An artificial mimic enzyme with antibacterial performance and a preparation method and application thereof

Abstract

This invention relates to the field of oral care products technology, specifically to an artificial enzyme with antibacterial properties, its preparation method, and its application. This technical solution employs a chemical reduction method, where a palladium source reacts in an environment containing surfactants and reducing agents to obtain palladium nanozymes. The obtained palladium nanozymes exhibit uniform morphology, controllable particle size, good dispersibility in water, and good biocompatibility; they have no significant toxic side effects on human gingival fibroblasts, possess highly efficient catalase activity, and demonstrate strong antibacterial and bactericidal effects against oral pathogens, significantly reducing bacterial drug resistance; they can inhibit acid production by oral bacteria, preventing tooth decay; and they retain their original catalytic activity even after multiple cycles of use. Applying the palladium nanozymes of this solution to the manufacture of oral care products can solve the technical problems of unsatisfactory safety and easy development of drug resistance in existing oral care products using antibacterial and anti-caries methods, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of oral care products technology, specifically to an artificial enzyme with antibacterial properties, its preparation method, and its application. Background Technology

[0002] Dental caries is a widespread chronic disease, especially prevalent among adolescents and children. Studies have shown that dental caries is mainly caused by the growth and acid production of cariogenic bacteria in the oral cavity, as well as their adhesion and accumulation on the tooth surface. For this chronic disease caused by bacteria, the most common methods are drug treatment and oral hygiene prevention.

[0003] Over the past few decades, numerous studies have been conducted on drugs for preventing and treating dental caries, resulting in the development of many medications. These include antibiotics such as penicillin, erythromycin, tetracycline, and spiramycin. While these drugs have some anti-caries effect, long-term use can cause oral and intestinal flora imbalance, leading to other diseases. Besides antibiotics, research on immunotherapy for dental caries has a 40-year history, accumulating a wealth of theoretical and practical results. Normal oral flora naturally contains specific antibodies against pathogens, but these naturally induced antibodies are insufficient to eliminate pathogens. Therefore, vaccination is needed to raise antibody levels to therapeutic or preventative levels, or specific antibodies can be directly administered to combat pathogens. Immunotherapy for dental caries faces challenges in enhancing immunogenicity and biological activity, potentially leading to antibiotic resistance in oral bacteria. In addition to the above two methods, the use of fluoride for caries prevention is currently the most popular method in oral care products. However, this method presents challenges such as the instability of fluoride, the toxic side effects of high concentrations, and the emergence of fluoride-resistant bacterial strains in the oral cavity. Therefore, overcoming the limitations of the above-mentioned caries prevention methods and finding a simple, economical, and effective method for caries prevention is the goal pursued by many caries researchers. There is an urgent need to find a safe and effective new caries prevention material to replace the above-mentioned caries prevention methods. Summary of the Invention

[0004] This invention aims to provide an application of an artificially simulated enzyme with antibacterial properties in oral care products, addressing the technical problems of unsatisfactory safety and easy development of drug resistance in existing oral care products that use antibacterial and anti-caries methods. This invention provides an ultrafine palladium nanoenzyme material that inhibits the growth of harmful bacteria in the oral cavity and suppresses bacterial acid production through catalase-like activity, thereby achieving the effect of preventing tooth decay.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The application of an artificial enzyme with antibacterial properties in the preparation of oral care products, wherein the artificial enzyme is prepared by the following method: palladium source is reacted in a reaction system containing surfactant and reducing agent to obtain palladium nanozyme.

[0007] This solution also provides a toothpaste containing artificially simulated enzymes, the formula of which, by weight, is: 41-80 parts of moisturizer, 10-30 parts of abrasive, 0.05-0.5 parts of palladium nanozyme, 0.1-1.5 parts of thickener, 0.5-1.5 parts of surfactant, 0.5-1.5 parts of fragrance, 1-6 parts of phosphate buffer, and the balance being water.

[0008] This solution also provides a method for preparing toothpaste containing artificially simulated enzymes. Palladium nanozymes and phosphate buffer are mixed and stirred at 50-150 rpm for 10-30 minutes to obtain an activated palladium nanozyme dispersion. A humectant and a thickener are mixed and homogenized, then a surfactant, the activated palladium nanozyme dispersion, and the remaining water are added and stirred evenly to obtain mixture A. An abrasive is added to mixture A, homogenized, and then vacuumed. A fragrance is added, and the mixture is stirred, homogenized, and degassed to obtain toothpaste.

