Disinfecting and bacteriostatic oral spray and preparation method thereof

Abstract

The invention belongs to the technical field of oral bacteriostasis, and particularly relates to a disinfection and bacteriostasis oral spray and a preparation method thereof. The disinfection and bacteriostasis oral spray comprises a liquid component and a solid component, the liquid component is prepared from the following components in parts by volume: 1 to 20 parts of PEG-40 hydrogenated castor oil, 1 to 20 parts of 1, 2-propylene glycol, 0.1 to 5 parts of chamomile essential oil, 0.1 to 5 parts of lavender essential oil, 0.1 to 5 parts of mint essential oil, 0.1 to 5 parts of tea tree essential oil and 20 to 90 parts of water; on the basis of the mass of 20-90 parts by volume of water, the solid component is prepared from the following components in parts by mass: 0.1-5 parts of tannic acid, 0.1-5 parts of L-arginine and 0.1-20 parts of a flavoring agent. The tannic acid and various plant essential oils can synergistically improve the antibacterial effect of the oral spray, and the diameter of the inhibition zone of the composite essential oil microemulsion added with the tannic acid is obviously larger than that of the single tannic acid solution and the single composite essential oil microemulsion.

Description

Technical Field

[0001] The invention belongs to the technical field of oral antibacterial agents, and in particular relates to a disinfecting and antibacterial oral spray and a preparation method thereof. Background Art

[0002] Oral health is an important part of overall health and one of the ten major standards of human health determined by the World Health Organization. Oral health is mainly manifested in dental caries, dental plaque, bleeding gums, bad breath, etc. Oral halitosis or bad breath is a common problem that affects people of all ages. With the development of the economy and the improvement of people's living standards, oral health issues are receiving unprecedented attention. Oral bacteria are the main pathogenic factors that cause oral problems. The oral cavity is one of the parts of the body with the highest density and the most types of microorganisms. Severe bad breath is mostly caused by oral diseases. The most common are bacterial plaques on the back of the tongue and teeth in the mouth, and infections and inflammations of the gums and periodontal tissues. Oral microbial flora and autoimmune disorders are important causes of oral diseases and even systemic diseases.

[0003] Antibiotics are one of the most widely used and frequently used drugs in clinical practice, helping to cure many infectious diseases caused by pathogenic microorganisms. However, with the abuse of antibiotics, a series of serious side effects have also emerged. People have found that pathogenic bacteria can evolve drug resistance and survive under the pressure of antibiotics. Over time, the drug resistance dose of pathogenic bacteria will expand to higher concentrations. Therefore, there is an urgent need to explore new strategies for effective prevention and treatment.

[0004] At present, the effective ingredients of oral disinfection and antibacterial care products on the domestic market are mostly chemical synthetic substances, and there are few natural antibacterial substances. Plant essential oils, as secondary metabolites of natural plants, are a general term for oily liquids with natural aromatic odors extracted from the roots, stems, and leaves of plants. They are considered to be one of the existing harmless natural substances in the world. The terpenoid compounds contained in natural plant essential oils are an excellent natural antibacterial substance, which has good microbial resistance to a variety of fungi and bacteria. At present, essential oils have been vigorously developed in preparations such as sterilization and preservation, and are widely used in food preservation and daily necessities industries. More and more researchers are looking for antibiotic substitutes from natural plants. However, as fat-soluble molecules, plant essential oils have poor water solubility, are easy to volatilize, and are easy to oxidize and degrade, resulting in low bioavailability, which seriously limits its application. Preparing plant essential oils into microemulsions can solve the problem of poor water solubility, but plant essential oil microemulsions still have the problem of poor antibacterial effect. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a disinfectant and antibacterial oral spray and a preparation method thereof. The disinfectant and antibacterial oral spray of the present invention has good antibacterial effect.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a disinfecting and antibacterial oral spray, comprising a liquid component and a solid component;

[0008] The liquid component includes the following components in parts by volume: 1 to 20 parts of PEG-40 hydrogenated castor oil, 1 to 20 parts of 1,2-propylene glycol, 0.1 to 5 parts of chamomile essential oil, 0.1 to 5 parts of lavender essential oil, 0.1 to 5 parts of peppermint essential oil, 0.1 to 5 parts of tea tree essential oil and 20 to 90 parts of water;

[0009] Based on 20 to 90 parts by volume of water, the solid component comprises the following components in parts by mass: 0.1 to 5 parts of tannic acid, 0.1 to 5 parts of L-arginine and 0.1 to 20 parts of flavoring agent.

[0010] Preferably, the volume ratio of the PEG-40 hydrogenated castor oil to 1,2-propylene glycol is 0.5 to 2:1.

[0011] Preferably, the volume ratio of the total volume of the chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil to the PEG-40 hydrogenated castor oil is 0.1-0.7:1.

[0012] Preferably, the volume ratio of the chamomile essential oil, lavender essential oil, peppermint essential oil, and tea tree essential oil is 1:1:1:1 to 1:1:4:4.

[0013] Preferably, 0.2 to 2 parts of tannic acid are included.

[0014] Preferably, it comprises 0.2 to 2 parts of L-arginine.

[0015] Preferably, the mass ratio of tannic acid to L-arginine is 1 to 10:1.

[0016] Preferably, the flavoring agent includes one or more of steviol glycosides, sorbitol and saccharin sodium.

[0017] Preferably, the disinfecting and antibacterial oral spray comprises the following components in parts by volume: 4 parts of PEG-40 hydrogenated castor oil, 4 parts of 1,2-propylene glycol, 0.4 parts of chamomile essential oil, 0.4 parts of lavender essential oil, 0.8 parts of peppermint essential oil, 0.4 parts of tea tree essential oil, and 90 parts of water; based on the mass of 90 parts by volume of water, it also includes the following components in parts by mass: 1 part of tannic acid, 0.5 parts of steviol glycosides, and 0.2 parts of L-arginine.

[0018] The present invention also provides a method for preparing the disinfecting and antibacterial oral spray described in the above technical solution, comprising the following steps:

[0019] Mixing a composite essential oil, PEG-40 hydrogenated castor oil, 1,2-propylene glycol and part of water to obtain a composite essential oil microemulsion; the composite essential oil includes chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil;

[0020] The composite essential oil microemulsion, tannic acid, L-arginine, flavoring agent and remaining water are mixed to obtain the disinfecting and antibacterial oral spray.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a disinfectant and antibacterial oral spray, comprising a liquid component and a solid component; the liquid component comprises the following components in parts by volume: 1 to 20 parts of PEG-40 hydrogenated castor oil, 1 to 20 parts of 1,2-propylene glycol, 0.1 to 5 parts of chamomile essential oil, 0.1 to 5 parts of lavender essential oil, 0.1 to 5 parts of peppermint essential oil, 0.1 to 5 parts of tea tree essential oil and 20 to 90 parts of water; based on the mass of 20 to 90 parts by volume of water, the solid component comprises the following components in parts by mass: 0.1 to 5 parts of tannic acid, 0.1 to 5 parts of L-arginine and 0.1 to 20 parts of flavoring agent. The disinfectant and antibacterial oral spray of the present invention contains tannic acid and natural plant essential oils, has no chemical synthetic substances, no preservatives, no alcohol addition, and is pure natural and non-toxic. Tannic acid and a variety of natural plant essential oils are applied to oral spray products, which can not only remove bad breath and penetrate into various parts of the oral cavity to achieve the purpose of cleaning the oral cavity, but also have disinfection, antibacterial and anti-inflammatory effects, achieving the purpose of oral health care and improving oral health. Tannic acid and a variety of plant essential oils can synergistically improve the antibacterial effect of disinfection and antibacterial oral sprays. The antibacterial zone diameter of the composite essential oil microemulsion with tannic acid added is significantly larger than the antibacterial diameter of the single tannic acid solution and the composite essential oil microemulsion.

