Composition containing probiotics and application thereof in oral cavity cleaning

Abstract

The invention provides a composition containing probiotics and application of the composition to oral cavity cleaning, more specifically, antibacterial peptide with specificity for inhibiting oral cavity pathogenic bacteria is obtained through screening from a peptide library, and the antibacterial peptide has a good bacterial plaque biological membrane inhibiting effect. After the antibacterial peptide and probiotics are combined for use, the oral microenvironment can be better improved, oral diseases can be better inhibited, the antibacterial peptide can be prepared into oral cleaning products such as toothpaste or mouth wash, and the antibacterial peptide has a good application prospect.

Description

Technical Field

[0001] The present application relates to the biological field and also to the oral care field, and is a composition containing probiotics and use thereof for oral cleaning. Background Art

[0002] Oral diseases are common, affecting more than 3.5 billion people worldwide, but oral health is often neglected in clinical practice. As a chronic disease that continues to develop, oral problems can affect a person's entire life and may also exacerbate the occurrence and development of other diseases and health problems. Ensuring oral health is essential for maintaining overall health and longevity. This is especially true in the context of the accelerated aging process around the world. Research results show that the probability of elderly people suffering from oral diseases is over 95%.

[0003] Oral diseases can be treated through general treatment, drug treatment, surgical treatment, laser treatment, ultrasonic cleaning, etc. However, if they can be detected and prevented early, a lot of costs will be saved. In recent years, scholars have successively discovered that a variety of probiotics can restrict the growth of oral pathogens, and play a role in the prevention and treatment of oral diseases such as caries, oral candidiasis, periodontal disease, and halitosis by inhibiting the adhesion and colonization of pathogens, releasing antibacterial substances, and regulating mucosal immunity. They may also be used in the treatment of oral tumors.

[0004] Streptococcus mutans is the main pathogen in the development of dental caries. Clinical studies have shown that probiotics such as Lactobacillus rhamnosus, Bifidobacterium, Lactobacillus acidophilus, Saccharomyces cerevisiae, Bacillus coagulans, and Streptococcus lactis have an inhibitory effect on cariogenic bacteria. Studies have shown that after 105 children were given yogurt containing Lactobacillus rhamnosus GG strain, the number of oral cariogenic bacteria decreased significantly. In another clinical trial, 40 adolescents ate ice cream containing Bifidobacterium Bb-12 and Lactobacillus acidophilus La-5, and the number of Streptococcus mutans in their saliva was significantly reduced. After 150 Indian children aged 7-14 were given two probiotic powders for 14 days, the number of Streptococcus mutans in the two probiotic groups containing Lactobacillus rhamnosus, Bifidobacterium longum and Saccharomyces cerevisiae and Bacillus coagulans was significantly reduced compared with the placebo group. Some scholars also believe that probiotics have no inhibitory effect on cariogenic bacteria, and some probiotics may even accelerate the progression of dental caries. In vitro studies have found that Lactobacillus paracasei F19 has no inhibitory effect on cariogenic bacteria such as Streptococcus mutans and Lactobacillus. Other studies have found that the number of cariogenic bacteria Streptococcus mutans and Lactobacillus did not decrease after children took Lactobacillus reuteri. LGG cannot inhibit the growth of Streptococcus mutans, but will accelerate the process of caries by causing tooth demineralization. This also fully demonstrates that the selection of specific probiotics is also crucial for the treatment of corresponding pathogens.

[0005] Oral candidiasis is the most common oral fungal infection in humans, and attempts to use probiotics to intervene in oral candidiasis have been a hot topic in recent years. Studies have found that co-culture of lactobacillus and Candida albicans can effectively inhibit the growth of Candida albicans. Studies have also shown that the use of Lactobacillus rhamnosus in mouse experiments can significantly reduce the incidence of candida albicans. Clinical studies have shown that probiotic intervention is significantly effective in the prevention and treatment of Candida albicans. 60 elderly people wearing dentures were divided into two groups. After the experimental group took cheese containing Lactobacillus acidophilus or Lactobacillus rhamnosus for 8 weeks, the Candida count in saliva decreased significantly, and the level of anti-Candida IgA increased significantly. There are also studies reporting that probiotics do not significantly affect the growth of oral Candida. Studies have found that after 7 elderly people wearing dentures consumed probiotic beverages for 4 weeks, there was no significant change in the Candida count in the mouth. This also fully demonstrates that the selection of specific probiotics is also crucial for the treatment of corresponding pathogens.

