Trifluoride fusion enzyme oral care preparation and preparation method thereof

Abstract

The invention relates to the technical field of oral care agents, in particular to a trifluoride fusion enzyme oral care preparation and a preparation method thereof. The trifluoride fusion enzyme oral care preparation is prepared from the following components in percentage by weight: 0.05 to 0.15 percent of trifluoride, 0.05 to 0.1 percent of fusion enzyme, 1 to 5 percent of hydroxyapatite, 0.05 to 0.2 percent of sodium hyaluronate, 15 to 20 percent of glycerol, 0.5 to 2 percent of zinc citrate trihydrate, 0.1 to 0.5 percent of peppermint essential oil, 20 to 30 percent of phosphate buffered solution, 0.1 to 0.3 percent of tea polyphenol and 41.75 to 66.05 percent of deionized water. The activity retention rate of the fusion enzyme in the fluoride can be remarkably improved by using a magnetic particle immobilized enzyme method, so that the oral cavity is deeply cleaned, meanwhile, the enamel remineralization efficiency can be remarkably improved by using the fluoride matched with the fusion enzyme method, and the dental enamel remineralization efficiency can be remarkably improved through the cooperation of the nano-hydroxyapatite and the fluoride. A compact repairing layer is formed on the tooth surface to accurately recover the tooth hardness, the gingival bleeding index is remarkably reduced, oral mucosa repairing is promoted, and the toothpaste is safe, non-irritant and suitable for various oral cavity problem patients to use.

Description

Technical Field

[0001] This invention relates to the field of formulation technology for oral care of teeth or dentures, specifically to a trifluoride fusion enzyme oral care formulation and its preparation method. Background Technology

[0002] With changing modern dietary habits, increased sugar intake, and inadequate oral hygiene, oral problems such as tooth decay, periodontal disease, mouth ulcers, and bleeding gums have gradually become global public health issues. Consumers' demand for oral care products is no longer limited to traditional toothpaste and toothbrushes. Many preparations for caring for teeth or dentures are now available, such as tooth powder or toothpaste mouthwash, dental floss, oral sprays, and mouthwashes. As consumers pay more attention to natural and harmless ingredients, the composition of oral care products is gradually shifting towards natural plant extracts and antibacterial ingredients.

[0003] Existing oral care products rely too heavily on chemical reagents, which can lead to bleeding gums and oral ulcers. Long-term use can weaken dental health. Furthermore, the teeth cleaning process relies on highly abrasive agents such as calcium carbonate, which can cause enamel wear, tooth sensitivity, and even accelerate dentin exposure with long-term use. At the same time, existing oral care products still have problems with poor cleaning effect and irritation to the oral mucosa.

[0004] To address the above problems, the present invention provides a solution. Summary of the Invention

[0005] The purpose of this invention is to provide a trifluoride fusion enzyme oral care preparation and its preparation method, which can effectively improve periodontal health and is safe and non-irritating.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A trifluoride fusion enzyme oral care preparation is composed of the following percentage components: 0.05-0.15% trifluoride, 0.05-0.1% fusion enzyme, 1-5% hydroxyapatite, 0.05-0.2% sodium hyaluronate, 15-20% glycerin, 0.5-2% zinc citrate trihydrate, 0.1-0.5% peppermint oil, 20-30% phosphate buffer solution, 0.1-0.3% tea polyphenols, and 41.75-66.05% deionized water;

[0007] The method for preparing the fusion enzyme includes the following steps:

[0008] A1: Weigh 25g of LB medium powder, dissolve it in pure water and bring the volume to 1L in a volumetric flask. After dissolving and bringing the volume to 1L, transfer the LB medium to an autoclave and sterilize it at 121℃ for 20min to obtain LB medium. Dispense the LB medium into clean Erlenmeyer flasks and inoculate them with Bacillus subtilis at a rate of 1 / 1000 of the LB medium mass. After inoculation, transfer the flasks to a constant temperature shaker. Set the temperature of the shaker to 37℃ and the shaking speed to 200rpm. Incubate the shaker for 12h to obtain the fermentation seed.