[0023] Essential oils are fat-soluble molecules with poor water solubility, easy volatility, and easy oxidation and degradation, resulting in low bioavailability. The present invention uses a specific emulsifier to prepare plant essential oils into a clear and transparent emulsion dispersion system, which can greatly increase the stability of the system.

[0024] The present invention uses tea tree essential oil, chamomile essential oil, lavender essential oil and peppermint essential oil in combination, which can play a synergistic role in the antibacterial effect and significantly improve the antibacterial efficiency of gram-negative bacteria. At the same time, the antibacterial spectrum of several essential oils can be increased by compounding them, thereby broadening their application range.

[0025] The present invention combines tannic acid with four essential oils, namely tea tree essential oil, chamomile essential oil, lavender essential oil and peppermint essential oil, which can also play a synergistic role in the antibacterial effect. On the basis of the original good inhibition of Gram-negative bacteria, the inhibition effect on Gram-positive bacteria and fungi is significantly enhanced.

[0026] Tannic acid is a kind of polyphenol, which will form water-insoluble precipitation with cations under weak acid or weak base conditions, thereby affecting its biological activity and antibacterial effect, and also affecting the appearance and quality of the product. The present invention adds L-arginine to the composite natural plant essential oil microemulsion containing tannic acid with precipitation, so that the precipitation disappears, the system is brown-yellow, clear and transparent, and antibacterial effect detection is performed, and it is found that the microemulsion system can still maintain good antibacterial activity.

[0027] The present invention tests the effect of a composite natural plant essential oil disinfectant and antibacterial oral spray (disinfectant and antibacterial oral spray) containing tannic acid on the oral microbiome of a test population, recruits volunteers after obtaining ethical approval, collects saliva samples of the volunteers after 8 days of use for 16S rRNA sequencing, detects changes in their oral flora, and finds that the microemulsion system can inhibit the composition of specific harmful bacterial genera in the oral flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 It is the pseudo-ternary phase diagram of orthogonal experimental essential oil microemulsion;

[0030] Figure 2 Characterization of antibacterial microemulsion; A is the appearance of essential oil microemulsion prepared at different Km values; B is the picture of tannic acid-essential oil microemulsion system before and after the addition of L-arginine; C is the Malvern particle size diagram of composite essential oil microemulsion with tannin added; D is the microemulsion photo under transmission electron microscope; E and F are the particle size and PDI change trend diagrams under storage conditions of 4°C and 40°C within 8 days; G and H are the stability investigations of 4 freeze-thaw cycles and heating-thaw cycles respectively;

[0031] Figure 3 This is a graph showing the determination of the antibacterial concentration of essential oil microemulsions, where A is the determination of the antibacterial concentration of Escherichia coli (E); B is the determination of the antibacterial concentration of Staphylococcus aureus (S); and C is the determination of the antibacterial concentration of Candida albicans (Ca).

[0032] Figure 4 A is a flow chart of the preparation of the essential oil microemulsion antibacterial spray in the embodiment; B is a diagram of the antibacterial circle of the essential oil microemulsion inhibiting bacteria;

[0033] Figure 5is the OTU analysis result, where A is the Shannon-Winner curve, the horizontal axis represents the sample size, the vertical axis represents the number of sample OTUs, and B is the Rank-Abundance curve, the horizontal axis represents the rank of OTUs according to abundance level, and the vertical axis represents the relative percentage of OTUs at this level in the sequence;

[0034] Figure 6 The difference analysis of oral flora diversity and abundance in each group, where A is the PCoA diagram, Note: The shapes under different colors represent samples in different groups; the horizontal and vertical axes are relative distance scales, and the distance represents the degree of similarity between samples; B is the Alpha diversity analysis diagram; the data are expressed as mean ± SD, "*" compared with the DSS group, *P < 0.05, **P < 0.01, ***P < 0.001;

[0035] Figure 7 LEfSe analysis of oral flora;

[0036] Figure 8 This is an analysis of the impact of representative oral flora on species and genera. DETAILED DESCRIPTION

[0037] The present invention provides a disinfecting and antibacterial oral spray, comprising a liquid component and a solid component;

[0038] The liquid component includes the following components in parts by volume: 1 to 20 parts of PEG-40 hydrogenated castor oil, 1 to 20 parts of 1,2-propylene glycol, 0.1 to 5 parts of chamomile essential oil, 0.1 to 5 parts of lavender essential oil, 0.1 to 5 parts of peppermint essential oil, 0.1 to 5 parts of tea tree essential oil and 20 to 90 parts of water;

[0039] Based on 20 to 90 parts by volume of water, the solid component comprises the following components in parts by mass: 0.1 to 5 parts of tannic acid, 0.1 to 5 parts of L-arginine and 0.1 to 20 parts of flavoring agent.

[0040] In the present invention, unless otherwise specified, the materials and equipment used are commercially available products in the art.

[0041] In the present invention, the volume fraction of water in the disinfecting and antibacterial oral spray is preferably 88 to 90 parts, more preferably 90 parts.

[0042] In the present invention, the water is preferably purified water.

[0043] Based on the volume fraction of water, the volume fraction of the PEG-40 hydrogenated castor oil is preferably 2 to 10 parts, more preferably 4 parts. The PEG-40 hydrogenated castor oil is an emulsifier. Compared with RH-40 and Tween-80, the essential oil microemulsion obtained when preparing the oral spray is more stable by using the PEG-40 hydrogenated castor oil as an emulsifier.

[0044] Based on the volume fraction of water, the volume fraction of the 1,2-propylene glycol is preferably 2 to 10 parts, more preferably 4 parts. The 1,2-propylene glycol is a cosolvent and a coemulsifier. Compared with glycerol and PEG-400, the essential oil microemulsion obtained when preparing the oral spray is more stable when the 1,2-propylene glycol is used as a cosolvent and a coemulsifier in the present invention.

[0045] In the present invention, the volume ratio of the PEG-40 hydrogenated castor oil to 1,2-propylene glycol is preferably 0.5 to 2:1, more preferably 1:1.

[0046] Based on the volume fraction of water, the volume fraction of the chamomile essential oil is preferably 0.2 to 2 parts, more preferably 0.4 parts. The chamomile essential oil has anti-inflammatory, antispasmodic, antifungal, anti-allergic and other effects, and can play a combined antibacterial, anti-inflammatory and fragrance role.