[0006] In addition, in the treatment of oral diseases, traditional antibiotics can easily induce bacterial gene mutations and produce drug resistance. Therefore, it is necessary to develop new drugs that can inhibit free or biofilm microorganisms. Antimicrobial peptides are the focus of recent oral microbiology research. Antimicrobial peptides have the characteristics of small molecular weight, good water solubility, strong heat resistance, non-immunogenicity, and a wide antibacterial spectrum. They are very likely to become a new antibacterial, antiviral, and anticancer drug with high efficiency, low toxicity, and no residual harm. The α-helical cationic antimicrobial peptide pleurocidin is isolated from the epidermis of American flounder and has become one of the most studied antimicrobial peptides in recent years, with a definite antibacterial effect. However, pleurocidin is 25 amino acids long, and the excessively long amino acids increase the cost of synthesis and reduce the ability to penetrate into bacterial biofilms. In addition, there are other polypeptides in the art for oral antibacterial. For example, the antimicrobial peptide PL-3301 has a high efficiency in killing various oral Candida and antibiotic-resistant Candida, and its antibacterial activity has been effectively demonstrated in preclinical in vitro tests and animal models. However, the efficacy of the antimicrobial peptide does not have broad-spectrum antibacterial properties, so it is even more necessary to develop new broad-spectrum antibacterial short peptides. Summary of the invention

[0007] The antimicrobial peptide provided by the present invention is named MS-N-17, and its sequence is

[0008] Ac-KKKSKKKNKKKFKAA-NH2.

[0009] The electrostatic binding of the antimicrobial peptide to the cell membrane is one of the first and key steps to combat invading pathogens. As the number of peptide molecules increases, the electrostatic attraction and permeation of the cell membrane are strengthened, and then the peptide molecules freely diffuse laterally and preassemble on the membrane surface. After the antimicrobial peptide is inserted into the cell membrane, the hydrophobic surface of the peptide binds to the non-polar fatty acyl chain region, while the hydrophilic surface remains in contact with the polar lipid head and the water phase, forming holes to lyse the cell membrane.

[0010] The antimicrobial peptides of the present invention may also be modified or conservatively substituted.

[0011] The polypeptide of the present invention may also be substituted by one or more basic amino acids. The basic amino acids are selected from Arg, Lys, His, preferably Arg and Lys; the hydrophobic amino acids are selected from Trp, Leu, Ile, Pro, Gly, Val, Ala or Met, preferably Ile, Gly, Trp, Pro and Leu.

[0012] Furthermore, the present invention also provides a composition for oral cleaning and sterilization, which comprises the antimicrobial peptide MS-N-17 of the present invention, and the sequence thereof is Ac-KKKSKKKNKKKFKAA-NH2.

[0013] Furthermore, the present invention also provides a composition containing antimicrobial peptides and probiotics.

[0014] The composition can be a toothpaste, and the toothpaste is stored at low temperature.

[0015] Furthermore, the probiotics are Lactobacillus plantarum N13 strain with a preservation number of CGMCC No.20496, Lactobacillus acidophilus LA85 strain with a preservation number of CGMCC No.1.12735,

[0016] The Lactobacillus paracasei LC86 strain of CGMCC No.1.12731 was cultured in an MRS liquid culture medium, and then the bacterial sludge was collected by centrifugation, and the bacterial sludges were mixed in a weight ratio of 1-5:1-5:1-5 to obtain a probiotic mixture.

[0017] The toothpaste is prepared by using the following components in mass fractions: 3-5% of probiotic mixture, 5-10% of silicon dioxide, 30-50% of sorbitol, 10-20% of glycerol, 1.5-5% of sodium lauryl sulfate, 0.5-2% of sodium hydroxymethyl cellulose, 0.8-2% of xanthan gum, 0.8-2% of sodium saccharin, 0.03-0.05% of preservative, 0.2-0.5% of tetrasodium pyrophosphate, 0.1-0.5% of sodium chloride, 2-5% of sodium lauroyl sarcosinate, 0.5-0.8% of flavor and 1-5% of MS-N-17 antimicrobial peptide, and the balance is deionized water.

[0018] The toothpaste preparation method comprises the following steps: S1, weighing the raw materials according to the aforementioned weight ratio; S2, mixing: adding the components weighed in S1 except the probiotics into a blender and stirring them uniformly first, and finally adding the probiotics and stirring them uniformly to obtain a mixture; S3, vacuum degassing treatment: vacuumizing the mixture in S2, and then degassing to obtain a paste, and grinding to obtain an antimicrobial peptide and probiotic toothpaste.

[0019] Furthermore, the present invention also provides a use of probiotics and antimicrobial peptides in preparing a pharmaceutical composition for inhibiting the proliferation of harmful oral bacteria.

