Composition for inhibiting Porphyromonas gingivalis and preparation method and application thereof

Abstract

The present invention provides a composition for inhibiting Porphyromonas gingivalis, and its preparation method and application, and the present invention belongs to the field of pet oral cleaning technology. The composition is composed of the following components in mass percentage: egg white lysozyme 0.1-2%, glucanase 0.1-2%, propolis extract 0.1-1%, persimmon extract 0.2-4%, sodium tripolyphosphate 0.2-4%, calcium glycerophosphate 0.1-1%, erythritol 0.5-5%, sorbitol 2-10%, vitamin C 0.2-3%, zinc gluconate 0.2-0.5%, sanguinarine 0.1-1%, PEG-40 castor oil 0.5-3%, peppermint oil 0.1-1%, propylene glycol 1-10%, glycerol 2-20%, and the balance is water. The composition prepared by the present invention has an inhibitory effect on the growth of Porphyromonas gingivalis and the formation of its biofilm, and also avoids the problem of oral microecological disorder.

Description

Technical Field

[0001] The invention relates to the technical field of pet oral cleaning, and in particular to a composition for inhibiting Porphyromonas gingivalis, and a preparation method and application thereof. Background Art

[0002] Oral disease is a common disease in pet clinics. Among dogs and cats aged 2 years and above, 80% of dogs and 70% of cats suffer from various types of periodontitis, which may cause pets to have symptoms such as bad breath, drooling, pain, and anorexia, seriously affecting the quality of life of animals.

[0003] Periodontitis is mainly caused by toxins and degradation products produced by pathogenic microorganisms in the periodontal pocket. Among them, Porphyromonas gingivalis is recognized as one of the important pathogens that cause periodontitis. Studies have reported that Porphyromonas is most enriched on the surface of pet teeth and in the periodontal pockets of periodontitis pets, and the biofilm formed by its attachment is an important cause of pet dental plaque formation. Porphyromonas gingivalis can produce endotoxin lipopolysaccharide, pili, gingipain, protease, and bacterial metabolites, which can cause immune damage and inflammatory response in the periapical tissue and destroy the cells of the periodontal tissue. Inhibition of Porphyromonas gingivalis is an important target for alleviating pet periodontitis.

[0004] Currently, pet oral cleaning products on the market mainly inhibit bacteria such as Staphylococcus aureus, Escherichia coli, and Candida albicans, rather than Porphyromonas gingivalis, the main pathogen of pet periodontitis. Some products inhibit the growth of oral bacteria through chemical antibacterial substances, which can easily damage the oral bacterial microecology, causing dysfunction of the normal function of the oral flora, and cannot prevent the occurrence of periodontitis in a targeted manner.

[0005] Therefore, developing an antibacterial spray based on natural ingredients can not only specifically inhibit the reproduction of Porphyromonas gingivalis, but also inhibit the formation of Porphyromonas gingivalis biofilm, and does not contain chemically synthesized antibacterial substances to ensure the health and safety of pets, which has important market value and social significance. Summary of the invention

[0006] The purpose of the present invention is to provide a composition for inhibiting Porphyromonas gingivalis, a preparation method and application thereof, so as to solve the technical problem that existing products do not target Porphyromonas gingivalis, an important pathogenic bacterium of periodontitis, and cannot fundamentally solve the occurrence of periodontitis.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides a composition for inhibiting Porphyromonas gingivalis. The composition is composed of the following components in percentage by mass: 0.1-2% of egg white lysozyme, 0.1-2% of glucanase, 0.1-1% of propolis extract, 0.2-4% of persimmon extract, 0.2-4% of sodium tripolyphosphate, 0.1-1% of calcium glycerophosphate, 0.5-5% of erythritol, 2-10% of sorbitol, 0.2-3% of vitamin C, 0.2-0.5% of zinc gluconate, 0.1-1% of sanguinarine, 0.5-3% of PEG-40 castor oil, 0.1-1% of peppermint oil, 1-10% of propylene glycol, 2-20% of glycerol, and the balance is water.