[0047] Based on the volume fraction of water, the volume fraction of the lavender essential oil is preferably 0.2 to 2 parts, more preferably 0.4 parts. The lavender essential oil has good antibacterial, anti-inflammatory, and anti-anxiety effects, and can play a combined antibacterial and anxiety-relieving role.

[0048] Based on the volume fraction of water, the volume fraction of the peppermint essential oil is preferably 0.2 to 2 parts, more preferably 0.8 parts. An appropriate amount of peppermint essential oil can shrink capillaries, relieve itching, inflammation and burns, and has a strong broad-spectrum antibacterial effect, and can play a combined antibacterial and refreshing role.

[0049] Based on the volume fraction of water, the volume fraction of the tea tree essential oil is preferably 0.2 to 2 parts, more preferably 0.4 parts. Tea tree essential oil has a broad-spectrum antimicrobial effect and health-care effect. It is a natural, excellent antibacterial agent that can be used to replace antibiotics and alleviate the current situation of microbial resistance. It is non-toxic and has a good therapeutic effect on treating skin and mucous membrane infections caused by molds, bacteria and fungi, and can play a combined antibacterial and antiviral role.

[0050] In the present invention, the volume ratio of the chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil is preferably 1:1:1:1 to 1:1:4:4, more preferably 1:1:2:1. The volume ratio of the present invention has a synergistic effect on the antibacterial effect of Gram-negative bacteria Escherichia coli.

[0051] In the present invention, the volume ratio of the total volume of the chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil to the PEG-40 hydrogenated castor oil is preferably 0.1-0.7:1, more preferably 0.25-0.5:1.

[0052] Based on the mass fraction of water, the mass fraction of the tannic acid is preferably 0.2 to 2 parts, more preferably 1 part. The tannic acid is an effective antibacterial component, and can synergistically improve the antibacterial effect of the disinfecting and antibacterial oral spray when combined with essential oils.

[0053] Based on the mass fraction of water, the mass fraction of the L-arginine is preferably 0.2 to 2 parts, more preferably 0.2 parts. The L-arginine is a cosolvent, which can increase the stability of tannic acid in the microemulsion.

[0054] In the present invention, the mass ratio of tannic acid to L-arginine is preferably 1 to 10:1, more preferably 5:1.

[0055] In the present invention, the flavoring agent preferably includes one or more of steviol glycoside, sorbitol and saccharin sodium, and more preferably steviol glycoside.

[0056] Based on the mass fraction of water, when the flavoring agent is preferably stevioside, the mass fraction of the flavoring agent is preferably 0.1 to 1 part, more preferably 0.5 part; when the flavoring agent is preferably sorbitol, the mass fraction of the flavoring agent is preferably 1 to 20 parts; when the flavoring agent is preferably saccharin sodium, the mass fraction of the flavoring agent is preferably 0.1 to 0.2 parts.

[0057] In the present invention, the disinfecting and antibacterial oral spray preferably includes the following components in parts by volume: 4 parts of PEG-40 hydrogenated castor oil, 4 parts of 1,2-propylene glycol, 0.4 parts of chamomile essential oil, 0.4 parts of lavender essential oil, 0.8 parts of peppermint essential oil, 0.4 parts of tea tree essential oil, and 90 parts of water; based on the mass of 90 parts by volume of water, it also includes the following components in parts by mass: 1 part of tannic acid, 0.5 parts of stevioside, and 0.2 parts of L-arginine.

[0058] The present invention also provides a method for preparing the disinfecting and antibacterial oral spray described in the above technical solution, comprising the following steps:

[0059] Mixing a composite essential oil, PEG-40 hydrogenated castor oil, 1,2-propylene glycol and part of water to obtain a composite essential oil microemulsion; the composite essential oil includes chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil;

[0060] The composite essential oil microemulsion, tannic acid, L-arginine, flavoring agent and remaining water are mixed to obtain the disinfecting and antibacterial oral spray.

[0061] The invention mixes compound essential oil, PEG-40 hydrogenated castor oil, 1,2-propylene glycol and part of water to obtain a compound essential oil microemulsion; the compound essential oil comprises chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil.

[0062] In the present invention, the composite essential oil, PEG-40 hydrogenated castor oil and 1,2-propylene glycol are preferably first mixed to obtain a mixed solution, and then the portion of water is added. In the present invention, the portion of water is preferably added dropwise to the mixed solution while stirring, the dropping rate is preferably 2 to 10 mL / min, more preferably 5 mL / min, and the stirring speed is preferably 400 to 2500 rpm.

[0063] In the present invention, the mass percentage of the partial water to the total water is preferably 10 to 100%, more preferably 44 to 78%.

[0064] After obtaining the composite essential oil microemulsion, the present invention mixes the composite essential oil microemulsion, tannic acid, L-arginine, flavoring agent and remaining water to obtain the disinfecting and antibacterial oral spray.

[0065] In the present invention, the tannic acid is preferably used in the form of a tannic acid aqueous solution, and the concentration of the tannic acid aqueous solution is preferably 50 mg / mL.

[0066] The present invention has no special requirements on the use form of the L-arginine and flavoring agent, and both the L-arginine and flavoring agent can be in the form of powder or aqueous solution.

[0067] When the disinfecting and antibacterial oral spray of the present invention is used, the nozzle is placed as close to the oral cavity as possible and aimed at the inside of the oral cavity, and 3 to 5 sprays are applied each time, 3 to 8 times a day.

[0068] In order to further illustrate the present invention, the disinfecting and antibacterial oral spray and the preparation method thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0069] The single essential oils in the embodiments are all provided by Xinjiang Yiparhan Spice Development Co., Ltd.

[0070] Example 1

[0071] Disinfectant and antibacterial oral spray prescriptions include:

[0072] Calculated by volume: 4 parts of PEG-40 hydrogenated castor oil, 4 parts of 1,2-propylene glycol, 0.4 parts of chamomile essential oil, 0.4 parts of lavender essential oil, 0.8 parts of peppermint essential oil, 0.4 parts of tea tree essential oil and 90 parts of purified water; based on the mass of 90 parts by volume of purified water (90 parts by mass), calculated by mass: 1 part of tannic acid, 0.2 parts of L-arginine, and 0.5 parts of steviol glycoside.

[0073] Preparation method:

[0074] 1. Evenly mix the oil phase chamomile essential oil, lavender essential oil, peppermint essential oil, tea tree essential oil, PEG-40 hydrogenated castor oil and 1,2-propylene glycol, slowly drop 40 parts of purified water thereto while stirring to obtain a composite essential oil microemulsion.

[0075] When the purified water is added dropwise, the stirring rate is 400 rpm, the system viscosity is low (1 to 10 parts of water), and an oil-in-water (W / O) type microemulsion is formed. As the amount of water increases (10 to 25 parts of water), the system viscosity gradually increases, and the stirring rate gradually increases to 2500 rpm. When purified water (greater than 25 parts of water) is continued to be added dropwise, the system suddenly becomes thinner from viscous, and the stirring rate gradually decreases to 1000 rpm. At this time, the W / O type microemulsion undergoes a phase transition to form a transparent water-in-oil (O / W) type composite essential oil microemulsion.

