate creaming time was 38 days; and no severe creaming was observed.

[0074] Example 3

[0075] After a mixture of eucalyptus globulus oil (4.2 g), peppermint oil (1.8 g), and Span 80 (0.6 g) was prepared into a first coarse emulsion with water (1.8 g), a first water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 170 MPa; after the first water-in-oil emulsion was mixed with the first aqueous solution (21.6 g), stirring was performed at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsification core, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsification core is 120 nm.

[0076] After a mixture of eucalyptus globulus oil (9.8 g), peppermint oil (4.2 g), and Span 80 (1.4 g) was prepared into a second coarse emulsion with water (4.2 g), a second water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 120 MPa; after the second water-in-oil emulsion was mixed with the first aqueous solution (50.4 g), stirring was performed at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsification core, and the D90 particle size of the inner aqueous phase of the second water-in-oil emulsification core is 285 nm.

[0077] After a mixture of carvacol (3 g), thymol (3 g), Span 60 (0.9 g), vitamin E (0.3 g), and MCT oil (3 g) was prepared into a third coarse emulsion with the second aqueous solution (2.7 g), a third water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 105 MPa; after the third water-in-oil emulsion was mixed with the first aqueous solution (37.1 g), stirring was performed at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsification core, and the D90 particle size of the inner aqueous phase of the third water-in-oil emulsification core is 255 nm.

[0078] A mixture of eucalyptol (3 g), thymol (3 g), Span 60 (0.9 g), vitamin E (0.3 g), ascorbyl palmitate (0.24 g), MCT oil (3 g) and the third aqueous solution (3.6 g) was prepared into a fourth coarse emulsion, and then a fourth water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (35.96 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the fourth water-in-oil emulsified core is 520 nm.

[0079] The four water-in-oil-in-water emulsions were mixed and cooled to form nanoemulsion 3.

[0080] The zeta potential of nanoemulsion 3 was -38 V; the repellent duration was 3.8 h; the 6-hour colony growth rate was -73%; the 20-hour colony growth rate was 280%; the preliminary oil separation time was 10 days; the moderate oil separation time was 32 days; and no severe oil separation was observed.

[0081] Example 4

[0082] A mixture of eucalyptus oil (8.4 g), peppermint oil (3.6 g), and Span 80 (1.2 g) and water (3.6 g) was prepared into a first coarse emulsion, and then a first water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with the first aqueous solution (43.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsified core is 120 nm.

[0083] A mixture of eucalyptus oil (5.6 g), peppermint oil (2.4 g), and Span 80 (0.8 g) and water (2.4 g) was prepared into a second coarse emulsion, and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (28.8 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the second water-in-oil emulsified core is 285 nm.

[0084] A mixture of carvacrol (3 g), thymol (3 g), Span 60 (0.9 g), vitamin E (0.3 g), MCT oil (3 g) and the second aqueous solution (2.7 g) was prepared into a third coarse emulsion, and then a third water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 105 MPa; the third water-in-oil emulsion was mixed with the first aqueous solution (37.1 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsion nucleus, and the D90 particle size of the inner water phase of the third water-in-oil emulsion nucleus is 255 nm.

[0085] A mixture of carvacrol (3 g), thymol (3 g), Span 60 (0.9 g), vitamin E (0.3 g), ascorbic acid palmitate (0.24 g), MCT oil (3 g) and the third aqueous solution (3.6 g) was prepared into a fourth coarse emulsion, and then a fourth water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (35.96 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsion nucleus, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsion nucleus is 520 nm.

[0086] The four water-in-oil-in-water emulsions were mixed and cooled to form nanoemulsion 4.

[0087] The zeta potential of nanoemulsion 4 was measured to be -37 V; the repellent duration was 8.9 h; the 6-hour colony growth rate was -75%; the 20-hour colony growth rate was 295%; the preliminary oil separation time was 12 days; the medium oil separation time was 36 days; no severe oil separation was observed.

[0088] Example 5

[0089] A mixture of blue eucalyptus oil (9.8 g), peppermint oil (4.2 g), Span 80 (1.4 g) and water (4.2 g) was prepared into a first coarse emulsion, and then a first water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with the first aqueous solution (50.4 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsion nucleus, and the D90 particle size of the inner water phase of the first water-in-oil emulsion nucleus is 120 nm.

[0090] A mixture of eucalyptus globulus oil (4.2 g), peppermint oil (1.8 g), Span 80 (0.6 g) and water (1.8 g) was prepared into a second coarse emulsion, and then a second water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (21.6 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsified core, and the D90 particle size of the inner water phase of the second water-in-oil emulsified core is 285 nm.

[0091] A mixture of carvacrol (3 g), thymol (3 g), Span 60 (0.9 g), vitamin E (0.3 g), MCT oil (3 g) and the second aqueous solution (2.7 g) was prepared into a third coarse emulsion, and then a third water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 105 MPa; the third water-in-oil emulsion was mixed with the first aqueous solution (37.1 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsified core, and the D90 particle size of the inner water phase of the third water-in-oil emulsified core is 255 nm.

[0092] A mixture of carvacrol (3 g), thymol (3 g), Span 60 (0.9 g), vitamin E (0.3 g), ascorbyl palmitate (0.24 g), MCT oil (3 g) and the third aqueous solution (3.6 g) was prepared into a fourth coarse emulsion, and then a fourth water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (35.96 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsified core, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsified core is 520 nm.

[0093] The four water-in-oil-in-water emulsions were mixed and cooled to form nanoemulsion 5.

[0094] The zeta potential of nanoemulsion 5 was measured to be -38 V; the repellent duration was 9.5 h; the 6-hour colony growth rate was -69%; the 20-hour colony growth rate was 315%; the preliminary oil separation time was 14 days; the medium oil separation time was 42 days; no severe oil separation was observed.

[0095] Example 6

[0096] A mixture of eucalyptus oil (5.6 g), peppermint oil (2.4 g), Span 80 (0.8 g) and water (2.4 g) was prepared into a second coarse emulsion, and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (28.8 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm.

[0097] A mixture of eucalyptus oil (5.6 g), peppermint oil (2.4 g), Span 80 (0.8 g) and water (2.4 g) was prepared into a second coarse emulsion, and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (28.8 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm.

[0098] A mixture of eucalyptus oil (5.6 g), peppermint oil (2.4 g), Span 80 (0.8 g) and water (2.4 g) was prepared into a second coarse emulsion, and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (28.8 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm.

[0099] A mixture of eucalyptus oil (5.6 g), peppermint oil (2.4 g), Span 80 (0.8 g) and water (2.4 g) was prepared into a second coarse emulsion, and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (28.8 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm.

[0100] The four water-in-oil-in-water emulsions were mixed and cooled to form nanoemulsion 6.

[0101] The zeta potential of the nanoemulsion 6 was measured to be -37 V; the repellent duration was 9.1 h; the 6-hour colony growth rate was -65%; the 20-hour colony growth rate was 105%; the preliminary creaming time was 12 days; the moderate creaming time was 38 days; no severe creaming was observed.

[0102] Example 7

[0103] After a mixture of eucalyptus globulus oil (8.4 g), peppermint oil (3.6 g), and Span 80 (1.2 g) was prepared into a first coarse emulsion with water (3.6 g), a first water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 170 MPa; after the first water-in-oil emulsion was mixed with the first aqueous solution (43.2 g), stirring was performed at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsification core, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsification core is 120 nm.