[0020] The compositions provided herein may further include one or more additional ingredients selected from abrasives, pH regulators, surfactants, foam regulators, thickeners, viscosity regulators, wetting agents, anticalculus agents or tartar control agents, sweeteners, flavoring agents, colorants and preservatives. These ingredients may also be considered as carrier materials. In one embodiment, the composition of the present invention comprises at least one abrasive that can be used as, for example, a polishing agent. Any orally acceptable abrasive can be used, but the type, fineness (particle size) and amount of the abrasive should be selected so that the enamel will not be excessively worn during the normal use of the composition. Suitable abrasives include, but are not limited to, silica gel, hydrated silica or precipitated silica, alumina, insoluble phosphates, calcium carbonate, resin abrasives such as silica in the form of urea-formaldehyde condensation products. Insoluble phosphates that can be used as abrasives are orthophosphates, polymetaphosphates and pyrophosphates. Illustrative examples are dihydrated dicalcium orthophosphate, calcium pyrophosphate, -calcium pyrophosphate, tricalcium phosphate, calcium polymetaphosphate and insoluble sodium polymetaphosphate.

[0021] In another embodiment, the composition of the present invention comprises at least one pH adjusting agent. These agents include acidulants that reduce pH, alkalizers that increase pH, and buffers that control pH within the desired range. For example, one or more compounds selected from acidulants, alkalizers, and buffers may be included to provide a pH of 2-10, or a pH of 2-8, 3-9, 4-8, 5-7, 6-10, or 7-9 in various illustrative embodiments. Any orally acceptable pH adjusting agent may be used, including but not limited to carboxylic acids, phosphoric acid, and sulfonic acids, acid salts (e.g., monosodium citrate, disodium citrate, monosodium malate), alkali metal hydroxides such as sodium hydroxide, carbonates such as sodium carbonate, bicarbonates, borates, silicates, phosphates (e.g., monosodium phosphate, trisodium phosphate, pyrophosphate), imidazoles, etc. One or more pH modifiers are optionally present in a total amount that effectively maintains the composition within an orally acceptable pH range.

[0022] In yet another embodiment, the composition of the present invention comprises at least one surfactant, which can be used, for example, to provide enhanced stability for the composition and the components contained therein, to help clean the tooth surface through detergent action, and to provide foam when stirred (for example, when brushing with the dentifrice composition of the present invention). Any orally acceptable surfactant can be used, including anionic, nonionic or amphoteric surfactants. Suitable anionic surfactants include, but are not limited to, water-soluble salts of CS-20 alkyl sulfates, sulfonated monoglycerides of CS-20 fatty acids, sarcosinates, taurates, etc. Suitable nonionic surfactants include, but are not limited to, poloxamers, polyoxyethylene sorbitan esters, fatty alcohol ethoxylates, alkylphenol ethoxylates, tertiary amine oxides, tertiary phosphine oxides, dialkyl sulfoxides, etc. Suitable amphoteric surfactants, but are not limited to derivatives of CS-20 aliphatic secondary amines and tertiary amines with anionic groups, such as carboxylates, sulfates, sulfonates, phosphates or phosphonates. A suitable example is cocamidopropyl betaine. One or more surfactants are optionally present in a total amount of 0.01 wt% to 10 wt%, such as 0.05 wt% to 5 wt% or 0.1 wt% to 2 wt% based on the total weight of the composition.

[0023] In yet another embodiment, the composition of the present invention comprises at least one flavoring agent, and this flavoring agent enhances the taste of the composition.Can use any natural or synthetic flavoring agent that is orally acceptable, including but not limited to vanillin, sage, Marjoram, parsley oil, spearmint oil, cinnamon oil, wintergreen oil (methyl salicylate), peppermint oil, clove oil, laurel oil, anise oil, eucalyptus oil, citrus oil, fruit oil and essence etc.Fragrance also comprises the component that provides fragrance and / or other sensory effects in oral cavity, including cooling or warming effect. These ingredients illustratively include menthol, menthyl acetate, menthyl lactate, camphor, eucalyptus oil, eugenol, cinnamon, -ionone, -ionone, thymol, linalool, benzaldehyde, cinnamaldehyde, N-ethyl-p-menthol-3-carboxyamide, N,2,3-trimethyl-2-isopropylbutyramide, 3-(1-menthoxy)-propane-1,2-diol, cinnamaldehyde glycerol acetal (CGA), menthone glycerol acetal (MGA), etc. One or more flavoring agents are optionally present in a total amount of 0.01 wt% to 5 wt% of the total weight of the composition. In another embodiment, the combination of the present invention The composition comprises at least one coloring agent. The coloring agent may have a variety of functions. These include providing a white or light-colored coating on the tooth surface, indicating the position on the tooth surface that has been effectively contacted by the composition, and / or changing the appearance of the composition to enhance the attractiveness to consumers. Any orally acceptable coloring agent may be used, including but not limited to talc, mica, magnesium carbonate, calcium carbonate, magnesium silicate, magnesium aluminum silicate, silicon dioxide, titanium dioxide, zinc oxide, iron oxide, ammonium ferrocyanide, manganese violet, titanium mica, bismuth oxychloride, etc. One or more coloring agents are optionally present in a total amount of 0.001% to 1% by weight of the total weight of the composition.