[0009] This solution also provides an artificial enzyme with antibacterial properties, which has a tetrahedral structure, a particle size of 40-80 nm, a hydrodynamic size of 62-106 nm, and a surface potential of -34±6 mV.

[0010] This solution also provides the application of an artificially mimicked enzyme with antibacterial properties in the preparation of reactive oxygen species scavenging agents, antibacterial agents, bacterial acid production inhibitors, or caries prevention agents.

[0011] This solution also provides a method for preparing an artificial enzyme with antibacterial properties, comprising the following steps performed sequentially:

[0012] S1: Disperse the surfactant and reducing agent in water to obtain a mixed solution;

[0013] S2: Stir and heat the mixed solution, then add the palladium source solution; react at 60-90℃ for 2-6 hours to obtain palladium nanozyme.

[0014] Furthermore, palladium nanozymes are obtained by reacting a palladium source in a reaction system containing a surfactant and a reducing agent; the reaction temperature is 60-90℃ and the reaction time is 2-6h.

[0015] Furthermore, the palladium source includes Pd-containing... 2+ Pd 3+ Pd 4+The surfactant comprises at least one cationic compound; the surfactant comprises at least one of polyvinylpyrrolidone, hydroxyethyl cellulose, and polyacrylamide; the reducing agent comprises at least one of L-ascorbic acid, potassium bromide (KBr), sodium borohydride, sodium citrate, potassium chloride, lithium aluminum hydride, and diborane.

[0016] Furthermore, the oral care products include toothpaste, mouthwash, tooth powder, and tooth gel.

[0017] Furthermore, the humectant includes at least one of polyethylene glycol, glycerin, sorbitol, and propylene glycol; the abrasive includes at least one of calcium carbonate, silica, calcium hydrogen phosphate, and glycerophosphate; the thickener includes at least one of sodium carboxymethyl cellulose, hydroxyethyl cellulose, xanthan gum, carrageenan, guar gum, and carbomer; and the surfactant includes at least one of amino acid surfactants, sodium lauryl sulfate, and alkyl glycosides.

[0018] In summary, the principle of this technical solution is as follows:

[0019] This invention provides an ultrafine palladium nanozyme material. This palladium nanozyme inhibits the growth of harmful bacteria in the oral cavity through catalase-like activity, suppressing bacterial acid production and thus preventing tooth decay. This technical solution adds the nanozyme to toothpaste, enhancing its inhibitory effect on common oral bacteria. Since this is the first time palladium nanozymes have been applied to toothpaste, ensuring the full effectiveness of the nanozyme in the product, maintaining the stability of the toothpaste after adding the nanozyme, and ensuring the compatibility of the nanozyme with other toothpaste components are problems that need to be solved in practical application. Extensive research by the inventors revealed that directly adding the nanozyme to toothpaste results in insufficient antibacterial activity. Activating the nanozyme with a specific buffer solution before adding it to the toothpaste is necessary to guarantee its antibacterial effect.

[0020] The beneficial effects of this technical solution are as follows:

[0021] (1) The palladium nanozyme has mild preparation conditions, simple method, easy particle size control, low cost, uniform and controllable morphology, good dispersibility in water, good biocompatibility, and no cytotoxicity. It is easily soluble in distilled water and can be used to make toothpaste and other oral care product compositions.

[0022] (2) The palladium nanozymes mentioned above can inhibit harmful cariogenic bacteria such as Streptococcus mutans, Lactobacillus rhamnosus, Streptococcus sanguinis, and Actinomyces colistii in the oral cavity, thus preventing tooth decay.

[0023] (3) Oral care products made from this nanoenzyme can effectively inhibit bacterial acid production, protect the oral cavity, and maintain the oral acid-base balance. Attached Figure Description

[0024] Figure 1 Transmission electron microscopy image of the palladium nanozyme prepared in Experiment Example 1.

[0025] Figure 2 The particle size distribution diagram is shown for the palladium nanozyme prepared in Experimental Example 1.

[0026] Figure 3 The Zeta potential diagram is for the palladium nanozyme prepared in Experiment Example 1.