[0104] After a mixture of eucalyptus globulus oil (5.6 g), peppermint oil (2.4 g), and Span 80 (0.8 g) was prepared into a second coarse emulsion with water (2.4 g), a second water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 120 MPa; after the second water-in-oil emulsion was mixed with the first aqueous solution (28.8 g), stirring was performed at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsification core, and the D90 particle size of the inner aqueous phase of the second water-in-oil emulsification core is 285 nm.

[0105] After a mixture of carvacol (1.8 g), thymol (1.8 g), Span 60 (0.54 g), vitamin E (0.18 g), and MCT oil (1.8 g) was prepared into a third coarse emulsion with the second aqueous solution (1.62 g), a third water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 105 MPa; after the third water-in-oil emulsion was mixed with the first aqueous solution (22.26 g), stirring was performed at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsification core, and the D90 particle size of the inner aqueous phase of the third water-in-oil emulsification core is 255 nm.

[0106] A mixture of eucalyptol (4.2 g), thymol (4.2 g), Span 60 (1.26 g), vitamin E (0.42 g), ascorbyl palmitate (0.336 g), MCT oil (4.2 g) and the third aqueous solution (5.04 g) were prepared into a fourth coarse emulsion, and then a fourth water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (50.344 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the fourth water-in-oil emulsified core is 520 nm.

[0107] The four water-in-oil-in-water emulsions were mixed and cooled to form nanoemulsion 7.

[0108] The zeta potential of nanoemulsion 7 was -38 V; the repellent duration was 8.9 h; the 6-hour bacterial colony growth rate was -53%; the 20-hour bacterial colony growth rate was 79%; the preliminary oil separation time was 9 days; the medium oil separation time was 26 days; and no severe oil separation was observed.

[0109] Example 8

[0110] A mixture of eucalyptus oil (8.4 g), peppermint oil (3.6 g), and Span 80 (1.2 g) was prepared into a first coarse emulsion with water (3.6 g), and then a first water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with the first aqueous solution (43.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsified core is 120 nm.

[0111] A mixture of eucalyptus oil (5.6 g), peppermint oil (2.4 g), and Span 80 (0.8 g) was prepared into a second coarse emulsion with water (2.4 g), and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (28.8 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the second water-in-oil emulsified core is 285 nm.

[0112] A mixture of carvacrol (3.6g), thymol (3.6g), Span 60 (1.08g), Vitamin E (0.36g), MCT oil (3.6g) and the second aqueous solution (3.24g) were combined to form a third coarse emulsion, which was then subjected to high pressure microfluidic nanoemulsification at 105 MPa to form a third water-in-oil emulsion; the third water-in-oil emulsion was combined with the first aqueous solution (44.52g) and stirred at 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion had a third water-in-oil emulsion nucleus, and the D90 particle size of the inner aqueous phase of the third water-in-oil emulsion nucleus was 255 nm.

[0113] A mixture of carvacrol (2.4g), thymol (2.4g), Span 60 (0.72g), Vitamin E (0.24g), ascorbyl palmitate (0.192g), MCT oil (2.4g) and the third aqueous solution (2.88g) were combined to form a fourth coarse emulsion, which was then subjected to high pressure microfluidic nanoemulsification at 50 MPa to form a fourth water-in-oil emulsion; the fourth water-in-oil emulsion was combined with the first aqueous solution (28.768g) and stirred at 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion had a fourth water-in-oil emulsion nucleus, and the D90 particle size of the inner aqueous phase of the fourth water-in-oil emulsion nucleus was 520 nm.

[0114] The four water-in-oil-in-water emulsions were mixed at a reduced temperature to form a nanoemulsion 8.

[0115] The zeta potential of the nanoemulsion 8 was measured to be -37 V; the repellent duration was 8.9 h; the 6-hour colony growth rate was -78%; the 20-hour colony growth rate was 425%; the preliminary oil separation time was 14 days; the moderate oil separation time was 34 days; no severe oil separation was observed.

[0116] Example 9

[0117] A mixture of bluegum oil (8.4g), peppermint oil (3.6g), Span 80 (1.2g) and water (3.6g) were combined to form a first coarse emulsion, which was then subjected to high pressure microfluidic nanoemulsification at 170 MPa to form a first water-in-oil emulsion; the first water-in-oil emulsion was combined with the first aqueous solution (43.2g) and stirred at 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion had a first water-in-oil emulsion nucleus, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsion nucleus was 120 nm.

[0118] A mixture of eucalyptus globulus oil (5.6g), peppermint oil (2.4g), Span 80 (0.8g) was prepared into a second coarse emulsion with water (2.4g), and a second water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at 120 MPa pressure; the second water-in-oil emulsion was mixed with the first aqueous solution (28.8g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm.

[0119] A mixture of carvacrol (4.2g), thymol (4.2g), Span 60 (1.26g), vitamin E (0.42g), MCT oil (4.2g) was prepared into a third coarse emulsion with the second aqueous solution (3.78g), and a third water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at 105 MPa pressure; the third water-in-oil emulsion was mixed with the first aqueous solution (51.94g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsion core, and the D90 particle size of the inner water phase of the third water-in-oil emulsion core is 255 nm.

[0120] A mixture of carvacrol (1.8g), thymol (1.8g), Span 60 (0.54g), vitamin E (0.18g), ascorbyl palmitate (0.144g), MCT oil (1.8g) was prepared into a fourth coarse emulsion with the third aqueous solution (2.16g), and a fourth water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at 50 MPa pressure; the fourth water-in-oil emulsion was mixed with the first aqueous solution (21.576g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsion core, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsion core is 520 nm.

[0121] The four water-in-oil-in-water emulsions were cooled and mixed to form nanoemulsion 9.

[0122] The zeta potential of nanoemulsion 9 was measured to be -38V; the repellent duration was 9h; the 6-hour colony growth rate was -85%; the 20-hour colony growth rate was 481%; the preliminary oil separation time was 14 days; the moderate oil separation time was 38 days; no severe oil separation was observed.

[0123] Example 10

[0124] A mixture of eucalyptus oil (8.4 g), peppermint oil (3.6 g), Span 80 (1.2 g) and water (3.6 g) was prepared into a first coarse emulsion, and then a first water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with a first aqueous solution (43.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsion core, and the D90 particle size of the inner water phase of the first water-in-oil emulsion core is 120 nm.

[0125] A mixture of eucalyptus oil (4.2 g), peppermint oil (1.8 g), Span 80 (0.6 g) and water (1.8 g) was prepared into a second coarse emulsion, and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with a first aqueous solution (21.6 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm.

[0126] A mixture of eucalyptus oil (4.2 g), peppermint oil (1.8 g), Span 80 (0.6 g) and water (1.8 g) was prepared into a second coarse emulsion, and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with a first aqueous solution (21.6 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm.

[0127] A mixture of eucalyptus oil (4.2 g), peppermint oil (1.8 g), Span 80 (0.6 g) and water (1.8 g) was prepared into a second coarse emulsion, and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with a first aqueous solution (21.6 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm.

[0128] The four water-in-oil-in-water emulsions were mixed and cooled to form a nanoemulsion 10.

[0129] The zeta potential of the nanoemulsion 10 was measured to be -38 V; the repellent duration was 8.5 h; the 6-hour colony growth rate was -79%; the 20-hour colony growth rate was 51%; the preliminary creaming time was 12 days; the moderate creaming time was 34 days; no severe creaming was observed.

[0130] Example 11

[0131] A mixture of Eucalyptus Globulus oil (8.4 g), Peppermint oil (3.6 g), Span 80 (1.2 g) was prepared into a first coarse emulsion with water (3.6 g), and then a first water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with the first aqueous solution (43.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsification core, and the D90 particle size of the inner water phase of the first water-in-oil emulsification core is 120 nm.