[0024] Beneficial Effects

[0025] The present invention provides a composition containing probiotics and its use for oral cleaning. More specifically, the present invention screens and obtains antibacterial peptides with specificity for inhibiting oral pathogens from a peptide library, and the antibacterial peptides have good inhibitory effects on plaque biofilm. The antibacterial peptides can be used in combination with probiotics to better improve the oral microenvironment and inhibit oral diseases, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Effect diagram of each group on the inhibition rate of plaque biofilm DETAILED DESCRIPTION

[0027] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all conventional methods, equipment, and materials in the art, and can be purchased from the market.

[0028] Example 1 Design of antimicrobial peptides

[0029] The inventors screened out a new type of artificially synthesized antimicrobial peptide from a polypeptide library, which has strong antimicrobial activity against harmful oral microorganisms. The antimicrobial peptide is named MS-N-17, and its sequence is

[0030] Ac-KKKSKKKNKKKFKAA-NH2.

[0031] The antimicrobial peptide screened by the present invention is cationic, has a net positive charge, is rich in hydrophilic amino acids at the N-terminus, and is rich in hydrophobic amino acids at the C-terminus, forming an amphiphilic molecular structure. Ac refers to acetylation at the N-terminus, and NH2 refers to amidation at the C-terminus.

[0032] The positive control was the antimicrobial peptide PL-3301 (Ac-FKKLKKLFSKLWNWK-NH2). The polypeptide was commissioned to be artificially synthesized by Peptide Valley Biotechnology, and the artificially synthesized antimicrobial peptide was resuspended in sterile double distilled water to a final concentration of 3 g / L, filtered through a pinhole filter, and stored at -20°C for later use.

[0033] Example 2 Identification of antimicrobial peptide activity

[0034] Streptococcus mutans ATCC25175 and Porphyromonas gingivalis ATCC33277 were cultured in BHI medium, Candida albicans ATCC10231 was cultured in YPD medium, Actinomyces naeslundii ATCC12140 was cultured in TPY medium, Candida albicans ATCC10691 was cultured in BA medium, and Escherichia coli ATCC43888 was cultured in LB medium. The culture temperature was 37 degrees Celsius and the shaking culture was 150r / min.

[0035] The experiment was divided into a positive control group (bacterial suspension culture medium containing PL-3301), a negative control group (bacterial suspension culture medium containing PBS), and an experimental group (culture medium containing different concentrations of MS-N-17 peptide). Three parallel groups were set up for each of the above groups. The MIC was determined using the liquid dilution method. The antimicrobial peptide experimental solution with a concentration of 3g / L was diluted in the liquid culture medium by multiple dilutions to make the final concentration of 2g·L -1 , 1g·L -1 , 500μg·mL -1 , 250μg·mL -1 , 125 μg·mL -1 、62.5μg·mL -1 , 31.3 μg·mL -1 , 15.6 μg·mL -1 The concentration is 2×10 6 CFU·mL -1 The bacterial suspension and each group of drug solution were inoculated in a sterile 96-well plate at a ratio of 1:1, 100 μL each, so that the final concentration of the bacterial suspension was 1×10 6 CFU·mL -1 , placed under appropriate culture conditions and cultured for 24-48 hours. Under a black background, the lowest drug concentration at which the liquid in the microwell is clear and has no turbidity or precipitation growth is the MIC of the antimicrobial peptide. The results are shown in Table 1.

[0036] Table 1 MIC values ​​of antimicrobial peptides against oral pathogens

[0037]

[0038]

[0039] As can be seen from Table 1, the antimicrobial peptide MS-N-17 of the present invention has a good MIC inhibitory effect on Streptococcus mutans ATCC25175, Porphyromonas gingivalis ATCC33277, Candida albicans ATCC10231, Actinomyces naeslundii ATCC12140, Candida albicans ATCC10691, and Escherichia coli ATCC43888, especially the inhibitory effect on Candida albicans, Porphyromonas gingivalis and Candida albicans is the most significant. The negative control group has no inhibitory effect on the bacteria.