[0009] Preferably, the enzymatic activity of the hen's egg white lysozyme is not less than 20,000 U / mg.

[0010] Preferably, the purity of the glucanase is not less than 100KDU-A / g.

[0011] The present invention also provides a method for preparing the above composition, comprising the following steps:

[0012] (1) mixing egg white lysozyme, glucanase, persimmon extract, sodium tripolyphosphate, calcium glycerophosphate, erythritol, sorbitol, vitamin C, zinc gluconate, sanguinarine with part of water, and then mixing with glycerol to obtain component 1;

[0013] (2) adding propylene glycol to the propolis extract, subjecting the mixture to ultrasonic treatment, and then mixing the mixture with PEG-40 castor oil and peppermint oil to obtain component 2;

[0014] (3) Component 1 and component 2 are mixed, and the remaining water is added. After filtering and sterilization, a composition for inhibiting Porphyromonas gingivalis is obtained.

[0015] Preferably, in step (1), the temperature when mixing with water is 20-30° C., the speed is 800-1200 rpm, and the time is 8-12 min; the temperature when mixing with glycerol is 20-30° C., the speed is 800-1200 rpm, and the time is 8-12 min.

[0016] Preferably, in the step (2), the temperature of the ultrasonic treatment is 35-40°C, the power is 180-220W, and the time is 3-7min; the temperature of the mixing is 35-40°C, the speed is 1800-2200rpm, and the time is 8-12min.

[0017] Preferably, the mixing temperature in step (3) is 22-27° C., the mixing rate is 2800-3200 rpm, and the mixing time is 8-12 min; and the pore size of the filter membrane used for the filtration sterilization is 0.22 μm.

[0018] The present invention also provides an application of the above combination or the combination prepared by the above preparation method in preparing a product for inhibiting Porphyromonas gingivalis.

[0019] Preferably, the product comprises a pet oral spray and a pet mouthwash.

[0020] The composition for inhibiting Porphyromonas gingivalis provided by the present invention has the following beneficial effects:

[0021] 1. The egg white lysozyme added in the present invention can hydrolyze the β-1,4-glycosidic bonds of peptidoglycan, decompose the cell wall protein layer, reduce the mechanical strength of the bacterial cell wall, and ultimately lead to bacterial death; glucanase can effectively catalyze the hydrolysis of glucan produced by oral pathogens into glucose, thereby inhibiting the formation of dental plaque. The combined use of the two can improve the inhibitory effect on pet periodontitis pathogens Porphyromonas gingivalis and biofilm formation.

[0022] 2. The present invention utilizes the synergistic effect of persimmon extract and hen egg white lysozyme. The hydrogen bonds and van der Waals forces between the persimmon extract and hen egg white lysozyme can form a stable structure, which can maintain the activity of hen egg white lysozyme and enable it to exert a long-lasting antibacterial effect.

[0023] 3. The present invention extracts the effective components of propolis, increases the solubility of propolis and reduces impurities such as wax, and does not require ethanol dissolution, making the product components purer and the system purer and more transparent.

[0024] 4. The sanguinarine added in the present invention is a natural plant antibacterial agent, which avoids the generation of drug resistance of pathogenic bacteria and is safe and highly effective in inhibiting oral pathogens in pets.

[0025] 5. The addition of calcium glycerophosphate in the present invention has the effect of antibacterial and anti-caries, can inhibit the demineralization of pet tooth enamel, change the acidity and alkalinity of the enamel surface, enhance its acid resistance, and play a role in protecting and repairing pet teeth. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram of the difference analysis of oral flora in Experimental Example 2;

[0027] Figure 2 This is the experimental diagram of the inhibition zone of Test Example 3;

[0028] Figure 3 This is the histogram of the diameter of the inhibition zone of Test Example 3;

[0029] Figure 4 The bar graph of the number of viable bacteria in Test Example 4;

[0030] Figure 5 This is a scanning electron microscope image of the bacteria in Experimental Example 5;

[0031] Figure 6 This is a scanning electron microscope image of the simulated tooth segment of Test Example 6. DETAILED DESCRIPTION

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example

[0034] A method for preparing an oral spray for inhibiting Porphyromonas gingivalis, comprising the following steps:

[0035] (1) Egg white lysozyme, glucanase, persimmon extract (purchased from Daqin Plant Essence Co., Ltd.), sodium tripolyphosphate, calcium glycerophosphate, erythritol, sorbitol, vitamin C, zinc gluconate, and sanguinarine were added to ultrapure water in sequence, stirred at 1000 rpm for 10 min at 25° C., and then glycerol was added and stirred at 1000 rpm for 10 min at 25° C. to obtain component 1.