[0132] A mixture of Eucalyptus Globulus oil (2.8 g), Peppermint oil (1.2 g), Span 80 (0.4 g) was prepared into a second coarse emulsion with water (1.2 g), and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (14.4 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsification core, and the D90 particle size of the inner water phase of the second water-in-oil emulsification core is 285 nm.

[0133] A mixture of Carvacol (3.6 g), Thymol (3.6 g), Span 60 (1.08 g), Vitamin E (0.36 g), MCT oil (3.6 g) was prepared into a third coarse emulsion with the second aqueous solution (3.24 g), and then a third water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 105 MPa; the third water-in-oil emulsion was mixed with the first aqueous solution (44.52 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsification core, and the D90 particle size of the inner water phase of the third water-in-oil emulsification core is 255 nm.

[0134] A mixture of carvacrol (3.6g), thymol (3.6g), Span 60 (1.08g), vitamin E (0.36g), ascorbyl palmitate (0.288g), MCT oil (3.6g) and the third aqueous solution (4.32g) were prepared into a fourth coarse emulsion, and then a fourth water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (43.152g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsion core, and the D90 particle size of the inner aqueous phase of the fourth water-in-oil emulsion core is 520 nm.

[0135] The four water-in-oil-in-water emulsions were mixed at low temperature to form a nanoemulsion 11.

[0136] The zeta potential of the nanoemulsion 11 was measured to be -38 V; the repellent duration was 7.2 h; the 6-hour bacterial colony growth rate was -88%; the 20-hour bacterial colony growth rate was 37%; the preliminary oil separation time was 14 days; the moderate oil separation time was 40 days; and no severe oil separation was observed.

[0137] Example 12

[0138] A mixture of eucalyptus globulus oil (8.4g), peppermint oil (3.6g), Span 80 (1.2g) and water (3.6g) were prepared into a first coarse emulsion, and then a first water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with the first aqueous solution (43.2g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsion core, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsion core is 120 nm.

[0139] A mixture of eucalyptus globulus oil (4.2g), peppermint oil (1.8g), Span 80 (0.6g) and water (1.8g) were prepared into a second coarse emulsion, and then a second water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (21.6g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner aqueous phase of the second water-in-oil emulsion core is 285 nm.

[0140] A mixture of carvacrol (3.6g), thymol (3.6g), Span 60 (1.08g), Vitamin E (0.36g), MCT oil (3.6g) was prepared into a third coarse emulsion with a second aqueous solution (3.24g), and a third water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 105 MPa; the third water-in-oil emulsion was mixed with the first aqueous solution (44.52g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsion nucleus, and the D90 particle size of the inner aqueous phase of the third water-in-oil emulsion nucleus is 255 nm.

[0141] A mixture of carvacrol (3.6g), thymol (3.6g), Span 60 (1.08g), Vitamin E (0.36g), ascorbic acid palmitate (0.288g), MCT oil (3.6g) was prepared into a fourth coarse emulsion with a third aqueous solution (4.32g), and a fourth water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (33.152g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsion nucleus, and the D90 particle size of the inner aqueous phase of the fourth water-in-oil emulsion nucleus is 520 nm.

[0142] The four water-in-oil-in-water emulsions were mixed at low temperature to form a nanoemulsion 12.

[0143] The zeta potential of the nanoemulsion 12 was measured to be -39V; the repellent duration was 8.4h; the 6-hour colony growth rate was -85%; the 20-hour colony growth rate was 59%; the preliminary oil separation time was 12 days; the medium oil separation time was 36 days; no severe oil separation was observed.

[0144] Example 13

[0145] A mixture of blue eucalyptus oil (9g), peppermint oil (4g), Span 80 (1.3g) was prepared into a first coarse emulsion with water (3.9g), and a first water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with the first aqueous solution (41.8g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsion nucleus, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsion nucleus is 120 nm.

[0146] A mixture of eucalyptus globulus oil (5 g), peppermint oil (2 g), Span 80 (0.7 g) was prepared into a second coarse emulsion with water (2.1 g), and a second water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at 120 MPa pressure; the second water-in-oil emulsion was mixed with the first aqueous solution (20.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsified core, and the D90 particle size of the inner water phase of the second water-in-oil emulsified core is 285 nm.

[0147] A mixture of carvacrol (3.6 g), thymol (3.6 g), Span 60 (1.08 g), vitamin E (0.36 g), MCT oil (3.6 g) was prepared into a third coarse emulsion with the second aqueous solution (3.24 g), and a third water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at 105 MPa pressure; the third water-in-oil emulsion was mixed with the first aqueous solution (44.52 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsified core, and the D90 particle size of the inner water phase of the third water-in-oil emulsified core is 255 nm.

[0148] A mixture of carvacrol (2.4 g), thymol (2.4 g), Span 60 (0.72 g), vitamin E (0.24 g), ascorbyl palmitate (0.192 g), MCT oil (2.4 g) was prepared into a fourth coarse emulsion with the third aqueous solution (2.88 g), and a fourth water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at 50 MPa pressure; the fourth water-in-oil emulsion was mixed with the first aqueous solution (38.768 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsified core, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsified core is 520 nm.

[0149] The four water-in-oil-in-water emulsions were cooled and mixed to form nanoemulsion 13.

[0150] The zeta potential of nanoemulsion 13 was measured to be -38 V; the repellent duration was 8.5 h; the 6-hour colony growth rate was -83%; the 20-hour colony growth rate was 46%; the preliminary oil separation time was 14 days; the medium oil separation time was 42 days; no severe oil separation was observed.

[0151] Example 14

[0152] A mixture of eucalyptus oil (9 g), peppermint oil (3 g), Span 80 (1.2 g) was prepared into a first coarse emulsion with water (3.6 g), and then a first water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with a first aqueous solution (43.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsion core, and the D90 particle size of the inner water phase of the first water-in-oil emulsion core is 120 nm.

[0153] A mixture of eucalyptus oil (5 g), peppermint oil (3 g), Span 80 (0.8 g) was prepared into a second coarse emulsion with water (2.4 g), and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with a first aqueous solution (18.8 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm.

[0154] A mixture of carvacrol (4 g), thymol (3 g), Span 60 (1.05 g), vitamin E (0.35 g), MCT oil (3.5 g) was prepared into a third coarse emulsion with a second aqueous solution (3.15 g), and then a third water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 105 MPa; the third water-in-oil emulsion was mixed with a first aqueous solution (44.95 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsion core, and the D90 particle size of the inner water phase of the third water-in-oil emulsion core is 255 nm.

[0155] A mixture of carvacrol (3 g), thymol (2 g), Span 60 (0.75 g), vitamin E (0.25 g), ascorbyl palmitate (0.2 g), MCT oil (2.5 g) was prepared into a fourth coarse emulsion with a third aqueous solution (3 g), and then a fourth water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with a first aqueous solution (38.3 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsion core, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsion core is 520 nm.

[0156] The four water-in-oil-in-water emulsions were mixed to form a nanoemulsion 14.

[0157] The zeta potential of the nanoemulsion 14 was measured to be -38 V; the repellent duration was 8.5 h; the 6-hour colony growth rate was -85%; the 20-hour colony growth rate was 49%; the preliminary creaming time was 14 days; the moderate creaming time was 38 days; no severe creaming was observed.

[0158] Example 15

[0159] A mixture of Eucalyptus Globulus oil (8 g), Peppermint oil (4 g), Span 80 (1.2 g) was prepared into a first coarse emulsion with water (3.6 g), and then a first water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with the first aqueous solution (43.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsification core, and the D90 particle size of the inner water phase of the first water-in-oil emulsification core is 120 nm.