[0009] A2: After mixing the LB medium evenly with a stirrer, transfer it to a fermenter. Then, inoculate the fermentation seed in the fermenter and add FMSF, dextran, sucrose and peptone. Set the fermenter temperature to 28-37℃. During fermentation, use saturated sodium hydroxide solution to adjust the pH to 5-7 and control the dissolved oxygen saturation to 60-70%. Ferment for 24 hours to obtain the fermentation broth.

[0010] A3: Transfer the fermentation broth to a high-speed centrifuge, centrifuge at 8000 rpm for 15 min to remove the cells, then transfer the supernatant to a clean centrifuge tube and add 60% ammonium sulfate solution. Let it stand at 4℃ for 12 h. After standing, centrifuge at 8000 rpm for 15 min to remove the supernatant. Then transfer the precipitate to 200 ml of Tris-HCl buffer to obtain the crude enzyme solution.

[0011] A4: Transfer the crude enzyme solution to a dialysis bag and clamp it tightly. Then transfer it to a saturated PEG-4000 solution and dialyze at 4°C for 12 hours to obtain the dialysate. Perfuse the DEAE-Sepharose column with 15 ml of deionized water. Then slowly inject the dialysate into the DEAE-Sepharose column and collect the column buffer. Repeat the column buffering twice. After the column buffering is completed, elute with 10 ml of 0.5 M sodium chloride solution. Repeat the elution twice and collect the eluent to obtain the first pre-enzyme.

[0012] A5: Cut the skin of immature papaya fruit, collect the white latex that flows out, and then immediately add 1% EDTA solution. Store at 4℃ to obtain papaya latex. Mix papaya latex with cysteine ​​and 1mM EDTA solution and place in an incubator at 25℃ for 1 hour. After incubation, transfer to a centrifuge and centrifuge at 10000rpm for 20 minutes. Take the supernatant to obtain the crude extract of papaya latex.

[0013] A6: Add 60% ammonium sulfate solution to the crude extract of papaya latex, precipitate at 4℃ for 12h to obtain salting-out solution, then perfuse a Phenyl-Sepharose column with 15ml of deionized water, and then slowly perfuse the Phenyl-Sepharose column with the salting-out solution. Collect the column buffer and repeat the column buffering twice. After the column buffering is completed, elute with 10ml of 0.5M sodium chloride solution, repeat the elution twice and collect the eluent to obtain the second pre-enzyme. Mix the first and second pre-enzymes at 4℃, add mannitol and freeze-dry for 8h to obtain the fusion enzyme.

[0014] Furthermore, the trifluoride is one of sodium fluoride, sodium monofluorophosphate, and olafluridine.

[0015] Furthermore, in step A2, the mass ratio of LB medium, PMSF, dextran, sucrose, and peptone is 100:1:1:9:10; in step A3, the mass ratio of supernatant to 60% ammonium sulfate solution is 1:1; in step A5, the mass ratio of white latex to 1% EDTA solution is 1:2; in step A5, the mass ratio of papaya latex, cysteine, and 1 mM / L EDTA solution is 100:1:2; in step A6, the mass ratio of crude papaya latex extract to 60% ammonium sulfate solution is 2:1; and in step A6, the mass ratio of the first pre-enzyme, the second pre-enzyme, and mannitol is 10:10:1.

[0016] Furthermore, a method for preparing a trifluoride fusion enzyme oral care preparation includes the following steps:

[0017] B1: Pre-cool the phosphate buffer solution to 4°C, then control the ambient temperature to 4°C, add iron oxide nanoparticles, stir repeatedly for 15 min, and after stirring, sonicate in an ice bath with the sonication power set to 120W for 10 min.

[0018] B2: After sonication, replace the ice to maintain the ice bath process, then add 0.5M epichlorohydrin dropwise, set the stirrer speed to 200 rpm, and stir slowly for 24 hours. After stirring, add the fusion enzyme and repeat the slow stirring for 12 hours with the speed unchanged. Maintain the ice bath throughout the stirring process. After repeated stirring, centrifuge to remove the supernatant, and wash the precipitate 3 times with phosphate buffer solution to obtain the immobilized enzyme.