[0036] (2) The propolis was crushed in a Chinese medicine grinder, and the propolis powder was collected through a 200-mesh sieve. Three volumes of 30% ethanol were added, and the mixture was stirred and extracted at a constant temperature of 37°C for 6 hours. The residue was filtered and the residue was retained. Six volumes of 70% ethanol were added, and the mixture was ultrasonically extracted at a power of 200W for 2 hours. The mixture was then stirred and extracted at a constant temperature of 50°C for 40 hours. The supernatant was filtered and the mixture was allowed to stand at 4°C for 12 hours. The supernatant was filtered and removed by suction, and the mixture was de-alcoholized under reduced pressure, and the mixture was allowed to stand for 12 hours. The mixture was centrifuged at 4500 rpm for 15 minutes to remove the precipitate. Three volumes of 95% ethanol were added to the precipitate, and the mixture was stirred and extracted at a constant temperature of 37°C for 36 hours. The supernatant was filtered and removed by suction. The mixture was allowed to stand at 4°C for 12 hours. The supernatant was filtered and removed by suction. The mixture was concentrated under reduced pressure and dried to obtain an alcohol-free water-soluble propolis extract.

[0037] (3) adding the propolis extract to propylene glycol, dissolving it by ultrasonication at 200 W power for 5 min at 37° C., adding PEG-40 castor oil and peppermint oil, stirring at 2000 rpm for 10 min at 37° C. to mix well, thereby obtaining component 2;

[0038] (4) Component 1 and component 2 are mixed, and finally ultrapure water is added to make up to 100 parts, and the mixture is mixed at a high speed of 3000 rpm at 25° C. for 10 minutes. The mixture is then sterilized by a sterile filtration system with a 0.22 μm filter membrane to obtain a composition for inhibiting Porphyromonas gingivalis, which is then dispensed into a sterile spray bottle to prepare an oral spray for inhibiting Porphyromonas gingivalis.

[0039] The amounts of the components used in the above preparation process are shown in Table 1 below, and Examples 1 to 5 were prepared respectively.

[0040] Table 1 Formulation composition of the oral spray of Examples 1 to 5 (%)

[0041]

[0042]

[0043] Test Example 1 Product Appearance and pH Measurement

[0044] Comparative Example 1 is a commercially available VK pet mouthwash, the main active ingredients of which are erythritol, inulin and pomegranate extract.

[0045] The product appearance and pH values ​​of the products of Examples 1 to 5 and Comparative Example 1 were measured and the results are shown in Table 2:

[0046] Table 2 Appearance and pH test results of Examples 1 to 4 and Comparative Example 1

[0047] Appearance pH Example 1 Clarity and transparency 6.4 Example 2 Clarity and transparency 6.5 Example 3 Clarity and transparency 6.7 Example 4 Clarity and transparency 6.5 Example 5 Clarity and transparency 6.6 Comparative Example 1 Clarity and transparency 6.5

[0048] As can be seen from Table 2, the oral sprays prepared in Examples 1 to 5 of the present application are clear and transparent in appearance, and their pH is close to 6.5, which meets the pH requirements for oral sprays in the product standards.

[0049] Experimental Example 2: Analysis of the Differences in Oral Microflora between Pet Cats with Good and Poor Oral Health

[0050] Oral swabs were taken from the periodontium of 7 pet cats kept in catteries and sent to Meiji Bio for 16s sequencing. The pets were tested for halitosis (the breath value detected by ShowNovo breath meter was greater than 0.5ppm) to assess periodontal health. Cats with halitosis values ​​≥ 0.5ppm were defined as the poor oral health group (4 cats), and cats with halitosis values ​​< 0.5ppm were defined as the good oral health group (3 cats). The Student's T test was used to analyze the differences in oral flora between the two groups at the genus level.