[0160] A mixture of Eucalyptus Globulus oil (5 g), Peppermint oil (3 g), Span 80 (0.8 g) was prepared into a second coarse emulsion with water (2.4 g), and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (18.8 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsification core, and the D90 particle size of the inner water phase of the second water-in-oil emulsification core is 285 nm.

[0161] A mixture of Carvacrol (4 g), Thymol (3 g), Span 60 (1.05 g), Vitamin E (0.35 g), MCT oil (3.5 g) was prepared into a third coarse emulsion with the second aqueous solution (3.15 g), and then a third water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 105 MPa; the third water-in-oil emulsion was mixed with the first aqueous solution (44.95 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsification core, and the D90 particle size of the inner water phase of the third water-in-oil emulsification core is 255 nm.

[0162] A mixture of eucalyptol (3 g), thymol (3 g), Span 60 (0.9 g), vitamin E (0.3 g), ascorbyl palmitate (0.24 g), MCT oil (3 g) and the third aqueous solution (3.6 g) was prepared into a fourth coarse emulsion, and then a fourth water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (35.96 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the fourth water-in-oil emulsified core is 520 nm.

[0163] The four water-in-oil-in-water emulsions were mixed at a low temperature to form a nanoemulsion 15.

[0164] The zeta potential of the nanoemulsion 15 was measured to be -40 V; the repellent duration was 8.4 h; the 6-hour bacterial colony growth rate was -84%; the 20-hour bacterial colony growth rate was 48%; the preliminary oil separation time was 12 days; the moderate oil separation time was 36 days; and no severe oil separation was observed.

[0165] Example 16

[0166] A mixture of eucalyptus oil (8 g), peppermint oil (3 g), and Span 80 (1.1 g) was prepared into a first coarse emulsion with water (3.3 g), and then a first water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with the first aqueous solution (44.6 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsified core is 120 nm.

[0167] A mixture of eucalyptus oil (5 g), peppermint oil (2 g), and Span 80 (0.7 g) was prepared into a second coarse emulsion with water (2.1 g), and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (20.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the second water-in-oil emulsified core is 285 nm.

[0168] A mixture of carvacrol (3 g), thymol (3 g), Span 60 (0.9 g), vitamin E (0.3 g), ascorbyl palmitate (0.24 g), MCT oil (3 g) and the third aqueous solution (3.6 g) was prepared into a fourth coarse emulsion, and then a fourth water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (35.96 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsified core, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsified core is 520 nm.

[0169] A mixture of carvacrol (3 g), thymol (3 g), Span 60 (0.9 g), vitamin E (0.3 g), ascorbyl palmitate (0.24 g), MCT oil (3 g) and the third aqueous solution (3.6 g) was prepared into a fourth coarse emulsion, and then a fourth water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (35.96 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsified core, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsified core is 520 nm.

[0170] The four water-in-oil-in-water emulsions were mixed at a low temperature to form a nanoemulsion 16.

[0171] The zeta potential of the nanoemulsion 16 was measured to be -38 V; the repellent duration was 8.6 h; the 6-hour bacterial colony growth rate was -83%; the 20-hour bacterial colony growth rate was 59%; the preliminary oil separation time was 14 days; the moderate oil separation time was 40 days; and no severe oil separation was observed.

[0172] Example 17

[0173] A mixture of eucalyptus globulus oil (9 g), peppermint oil (4 g), Span 80 (1.3 g) and water (3.9 g) was prepared into a first coarse emulsion, and then a first water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 150 MPa; the first water-in-oil emulsion was mixed with the first aqueous solution (41.8 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsified core, and the D90 particle size of the inner water phase of the first water-in-oil emulsified core is 100 nm.

[0174] A mixture of eucalyptus globulus oil (5 g), peppermint oil (2 g), Span 80 (0.7 g) was prepared into a second coarse emulsion with water (2.1 g), and a second water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at 120 MPa pressure; the second water-in-oil emulsion was mixed with the first aqueous solution (20.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsified core, and the D90 particle size of the inner water phase of the second water-in-oil emulsified core is 285 nm.

[0175] A mixture of carvacrol (3.6 g), thymol (3.6 g), Span 60 (1.08 g), vitamin E (0.36 g), MCT oil (3.6 g) was prepared into a third coarse emulsion with the second aqueous solution (3.24 g), and a third water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at 105 MPa pressure; the third water-in-oil emulsion was mixed with the first aqueous solution (44.52 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsified core, and the D90 particle size of the inner water phase of the third water-in-oil emulsified core is 255 nm.

[0176] A mixture of carvacrol (2.4 g), thymol (2.4 g), Span 60 (0.72 g), vitamin E (0.24 g), ascorbyl palmitate (0.192 g), MCT oil (2.4 g) was prepared into a fourth coarse emulsion with the third aqueous solution (2.88 g), and a fourth water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at 50 MPa pressure; the fourth water-in-oil emulsion was mixed with the first aqueous solution (38.768 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsified core, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsified core is 520 nm.

[0177] The four water-in-oil-in-water emulsions were cooled and mixed to form nanoemulsion 17.

[0178] The zeta potential of nanoemulsion 17 was measured to be -38 V; the repellent duration was 8.2 h; the 6-hour colony growth rate was -85%; the 20-hour colony growth rate was 72%; the preliminary oil separation time was 14 days; the medium oil separation time was 42 days; no severe oil separation was observed.

[0179] Example 18

[0180] A mixture of Eucalyptus Globulus oil (9 g), Peppermint oil (4 g), Span 80 (1.3 g) was prepared into a first coarse emulsion with water (3.9 g), and then a first water-in-oil emulsion was prepared by high pressure microfluidic nano-emulsification technology at a pressure of 200 MPa; the first water-in-oil emulsion was mixed with a first aqueous solution (41.8 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsion core, and the D90 particle size of the inner water phase of the first water-in-oil emulsion core is 150 nm.

[0181] A mixture of Eucalyptus Globulus oil (5 g), Peppermint oil (2 g), Span 80 (0.7 g) was prepared into a second coarse emulsion with water (2.1 g), and then a second water-in-oil emulsion was prepared by high pressure microfluidic nano-emulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with a first aqueous solution (20.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm.

[0182] A mixture of Carvacrol (3.6 g), Thymol (3.6 g), Span 60 (1.08 g), Vitamin E (0.36 g), MCT oil (3.6 g) was prepared into a third coarse emulsion with a second aqueous solution (3.24 g), and then a third water-in-oil emulsion was prepared by high pressure microfluidic nano-emulsification technology at a pressure of 105 MPa; the third water-in-oil emulsion was mixed with a first aqueous solution (44.52 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsion core, and the D90 particle size of the inner water phase of the third water-in-oil emulsion core is 255 nm.

[0183] A mixture of Carvacrol (2.4 g), Thymol (2.4 g), Span 60 (0.72 g), Vitamin E (0.24 g), Ascorbyl Palmitate (0.192 g), MCT oil (2.4 g) was prepared into a fourth coarse emulsion with a third aqueous solution (2.88 g), and then a fourth water-in-oil emulsion was prepared by high pressure microfluidic nano-emulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with a first aqueous solution (38.768 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsion core, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsion core is 520 nm.

[0184] The four water-in-oil-in-water emulsions were mixed at a low temperature to form a nanoemulsion 18.

[0185] The zeta potential of the nanoemulsion 18 was measured to be -37 V; the repellent duration was 8.6 h; the 6-hour colony growth rate was -85%; the 20-hour colony growth rate was 59%; the preliminary creaming time was 12 days; the moderate creaming time was 38 days; no severe creaming was observed.