[0027] Figure 4 The statistical graph shows the bacterial acid production inhibition effect of the palladium nanozyme-containing toothpaste prepared in Experiment Example 2.

[0028] Figure 5 The results of the cytotoxicity test of the palladium nanozyme prepared in Experiment Example 3 are shown. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0030] Example 1: Preparation of palladium nanozymes

[0031] The general preparation process of palladium nanozymes is as follows:

[0032] S1: Add 5-40 mL of deionized water to a round-bottom flask, then add 40-80 mg of surfactant and 440-880 mg of reducing agent. Under ultrasonic assistance, promote the dissolution of the above solutes to obtain a mixed solution.

[0033] S2: Transfer the mixed solution to an oil bath and heat it to 60-100℃ while stirring at 150-350 rpm. Quickly add 1-6 mL of a palladium source solution with a solute concentration of 10-40 mg / mL to the mixed solution to obtain the reaction system. Reflux the reaction system at 60-90℃ for 2-6 hours, then stop the reaction. Wash the precipitate several times with deionized water, then collect the precipitate by centrifugation. Disperse the precipitate in ultrapure water at a concentration of 0.001-10 mg / mL to obtain a palladium nanozyme dispersion, which is then refrigerated for subsequent use.

[0034] Among them, palladium sources include those containing Pd 2+ Pd 3+ Pd 4+ The compound containing at least one cation is preferably at least one of sodium chloropalladium (Na2PdCl4), PdCl2, and Pd(NH3)4Cl2;

[0035] Surfactants include at least one of polyvinylpyrrolidone, hydroxyethyl cellulose, and polyacrylamide;

[0036] The reducing agent includes at least one of L-ascorbic acid, potassium bromide (KBr), sodium borohydride, sodium citrate, potassium chloride, lithium aluminum hydride, and diborane.

[0037] Example 2: Preparation of toothpaste containing palladium nanozymes

[0038] The toothpaste is formulated by weighing the following parts by weight: humectant, abrasive, palladium nanozyme, thickener, surfactant, and fragrance. The toothpaste has anti-inflammatory properties, inhibits bacterial acid production, and kills harmful oral bacteria. The composition is as follows: humectant 41-80 parts, abrasive 10-30 parts, palladium nanozyme 0.05-0.5 parts (specifically, the palladium nanozyme solution (palladium nanozyme dispersion) prepared in Example 1 can be used, for example: a 0.001-10 mg / ml palladium nanozyme solution; in this formulation, the mass of palladium nanozyme is calculated based on the mass of the palladium nanozyme solution), thickener 0.1-1.5 parts, surfactant 0.5-1.5 parts, fragrance 0.5-1.5 parts, and water balance (total 100 parts). In addition to the above ingredients, the palladium nanozyme-containing toothpaste prepared using this method requires the addition of a specific pH buffer to ensure the antibacterial activity of the palladium nanozyme. The buffer type is phosphate buffer (pH 6.6), and the buffer volume is 1-6 parts buffer for every 0.05-0.5 parts palladium nanozyme. This phosphate buffer is prepared as follows: dissolve 1.74g of sodium dihydrogen phosphate, 2.7g of disodium hydrogen phosphate, and 1.7g of sodium chloride in distilled water and bring the volume to 400ml. Adjust the pH to 6.6.

[0039] The moisturizer includes at least one of polyethylene glycol, glycerin, sorbitol and propylene glycol.

[0040] The abrasive includes at least one of silica, dicalcium phosphate, calcium carbonate, and glycerol phosphate.

[0041] Thickeners include at least one of sodium carboxymethyl cellulose, hydroxyethyl cellulose, xanthan gum, carrageenan, guar gum, and carbomer.

[0042] Surfactants include at least one of amino acid surfactants, sodium dodecyl sulfate, and alkyl glycosides.

[0043] In subsequent experimental studies, the specific toothpaste formula used was as follows:

[0044] Sorbitol 40-70 parts, polyethylene glycol (PEG) 1-10 parts, silica 10-30 parts, palladium nanozyme 0.05-0.5 parts, sodium carboxymethyl cellulose 0.1-1.5 parts, sodium dodecyl sulfate (K12) 0.5-1.5 parts, peppermint flavor 0.5-1.5 parts, buffer solution 1-6 parts.