[0051] The differences in oral flora between the two groups at the genus level are shown in Figure 1 As shown, Porphyromonas is the genus with the highest proportion in the oral cavity of pet cats in the poor oral health group, accounting for up to 34.5%, while the oral health group is only 11.63%, and there is a significant difference between the two. It can be seen that Porphyromonas is a signature bacterium for pet oral health, and the lower its content, the healthier the oral cavity. It is scientific to subsequently select the standard strain Porphyromonas gingivalis to evaluate the effect of the present invention on pet oral health.

[0052] Test Example 3: Inhibition Zone Experiment

[0053] Bacterial culture medium preparation

[0054] Preparation of BHI blood plate (100mL): 3.6g BHI medium powder + 1.5g agar powder (both purchased from Haibo Biotechnology), add 100mL water to dissolve and sterilize at 121℃ for 15min. When the temperature drops to a level that is not too hot to touch, add 5mL 5% sterile defibrinated sheep blood, 500μL hemin chloride, 500μL vitamin K1, mix well and pour on the plate.

[0055] Preparation of BHI liquid culture medium (100 mL): 3.6 g of BHI culture medium powder was dissolved in 100 mL of water and dispensed into screw-capped anaerobic culture tubes in an amount of 10 ml / tube. After filling with anaerobic gas (a mixture of 80% N2, 10% H2 and 10% CO2), the tube was sterilized at 121°C for 15 min. When the temperature dropped to a level that was not too hot to touch, 0.5 mL of 5% sterile defibrinated sheep blood, 50 μL of hemin chloride, and 50 μL of vitamin K1 were added to each tube.

[0056] Bacterial culture: The primary revived Porphyromonas gingivalis (strain number ATCC BAA-308, purchased from Guangdong Microbial Culture Collection Center) was transferred to BHI liquid medium and cultured at 37°C under anaerobic conditions (80% N2, 10% H2 and 10% CO2) for 48 h. After three generations, the strain reached a stable state and was used for subsequent experiments.

[0057] Determination of the diameter of the inhibition zone: The antibacterial activity of the oral sprays prepared in different embodiments against Porphyromonas gingivalis was determined by the punching method. In a sterile operating table, the bacterial culture solution was diluted to 10 with physiological saline. 7 CFU / mL, take 100 μL of diluted bacterial solution on BHI blood plate, use sterile coating rod to spread evenly, and then use sterile puncher to make round holes evenly on the plate. Take 100 μL of each of the five examples and comparative example 1, and inject them into the corresponding marked holes respectively. At the same time, take an equal amount of sterile saline as blank control. Place the culture dish at 37℃ for 48h, observe and measure the diameter of the inhibition zone, and the results are as follows Figure 2 The experiment was repeated three times independently, and the average value was taken as the measurement result. Figure 3 shown.

[0058] The size of the inhibition zone represents the ability of the antibacterial substance to inhibit specific bacteria. Figure 3 It can be seen from the results of the inhibition zone diameter that, compared with Comparative Example 1, the inhibition zone diameters of Example 1, Example 2 and Example 3 are significantly higher than that of Comparative Example 1, indicating that the present invention has a significant in vitro inhibitory effect on Porphyromonas gingivalis and is superior to similar products on the market, among which the antibacterial activity of Example 1 is the most prominent.

[0059] In order to explore the effect of persimmon extract content on the antibacterial effect, Examples 1, 4, and 5 were set to have the same egg white lysozyme content (2%), while the persimmon extract content decreased. The results showed that the size of the inhibition zone also decreased, indicating that the coordinated effect of persimmon extract and lysozyme promoted the antibacterial effect.