[0019] B3: The immobilized enzyme was transferred to PBS buffer for resuspension, then glycerol was added and the mixture was stirred at 4°C for 15 min. Sodium fluoride, hydroxyapatite, sodium hyaluronate and zinc citrate trihydrate were then added and stirred at 4°C for 30 min. After repeated stirring, deionized water, tea polyphenols and peppermint oil were added. The homogenizer was pre-cooled to 4°C and homogenized for 30 min to obtain the trifluoride fusion enzyme oral care preparation.

[0020] Furthermore, the iron oxide nanoparticles mentioned in step B1 have a particle size of 20 nm, and the mass ratio of the phosphate buffer solution to the iron oxide nanoparticles is 100:1; the mass ratio of the 0.5 M epichlorohydrin to the fusion enzyme mentioned in step B2 is 10:1.

[0021] In summary, due to the adoption of the above technical solutions, the beneficial effects of this invention are as follows: the method of using magnetic particles to solidify enzymes can significantly improve the activity retention rate of fusion enzymes in fluoride, thereby deeply cleaning the oral cavity; at the same time, the method of using fluoride in combination with fusion enzymes can significantly improve the remineralization efficiency of tooth enamel; through the synergy of nano-hydroxyapatite and fluoride, a dense repair layer is formed on the tooth surface to accurately restore tooth hardness, significantly reduce the gingival bleeding index and promote oral mucosal repair, and is safe and non-irritating, suitable for patients with various oral problems. Detailed Implementation

[0022] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0024] Example 1

[0025] 1. Weigh 25g of LB medium powder, dissolve it in pure water and bring the volume to 1L in a volumetric flask. After dissolving and bringing the volume to 1L, transfer the flask to an autoclave and sterilize it at 121℃ for 20min to obtain LB medium. Dispense the LB medium into 5 clean Erlenmeyer flasks, 200g in each flask, and then inoculate each flask with 0.2g of Bacillus subtilis. After inoculation, transfer the flask to a constant temperature shaker. Set the temperature of the shaker to 37℃ and the shaking speed to 200rpm. Incubate at a constant temperature for 12h to obtain the fermentation seed.

[0026] 2: After mixing 1 kg of LB medium evenly with a stirrer, transfer it to a fermenter. Then, inoculate the fermentation seed in the fermenter and add 10 g FMSF, 10 g dextran, 90 g sucrose and 100 g peptone. Set the fermenter temperature to 37℃. During fermentation, use saturated sodium hydroxide solution to adjust the pH to 7 and control the dissolved oxygen saturation to 70%. Ferment for 24 hours to obtain the fermentation broth.

[0027] 3: Transfer the fermentation broth to a high-speed centrifuge, centrifuge at 8000 rpm for 15 min to remove the cells, then transfer 800 g of supernatant to a clean centrifuge tube and add 800 g of 60% ammonium sulfate solution. Let it stand at 4℃ for 12 h. After standing, centrifuge at 8000 rpm for 15 min to remove the supernatant. Then transfer the precipitate to 200 ml of Tris-HCl buffer to obtain the crude enzyme solution.

[0028] 4: Transfer the crude enzyme solution to a dialysis bag and clamp it tightly. Then transfer it to a saturated PEG-4000 solution and dialyze at 4°C for 12 hours to obtain the dialysate. Perfuse the DEAE-Sepharose column with 15 ml of deionized water, and then slowly inject the dialysate into the DEAE-Sepharose column. Collect the column buffer and repeat the column buffering twice. After the column buffering is completed, elute with 10 ml of 0.5 M sodium chloride solution. Repeat the elution twice and collect the eluent to obtain 12 g of the first pre-enzyme.

[0029] 5: Cut the skin of unripe papaya fruit and collect 500g of white latex. Immediately add 1kg of 1% EDTA solution and store at 4℃ to obtain 1.2kg of papaya latex. Mix the papaya latex with 12g of cysteine ​​and 24g of 1mM EDTA solution and place in an incubator at 25℃ for 1 hour. After incubation, transfer to a centrifuge and centrifuge at 10000rpm for 20 minutes. Take the supernatant to obtain 1kg of crude papaya latex extract.