[0076] 2. Slowly add tannic acid aqueous solution (50 mg / mL) to the prepared composite essential oil microemulsion, then add L-arginine, steviol glycoside and remaining water, continue stirring to obtain a brown-yellow transparent emulsion, which is an oral spray.

[0077] With the addition of tannic acid aqueous solution, the system began to change from clear to turbid, and a light brown precipitate appeared. After adding L-amino acid, the system became clear and transparent again.

[0078] Comparative Example 1 Preparation of four single essential oil microemulsions

[0079] The oil phase tea tree essential oil, chamomile essential oil, lavender essential oil, and peppermint essential oil (2 parts) were respectively mixed evenly with 4 parts of PEG-40 hydrogenated castor oil and 4 parts of 1,2-propylene glycol in proportion, and 90 parts of purified water were slowly added dropwise thereto while stirring to obtain one part each of tea tree essential oil, chamomile essential oil, lavender essential oil, and peppermint essential oil microemulsions.

[0080] Comparative Example 2 Preparation of composite essential oil microemulsion

[0081] 0.4 parts of oil phase chamomile essential oil, 0.4 parts of lavender essential oil, 0.8 parts of peppermint essential oil, 0.4 parts of tea tree essential oil, 4 parts of PEG-40 hydrogenated castor oil and 4 parts of 1,2-propylene glycol were uniformly mixed in proportion, and 90 parts of purified water were slowly added dropwise while stirring to obtain a composite essential oil microemulsion 1.

[0082] In Comparative Examples 1 to 2, the viscosity of the system was low at the beginning of the addition of purified water, and water-in-oil (W / O) microemulsions were formed. As the amount of water increased, the viscosity of the system gradually increased. When distilled water was continued to be added, the system suddenly became thinner from viscous. At this time, the W / O microemulsion phase transition occurred, and a transparent oil-in-water (O / W) essential oil microemulsion was formed.

[0083] Comparative Example 3 Preparation of composite essential oil microemulsions with different essential oil ratios

[0084] 0.35 parts of oil phase chamomile essential oil, 0.35 parts of lavender essential oil, 0.65 parts of peppermint essential oil, 0.65 parts of tea tree essential oil, 4 parts of PEG-40 hydrogenated castor oil, and 4 parts of 1,2-propylene glycol were uniformly mixed in proportion, and 90 parts of purified water were slowly added dropwise while stirring to obtain composite essential oil microemulsion 2.

[0085] Comparative Example 4 Preparation of composite essential oil aqueous suspension

[0086] Directly dissolve 0.4 parts of oil phase chamomile essential oil, 0.4 parts of lavender essential oil, 0.8 parts of peppermint essential oil and 0.4 parts of tea tree essential oil in 98 parts of water, stir evenly to obtain a composite essential oil water suspension.

[0087] Test Example 1 Prescription Screening of Transparent Essential Oil Microemulsion

[0088] In order to obtain a clear essential oil microemulsion with a transparent appearance, an orthogonal experiment was set up. The composite oil (the volume ratio of chamomile essential oil, lavender essential oil, peppermint essential oil, and tea tree essential oil was 1:1:2:1) was used as the oil phase, Tween-80, PEG-40 hydrogenated castor oil (CO-40) and RH-40 (polyoxyethylene hydrogenated castor oil) were used as surfactants, and glycerol, PEG-400, and 1,2-propylene glycol were used as cosolvents. The surfactants and cosolvents were mixed at a certain ratio (Km value) to form a mixed surfactant, and the Km values ​​were 0.5, 1, or 2, respectively; the essential oil microemulsions were prepared with the volume ratios of the oil phase to the mixed surfactants of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, and the microemulsion formation was observed. The pseudo-ternary phase diagram was drawn using the origin software and the area was calculated.

[0089] Experiment 1: Essential oil microemulsion was prepared with composite oil (the volume ratio of chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil was 1:1:2:1) as the oil phase, Tween-80 as the surfactant and glycerol as the cosolvent. The Km value was 0.5.

[0090] Experiment 2: The essential oil microemulsion was prepared with composite oil (the volume ratio of chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil was 1:1:2:1) as the oil phase, Tween-80 as the surfactant and PEG-400 as the cosolvent. The Km value was 1.

[0091] Experiment 3: The essential oil microemulsion was prepared with composite oil (the volume ratio of chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil was 1:1:2:1) as the oil phase, Tween-80 as the surfactant and 1,2-propylene glycol as the cosolvent. The Km value was 2.

[0092] Experiment 4: Essential oil microemulsion was prepared with composite oil (the volume ratio of chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil was 1:1:2:1) as the oil phase, PEG-40 hydrogenated castor oil as the surfactant and glycerol as the cosolvent. The Km value was 1.

[0093] Experiment 5: Essential oil microemulsion was prepared with composite oil (the volume ratio of chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil was 1:1:2:1) as the oil phase, PEG-40 hydrogenated castor oil as the surfactant and PEG-400 as the cosolvent. The Km value was 2.

[0094] Experiment 6: Essential oil microemulsion was prepared with composite oil (the volume ratio of chamomile essential oil, lavender essential oil, peppermint essential oil, and tea tree essential oil was 1:1:2:1) as the oil phase, PEG-40 hydrogenated castor oil as the surfactant, and 1,2-propylene glycol as the cosolvent. The Km value was 0.5.

[0095] Experiment 7: Essential oil microemulsion was prepared with composite oil (the volume ratio of chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil was 1:1:2:1) as the oil phase, RH-40 as the surfactant and glycerol as the cosolvent. The Km value was 2.

[0096] Experiment 8: Essential oil microemulsion was prepared with composite oil (the volume ratio of chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil was 1:1:2:1) as the oil phase, RH-40 as the surfactant and PEG-400 as the cosolvent with a Km value of 0.5.

[0097] Experiment 9: Essential oil microemulsion was prepared with composite oil (the volume ratio of chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil was 1:1:2:1) as the oil phase, RH-40 as the surfactant and 1,2-propylene glycol as the cosolvent. The Km value was 1.

[0098] Taking the area percentage of the microemulsion region in the pseudo-ternary phase diagram as the reference index, surfactant, cosolvent, Km value (volume ratio of surfactant to cosolvent) and preparation temperature as the investigation factors, a 4-factor 3-level orthogonal experiment was designed (Table 1), and the optimal preparation conditions of essential oil microemulsion were obtained through range analysis. The analysis results are shown in Table 2.