[0186] Example 19

[0187] After a mixture of Eucalyptus Globulus oil (9 g), Peppermint oil (4 g), Span 80 (1.3 g) and water (3.9 g) was prepared into a first coarse emulsion, a first water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 150 MPa; after the first water-in-oil emulsion was mixed with the first aqueous solution (41.8 g) and stirred at a speed of 5000 rpm for 2 minutes, a first water-in-oil-in-water emulsion was formed; the first water-in-oil-in-water emulsion has a first water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsified core is 100 nm.

[0188] After a mixture of Eucalyptus Globulus oil (5 g), Peppermint oil (2 g), Span 80 (0.7 g) and water (2.1 g) was prepared into a second coarse emulsion, a second water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 100 MPa; after the second water-in-oil emulsion was mixed with the first aqueous solution (20.2 g) and stirred at a speed of 5000 rpm for 2 minutes, a second water-in-oil-in-water emulsion was formed; the second water-in-oil-in-water emulsion has a second water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the second water-in-oil emulsified core is 250 nm.

[0189] After a mixture of Carvacrol (3.6 g), Thymol (3.6 g), Span 60 (1.08 g), Vitamin E (0.36 g), MCT oil (3.6 g) and the second aqueous solution (3.24 g) was prepared into a third coarse emulsion, a third water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 105 MPa; after the third water-in-oil emulsion was mixed with the first aqueous solution (44.52 g) and stirred at a speed of 5000 rpm for 2 minutes, a third water-in-oil-in-water emulsion was formed; the third water-in-oil-in-water emulsion has a third water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the third water-in-oil emulsified core is 255 nm.

[0190] A mixture of eucalyptol (2.4 g), thymol (2.4 g), Span 60 (0.72 g), vitamin E (0.24 g), ascorbyl palmitate (0.192 g), MCT oil (2.4 g) and the third aqueous solution (2.88 g) were used to prepare a fourth coarse emulsion, which was then used to prepare a fourth water-in-oil emulsion using high-pressure microfluidic nanoemulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (38.768 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion had a fourth water-in-oil emulsion nucleus, and the D90 particle size of the inner aqueous phase of the fourth water-in-oil emulsion nucleus was 520 nm.

[0191] The four water-in-oil-in-water emulsions were mixed and cooled to form a nanoemulsion 19.

[0192] The zeta potential of the nanoemulsion 19 was -38 V; the repellent duration was 8.2 h; the 6-hour colony growth rate was -84%; the 20-hour colony growth rate was 53%; the preliminary oil separation time was 14 days; the moderate oil separation time was 32 days; and no severe oil separation was observed.

[0193] Example 20

[0194] A mixture of eucalyptus oil (9 g), peppermint oil (4 g), and Span 80 (1.3 g) was prepared into a first coarse emulsion with water (3.9 g), and then a first water-in-oil emulsion was prepared using high-pressure microfluidic nanoemulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with the first aqueous solution (41.8 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion had a first water-in-oil emulsion nucleus, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsion nucleus was 120 nm.

[0195] A mixture of eucalyptus oil (5 g), peppermint oil (2 g), and Span 80 (0.7 g) was prepared into a second coarse emulsion with water (2.1 g), and then a second water-in-oil emulsion was prepared using high-pressure microfluidic nanoemulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (20.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion had a second water-in-oil emulsion nucleus, and the D90 particle size of the inner aqueous phase of the second water-in-oil emulsion nucleus was 285 nm.

[0196] A mixture of carvacrol (3.6g), thymol (3.6g), Span 60 (1.08g), Vitamin E (0.36g), MCT oil (3.6g) was prepared into a third coarse emulsion with a second aqueous solution (3.24g), and a third water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 90 MPa; the third water-in-oil emulsion was mixed with the first aqueous solution (44.52g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsion nucleus, and the D90 particle size of the inner water phase of the third water-in-oil emulsion nucleus is 210 nm.

[0197] A mixture of carvacrol (2.4g), thymol (2.4g), Span 60 (0.72g), Vitamin E (0.24g), ascorbic acid palmitate (0.192g), MCT oil (2.4g) was prepared into a fourth coarse emulsion with a third aqueous solution (2.88g), and a fourth water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 50 MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (38.768g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsion nucleus, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsion nucleus is 520 nm.

[0198] The four water-in-oil-in-water emulsions were mixed at low temperature to form a nanoemulsion 20.

[0199] The zeta potential of the nanoemulsion 20 was measured to be -38V; the mosquito repellent duration was 8.1h; the 6-hour bacterial colony growth rate was -86%; the 20-hour bacterial colony growth rate was 68%; the preliminary oil separation time was 12 days; the medium oil separation time was 36 days; no severe oil separation was observed.

[0200] Example 21

[0201] A mixture of blue eucalyptus oil (9g), peppermint oil (4g), Span 80 (1.3g) was prepared into a first coarse emulsion with water (3.9g), and a first water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with the first aqueous solution (41.8g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsion nucleus, and the D90 particle size of the inner water phase of the first water-in-oil emulsion nucleus is 120 nm.

[0202] A mixture of eucalyptus globulus oil (5 g), peppermint oil (2 g), Span 80 (0.7 g) was prepared into a second coarse emulsion with water (2.1 g), and then a second water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with the first aqueous solution (20.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsified core, and the D90 particle size of the inner water phase of the second water-in-oil emulsified core is 285 nm.

[0203] A mixture of carvacol (3.6 g), thymol (3.6 g), Span 60 (1.08 g), vitamin E (0.36 g), MCT oil (3.6 g) was prepared into a third coarse emulsion with the second aqueous solution (3.24 g), and then a third water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 90 MPa; the third water-in-oil emulsion was mixed with the first aqueous solution (44.52 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsified core, and the D90 particle size of the inner water phase of the third water-in-oil emulsified core is 210 nm.

[0204] A mixture of carvacol (2.4 g), thymol (2.4 g), Span 60 (0.72 g), vitamin E (0.24 g), ascorbyl palmitate (0.192 g), MCT oil (2.4 g) was prepared into a fourth coarse emulsion with the third aqueous solution (2.88 g), and then a fourth water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 70 MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (38.768 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsified core, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsified core is 700 nm.

[0205] The four water-in-oil-in-water emulsions were cooled and mixed to form nanoemulsion 21.

[0206] The zeta potential of nanoemulsion 21 was measured to be -38 V; the repellent duration was 8.3 h; the 6-hour colony growth rate was -84%; the 20-hour colony growth rate was 51%; the preliminary oil separation time was 9 days; the moderate oil separation time was 28 days; and the severe oil separation time was 48 days.

[0207] Example 22

[0208] A mixture of eucalyptus oil (9 g), peppermint oil (4 g), Span 80 (1.3 g) was prepared into a first coarse emulsion with water (3.9 g), and then a first water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with a first aqueous solution (41.8 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsion core, and the D90 particle size of the inner water phase of the first water-in-oil emulsion core is 120 nm.

[0209] A mixture of eucalyptus oil (5 g), peppermint oil (2 g), Span 80 (0.7 g) was prepared into a second coarse emulsion with water (2.1 g), and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 120 MPa; the second water-in-oil emulsion was mixed with a first aqueous solution (20.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsion core, and the D90 particle size of the inner water phase of the second water-in-oil emulsion core is 285 nm.