[0030] 6: Add 500g of 60% ammonium sulfate solution to the crude extract of papaya latex, place it in a refrigerator at 4℃ for 12h to precipitate and obtain salting-out solution. Then, perfuse a Phenyl-Sepharose column with 15ml of deionized water, and then slowly perfuse the Phenyl-Sepharose column with the salting-out solution. Collect the column pass solution and repeat the column pass solution twice. After the column pass solution is completed, elute with 10ml of 0.5M sodium chloride solution. Repeat the elution twice and collect the eluent to obtain 14g of the second pre-enzyme. Mix 12g of the first pre-enzyme and 12g of the second pre-enzyme in a 4℃ environment, add 1.2g of mannitol, and freeze-dry in a freeze dryer for 8h to obtain 5g of the fusion enzyme prepared in Example 1.

[0031] Table 1. Reagent parameters used in Example 1

[0032] Drug Name source model parameter FMSF Wuhan Huaxiang Kejie Biotechnology Co., Ltd. 329-98-6 100g beta-glucan Xi'an Tianguangyuan Biotechnology Co., Ltd. 210544 500g peptone Jinan Qingyuyuan New Materials Co., Ltd. 202308 500g Cysteine Anhui Weimao Biotechnology Co., Ltd. HY-L 500g ammonium sulfate Tianjin Jinhui Taiya Chemical Reagent Co., Ltd. 1127 500g Mannitol Henan Zhuosheng Food Ingredients Co., Ltd. QN-D 500g

[0033] Example 2

[0034] 1. Weigh 25g of LB medium powder, dissolve it in pure water and bring the volume to 1L in a volumetric flask. After dissolving and bringing the volume to 1L, transfer the flask to an autoclave and sterilize it at 121℃ for 20min to obtain LB medium. Dispense the LB medium into 5 clean Erlenmeyer flasks, 200g in each flask, and then inoculate each flask with 0.2g of Bacillus subtilis. After inoculation, transfer the flask to a constant temperature shaker. Set the temperature of the shaker to 37℃ and the shaking speed to 200rpm. Incubate at a constant temperature for 12h to obtain the fermentation seed.

[0035] 2: After mixing 1 kg of LB medium evenly with a stirrer, transfer it to a fermenter. Then, inoculate the fermentation seed in the fermenter and add 10 g FMSF, 10 g dextran, 90 g sucrose and 100 g peptone. Set the fermenter temperature to 28℃. During the fermentation process, use saturated sodium hydroxide solution to adjust the pH to 5 and control the dissolved oxygen saturation to 60%. Ferment for 24 hours to obtain the fermentation broth.

[0036] 3: Transfer the fermentation broth to a high-speed centrifuge, centrifuge at 8000 rpm for 15 min to remove the bacterial cells, then transfer 820 g of supernatant to a clean centrifuge tube and add 820 g of 60% ammonium sulfate solution. Let it stand at 4℃ for 12 h. After standing, centrifuge at 8000 rpm for 15 min to remove the supernatant. Then transfer the precipitate to 200 ml of Tris-HCl buffer to obtain the crude enzyme solution.

[0037] 4: Transfer the crude enzyme solution to a dialysis bag and clamp it tightly. Then transfer it to a saturated PEG-4000 solution and dialyze at 4°C for 12 hours to obtain the dialysate. Perfuse the DEAE-Sepharose column with 15 ml of deionized water, and then slowly inject the dialysate into the DEAE-Sepharose column. Collect the column buffer and repeat the column buffering twice. After the column buffering is completed, elute with 10 ml of 0.5 M sodium chloride solution. Repeat the elution twice and collect the eluent to obtain 15 g of the first pre-enzyme.