[0099] Table 1 Orthogonal experiment table

[0100]

[0101]

[0102] Table 2 Orthogonal experiment results

[0103] Experiment No. A B C D Microemulsion area 1 1 1 1 1 26.66 2 1 2 2 2 419.3 3 1 3 3 3 476.5 4 2 1 2 3 148 5 2 2 3 1 358 6 2 3 1 2 423.9 7 3 1 3 2 65.4 8 3 2 1 3 188.2 9 3 3 2 1 526.9 K1 307.467 80.000 212.900 303.833 K2 309.967 321.833 364.733 302.864 K3 260.167 475.767 299.967 270.900 Range R 49.800 395.767 151.833 32.933 Best solution A2 B3 C2 D1

[0104] From the range analysis in Table 2, we can see that R B >R C >R A >R D , the four influencing factors are in order of significance: cosolvent, Km value, surfactant, and temperature, that is, among the selected influencing factors, the cosolvent has the greatest impact on the microemulsion area, followed by the Km value, the three selected surfactants, and the temperature has the smallest impact. Therefore, the optimal solution is A2B3C2D1, that is, CO-40 is selected as the surfactant, 1,2-propylene glycol is selected as the cosolvent, the Km value is 1, and the preparation temperature is 20℃, which is the best preparation condition for the composite essential oil microemulsion, and the microemulsion is most stable at this time. Figure 1 This is the pseudo-ternary phase diagram of the orthogonal experimental essential oil microemulsion, and the black part in the figure is the microemulsion area.

[0105] According to the results of orthogonal experiments, composite oil (the volume ratio of chamomile essential oil, lavender essential oil, peppermint essential oil, and tea tree essential oil is 1:1:2:1) was used as the oil phase, PEG-40 hydrogenated castor oil was used as the surfactant, and 1,2-propylene glycol was used as the cosolvent. The surfactant and the cosolvent were mixed at Km=1 to form a mixed surfactant. The volume ratios of the oil phase and the mixed surfactant were 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, respectively, to prepare essential oil microemulsions, and the formation of microemulsions was observed. The results are as follows Figure 2 Middle A.

[0106] When the Km value is 1, a pseudo-ternary phase diagram is drawn with PEG-40 hydrogenated castor oil as a surfactant, 1,2-propylene glycol as a cosolvent, and the composite essential oil as the oil phase. When the Km value is 1 and the ratio of the composite essential oil to PEG-40 hydrogenated castor oil is 1:4, the prepared transparent microemulsion is the clearest and has the best encapsulation effect for the next experiment.

[0107] Test Example 2 Antibacterial Experiment

[0108] In vitro antibacterial comparison of the single essential oil microemulsion, compound essential oil microemulsion and aqueous suspension prepared in Comparative Examples 1 to 4

[0109] The in vitro minimum inhibitory concentration test (MIC) and minimum bactericidal concentration test (MBC) were used to examine the antibacterial ability of the test plant essential oils against Staphylococcus aureus, Escherichia coli, and Candida albicans at different concentrations. The antibacterial ability and antibacterial spectrum screening of the test substances were analyzed to provide preclinical experimental data for the study of the test essential oils and their disinfection and antibacterial oral sprays.

[0110] The antibacterial test of the single essential oil microemulsion, composite essential oil microemulsion and aqueous suspension prepared in Comparative Examples 1 to 4 on Staphylococcus aureus, Escherichia coli and Candida albicans, the results of the inhibition effect are shown in Table 3:

[0111] Table 3 Antibacterial effects of different essential oil microemulsions or suspensions

[0112]

[0113] Note: “-” in Table 3 indicates that the concentration is greater than 64 μL / mL.

[0114] The results showed that compared with the composite essential oil water suspension, the composite essential oil microemulsion had a more ideal inhibitory effect on Escherichia coli. Compared with the single essential oil microemulsions, the composite essential oil microemulsion had a stronger inhibitory effect on Escherichia coli than chamomile and lavender essential oil microemulsions, but not as good as tea tree and peppermint essential oil microemulsions. In summary, the composite essential oil group had a certain synergistic effect on the antibacterial effect of Gram-negative bacteria Escherichia coli. However, the inhibitory effect on Staphylococcus aureus and Candida albicans was not obvious.

[0115] The composite essential oil microemulsion prepared in the comparative example has an unsatisfactory inhibitory effect on gram-positive bacteria and fungi, and other antibacterial ingredients need to be added. The inventors considered adding tannic acid.

[0116] Test Example 3 Preparation of a clarified essential oil microemulsion system containing tannic acid by adding L-amino acid

[0117] According to the method of step 1 of Example 1, a 50 mg / mL composite essential oil microemulsion (the mass ratio of chamomile essential oil, lavender essential oil, peppermint essential oil, and tea tree essential oil is 1:1:2:1, the concentration of the total amount of essential oil) was prepared, and then tannic acid (concentration of 10 mg / mL) was added, and the microemulsion system became turbid; then L-arginine (concentration of 10 mg / mL) was added, and the system became transparent again, showing a brown-yellow clear color; it is speculated that the carboxyl group in the L-amino acid reacts with the phenolic hydroxyl group in the tannin, thereby increasing the stability of tannic acid in the microemulsion. Figure 2 Middle B is a picture of the tannic acid-essential oil microemulsion system before and after the improvement by adding L-arginine.

[0118] Test Example 4 Physical and Chemical Properties

[0119] Based on the microemulsion preparation process with the optimal prescription, the physicochemical properties and stability of the composite essential oil microemulsion were investigated. The physicochemical properties included appearance morphology, type identification, particle size, PDI, Zeta potential, and stability.

[0120] 1) Appearance: The appearance of the prepared sample is as follows Figure 2 As shown in Figure B, the composite essential oil microemulsion with tannic acid added is a brown-yellow transparent liquid with no precipitation or flocculation. Figure 2In the figure, D, the microemulsion particles have uniform particle size and good dispersion.

[0121] 2) Type identification: Take the same group of prepared composite essential oil microemulsions and add Sudan III dye solution and 0.1% methylene blue dye solution at the same time. Sudan III dye solution is an oil-soluble dye, while methylene blue dye solution is a water-soluble dye. Observe the dispersion speed of the two to determine the type of microemulsion. The results show that the composite essential oil microemulsion is O / W type.

[0122] 3) Particle size, PDI and Zeta potential: The particle size, polydispersity index PDI and Zeta potential of the composite essential oil microemulsion were measured three times using a Malvern nanoparticle size analyzer. Figure 2 In C, the data are shown in Table 4, showing that the average particle size is 42.86nm, the PDI is 0.194, and the Zeta potential is -9.33mV. The PDI value is an important indicator reflecting the uniformity of the microemulsion. The smaller the PDI, the better the uniformity of the system and the easier it is to maintain stability. The microemulsion prepared by the present invention has a small particle size and is evenly distributed, and the composite essential oil microemulsion has good stability.

[0123] Table 4 Particle size, PDI and Zeta potential of composite essential oil microemulsion

[0124] sample Particle size / nm PDI Zeta potential (mV) 1 42.45 0.186 -7.58 2 42.91 0.196 -11.2 3 43.22 0.199 -9.22 average value 42.86 0.194 -9.33

[0125] 4) The cold and heat resistance of microemulsion is part of the stability test, so it is necessary to conduct multiple heating and freezing cycles of the composite essential oil microemulsion with particle size and PDI as indicators. The three batches of composite essential oil microemulsion prepared were placed in a 4℃ and 40℃ constant temperature box, and their particle size and PDI were measured within 8 days. The results are as follows Figure 2 In addition, in the stability study of the heating-melting cycle, the three batches of composite essential oil microemulsions were placed in a 4°C thermostat and a 40°C thermostat, respectively, and placed at room temperature after 24 hours. After standing for 24 hours, the particle size and PDI were measured. The cycle was repeated three times. The results are as follows Figure 2 G and H in.