[0060] Test Example 4: Live Bacteria Count Experiment

[0061] Take 10ml of the oral spray of Examples 1 to 3 and Comparative Example 1, take 10ml of distilled water as the control group, add 3.6g of BHI culture medium powder, 0.5mL of 5% sterile defibrinated sheep blood, 50μL of hemin chloride, and 50μL of vitamin K1, dissolve and filter with a 0.22μm filter membrane to sterilize and prepare a liquid culture medium for standby use. In each culture medium, 2% volume fraction of activated Porphyromonas gingivalis is inoculated and cultured in a screw-mouth anaerobic tube at 37°C for 48h. Take the cultured bacterial solution, apply it to the BHI blood plate by the plate dilution coating method, and invert the blood plate at 37°C anaerobic conditions (80% N2, 10% H2 and 10% CO2) for 24h, observe and count the number of live bacteria. The experiment was repeated three times independently, and the counting principle was based on the national standard GB4789.2-2016. The colony count was selected between 30CFU and 300CFU for colony counting, and the results were expressed as log 10 CFU / mL, such as Figure 4 The antibacterial rate of each embodiment and comparative example against Porphyromonas gingivalis was calculated using the following formula: antibacterial rate = (number of live bacteria in the blank control - number of live bacteria in this group) / number of live bacteria in the blank control × 100%. The results are shown in Table 3.

[0062] Table 3 Antibacterial rate of Examples 1 to 3 and Comparative Example 1 against Porphyromonas gingivalis

[0063] Antibacterial rate Example 1 99.5％ Example 2 92.8％ Example 3 84％ Comparative Example 1 60％

[0064] Depend on Figure 4 It can be seen that the number of viable bacteria in the blank control group, Example 1, Example 2, Example 3 and Comparative Example 1 was 7.5×10 7 CFU / mL, 3.9×10 6 CFU / mL, 5.4×10 6 CFU / mL, 1.2×10 7 CFU / mL and 3.0×10 7CFU / mL; As can be seen from Table 3, the antibacterial rates of Example 1, Example 2, and Example 3 are 99.5%, 92.8%, and 84%, respectively, while the antibacterial effect of Comparative Example 1 is very limited, with an antibacterial rate of only 60%. The inhibitory effects of Examples 1, 2, and 3 of the present invention on live bacteria are significantly better than those of Comparative Example 1. The above results show that the present invention can effectively inhibit the growth of Porphyromonas gingivalis, and can reduce the relative abundance of Porphyromonas gingivalis in the oral flora when used in pets.

[0065] Experimental Example 5 Bacteria Scanning Electron Microscope

[0066] Porphyromonas gingivalis cultured to the logarithmic phase in ordinary BHI liquid culture medium was inoculated at an inoculation ratio of 2% in ordinary sterile water (blank control), Example 1 and Comparative Example 1 BHI culture medium configured as a liquid matrix, and cultured in a screw-mouth anaerobic jar at 37°C for 48h. After culture, 1mL of culture solution was taken, centrifuged at 4500g for 15min, the bacteria were collected, fixed with 2.5% glutaraldehyde at 4°C in the dark for 12h, and the samples were dehydrated with different concentrations of ethanol aqueous solution at a concentration gradient of 30%, 50%, 70%, 80% and 90%, and then dehydrated twice in 100% ethanol, and then replaced once with ethanol: tert-butanol (2:1, 1:1) and 100% tert-butanol respectively, and finally the bacterial tert-butanol suspension was aspirated and dropped on aluminum foil, pre-cooled at -80°C, and then placed in a freeze dryer for freeze drying. The dried sample was fixed on aluminum foil and sprayed with gold, and the bacterial morphology was observed with a scanning electron microscope and photographed. The results are as follows Figure 5 shown.

[0067] Depend on Figure 5 It can be seen that compared with the blank control group, the bacterial cells in the comparative example 1 partially underwent morphological changes, while the bacterial cells in the example 1 group underwent obvious morphological changes as a whole. The cell membrane surface of Porphyromonas gingivalis in the blank control group was neat, smooth, dense, and the chain structure was complete. A small part of the cells in the comparative example 1 group was damaged and broken; however, all the bacterial cells in the example 1 group showed obvious abnormal cell membrane shape, which became irregular, broken and wrinkled, and there was no complete cell. The degree of cell damage was very obvious, which was consistent with the results of the inhibition zone test and the viable bacteria count test.