[0038] A5: Cut the skin of immature papaya fruit and collect 540g of white latex. Immediately add 1080g of 1% EDTA solution and store at 4℃ to obtain 1.4kg of papaya latex. Mix the papaya latex with 14g of cysteine ​​and 28g of 1mM EDTA solution and incubate at 25℃ for 1 hour. After incubation, transfer to a centrifuge and centrifuge at 10000rpm for 20 minutes. Take the supernatant to obtain 1.2kg of crude papaya latex extract.

[0039] A6: Add 600g of 60% ammonium sulfate solution to the crude extract of papaya latex, place it in a refrigerator at 4℃ for 12h to precipitate and obtain salting-out solution. Then, perfuse a Phenyl-Sepharose column with 15ml of deionized water. Then, slowly perfuse the Phenyl-Sepharose column with the salting-out solution and collect the column buffer. Repeat the column buffering twice. After the column buffering is completed, elute with 10ml of 0.5M sodium chloride solution. Repeat the elution twice and collect the eluent to obtain 18g of the second pre-enzyme. Mix 15g of the first pre-enzyme and 15g of the second pre-enzyme at 4℃, add 1.5g of mannitol and freeze-dry in a freeze dryer for 8h to obtain 10g of the fusion enzyme prepared in Example 2.

[0040] Table 2, Reagent Parameters Used in Example 2

[0041]

[0042]

[0043] Example 3

[0044] 1. Pre-cool 500ml of phosphate buffer solution to 4℃, then control the ambient temperature at 4℃, add 5g of 20nm iron oxide nanoparticles, stir repeatedly for 15min, and after stirring, sonicate in an ice bath with the sonication power set to 120W for 10min.

[0045] 2: After sonication, replace the ice to maintain the ice bath process. Then, add 50g of 0.5M epichlorohydrin dropwise, set the stirrer speed to 200rpm, and stir slowly for 24h. After stirring, add 5g of the fusion enzyme prepared in Example 1 and repeat the slow stirring for 12h at the same speed. The ice bath is maintained throughout the stirring process. After repeated stirring, centrifuge to remove the supernatant, and wash the precipitate three times with phosphate buffer solution to obtain the immobilized enzyme.

[0046] 3: The immobilized enzyme was transferred to 2 kg PBS buffer for resuspension, followed by the addition of 2.5 kg glycerol. The mixture was stirred at 4°C for 15 min, then 5 g sodium fluoride, 100 g hydroxyapatite, 5 g sodium hyaluronate, and 50 g zinc citrate trihydrate were added. The mixture was stirred at 4°C for 30 min. After repeated stirring, 6.605 kg deionized water, 10 g tea polyphenols, and 10 g peppermint oil were added. The homogenizer was pre-cooled to 4°C and homogenized for 30 min to obtain the trifluoride fusion enzyme oral care preparation prepared in Example 3.

[0047] Table 3. Reagent parameters used in Example 3

[0048]

[0049]

[0050] Example 4

[0051] 1. Pre-cool 500ml of phosphate buffer solution to 4℃, then control the ambient temperature at 4℃, add 5g of 20nm iron oxide nanoparticles, stir repeatedly for 15min, and after stirring, sonicate in an ice bath with the sonication power set to 120W for 10min.

[0052] 2: After sonication, replace the ice to maintain the ice bath process. Then, add 100g of 0.5M epichlorohydrin dropwise, set the stirrer speed to 200rpm, and stir slowly for 24h. After stirring, add 10g of the fusion enzyme prepared in Example 2 and repeat the slow stirring for 12h at the same speed. The ice bath is maintained throughout the stirring process. After repeated stirring, centrifuge to remove the supernatant, and wash the precipitate three times with phosphate buffer solution to obtain the immobilized enzyme.

[0053] 3: The immobilized enzyme was transferred to 3 kg of PBS buffer for resuspension, followed by the addition of 2 kg of glycerol. The mixture was stirred at 4°C for 15 min, then 15 g of olaflumethrin, 300 g of hydroxyapatite, 20 g of sodium hyaluronate and 200 g of zinc citrate trihydrate were added. The mixture was stirred at 4°C for 30 min. After repeated stirring, 4.175 kg of deionized water, 30 g of tea polyphenols and 50 g of peppermint oil were added. The homogenizer was pre-cooled to 4°C and homogenized for 30 min to obtain the trifluoride fusion enzyme oral care preparation prepared in Example 4.