[0045] The specific manufacturing process of toothpaste is as follows:

[0046] SS1: Mix palladium nanozyme (e.g., 1 mg / ml palladium nanozyme solution) and buffer solution, and stir at 50-150 rpm for 10-30 min to obtain activated palladium nanozyme dispersion.

[0047] SS2: Mix the humectant and thickener homogenized, then add the surfactant, activated palladium nanozyme dispersion, and the remaining water, and stir evenly to obtain mixture A.

[0048] SS3: Add 60% by mass of abrasive to mixture A, mix and stir evenly, then add the remaining abrasive and stir evenly again before vacuuming; add fragrance, stir to homogenize, degas, and obtain toothpaste.

[0049] Experimental Example 1: MIC value of palladium nanozyme in inhibiting harmful bacteria in the oral cavity

[0050] (I) Preparation of palladium nanozymes

[0051] The palladium nanozyme in this experiment was prepared using the method described in Example 1. For specific parameter selection, please refer to Table 1.

[0052] Table 1: Selection of Process Parameters for Palladium Nanozymes

[0053]

[0054] Taking sample 1 as an example, a transmission electron microscope image of palladium nanozyme is shown. Figure 1 ), Particle size distribution map ( Figure 2 ), Zeta potential diagram ( Figure 3 The palladium nanozymes have a uniform morphology, a tetrahedral structure, a particle size of 40-80 nm, a hydrodynamic size of 62-106 nm, and a surface potential of -34±6 mV.

[0055] (II) Experimental Methods

[0056] (1) The method refers to the "Disinfection Technical Specifications" --- Minimum Inhibitory Concentration Determination Test (Nutritional Broth Dilution Method).

[0057] (2) Target strains: Streptococcus mutans ATCC 25175, Streptococcus mutans ATCC700610, Lactobacillus rhamnosus ATCC416, Streptococcus sanguinis ATCC10556, and Actinomyces viscous ATCC15987.

[0058] (3) Sample processing:

[0059] Sample 1 was first prepared with sterile distilled water to concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and 0.0625 mg / mL. When using the sample solution, it was dissolved in TSB / BHI liquid culture medium by a 2-fold dilution method to achieve a final concentration of 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, and 0.03125 mg / mL.

[0060] (4) Preparation of bacterial suspension

[0061] Streptococcus mutans (ATCC25175), Streptococcus sanguinis (ATCC10556), Actinomyces viscosus (ATCC15987), and Lactobacillus rhamnosus (ATCC416) were purchased from Beina Biotechnology Co., Ltd. Streptococcus mutans and Lactobacillus rhamnosus were anaerobically revived on TSA solid medium for 48 h, while Streptococcus sanguinis and Actinomyces viscosus were anaerobically revived on BHI solid medium for 48 h. Single colonies were picked and anaerobically expanded in TSA and BHI liquid media for 24 h, respectively. The bacterial concentration was adjusted to approximately 1 × 10⁻⁶ using physiological saline via turbidimetric assay. 8 CFU / mL available for use.

[0062] (5) Determination of MIC value of sample against bacterial culture

[0063] Add 400 μL of dual-material culture medium, 400 μL of sample solution, and 16 μL of bacterial suspension to a 48-well plate as the test sample. Inoculate the culture medium and bacteria without the sample using the same method as the positive control. Add only culture medium as the negative control. Place the test sample, negative control, and positive control in a 37°C incubator for anaerobic incubation for 48 hours and observe the results.

[0064] (III) Experimental Results

[0065] Table 2: MIC value determination results for sample 1

[0066]

[0067]

[0068] Experimental results showed that palladium nanozymes had a significant inhibitory effect on the growth of five common harmful bacteria in the oral cavity. The MIC values ​​of palladium nanozymes against Streptococcus mutans (ATCC700610) were 0.125 mg / mL, against Streptococcus mutans (ATCC25175) were 0.25 mg / mL, against Lactobacillus rhamnosus (ATCC416) were 0.25 mg / mL, against Streptococcus sanguinis (ATCC15987) were 0.125 mg / mL, and against Actinomyces viscosus (ATCC10556) were 0.125 mg / mL.