[0068] It can be seen from the above examples that the composition for inhibiting Porphyromonas gingivalis provided by the present invention can exert an antibacterial effect by destroying the bacterial structure of Porphyromonas gingivalis, inhibiting the growth and reproduction of Porphyromonas gingivalis, thereby alleviating pet periodontitis caused by Porphyromonas gingivalis.

[0069] Experimental Example 6 Bacteria Scanning Electron Microscope

[0070] Preparation and construction of simulated pet tooth slices:

[0071] Preparation of potassium chloride buffer: 1mmol / L potassium dihydrogen phosphate, 0.05mol / L potassium chloride, 0.1mmol / L magnesium chloride, 1mmol / L calcium chloride, 1mmol / L potassium dihydrogen phosphate, pH=6.0.

[0072] Preparation of collagen solution: Dilute the bovine type I collagen solution with a mass concentration of 20 g / L with distilled water to a mass concentration of 250 mg / L, and store it in a 4°C refrigerator for later use.

[0073] The HA disc (purchased from Kunshan Menghua Technology Co., Ltd.) that had been disinfected and soaked in potassium chloride buffer overnight was immersed in a bovine type I collagen solution for 1 hour to form a collagen-coated hydroxyapatite disc on its surface. It was then immersed in bovine serum albumin-potassium chloride buffer and allowed to stand for 0.5 hours before being washed three times with potassium chloride buffer and dried to make a simulated pet tooth disc.

[0074] Construction of Porphyromonas gingivalis biofilm: Take 10 ml of Example 1, Comparative Example 1, and distilled water (control group), add 3.6 g of BHI culture medium powder, 0.5 mL of 5% sterile defibrinated sheep blood, 50 μL of hemin chloride, and 50 μL of vitamin K1, dissolve and filter with a 0.22 μm filter membrane to sterilize and prepare BHI liquid culture medium for standby use.

[0075] The prepared simulated tooth pieces were placed in a 24-well plate, 500 μL of BHI liquid culture medium containing the example, comparative example, and blank control were added to each well, and 2% volume fraction of activated Porphyromonas gingivalis was inoculated respectively, and cultured at 37°C in an anaerobic environment for 48 hours to form a Porphyromonas gingivalis biofilm on the surface of the simulated tooth pieces.

[0076] After the culture was completed, the simulated tooth piece containing the Porphyromonas gingivalis biofilm was taken out, fixed with 1 ml of 2.5% glutaraldehyde at 4°C in the dark for 12 hours, and dehydrated with different concentrations of ethanol aqueous solution at a concentration gradient of 30%, 50%, 70%, 80% and 90%. The sample was dehydrated twice in 100% ethanol, and then replaced once with ethanol: tert-butanol (2:1, 1:1) and 100% tert-butanol, respectively. The simulated tooth piece was placed on aluminum foil, pre-cooled at -80°C, and then placed in a freeze dryer for freeze drying. Finally, the dried sample was fixed on aluminum foil and sprayed with gold. The morphology of the Porphyromonas gingivalis biofilm on the surface of the simulated tooth piece was observed with a scanning electron microscope and photographed. The results are as follows: Figure 6 shown.