[0054] Table 4. Reagent parameters used in Example 4

[0055]

[0056]

[0057] Comparative Example 1

[0058] Example 1 of Chinese Patent Publication No. CN120093610A was selected as Comparative Example 1.

[0059] Comparative Example 2

[0060] Example 1 of Chinese Patent Publication No. CN1778288A was selected as Comparative Example 2.

[0061] MTT toxicity test

[0062] The compositions obtained in Examples 3, 4, Comparative Examples 1 and 2 were dissolved in pure water and then diluted to a 1% concentration using pure water. Human oral epithelial cells-HOK cells were then revived and cultured. Logarithmic 293T cells were collected after 1 day, and the cell suspension concentration was adjusted and distributed into 96-well plates, 180 μL per well, 10,000 cells / well.

[0063] The 96-well plate was incubated at 37°C with 5% CO2 for 6 hours. Then, 10 μL of diluted drug solution was added to each well. After removing the supernatant, 90 μL of fresh culture medium was added, followed by 10 μL of MTT solution. The plate was incubated for another 4 hours. After removing the supernatant, 110 μL of Formazan solution was added to each well. The plate was then shaken on a shaker at low speed for 10 minutes to fully dissolve the crystals. After shaking, the 96-well plate was transferred to a microplate reader and the absorbance of each well was measured at 490 nm. Simultaneously, blanks (culture medium, MTT solution, and Formazan solution) were set up, with 3 replicates per well to detect cytotoxicity.

[0064] Table 5. MTT toxicity test results

[0065]

[0066]

[0067] As can be seen from the analysis of Table 5, the oral care preparations prepared using Examples 3 and 4 showed virtually no cytotoxicity in the cytotoxicity test. Among them, the oral care preparation prepared using Example 3 had the lowest cytotoxicity and the highest cell activity, indicating that the oral care preparations prepared using Examples 3 and 4 would not cause any harm or irritation to the oral cavity during use.

[0068] Remineralization capacity test

[0069] Healthy bovine teeth were selected, rinsed with physiological saline, and then ultrasonically cleaned at 80W for 5 minutes. Soft tissue was then removed, and the crowns were cut into 4×4mm enamel slices with a thickness of 2mm using a diamond microtome. The enamel slices were polished to a mirror finish with graded wet sandpaper, ultrasonically cleaned to remove debris, and then placed on a Shimadzu HMV-2 microhardness tester. A load of 50gf was applied for 10 seconds, and five points were taken from each enamel slice, with the average value used as the initial hardness.

[0070] A demineralization solution was prepared using 2.2 mM calcium chloride, 2.2 mM sodium hydrogen phosphate, and 0.05 M acetate buffer. Enamel sheets were immersed in the demineralization solution and shaken at 37°C and 120 rpm for 24 hours. After shaking, enamel sheets with a demineralization rate of 42% were obtained. The enamel surface was then observed using a Hitachi SU8010 scanning electron microscope. Oral care preparations prepared in Examples 3, 4, Comparative Example 1, and Comparative Example 2 were used to simulate the brushing process, each time for 1 minute, twice daily. During the simulated brushing process, the sheets were immersed in a solution containing (pH 6.8, Ca...) 2+ / PO4 3-The hardness of the treated glaze was measured in artificial saliva after 28 days of repeated simulation to obtain the remineralization rate. Then, the changes in surface porosity of the glaze were observed using a Hitachi SU8010 scanning electron microscope.

[0071] Table 6. Remineralization Capacity Test

[0072]

[0073]

[0074] As can be seen from the analysis of Table 6, the oral care preparations prepared in Examples 3 and 4 can significantly improve the remineralization rate of bovine teeth, and can also effectively protect acid-eroded teeth and improve caries by covering the enamel surface with a dense mineral layer.