[0210] A mixture of carvacrol (3.6 g), thymol (3.6 g), Span 60 (1.08 g), vitamin E (0.36 g), MCT oil (3.6 g) was prepared into a third coarse emulsion with a second aqueous solution (3.24 g), and then a third water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 130 MPa; the third water-in-oil emulsion was mixed with a first aqueous solution (44.52 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsion core, and the D90 particle size of the inner water phase of the third water-in-oil emulsion core is 300 nm.

[0211] A mixture of carvacrol (2.4 g), thymol (2.4 g), Span 60 (0.72 g), vitamin E (0.24 g), ascorbyl palmitate (0.192 g), MCT oil (2.4 g) was prepared into a fourth coarse emulsion with a third aqueous solution (2.88 g), and then a fourth water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology at a pressure of 65 MPa; the fourth water-in-oil emulsion was mixed with a first aqueous solution (38.768 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsion core, and the D90 particle size of the inner water phase of the fourth water-in-oil emulsion core is 640 nm.

[0212] The four water-in-oil-in-water emulsions were mixed at a low temperature to form a nanoemulsion 22.

[0213] The zeta potential of the nanoemulsion 22 was measured to be -38 V; the repellent duration was 8.4 h; the 6-hour colony growth rate was -83%; the 20-hour colony growth rate was 63%; the preliminary creaming time was 8 days; the moderate creaming time was 28 days; and the severe creaming time was 42 days.

[0214] Experimental Example 23

[0215] A mixture of eucalyptus globulus oil (14 g), peppermint oil (6 g), and Span 80 (2 g) was prepared into a first coarse emulsion with water (6 g), and then a first water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 170 MPa; the first water-in-oil emulsion was mixed with a first aqueous solution (72 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsification core, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsification core is 120 nm.

[0216] A mixture of eucalyptus globulus oil (0 g), peppermint oil (0 g), and Span 80 (0 g) was prepared into a second coarse emulsion with water (0 g), and then a second water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of MPa; the second water-in-oil emulsion was mixed with a first aqueous solution (0 g) and stirred at a speed of 5000 rpm for 2 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsification core, and the D90 particle size of the inner aqueous phase of the second water-in-oil emulsification core is between nm.

[0217] A mixture of carvacol (6 g), thymol (6 g), Span 60 (1.8 g), vitamin E (0.6 g), and MCT oil (6 g) was prepared into a third coarse emulsion with a second aqueous solution (5.4 g), and then a third water-in-oil emulsion was prepared by high-pressure microfluidic nanoemulsification technology at a pressure of 105 MPa; the third water-in-oil emulsion was mixed with a first aqueous solution (74.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsification core, and the D90 particle size of the inner aqueous phase of the third water-in-oil emulsification core is between 255 nm.

[0218] A mixture of carvacrol (0 g), thymol (0 g), Span 60 (0 g), vitamin E (0 g), ascorbyl palmitate (0 g), MCT oil (0 g) and the third aqueous solution (0 g) were prepared into a fourth coarse emulsion, and then a fourth water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology under a pressure of MPa; the fourth water-in-oil emulsion was mixed with the first aqueous solution (0 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the fourth water-in-oil emulsified core is between 100 nm and 200 nm.

[0219] The two water-in-oil-in-water emulsions were mixed and cooled to form a nanoemulsion 23.

[0220] The zeta potential of the nanoemulsion 23 was -38 V; the mosquito repellent duration was 4.5 h; the 6-hour bacterial colony growth rate was -82%; the 15-hour bacterial colony growth rate was 782%; the preliminary oil separation time was 12 days; the medium oil separation time was 46 days; and no serious emulsification was observed.

[0221] Experimental Example 24

[0222] A mixture of eucalyptus oil (14 g), peppermint oil (6 g), and Span 80 (2 g) and water (6 g) were prepared into a first coarse emulsion, and then a first water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology under a pressure of 200 MPa; the first water-in-oil emulsion was mixed with the first aqueous solution (72 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsified core is 150 nm.

[0223] A mixture of carvacrol (6 g), thymol (8 g), Span 60 (2.1 g), vitamin E (0.7 g), and MCT oil (7 g) and the second aqueous solution (6.3 g) were prepared into a third coarse emulsion, and then a third water-in-oil emulsion was prepared by high-pressure microfluidic nano-emulsification technology under a pressure of 130 MPa; the third water-in-oil emulsion was mixed with the first aqueous solution (69.9 g) and stirred at a speed of 5000 rpm for 2 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the third water-in-oil emulsified core is between 100 nm and 200 nm.

[0224] The two water-in-oil-in-water emulsions were mixed and cooled to form a nanoemulsion 24.

[0225] The zeta potential of the nanoemulsion 24 was measured to be -38 V; the repellent duration was 5.2 h; the 6-hour colony growth rate was -83%; the 20-hour colony growth rate was 904%; the preliminary creaming time was 18 days; the moderate creaming time was 42 days; and no severe creaming was observed.

[0226] Experimental Example 25

[0227] After a mixture of eucalyptus globulus oil (12 g), peppermint oil (8 g), and Span 80 (2 g) was prepared into a first coarse emulsion with water (6 g), a first water-in-oil emulsion was prepared at a pressure of 200 MPa by high-pressure microfluidic nanoemulsification technology; after the first water-in-oil emulsion was mixed with a first aqueous solution (72 g) and stirred at a speed of 5000 rpm for 2 minutes, a first water-in-oil-in-water emulsion was formed; the first water-in-oil-in-water emulsion has a first water-in-oil emulsification core, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsification core is 150 nm.

[0228] After a mixture of carvacol (8 g), thymol (6 g), Span 60 (2.1 g), vitamin E (0.7 g), ascorbyl palmitate (0.56 g), and MCT oil (7 g) was prepared into a fourth coarse emulsion with a third aqueous solution (8.4 g), a fourth water-in-oil emulsion was prepared at a pressure of 50 MPa by high-pressure microfluidic nanoemulsification technology; after the fourth water-in-oil emulsion was mixed with a first aqueous solution (67.24 g) and stirred at a speed of 5000 rpm for 2 minutes, a fourth water-in-oil-in-water emulsion was formed; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsification core, and the D90 particle size of the inner aqueous phase of the fourth water-in-oil emulsification core is between 520 nm.

[0229] After the first water-in-oil-in-water emulsion, the second water-in-oil-in-water emulsion, the third water-in-oil-in-water emulsion, and the fourth water-in-oil-in-water emulsion were all cooled to 10-15°C, they were mixed and stirred at a speed of 2500 rpm for 2 minutes to form a nanoemulsion 36.

[0230] The zeta potential of the nanoemulsion 25 was measured to be -37 V; the repellent duration was 5.3 h; the 6-hour colony growth rate was -74%; the 20-hour colony growth rate was 139%; the preliminary creaming time was 8 days; the moderate creaming time was 24 days; and the severe creaming time was 36 days.

[0231] Experimental Example 26

[0232] A mixture of eucalyptus globulus oil (14 g), peppermint oil (8 g), Span 80 (2.2 g), and water (6.6 g) was prepared into a first coarse emulsion, and then a first water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 200 MPa; the first water-in-oil emulsion was mixed with a first aqueous solution (69.2 g) and stirred at a speed of 5000 rpm for 2 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsion core, and the D90 particle size of the inner aqueous phase of the first water-in-oil emulsion core is 150 nm.

[0233] A mixture of carvacol (8 g), thymol (8 g), Span 60 (2.4 g), vitamin E (0.8 g), ascorbyl palmitate (0.64 g), MCT oil (8 g), and a third aqueous solution (9.6 g) was prepared into a fourth coarse emulsion, and then a fourth water-in-oil emulsion was prepared by high pressure microfluidic nanoemulsification technology at a pressure of 70 MPa; the fourth water-in-oil emulsion was mixed with a first aqueous solution (62.56 g) and stirred at a speed of 5000 rpm for 2 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsion core, and the D90 particle size of the inner aqueous phase of the fourth water-in-oil emulsion core is between 700 nm.