[0126] The experimental results show that after multiple freeze-thaw / heating-cooling cycles, the composite essential oil microemulsion has a clear and transparent appearance, without stratification, flocculation, precipitation, etc. The particle size decreased in the heating-cooling / freezing-thaw cycle experiment, but the particle size remained stable between 40±10nm, and the PDI value remained below 0.3, indicating that the composite essential oil microemulsion has good thermodynamic stability.

[0127] Test Example 5 Antibacterial experiment of essential oil microemulsion with added tannin

[0128] The minimum inhibitory concentration (MIC) refers to the lowest drug concentration that can inhibit the growth of pathogens in the culture medium after 18 to 24 hours of in vitro bacterial culture. It is an indicator for measuring the antibacterial activity of antibacterial drugs. The minimum bactericidal concentration (MBC) refers to the lowest drug concentration required to kill 99.9% (reduced by 3 orders of magnitude) of the test microorganisms.

[0129] MIC and MBC are important indicators of the resistance of antibiotics to bacteria in diagnostic laboratories. At the same time, MIC and MBC are also very important bases for verifying the effects of new antibiotics. The lower the MIC and MBC, the better the effect on bacteria.

[0130] To further verify the antibacterial effect of essential oil microemulsion with added tannin, four groups were set up, including blank group (containing only bacterial solution and culture medium), essential oil microemulsion group (EOs), tannin group (TA), and essential oil microemulsion + tannin group (EOs+TA), with three parallel groups in each group.

[0131] Prepare the sample to be tested to a concentration of 80 mg / mL. After adding 100 μL of culture medium to each well of the 96-well plate B2-G10, add 100 μL of 80 mg / mL sample to be tested to the 96-well plate B2-G2 (the concentration of the B2-G2 well is 40 mg / mL at this time) and mix well. Then aspirate 100 μL from the B2-G2 well and add it to the B3-G3 well and mix well. Then aspirate 100 μL from the B3-G3 well and add it to the B4-G4 well and mix well. And so on until the B10-G10 well. At this time, the concentrations of the sample solutions in wells 2-9 from left to right in the 96-well plate are 40 mg / mL-0.312 mg / mL from high to low.

[0132] Take the cultured bacterial solution and adjust the concentration to OD 600 =0.8~1.2 and the concentration is 1×10 9 CFU / mL, diluted to 1×10 5 CFU / mL, add 100 μL of 1×10 5 CFU / mL bacterial solution. At this time, the concentrations of the sample solutions in wells 2 to 9 in the 96-well plate from left to right are 20 mg / mL to 0.15625 mg / mL, respectively. The 10th well was not added with bacteria as a sterile control.

[0133] Bacteria grow in MH medium in a 96-well plate, and the minimum inhibitory concentration can be measured using a small amount of sample. Since large bacterial growth will produce turbidity or precipitation, it is easy to distinguish whether there is colony growth. The antibacterial preparation to be tested is diluted in multiples, and after adding bacterial suspension and bacterial culture solution for a period of time, the last well without precipitation is the MIC concentration.

[0134] After multiple dilutions in a 96-well plate, the minimum inhibitory concentration (MIC) was determined based on the clarity and absorbance values. The minimum bactericidal concentration (MBC) was determined by coating the plate. The results are shown in the table below. Figure 3 , from left to right, the concentration of the antibacterial agent is diluted in multiples, while the concentration of the bacterial solution remains unchanged. Figure 3 A in the figure is the inhibitory concentration determination of Escherichia coli (E). The bacteria in the blank group grew vigorously, while the TA group, EOs group, and TA+EOs group could effectively inhibit the growth of bacteria. Among them, the TA+EOs group had the best inhibitory effect, followed by the TA group, and the EOs group was weaker. Figure 3 B is the antibacterial concentration determination of Staphylococcus aureus (S). The bacteria in the blank group grew vigorously. The TA group, EOs group, and TA+EOs group could effectively inhibit the growth of bacteria. Among them, the TA+EOs group had the best inhibitory effect, followed by the TA group, and the EOs group was weaker. Figure 3 C in the figure is the antibacterial concentration determination of Candida albicans (Ca.). The bacteria in the blank group grew vigorously. The TA group, EOs group, and TA+EOs group could effectively inhibit the growth of bacteria. Among them, the TA+EOs group had the best inhibitory effect, followed by the EOs group, and the TA group was weaker. The antibacterial concentrations are shown in Table 5.

[0135] Table 5 Antibacterial effect of compound essential oil

[0136]

[0137] Test Example 6 Antibacterial Zone Experiment of Essential Oil Microemulsion with Added Tannin

[0138] Prepare LB and YPD liquid culture medium and solid culture medium respectively, take the frozen bacterial solution, streak the colony on the solid culture medium, wait for the colony to grow (1-2 days), pick a single colony, place it in the liquid culture medium, shake and culture (37℃, 140r / min) until the logarithmic phase (12-20h), and use a microplate reader to measure the OD 600 Adjust the bacterial concentration to OD 600 The value is in the range of 0.8 to 1.2 and the bacterial concentration is 1×10 9 CFU / mL, dilute to 1×10 7 CFU / mL and then evenly spread on a sterile solid culture dish.

[0139] After soaking the 6 mm drug-sensitive paper in the essential oil solution prepared in Comparative Examples 1 to 4 for the same time, it was placed in an agar surface dish inoculated with Escherichia coli, Staphylococcus aureus and Candida albicans. The drug solution diffused along the paper and gradually formed a concentration gradient. Bacteria that came into contact with the drug solution could not grow, so a transparent antibacterial zone appeared around the paper. The sensitivity of the bacteria to the drug solution was determined according to the size of the antibacterial zone.

[0140] The drug sensitivity inhibition zone experiment was carried out on the most preferred composite essential oil microemulsion with tannin added, tannin at the same concentration, composite essential oil microemulsion at the same concentration, and L-arginine at the same concentration, and PBS solution was set as blank. Figure 4 B in the figure is the inhibition zone diagram of the essential oil microemulsion with added tannic acid to inhibit bacteria, and the diameter of the inhibition zone is shown in Table 6.

[0141] Table 6 Antibacterial zone diameter of composite essential oil microemulsion

[0142]

[0143] In the culture dishes of Staphylococcus aureus and Escherichia coli, the diameter of the antibacterial zone of the composite essential oil microemulsion with tannin added is significantly larger than the antibacterial diameter of the tannin solution and the composite essential oil microemulsion alone, indicating that the composite essential oil microemulsion with tannin added has a significant inhibitory effect on Staphylococcus aureus and also has a certain inhibitory effect on Escherichia coli. And the inhibitory effect on both bacteria is better than that of the single component, showing a synergistic effect. The addition of tannin in the present invention can significantly improve the antibacterial effect of the essential oil microemulsion.