[0069] Experimental Example 2: Study on the Inhibitory Acid Production Performance of Toothpaste Containing Palladium Nanozymes

[0070] (I) Experimental Methods

[0071] (1) Samples: The toothpaste containing palladium nanozyme was prepared and provided by our company, with the product numbers LY211201 and LY211202. The palladium nanozyme content was 500 ppm and 5000 ppm, respectively. The two toothpastes are identical in all components except for the palladium nanozyme content.

[0072] The specific formulation of LY211201 is as follows: 62 parts sorbitol, 4 parts polyethylene glycol (PEG), 20 parts silica, 0.05 parts palladium nanozyme (using sample 1 prepared in Experiment 1, and preparing a 1 mg / ml palladium nanozyme solution; all samples below use this nanozyme), 0.65 parts sodium carboxymethyl cellulose, 0.9 parts sodium dodecyl sulfate (K12), 1.0 part peppermint flavor, 4 parts buffer solution, and the remainder is water.

[0073] The specific formulation of LY211202 is as follows: 62 parts sorbitol, 4 parts polyethylene glycol (PEG), 20 parts silica, 0.5 parts palladium nanozyme, 0.65 parts sodium carboxymethyl cellulose, 0.9 parts sodium dodecyl sulfate (K12), 1.0 part peppermint flavor, 4 parts buffer solution, and the remainder is water. The toothpaste preparation method is described in Example 2, wherein the process for obtaining the activated palladium nanozyme dispersion is as follows: the palladium nanozyme and buffer solution are mixed and stirred at 100 rpm for 20 min.

[0074] (2) Strains: Streptococcus mutans (S. mutans) is the international standard strain ATCC700610.

[0075] (3) Preparation of bacterial culture: The frozen strains were revived, passaged, and identified. Then, 2-3 colonies were picked and inoculated into TSB liquid medium, incubated at 37℃ for 8 hours, centrifuged, smeared, and the bacterial concentration was adjusted to 1.0×10⁻⁶ with physiological saline. 8 CFU / mL.

[0076] (4) Sample solution preparation: Mix the sample toothpaste with TSB liquid culture medium containing 1% sucrose at a mass ratio of 1:3 and set aside.

[0077] (5) Culture medium preparation: TSB liquid culture medium containing 1% sucrose, 30g of culture medium powder and 10g of sucrose are added to 1000ml of distilled water.

[0078] (6) Operating procedures: Taking LY211201 as an example, add 0.5 ml of bacterial culture to 5 ml of sample solution and mix well. Set up one replicate, for a total of 6 groups. Incubate anaerobically at 37℃. Measure the pH of the supernatant after centrifugation at 1 h, 5 h, 7 h, 9 h, 24 h, and 48 h. Centrifugation conditions are 5000 rpm for 3 min. For LY211202, follow the same setup as LY211201. Add distilled water as the negative control.

[0079] (II) Experimental Results

[0080] Experimental results demonstrate that, in the presence of 1% sucrose, compared to the control group, toothpaste with added nanozymes significantly inhibited acid production by Streptococcus mutans and balanced pH levels within the experimental period (5h, 9h, 24h, 48h). See the experimental results for details. Figure 4 .

[0081] Experimental Example 3: Cytotoxicity Study of Palladium Nanozymes

[0082] Gingival fibroblasts were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. The culture was maintained at 37°C and 5% CO2. Cells were passaged 1 to 3 times per pass, with a passage interval of approximately two days. Gingival fibroblasts in the logarithmic growth phase were cultured at a concentration of 5 × 10⁶ cells / year. 4 Cells were seeded at a density of 1 / 2 well in 96-well plates and co-incubated with different concentrations of palladium nanozyme (100 μg / ml, 200 μg / ml, 400 μg / ml) for 24 h and 48 h. After removing the old culture medium from the 96-well plates, the cells were washed twice with PBS, and 120 μL of CCK8 reagent diluted with culture medium and lactate were added. The plates were incubated at 37°C in the dark for 2 h, and the absorbance was measured at 450 nm and 490 nm using a microplate reader. Six replicates were set up for each group, with the PBS-treated cell group serving as a blank control. The average cell viability (%) was calculated. The results showed that the palladium nanozyme did not significantly inhibit the activity of gingival fibroblasts; after 48 h, the activity of gingival fibroblasts still reached 90%. This indicates that the palladium nanozyme in this protocol has low cytotoxicity, good biocompatibility, and almost no toxicity to gingival fibroblasts.