[0234] The two water-in-oil-in-water emulsions were mixed at a low temperature to form a nanoemulsion 26.

[0235] The zeta potential of the nanoemulsion 26 was measured to be -38 V; the repellent duration was 4.8 h; the 6-hour bacterial colony growth rate was -76%; the 20-hour bacterial colony growth rate was 157%; the preliminary oil separation time was 6 days; the moderate oil separation time was 20 days; and the severe oil separation time was 32 days.

[0236] Experimental Example 27

[0237] The preparation process of Example 27 was basically the same as that of Example 1, except that in Example 27, the oil phase of the fourth water-in-oil emulsion core did not contain ascorbyl palmitate. The nanoemulsion prepared in Example 27 was marked as nanoemulsion 27.

[0238] The zeta potential of the nanoemulsion 27 was measured to be -37 V; the repellent duration was 8.6 h; the 6-hour bacterial colony growth rate was -62%; the 20-hour bacterial colony growth rate was 635%; the preliminary oil separation time was 14 days; the moderate oil separation time was 44 days; and no severe emulsification was observed.

[0239] Experimental Example 28

[0240] The preparation process of Example 28 was basically the same as that of Example 1, except that in Example 28, the inner aqueous phase of the fourth water-in-oil emulsion core did not contain hydroxypropyl-β-cyclodextrin. The nanoemulsion prepared in Example 28 was marked as nanoemulsion 28.

[0241] The zeta potential of the nanoemulsion 28 was -38 V; the repellent duration was 8.3 h; the 6-hour colony growth rate was -90%; the 20-hour colony growth rate was 791%; the preliminary creaming time was 12 days; the moderate creaming time was 38 days; and no severe creaming was observed.

[0242] The test results of the various nanoemulsions are shown in Table 1 below.

[0243] Table 1: Test results of the nanoemulsions

[0244]

[0245]

[0246] In which, “-” means that no severe creaming was observed until the fiftieth day.

[0247] The nanoemulsions 1-22 are applicable nanoemulsions provided by the embodiments of the present application, most of which can have a longer repellent duration, a longer bacteriostatic and bactericidal duration, and higher stability.

[0248] From the nanoemulsions 1-22, it can be seen that the adjustment of the ratio of small-particle-size emulsion nuclei and large-particle-size emulsion nuclei has an effect on the duration of the effect of the nanoemulsion. For example, in the nanoemulsions 2 and 3, the proportion of the first water-in-oil emulsion nuclei is lower than that of the other nanoemulsions, and the proportion of the second water-in-oil emulsion nuclei is higher than that of the other nanoemulsions. The repellent duration of the nanoemulsions 2 and 3 is insufficient. This can be related to the fact that the nanoemulsions 2 and 3 cannot quickly form a high concentration of volatile essential oil to quickly repel mosquitoes in the initial stage of spray application, resulting in insufficient repellency (individual mosquitoes staying on the drug-treated gauze) in a short time after application. For another example, in the nanoemulsion 7, the proportion of the third water-in-oil emulsion nuclei is lower than that of the other nanoemulsions, and the proportion of the fourth water-in-oil emulsion nuclei is higher than that of the other nanoemulsions. According to the test results, it can be seen that the bacteriostatic and bactericidal effect of the nanoemulsion 7 is not outstanding within 6 hours, but is more outstanding within 18 hours, showing obvious long-acting bacteriostatic characteristics.

[0249] From the nanoemulsions 1-22, it can be seen that the adjustment of the ratio of small-particle-size emulsion nuclei and large-particle-size emulsion nuclei, as well as the adjustment of the particle size of each emulsion nucleus, has a certain effect on the stability of the nanoemulsion. Generally, the more small-particle-size emulsion nuclei there are, and the smaller the small-particle-size emulsion nuclei are, the higher the stability of the nanoemulsion is. For example, the nanoemulsions 21 and 22 have larger emulsion nuclei, which results in a decrease in stability.

[0250] Experimental Examples 23-26 are exploratory experimental examples of the present application in the research and development stage, which only set two kinds of emulsified cores. It can be seen from Experimental Examples 23-26 that the long-acting mosquito repellent effects of these nanoemulsions are not ideal, and there is a clear conflict between the target of long-acting bactericidal / inhibitory and the target of long-term stability.

[0251] Experimental Example 27 is a typical example of a plurality of experimental examples of the present application in the research and development stage, which does not add ascorbic acid palmitate to the oil phase of the fourth water-in-oil emulsion. It can be seen from Table 1 that when ascorbic acid palmitate is not added to the oil phase, the 6-hour colony growth is -62%, and the 20-hour colony growth rate reaches 635%. This indicates that the bactericidal effect of nanoemulsion 27 is initially weak, and the sustained bactericidal time, the sustained inhibition of bacteria, etc. are all weak. This may be because ascorbic acid palmitate, together with vitamin E, can more effectively provide antioxidant protection for carvacol and thymol, reducing the oxidative loss of carvacol and thymol during long-term sustained release.

[0252] Experimental Example 28 is a typical example of a plurality of experimental examples of the present application in the research and development stage, which does not add hydroxypropyl-β-cyclodextrin to the internal water phase of the fourth water-in-oil emulsion. It can be seen from Table 1 that when hydroxypropyl-β-cyclodextrin is not added to the internal water phase, the 6-hour colony growth rate is as low as -90%, indicating that about 90% of the bacteria have lost activity, showing very good rapid killing effect. However, the 20-hour colony growth rate reached 791%, indicating that this nanoemulsion does not have a long-term inhibition of bacteria. The water phase of the fourth water-in-oil emulsion core of nanoemulsion 1-nanoemulsion 22 all contains hydroxypropyl-β-cyclodextrin, and the 20-hour colony growth rate is lower than that of Experimental Example 28. This may be because if hydroxypropyl-β-cyclodextrin is added to the internal water phase, carvacol and thymol can be partially combined with and protected by hydroxypropyl-β-cyclodextrin; after the droplets of the nanoemulsion volatilize in the water phase, carvacol and thymol in the oil phase play a rapid bacteriostatic and bactericidal role. The carvacol and thymol combined with hydroxypropyl-β-cyclodextrin are slowly released under the wrapping of sodium alginate, thereby enabling the surrounding of the bacteria to continuously maintain a low concentration of carvacol and thymol, which may inhibit or interfere with the proliferation of bacteria, thereby slowing down the rapid rebound of bacteria after killing.

[0253] Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the application being indicated by the following claims.

Claims

1. A nanoemulsion for preventing and treating respiratory disease in swine, characterized in that, The emulsion comprises an external water phase and a first water-in-oil emulsion core, a second water-in-oil emulsion core, a third water-in-oil emulsion core and a fourth water-in-oil emulsion core dispersed in the external water phase; The oil phase of the first water-in-oil emulsion core and the second water-in-oil emulsion core is a mixture of eucalyptus oil and peppermint oil, Span 80, and the internal water phase of the first water-in-oil emulsion core and the second water-in-oil emulsion core is water; the D90 particle size of the internal water phase of the first water-in-oil emulsion core is between 100nm and 150nm; the D90 particle size of the internal water phase of the second water-in-oil emulsion core is between 250nm and 300nm; The oil phase of the third water-in-oil emulsion core is a mixture of carvacol, thymol, Span 60, vitamin E and MCT oil, and the internal water phase of the third water-in-oil emulsion core is a sodium alginate aqueous solution; the D90 particle size of the internal water phase of the third water-in-oil emulsion core is between 200nm and 300nm; The oil phase of the fourth water-in-oil emulsion core is a mixture of carvacol, thymol, Span 60, vitamin E, ascorbic acid palmitate and MCT oil, and the internal water phase of the fourth water-in-oil emulsion core is a sodium alginate and hydroxypropyl-β-cyclodextrin aqueous solution; the D90 particle size of the internal water phase of the fourth water-in-oil emulsion core is between 500nm and 700nm; The external water phase is a water solution of Tween 80, xanthan gum, sodium caseinate and sodium benzoate.