[0144] Test Example 7: Experiment on the influence of oral flora on human body

[0145] In order to understand the effect of tannin-added essential oil microemulsion on the oral microbiome of the subjects, chlorhexidine mouthwash, which is widely used in clinical practice, was used as the positive control group, and normal saline was used as the placebo group. Thirty subjects were recruited for 8 days and divided into 3 groups, with 10 subjects in each group. The height and weight of each subject were recorded. Before the formal experiment, each subject rinsed his mouth with a tube of 10mL sterile water for 1min, and then collected 2mL of saliva sample as the initial sample; in the formal experiment, each of the three groups of subjects will be randomly given a tube containing 10mL placebo (recorded as PBO group), tannin-added essential oil microemulsion (recorded as EOs group) or chlorhexidine mouthwash (recorded as CHX group). The subjects rinsed their mouths for 1min and could not swallow the mouthwash, once in the morning and evening, for a total of 7 days. The second batch of saliva samples of 2mL were collected at 10 am on the 7th day. For standardization, the subjects used the same toothpaste and toothbrush throughout the study, and arrived at the laboratory at 10 am and 10 pm every day, fasting overnight. In addition, the subjects will receive written instructions via email, asking them not to drink coffee or do strenuous exercise before the test. The 16S rRNA sequencing technology will be used to analyze the changes in the oral microbial flora of the subjects before and after the experiment.

[0146] After collecting saliva samples, the Pacbio Sequel third-generation sequencing platform was used to perform SMRT (Single Molecule Real-Time) sequencing on the community DNA fragments. The bam files after being downloaded from the machine were converted into FASTQ format files using CCS v 4.0.0 (Generate Highly Accurate Single-Molecule Consensus Reads (HiFi Reads)) software (setting parameters --min-length 500, --max-length 3000, and other default parameters) and saved. The samples were divided according to the barcode sequences at both ends of the sequence using a proprietary perl script, and the barcodes were removed, and the reverse complementary sequences were transposed to forward sequences according to the primer sequences.

[0147] OTU result analysis:

[0148] The reads of 16s rRNA sequencing results were directly divided into groups with a similarity of 97%. One group was one OTU. One OTU could be annotated to one species, and one species could be annotated by multiple OUTs.

[0149] The Shannon-Winner curve is constructed using the microbial diversity index of each sample at different sequencing depths, which can reflect the microbial diversity of each sample at different sequencing quantities. Figure 5 A in the middle shows that as the sample size increases, the curves of each group rise, among which the CHX-Day7 group has the smallest increase, indicating the lowest microbial diversity. The curve flattens when the sample size reaches 50,000, indicating that the amount of sequencing data is large enough to reflect the information of most microbial species in the sample.

[0150] The length of the horizontal axis of the Rank-Abundance curve can reflect the richness of species. The longer the horizontal axis, the richer the species composition. The shape of the curve can reflect the uniformity of the species composition. The flatter the curve, the higher the uniformity of the species composition. The smoother the curve drops, the higher the species diversity of the sample. A rapid and steep drop in the curve indicates that the dominant bacterial community in the sample accounts for a high proportion and the diversity is low. Figure 5 Figure B in the middle shows that the curve has a wide range on the horizontal axis and the curve declines relatively smoothly, indicating that the abundance of oral microbial species in each group is high and the distribution is relatively uniform.

[0151] In summary, the Shannon-Winner curve and Rank-Abundance curve show that the OUT results obtained by sequencing meet the requirements of sequencing analysis.

[0152] Analysis of oral flora diversity and abundance differences:

[0153] PCoA (principal co-ordinates analysis) is a visualization method for studying data similarities or differences. It reflects the relative position relationship between sample points by arranging a series of eigenvalues ​​and eigenvectors. Figure 6 Figure A shows that on day 0, the distances between the three groups in the coordinate diagram almost overlap, revealing that the three groups had similar oral flora structures on day 0. On day 7, obvious separation between the groups can be observed, revealing that after 7 days of administration, different oral flora structures appeared between the groups. Among them, the flora structure of the PBO group was still similar to that on day 0, indicating that physiological saline had almost no effect on the oral flora structure. Among the three groups, the CHX and PBO groups had the largest distance in the coordinate diagram, indicating that there was a big gap in the oral flora composition of the two groups, and the EOs group was between the CHX and PBO groups, indicating that the effect of the composite essential oil microemulsion with the addition of tannic acid on the saliva oral flora was between the CHX group and the PBO group.

[0154] Alpha diversity refers to the diversity within a specific area or ecosystem. The metrics used in this invention include Chao1 index, where a larger value indicates a higher abundance of the bacterial community; Shannon and Simpson indexes, where a larger value indicates a higher diversity of the community; and Good's coverage index, which is used to determine the coverage of each sample library. The closer its value is to 1, the more it represents the true situation of the microorganisms in the sample. The alpha diversity index is used to measure the abundance and diversity of microorganisms, and is expressed as Figure 6 As shown in Figure B, the Good's coverage index results of each group were all greater than 0.98, indicating that the data can well represent the actual situation of the microorganisms in the samples. In addition, the Chao1, Shannon, and Simpson index results show that compared with the PBO group, the CHX group significantly reduced the types and abundance of oral microorganisms and changed the diversity of oral microorganisms after 7 days of administration, while the types and abundance of oral microorganisms in the EOs group also decreased, but the degree of decrease was not as high as that in the CHX group.

[0155] LEfSe analysis of oral flora:

[0156] In the LEfSe analysis, the ordinate of the bar graph is the taxonomic units with significant differences between groups, and the abscissa uses a bar graph to intuitively display the LDA analysis logarithmic score of each taxonomic unit. Taxonomic units are sorted according to the size of the score value to describe their specificity in the sample grouping. The longer the length, the more significant the difference of the taxonomic unit, and the color of the bar graph indicates the sample group with the highest abundance corresponding to the taxonomic unit. The taxonomic branch diagram shows the taxonomic hierarchy relationship of the main taxonomic units in the sample community from phylum to genus (from inner circle to outer circle). The node size corresponds to the average relative abundance of the taxonomic unit; hollow nodes represent taxonomic units with no significant differences between groups, while nodes of other colors indicate that these taxonomic units show significant differences between groups and are more abundant in the samples represented by the group represented by the color. Letters indicate the names of taxonomic units with significant differences between groups.

[0157] like Figure 7 LEfSe analysis (LDA score>4.0) was used to determine the specific flora changes in each group before and after 7 days of administration. The results are shown in the figure. Before and after 7 days of administration, the oral flora of the PBO group changed the least. Since the subjects were required to eat a healthy and regular diet and perform daily oral care during the administration period, the abundance of a small number of Firmicutes flora increased 7 days after administration; the oral flora of the CHX group changed greatly, mainly manifested in a decrease in the abundance of some flora of the Firmicutes phylum, and an increase in the abundance of some flora of the Proteobacteria and Flavobacteria phylum; the oral flora of the EOs group showed a decrease in the abundance of some flora of the Proteobacteria phylum and an increase in the abundance of some flora of the Firmicutes phylum before and after 7 days of administration.