[0083] Experiment Example 4: Study on the Addition Method of Palladium Nanozyme in Toothpaste

[0084] (1) The method refers to the "Disinfection Technical Specifications" --- inhibition zone test.

[0085] (2) Target strain: Streptococcus mutans ATCC 25175.

[0086] (3) Sample processing:

[0087] The toothpaste to be tested is diluted with physiological saline at a ratio of 1:20 to form a homogeneous test solution in a beaker. Using tweezers, a sterile filter paper disc is immersed in the toothpaste test solution for 2–3 seconds. After absorbing the solution, the disc is removed, drained, and placed in a sterile Petri dish. The dish is then left uncovered and allowed to air dry in a laminar flow hood. For the negative control, a sterile filter paper disc is immersed in sterile distilled water for 2–3 seconds. After absorbing the water, the disc is removed, drained, and placed in a sterile Petri dish. The dish is then left uncovered and allowed to air dry in a laminar flow hood.

[0088] The toothpaste samples to be tested are as follows:

[0089] Toothpaste Sample 1:

[0090] The formula is as follows: 55 parts sorbitol, 6 parts polyethylene glycol (PEG), 15 parts silica, 0.05 parts palladium nanozyme (using sample 1 prepared in Experiment 1, and preparing a 1 mg / ml palladium nanozyme solution; all samples below use this nanozyme), 1.5 parts sodium carboxymethyl cellulose, 1.5 parts sodium dodecyl sulfate (K12), 1.5 parts peppermint flavoring, 4 parts buffer solution, and the remainder is water, for a total of 100 parts.

[0091] The preparation method of palladium nanozyme is described in Example 1, Sample 1. The preparation method of toothpaste is described in Example 2, wherein the process of obtaining the activated palladium nanozyme dispersion is as follows: the palladium nanozyme and buffer solution are mixed and stirred at 100 rpm for 20 min.

[0092] Toothpaste sample 2:

[0093] The mixture consists of 40 parts sorbitol, 10 parts polyethylene glycol (PEG), 30 parts silica, 0.05 parts palladium nanozyme, 0.1 parts sodium carboxymethyl cellulose, 0.5 parts sodium dodecyl sulfate (K12), 0.5 parts peppermint flavor, 1 part buffer solution, and the remainder is water, for a total of 100 parts.

[0094] The preparation method of palladium nanozyme is described in Example 1, Sample 1. The preparation method of toothpaste is described in Example 2, wherein the process of obtaining the activated palladium nanozyme dispersion is as follows: the palladium nanozyme and buffer solution are mixed and stirred at 150 rpm for 30 min.

[0095] Toothpaste sample 3:

[0096] The mixture consists of 70 parts sorbitol, 1 part polyethylene glycol (PEG), 10 parts silica, 0.5 parts palladium nanozyme, 0.5 parts sodium carboxymethyl cellulose, 0.5 parts sodium dodecyl sulfate (K12), 0.5 parts peppermint flavor, 6 parts buffer solution, and the remainder is water, for a total of 100 parts.

[0097] The preparation method of palladium nanozyme is described in Example 1, Sample 1. The preparation method of toothpaste is described in Example 2, wherein the process of obtaining the activated palladium nanozyme dispersion is as follows: the palladium nanozyme and buffer solution are mixed and stirred at 50 rpm for 10 min.

[0098] Compared to toothpaste sample 1: The formula is basically the same as toothpaste sample 1, except that palladium nanozyme and buffer solution are not added. The preparation process has also been adjusted accordingly, as follows: The humectant and thickener are mixed and homogenized, then the surfactant and remaining water are added and stirred evenly to obtain mixture A. 60% by weight of the abrasive is added to mixture A, mixed and stirred evenly, then the remaining abrasive is added and stirred evenly before vacuuming; the fragrance is added, stirred and homogenized, degassed, and the toothpaste is obtained.