[0077] Depend on Figure 6The results show that after the blank control group was inoculated with Porphyromonas gingivalis, a Porphyromonas gingivalis biofilm was successfully constructed, and more bacteria were attached to the surface of the simulated pet tooth piece; while the amount of Porphyromonas gingivalis attached to the commercially available product comparative example 1 was reduced, indicating that the comparative example had a certain effect in inhibiting the formation of bacterial biofilm; after the bacteria were treated in Example 1, their attachment to the simulated tooth piece was significantly reduced, and the bacteria showed wrinkled deformation, indicating that Example 1 can significantly reduce the formation of biofilm by Porphyromonas gingivalis on the surface of the simulated pet tooth piece, and the effect is better than that of commercially available products. From the above results, it can be seen that the formula of Example 1 can inhibit the adhesion of Porphyromonas gingivalis on the tooth surface, inhibit the formation of bacterial biofilm, and alleviate the formation of dental plaque by Porphyromonas gingivalis, thereby exerting the effect of relieving pet periodontitis.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition for inhibiting Porphyromonas gingivalis, characterized in that: The invention is composed of the following components in percentage by mass: 0.1-2% of egg white lysozyme, 0.1-2% of glucanase, 0.1-1% of propolis extract, 0.2-4% of persimmon extract, 0.2-4% of sodium tripolyphosphate, 0.1-1% of calcium glycerophosphate, 0.5-5% of erythritol, 2-10% of sorbitol, 0.2-3% of vitamin C, 0.2-0.5% of zinc gluconate, 0.1-1% of sanguinarine, 0.5-3% of PEG-40 castor oil, 0.1-1% of peppermint oil, 1-10% of propylene glycol, 2-20% of glycerol, and the balance is water; The enzyme activity of the egg white lysozyme is not less than 20000U / mg; The purity of the glucanase is not less than 100KDU-A / g; The preparation method of the propolis extract is: (1) crushing propolis and passing it through a 200-mesh sieve to obtain propolis powder; (2) adding 3 to 4 times the volume of 30 to 35% ethanol aqueous solution to the propolis powder, extracting at 35 to 40° C. for 5 to 6 hours, filtering and collecting the residue; (3) adding 6 to 7 times the volume of the residue to an ethanol aqueous solution with a concentration of 70 to 75%, ultrasonically treating the residue at a power of 200 to 220 W for 2 to 3 hours, extracting the residue at 50 to 55° C. for 40 to 45 hours, filtering the residue, and obtaining the supernatant; (4) the supernatant is allowed to stand at 4-5° C. for 10-12 hours, the supernatant is filtered out and de-alcoholized under reduced pressure, and after standing for 10-12 hours, the supernatant is centrifuged at 4500 rpm for 15-20 minutes to obtain a precipitate; (5) Add 3 to 4 times the volume of 90-95% ethanol aqueous solution to the precipitate, extract at 35-40° C. for 35-38 hours, filter out the supernatant, let it stand at 4-5° C. for 10-12 hours, filter out the supernatant, concentrate under reduced pressure and dry to obtain the propolis extract.

2. A method for preparing the composition according to claim 1, characterized in that: The steps include: (1) mixing egg white lysozyme, glucanase, persimmon extract, sodium tripolyphosphate, calcium glycerophosphate, erythritol, sorbitol, vitamin C, zinc gluconate, sanguinarine with part of water, and then mixing with glycerol to obtain component 1; (2) adding propylene glycol to the propolis extract, subjecting the mixture to ultrasonic treatment, and then mixing the mixture with PEG-40 castor oil and peppermint oil to obtain component 2; (3) Component 1 and component 2 are mixed, and the remaining water is added. After filtering and sterilization, a composition for inhibiting Porphyromonas gingivalis is obtained.

3. The preparation method according to claim 2, characterized in that: In step (1), the temperature when mixing with water is 20-30° C., the speed is 800-1200 rpm, and the time is 8-12 min; the temperature when mixing with glycerol is 20-30° C., the speed is 800-1200 rpm, and the time is 8-12 min.

4. The preparation method according to claim 3, characterized in that: In step (2), the temperature of the ultrasonic treatment is 35-40° C., the power is 180-220 W, and the time is 3-7 min; the temperature of the mixing is 35-40° C., the speed is 1800-2200 rpm, and the time is 8-12 min.

5. The preparation method according to claim 4, characterized in that: The mixing temperature in step (3) is 22-27° C., the mixing rate is 2800-3200 rpm, and the mixing time is 8-12 min. The pore size of the filter membrane used for the filtration sterilization is 0.22 μm-0.45 μm.

6. Use of the composition according to claim 1 or the composition prepared by the preparation method according to any one of claims 2 to 5 in preparing a product for inhibiting Porphyromonas gingivalis.

7. The use according to claim 6, characterized in that The products include pet oral sprays and pet mouthwashes.

Images