[0158] Analysis of the impact of representative oral flora by species and genus:

[0159] Figure 8(a) to (c) are beneficial oral bacteria that changed before and after administration, namely Blautia wexlerae, Streptococcus thermophilus and Streptococcus mitis. There was no significant change in the abundance of Blautia wexlerae in the PBO group before and after 7 days of administration, while the abundance of Streptococcus thermophilus and Streptococcus mitis increased slightly. There was no significant change in the abundance of Blautia wexlerae in the CHX group, while the abundance of Streptococcus thermophilus and Streptococcus mitis decreased significantly. The abundance of the three bacteria in the EOs group increased significantly. Among them, (a) is Blautia wexlerae in the human oral cavity, which is generally considered to have the potential to improve host health and prevent certain diseases. It helps to alleviate inflammatory diseases, inhibit pathogen colonization, improve obesity and type 2 diabetes, and improve lipid metabolism through the production of short-chain fatty acids (SCFAs) and other metabolites, such as acetate. (b) and (c) are Streptococcus thermophilus and Streptococcus mitis in the human oral cavity, respectively. They help maintain oral health and prevent certain diseases, and have multiple beneficial effects in the human oral cavity.

[0160] Figure 8(d) to (f) are oral pathogenic bacteria related to oral diseases that have changed before and after administration, namely Prevotella, Veillonella and Pasteurella multocida. There was no significant change in the abundance of the three bacteria in the PBO group before and after 7 days of administration, while the abundance of the three bacteria in the CHX and EOs groups decreased significantly, and the abundance of the CHX group decreased more significantly. (d) and (e) are Prevotella and Veillonella in the human oral cavity, respectively. Prevotella has multiple pathogenicity in the oral cavity, and some species of Prevotella are closely related to the occurrence and development of periodontal disease, dental caries, pulp infection, oral inflammation, etc. Veillonella has a symbiotic relationship with Streptococcus mutans, the main cariogenic bacteria in the oral cavity. It helps the adhesion of Streptococcus mutans and can decompose the lactic acid produced by the metabolism of streptococci, thereby promoting the formation of caries. Veillonella provides adhesion sites for Porphyromonas gingivalis and can participate in the occurrence and development of periodontal disease by promoting immune response. (f) Pasteurella multocida in the oral cavity can cause local infections in the oral cavity, such as periodontitis and oral mucosal infections, and may also enter the blood circulation and cause systemic infections, such as bacteremia, endocarditis, liver abscesses, etc.

[0161] Figure 8 (g) to (i) are three conditional pathogens detected in the oral cavity, namely Neisseria weaveri, Flavobacterium nucleatum, and Granulicatellaadiacens. It can be found that there was no significant change in the abundance of the PBO group and the EOs group before and after 7 days of administration, while the abundance of these three conditional pathogens in the CHX group increased significantly before and after administration. They are considered to be part of the normal flora, but they may also become conditional pathogens under certain conditions. In some cases, Neisseria weaveri may be related to oral diseases, such as periodontal disease and dental caries, and may also cause systemic infection when the bacteria enter the blood circulation. Granulicatellaadiacens is most famous for its pathogenicity, which can cause bacterial endocarditis (IE). Similarly, Flavobacterium nucleatum may become a pathogenic bacterium under reduced host immunity or other susceptible conditions, causing oral diseases such as periodontal disease and dental caries.

[0162] In summary, from the results, both the CHX group and the EOs group have a good inhibitory effect on oral pathogenic bacteria. Although the CHX group has a slightly higher degree of inhibition on oral pathogens than the EOs group, CHX will greatly change the composition of oral flora, resulting in a decrease in the abundance of some beneficial oral bacteria, while greatly increasing the abundance of conditional pathogens, which can easily cause infection risks. This shows that without subverting the composition of oral microorganisms, EOs can increase the abundance of beneficial oral flora and reduce the abundance of harmful flora. In summary, EOs treatment can reduce the abundance of oral pathogenic flora while increasing the abundance of beneficial oral flora. While inhibiting oral pathogens, it will not greatly subvert the composition of oral flora, and can avoid the risk of oral dysbiosis.

[0163] Although the above-mentioned embodiments have made a detailed description of the present invention, they are only some embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on the embodiments of the present invention without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A disinfecting and antibacterial oral spray, characterized in that: including a liquid component and a solid component; The liquid component includes the following components in parts by volume: 1 to 20 parts of PEG-40 hydrogenated castor oil, 1 to 20 parts of 1,2-propylene glycol, 0.1 to 5 parts of chamomile essential oil, 0.1 to 5 parts of lavender essential oil, 0.1 to 5 parts of peppermint essential oil, 0.1 to 5 parts of tea tree essential oil and 20 to 90 parts of water; Based on 20 to 90 parts by volume of water, the solid component comprises the following components in parts by mass: 0.1 to 5 parts of tannic acid, 0.1 to 5 parts of L-arginine and 0.1 to 20 parts of flavoring agent.

2. The disinfecting and antibacterial oral spray according to claim 1, characterized in that: The volume ratio of the PEG-40 hydrogenated castor oil to 1,2-propylene glycol is 0.5-2:

1.

3. The disinfecting and antibacterial oral spray according to claim 1 or 2, characterized in that: The volume ratio of the total volume of the chamomile essential oil, the lavender essential oil, the peppermint essential oil and the tea tree essential oil to the PEG-40 hydrogenated castor oil is 0.1-0.7:

1.

4. The disinfecting and antibacterial oral spray according to claim 1, characterized in that: The volume ratio of the chamomile essential oil, the lavender essential oil, the peppermint essential oil and the tea tree essential oil is 1:1:1:1 to 1:1:4:

4.

5. The disinfecting and antibacterial oral spray according to claim 1, characterized in that: Includes 0.2 to 2 parts of tannic acid.

6. The disinfecting and antibacterial oral spray according to claim 1, characterized in that: Includes 0.2 to 2 portions of L-arginine.

7. The disinfecting and antibacterial oral spray according to claim 1, characterized in that: The mass ratio of the tannic acid to L-arginine is 1 to 10:

1.

8. The disinfecting and antibacterial oral spray according to claim 1, characterized in that: The flavoring agent includes one or more of steviol glycoside, sorbitol and saccharin sodium.

9. The disinfecting and antibacterial oral spray according to claim 1, characterized in that: The disinfecting and antibacterial oral spray comprises the following components in parts by volume: 4 parts of PEG-40 hydrogenated castor oil, 4 parts of 1,2-propylene glycol, 0.4 parts of chamomile essential oil, 0.4 parts of lavender essential oil, 0.8 parts of peppermint essential oil, 0.4 parts of tea tree essential oil, and 90 parts of water; based on the mass of 90 parts by volume of water, the spray also comprises the following components in parts by mass: 1 part of tannic acid, 0.5 parts of stevioside, and 0.2 parts of L-arginine.

10. The method for preparing the disinfecting and antibacterial oral spray according to any one of claims 1 to 9, characterized in that: The following steps are involved: Mixing a composite essential oil, PEG-40 hydrogenated castor oil, 1,2-propylene glycol and part of water to obtain a composite essential oil microemulsion; the composite essential oil includes chamomile essential oil, lavender essential oil, peppermint essential oil and tea tree essential oil; The composite essential oil microemulsion, tannic acid, L-arginine, flavoring agent and remaining water are mixed to obtain the disinfecting and antibacterial oral spray.

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