[0099] Comparison with toothpaste sample 2: The formula is basically the same as toothpaste sample 1, except that buffer solution is not added. The preparation process has also been adjusted accordingly, as follows: The humectant and thickener are mixed and homogenized, then the surfactant, palladium nanozyme, and the remaining water are added and stirred evenly to obtain mixture A. 60% by mass of abrasive is added to mixture A, mixed and stirred evenly, then the remaining abrasive is added and stirred evenly before vacuuming; fragrance is added, stirred and homogenized, degassed, and the toothpaste is obtained.

[0100] Comparison with toothpaste sample 3: The formula is basically the same as toothpaste sample 1, except that no buffer solution was added. The preparation process was also adjusted accordingly, as follows: Palladium nanozyme and water (4 parts) were mixed and stirred at 100 rpm for 20 minutes to obtain an aqueous dispersion of palladium nanozyme. The humectant and thickener were mixed and homogenized, then the surfactant, the aqueous dispersion of palladium nanozyme, and the remaining water were added and stirred evenly to obtain mixture A. 60% by weight of abrasive was added to mixture A, mixed and stirred evenly, then the remaining abrasive was added and stirred evenly before vacuuming; the fragrance was added, stirred and homogenized, degassed, and the toothpaste was obtained.

[0101] Comparison with toothpaste sample 4: The formula is basically the same as toothpaste sample 1, except that the type of buffer solution is replaced with HBSS buffer (commercially available Hank's balanced salt solution). HBSS buffer is prepared as follows: 8g NaCl, 0.4g KCl, 1g glucose, 60mg KH2PO4, 47.5mg Na2HPO4, diluted with distilled water to 1000ml, and the pH is adjusted to 7.2.

[0102] Comparison with toothpaste sample 5: The formula is basically the same as toothpaste sample 1, except that the type of buffer solution is replaced with HEPES buffer. HEPES buffer is prepared as follows: Take 119.15g of HEPES, dilute to 500ml with distilled water, and adjust the pH to 6.8.

[0103] Comparison with toothpaste sample 6: The formula is basically the same as toothpaste sample 1, except that the pH value of the phosphate buffer is adjusted to 7.4, and the other preparation process of the phosphate buffer is the same as in Example 2.

[0104] Comparison with toothpaste sample 7: The formula is basically the same as toothpaste sample 1, except that the pH value of the phosphate buffer is adjusted to 5.8, and the other preparation process of the phosphate buffer is the same as in Example 2.

[0105] (4) Preparation of bacterial suspension

[0106] Streptococcus mutans (ATCC25175) was purchased from Beina Biotechnology Co., Ltd. Streptococcus mutans and Lactobacillus rhamnosus were anaerobically revived on TSA solid medium for 48 h, while Streptococcus sanguinis and Actinomyces viscous were anaerobically revived on BHI solid medium for 48 h. Single colonies were picked and anaerobically expanded in TSA and BHI liquid media for 24 h, respectively. The bacterial concentration was adjusted to approximately 1 × 10⁻⁶ using physiological saline via turbidimetric assay. 6 CFU / mL available for use.

[0107] (5) Inoculation of test bacteria:

[0108] Use a sterile cotton swab to collect a solution with a concentration of 1×10⁻⁶. 6 Spread the CFU / ml bacterial suspension evenly three times on the surface of a TSA solid medium plate. Rotate the plate 60° after each spread, and finally swab the plate around its edge. Cover the plate and incubate at room temperature in an anaerobic environment for 5 minutes to dry.

[0109] Placement of antibacterial agent samples: For each test, use one contaminated plate, with 4 test samples and 1 negative control sample on each plate, for a total of 5 samples. Use sterile forceps to place the samples onto the plate surface. The centers of each sample should be at least 25 mm apart, and the sample should be at least 15 mm away from the periphery of the plate. After placement, gently press the sample with sterile forceps to ensure it adheres tightly to the plate surface. Cover the plate and incubate at 37℃ for anaerobic incubation for 16-18 hours, observing the results. Measure and record the diameter of the inhibition zone (including the sample) using calipers. The test is repeated 3 times. The inhibition zone diameter value for each replication is the average of the 4 test samples. The test results are shown in Table 3.