[0075] Periodontal health assessment

[0076] Fifty volunteers aged 18-65 years with mild gingivitis (gingival index GI = 1-2) were recruited and divided into 5 groups. The volunteers used toothpaste and mouthwash prepared with the oral care formulations prepared in Examples 3, 4, 1, and 2, respectively, for 28 days. They brushed their teeth twice daily for 2 minutes each time and rinsed their mouths with 10ml of mouthwash twice daily for 30 seconds each time. The oral care formulations in the toothpaste and mouthwash contained 0.12% of the formulation. The control group used regular toothpaste and regular mouthwash.

[0077] Before the experiment, the gingival sulcus of the volunteers was gently probed for 30 seconds, and the number of bleeding points / total number of points detected was recorded within 30 seconds. Then, [the procedure was performed]. The plaque index was assessed using a method; after the experiment was completed, the plaque reduction rate and gingival bleeding improvement rate were recorded repeatedly.

[0078] Table 7. Results of Periodontal Health Assessment

[0079] Grouping Plaque reduction rate Improvement rate of gingival bleeding Example 3 62.48％ 75.66％ Example 4 62.37％ 74.89％ Comparative Example 1 54.79％ 59.11％ Comparative Example 2 41.43％ 42.38％ control group 40.47％ 35.69％

[0080] Analysis of Table 7 shows that the oral care preparations prepared in Examples 3 and 4 can significantly improve the plaque reduction rate and gingival bleeding improvement rate in patients with mild gingivitis.

[0081] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A trifluoride fusion enzyme oral care preparation, characterized in that, It consists of the following percentage components: 0.05-0.15% trifluoride, 0.05-0.1% fusion enzyme, 1-5% hydroxyapatite, 0.05-0.2% sodium hyaluronate, 15-20% glycerin, 0.5-2% zinc citrate trihydrate, 0.1-0.5% peppermint oil, 20-30% phosphate buffer solution, 0.1-0.3% tea polyphenols, and 41.75-66.05% deionized water; The method for preparing the fusion enzyme includes the following steps: A1: Weigh 25g of LB medium powder, dissolve it in pure water and bring the volume to 1L in a volumetric flask. After dissolving and bringing the volume to 1L, transfer the LB medium to an autoclave and sterilize it at 121℃ for 20min to obtain LB medium. Dispense the LB medium into clean Erlenmeyer flasks and inoculate them with Bacillus subtilis at a rate of 1 / 1000 of the LB medium mass. After inoculation, transfer the flasks to a constant temperature shaker. Set the temperature of the shaker to 37℃ and the shaking speed to 200rpm. Incubate the shaker for 12h to obtain the fermentation seed. A2: After mixing the LB medium evenly with a stirrer, transfer it to a fermenter. Then, inoculate the fermentation seed in the fermenter and add FMSF, dextran, sucrose and peptone. Set the fermenter temperature to 28-37℃. During fermentation, use saturated sodium hydroxide solution to adjust the pH to 5-7 and control the dissolved oxygen saturation to 60-70%. Ferment for 24 hours to obtain the fermentation broth. A3: Transfer the fermentation broth to a high-speed centrifuge, centrifuge at 8000 rpm for 15 min to remove the cells, then transfer the supernatant to a clean centrifuge tube and add 60% ammonium sulfate solution. Let it stand at 4℃ for 12 h. After standing, centrifuge at 8000 rpm for 15 min to remove the supernatant. Then transfer the precipitate to 200 ml of Tris-HCl buffer to obtain the crude enzyme solution. A4: Transfer the crude enzyme solution to a dialysis bag and clamp it tightly. Then transfer it to a saturated PEG-4000 solution and dialyze at 4°C for 12 hours to obtain the dialysate. Perfuse the DEAE-Sepharose column with 15 ml of deionized water. Then slowly inject the dialysate into the DEAE-Sepharose column and collect the column buffer. Repeat the column buffering twice. After the column buffering is completed, elute with 10 ml of 0.5 M sodium chloride solution. Repeat the elution twice and collect the eluent to obtain the first pre-enzyme. A5: Cut the skin of immature papaya fruit, collect the white latex that flows out, and then immediately add 1% EDTA solution. Store at 4℃ to obtain papaya latex. Mix papaya latex with cysteine ​​and 1mM EDTA solution and place in an incubator at 25℃ for 1 hour. After incubation, transfer to a centrifuge and centrifuge at 10000rpm for 20 minutes. Take the supernatant to obtain the crude extract of papaya latex. A6: Add 60% ammonium sulfate solution to the crude extract of papaya latex, precipitate at 4℃ for 12h to obtain salting-out solution, then perfuse a Phenyl-Sepharose column with 15ml of deionized water, and then slowly perfuse the Phenyl-Sepharose column with the salting-out solution. Collect the column buffer and repeat the column buffering twice. After the column buffering is completed, elute with 10ml of 0.5M sodium chloride solution, repeat the elution twice and collect the eluent to obtain the second pre-enzyme. Mix the first and second pre-enzymes at 4℃, add mannitol and freeze-dry for 8h to obtain the fusion enzyme.