2. The nanoemulsion for preventing and treating swine respiratory disease according to claim 1, characterized by, The mass ratio of the first water-in-oil emulsion core to the second water-in-oil emulsion core is in the range of 7:3 to 3:7; The mass ratio of the third water-in-oil emulsion core to the fourth water-in-oil emulsion core is in the range of 7:3 to 3:

7.

3. The nanoemulsion for preventing and treating swine respiratory disease according to claim 1, characterized by, The mass ratio of the first water-in-oil emulsion core to the second water-in-oil emulsion core is 2:1; The mass ratio of the third water-in-oil emulsion core to the fourth water-in-oil emulsion core is 6:

5.

4. The nanoemulsion for preventing and treating swine respiratory disease according to claim 1, characterized by, The mass of the eucalyptus oil in the first water-in-oil emulsion core is 2.8% to 4.5% of the total mass of the nanoemulsion; The mass of the eucalyptus oil in the second water-in-oil emulsion core is 2.5% to 4.1% of the total mass of the nanoemulsion; The mass of the peppermint oil in the first water-in-oil emulsion core is 1.2% to 2.1% of the total mass of the nanoemulsion; The mass of the peppermint oil in the second water-in-oil emulsion core is 0.9% to 1.8% of the total mass of the nanoemulsion.

5. The nanoemulsion for preventing and treating swine respiratory disease according to claim 1, characterized by, The mass of the carvacol in the third water-in-oil emulsion core is 1.5% to 2.1% of the total mass of the nanoemulsion; The mass of the thymol in the third water-in-oil emulsion core is 1.5% to 2.1% of the total mass of the nanoemulsion; The mass of the carvacol in the fourth water-in-oil emulsion core is 0.9% to 1.8% of the total mass of the nanoemulsion; The mass of the thymol in the fourth water-in-oil emulsion core is 0.9% to 1.8% of the total mass of the nanoemulsion.

6. The nanoemulsion for preventing and treating swine respiratory disease according to any one of claims 1 to 5, characterized by, The D90 particle size of the internal water phase of the first water-in-oil emulsion core is between 115nm and 125nm; The D90 particle size of the internal water phase of the second water-in-oil emulsion core is between 280nm and 290nm; The D90 particle size of the internal water phase of the third water-in-oil emulsion core is between 250nm and 260nm; The D90 particle size of the internal water phase of the fourth water-in-oil emulsion core is between 500nm and 700nm. The D90 particle size of the inner water phase of the fourth water-in-oil emulsified core is between 500 nm and 550 nm.

7. The nanoemulsion for preventing and treating swine respiratory disease according to any one of claims 1 to 5, characterized by, In the third water-in-oil emulsified core, the mass of vitamin E is 3% to 7% of the total mass of carvacol and thymol; In the fourth water-in-oil emulsified core, the mass of vitamin E is 3% to 7% of the total mass of carvacol and thymol, and the mass of ascorbyl palmitate is 3% to 5% of the total mass of carvacol and thymol.

8. The nanoemulsion for preventing and treating swine respiratory disease according to any one of claims 1 to 5, characterized by, In the fourth water-in-oil emulsified core, the mass content of hydroxypropyl-β-cyclodextrin in the inner water phase is 0.1% to 0.3%.

9. The nanoemulsion for preventing and treating swine respiratory disease according to any one of claims 1 to 5, characterized by, In the nanoemulsion, the mass content of the bluegum oil is 6.3% to 7.5%, the mass content of the peppermint oil is 2.7% to 3.5%, the mass content of the carvacol is 2.5% to 3.6%, and the mass content of the thymol is 2.5% to 3.6%.

10. The method for preparing the nanoemulsion for preventing and treating swine respiratory disease according to any one of claims 1 to 9, characterized in that, Comprise: Dissolve Tween 80, xanthan gum, sodium caseinate, and sodium benzoate in water to form a first aqueous solution; Prepare a first coarse emulsion by mixing the bluegum oil, the peppermint oil, and the Span 80 with water, and then prepare a first water-in-oil emulsion by high-pressure microfluidic nanoemulsification technology at a pressure of 150 MPa to 200 MPa; mix the first water-in-oil emulsion with part of the first aqueous solution, and stir at a speed of 4000 rpm to 6000 rpm for 1 to 3 minutes to form a first water-in-oil-in-water emulsion; the first water-in-oil-in-water emulsion has a first water-in-oil emulsified core, and the D90 particle size of the inner water phase of the first water-in-oil emulsified core is between 100 nm and 150 nm; Prepare a second coarse emulsion by mixing the bluegum oil, the peppermint oil, and the Span 80 with water, and then prepare a second water-in-oil emulsion by high-pressure microfluidic nanoemulsification technology at a pressure of 100 MPa to 130 MPa; mix the second water-in-oil emulsion with part of the first aqueous solution, and stir at a speed of 4000 rpm to 6000 rpm for 1 to 3 minutes to form a second water-in-oil-in-water emulsion; the second water-in-oil-in-water emulsion has a second water-in-oil emulsified core, and the D90 particle size of the inner water phase of the second water-in-oil emulsified core is between 250 nm and 300 nm; Dissolve sodium alginate in water to form a second aqueous solution; Dissolve sodium alginate and hydroxypropyl-β-cyclodextrin in water to form a third aqueous solution; Prepare a third coarse emulsion by mixing the carvacol, the thymol, the Span 60, the vitamin E, and the MCT oil with the second aqueous solution, and then prepare a third water-in-oil emulsion by high-pressure microfluidic nanoemulsification technology at a pressure of 90 MPa to 130 MPa; mix the third water-in-oil emulsion with part of the first aqueous solution, and stir at a speed of 4000 rpm to 6000 rpm for 1 to 3 minutes to form a third water-in-oil-in-water emulsion; the third water-in-oil-in-water emulsion has a third water-in-oil emulsified core, and the D90 particle size of the inner water phase of the third water-in-oil emulsified core is between 200 nm and 300 nm; The mixture of carvacol, thymol, Span 60, vitamin E, ascorbic acid palmitate, MCT oil and the third aqueous solution are prepared into a fourth coarse emulsion, and then the fourth water-in-oil emulsion is prepared by high-pressure microfluidic nano-emulsification technology under the pressure of 50MPa-70MPa; the fourth water-in-oil emulsion is mixed with part of the first aqueous solution and stirred at the speed of 4000rpm-6000rpm for 1-3 minutes to form a fourth water-in-oil-in-water emulsion; the fourth water-in-oil-in-water emulsion has a fourth water-in-oil emulsified core, and the D90 particle size of the inner aqueous phase of the fourth water-in-oil emulsified core is between 500nm-700nm; The first water-in-oil-in-water emulsion, the second water-in-oil-in-water emulsion, the third water-in-oil-in-water emulsion and the fourth water-in-oil-in-water emulsion are all cooled to 10℃-15℃, then mixed and stirred at the speed of 2000rpm-3000rpm for 1-3 minutes to form the nanoemulsion.

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