[0110] Table 3: Statistical analysis of antibacterial test results for each group of toothpaste samples and the control toothpaste samples (* indicates that the experimental group and toothpaste sample 1 were compared by a T-test, p < 0.05)

[0111]

[0112] The experimental results above show that toothpaste samples 1-3 prepared using this method can effectively inhibit bacteria. The higher the amount of palladium nanozyme added, the more obvious the antibacterial effect. If nanozyme is not added to the toothpaste (toothpaste comparison sample 1), the antibacterial performance of the toothpaste decreases significantly. In addition, the inventors also found that the effect of nanozyme is related to the preparation method. Nanozyme needs to be mixed with phosphate buffer in advance to ensure the ideal antibacterial effect; otherwise, the antibacterial performance of the toothpaste will decrease significantly (toothpaste comparison sample 2). For example, in the preparation of toothpaste comparison sample 2, phosphate buffer was not added. The humectant and thickener were mixed and homogenized, and then the surfactant, palladium nanozyme, and the remaining water were added and stirred evenly. In the preparation of toothpaste comparison sample 3, phosphate buffer was not added. The palladium nanozyme was mixed with some water and stirred at 100 rpm for 20 minutes to obtain an aqueous dispersion of palladium nanozyme. Mix the humectant and thickener homogenized, then add the surfactant, an aqueous dispersion of palladium nanozyme, and the remaining water, and stir evenly; all of the above operations will reduce the antibacterial ability of the toothpaste.

[0113] In addition, the type and pH of the buffer solution also have a crucial impact on the activation of nanozymes. For example, replacing the phosphate buffer with HEPES buffer or Hank's balanced salt solution will not achieve effective activation of nanozymes. Adjusting the pH of the phosphate buffer, whether too high or too low, will also cause significant differences in the activation effect of nanozymes.

[0114] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. The application of an artificial enzyme with antibacterial properties in the preparation of oral care products, characterized in that, The artificial enzyme is a palladium nanozyme, prepared by the following method: Surfactant 1 and reducing agent are dispersed in water to obtain a mixed solution; the mixed solution is stirred and heated, and then a palladium source solution is added; the reaction is carried out at 60-90℃ for 2-6 hours to obtain the palladium nanozyme; the palladium nanozyme is used to inhibit the growth of harmful bacteria, including Streptococcus mutans, Lactobacillus rhamnosus, Streptococcus sanguinis, and Actinomyces viscous; the palladium source is a Pd-containing... 2+ The compound is a cationic compound; the surfactant includes at least one of polyvinylpyrrolidone, hydroxyethyl cellulose, and polyacrylamide; the reducing agent includes at least one of L-ascorbic acid, potassium bromide, sodium borohydride, sodium citrate, potassium chloride, lithium aluminum hydride, and diborane. The oral care product mentioned is toothpaste; The toothpaste formula, by weight, comprises: 41-80 parts humectant, 10-30 parts abrasive, 0.05-0.5 parts palladium nanozyme, 0.1-1.5 parts thickener, 0.5-1.5 parts surfactant, 0.5-1.5 parts fragrance, 1-6 parts phosphate buffer, and the balance being water; in the phosphate buffer, the mass ratio of sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride is 1.74:2.7:1.7, and the pH is 6.

6. The toothpaste is prepared by the following method: Palladium nanozyme and phosphate buffer are mixed and stirred at 50-150 rpm for 10-30 min to obtain an activated palladium nanozyme dispersion; humectant and thickener are mixed and homogenized, then surfactant II, the activated palladium nanozyme dispersion, and the remaining water are added and stirred evenly to obtain mixture A; an abrasive is added to mixture A, homogenized, and then vacuumed; then fragrance is added, stirred, homogenized, and degassed to obtain toothpaste.

2. The application according to claim 1, characterized in that: The humectant includes at least one of polyethylene glycol, glycerin, sorbitol, and propylene glycol; the abrasive includes at least one of calcium carbonate, silica, dicalcium phosphate, and glycerophosphate; the thickener includes at least one of sodium carboxymethyl cellulose, hydroxyethyl cellulose, xanthan gum, carrageenan, guar gum, and carbomer; and the surfactant includes at least one of amino acid surfactants, sodium dodecyl sulfate, and alkyl glycosides.

3. The application of the artificial enzyme with antibacterial properties according to claim 2 in the preparation of oral care products, characterized in that: The artificially simulated enzyme has a tetrahedral structure, a particle size of 40-80 nm, a hydrodynamic size of 62-106 nm, and a surface potential of -34±6 mV.

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