2. The trifluoride fusion enzyme oral care preparation according to claim 1, characterized in that, The trifluoride mentioned is one of sodium fluoride, sodium monofluorophosphate, and olafluridine.

3. The trifluoride fusion enzyme oral care preparation according to claim 1, characterized in that, The mass ratio of LB medium, PMSF, dextran, sucrose and peptone in step A2 is 100:1:1:9:

10.

4. The trifluoride fusion enzyme oral care preparation according to claim 1, characterized in that, The mass ratio of the supernatant to the 60% ammonium sulfate solution mentioned in step A3 is 1:

1.

5. The trifluoride fusion enzyme oral care preparation according to claim 1, characterized in that, The mass ratio of the white emulsion to the 1% EDTA solution mentioned in step A5 is 1:

2.

6. The trifluoride fusion enzyme oral care preparation according to claim 1, characterized in that, The mass ratio of papaya latex, cysteine, and 1 mM / L EDTA solution described in step A5 is 100:1:

2.

7. The trifluoride fusion enzyme oral care preparation according to claim 1, characterized in that, The mass ratio of the papaya latex crude extract and the 60% ammonium sulfate solution in step A6 is 2:1; the mass ratio of the first pre-enzyme, the second pre-enzyme, and mannitol in step A6 is 10:10:

1.

8. The method for preparing a trifluoride fusion enzyme oral care preparation according to claim 1, characterized in that, Includes the following steps: B1: Pre-cool the phosphate buffer solution to 4°C, then control the ambient temperature to 4°C, add iron oxide nanoparticles, stir repeatedly for 15 min, and after stirring, sonicate in an ice bath with the sonication power set to 120W for 10 min. B2: After sonication, replace the ice to maintain the ice bath process, then add 0.5M epichlorohydrin dropwise, set the stirrer speed to 200 rpm, and stir slowly for 24 hours. After stirring, add the fusion enzyme and repeat the slow stirring for 12 hours with the speed unchanged. Maintain the ice bath throughout the stirring process. After repeated stirring, centrifuge to remove the supernatant, and wash the precipitate 3 times with phosphate buffer solution to obtain the immobilized enzyme. B3: The immobilized enzyme was transferred to PBS buffer for resuspension, then glycerol was added and the mixture was stirred at 4°C for 15 min. Sodium fluoride, hydroxyapatite, sodium hyaluronate and zinc citrate trihydrate were then added and stirred at 4°C for 30 min. After repeated stirring, deionized water, tea polyphenols and peppermint oil were added. The homogenizer was pre-cooled to 4°C and homogenized for 30 min to obtain the trifluoride fusion enzyme oral care preparation.

9. The method for preparing a trifluoride fusion enzyme oral care preparation according to claim 8, characterized in that, The iron oxide nanoparticles mentioned in step B1 have a particle size of 20 nm, and the mass ratio of the phosphate buffer solution to the iron oxide nanoparticles is 100:

1.

10. The method for preparing a trifluoride fusion enzyme oral care preparation according to claim 8, characterized in that, The mass ratio of 0.5M epichlorohydrin to fusion enzyme mentioned in step B2 is 10:1.

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