[0040] Example 3 Effect of antimicrobial peptides on plaque biofilm

[0041] Supragingival plaque was collected from the proximal surface of the mandibular second molar of a healthy adult volunteer using dental floss. The collected supragingival plaque was added to fresh BHI culture medium to prepare a plaque suspension. The absorbance (A) value of the bacterial suspension was detected in a 96-well plate using a multifunctional microplate reader. 150 μL of the bacterial suspension was added to each well, and the OD600 was adjusted to 0.1 as the standardized density of the bacterial suspension. The pretreated HA disc (HA disc was used as a biofilm formation carrier. The HA disc was sterilized by high-pressure steam at 121°C and placed in a drying oven overnight. The HA disc was soaked in 1 mL of type I collagen solution and the pretreated HA disc was obtained at 4°C overnight) was placed in a 24-well culture plate, 1.8 mL of BHI culture medium and 0.2 mL of bacterial suspension were added to each well, and cultured under anaerobic conditions at 37°C for 3 days to form a multi-species oral biofilm.

[0042] Prepare 3d dental plaque biofilm HA discs and soak them in 1mL PBS for 1min. Slowly drip 100μL of different antimicrobial peptide concentrations on the HA discs for 3min, and divide them into the following groups (10 replicates per group):

[0043] (1) Sterile water;

[0044] (2) 2% PL-3301 positive control peptide solution;

[0045] (3) 1% MS-N-17 peptide solution;

[0046] (4) 2% MS-N-17 peptide solution;

[0047] The HA discs treated with sterile water were used as the blank group, and the HA discs treated with 2% PL-3301 peptide were used as the positive control group. After the treatment, they were soaked in 1 mL PBS for 1 min, and the LIVE / DEAD TM BacLight TM The bacterial activity detection kit was used for quantitative analysis and the results were as follows Figure 1 shown.

[0048] from Figure 1 It can be seen that compared with sterile water, the proportion of dead bacteria in the biofilm of each experimental group was significantly increased (P<0.05). At the same concentration, the MS-N-17 antimicrobial peptide group of the present invention has the strongest bactericidal effect on dental plaque biofilm, with a bactericidal rate of (64.8±1.5)%.

[0049] Example 4 Preparation and efficacy testing of antimicrobial peptide mouthwash

[0050] Prepare mouthwash according to the following proportions: take 100 mL of deionized water, add 5 mg of polyvinyl pyrrolidone, 12.5 mg of gelatin and 5 mg of vitamin C in sequence, stir to dissolve evenly, add 500 mg of MS-N-17 antimicrobial peptide, continue stirring for half an hour until the system becomes clear and transparent, and obtain a mouthwash containing antimicrobial peptides.

[0051] A control mouthwash was prepared according to the following proportions: 100 mL of deionized water was taken, 5 mg of polyvinyl pyrrolidone, 12.5 mg of gelatin and 5 mg of vitamin C were added in sequence, stirred to dissolve evenly, 500 mg of PL-3301 antimicrobial peptide was added, and stirring was continued for half an hour until the system became clear and transparent, thereby obtaining a control mouthwash.

[0052] Evaluation of oral bacteria removal effect

[0053] (1) Experimental groups

[0054] Group A: gargle with antimicrobial peptide mouthwash for 1 min, 10 mL each time;

[0055] Group B: gargle with the control mouthwash for 1 min, 10 mL each time;

[0056] Group C: rinse with clean water for 1 min, 10 mL each time.

[0057] Each group included 5 men and 5 women, with an average age of 30-35 years old, all of whom were orthodontic patients. Oral hygiene maintenance was the same, that is, brushing teeth once in the morning and evening, using the same type of toothpaste, each time for 5 minutes. The experiment lasted for 3 months in each group.

[0058] (2) Bacteria collection method: Before and after gargling, use sterile filter paper to randomly and symmetrically collect bacteria on the left and right sides of the horizontal mucosa of the palate molars. The collection area is 1.0 cm 2 The filter paper was placed in the bacterial collection area for 30 seconds, then taken out and placed in a centrifuge tube with 1 mL of PBS solution. The mixture was thoroughly mixed in a vortex mixer, diluted 10 times, and 10 μL was inoculated in an agar culture dish, and cultured at 37°C for 48 hours. The colony count (CFU / mL) was performed to calculate the total amount of oral bacteria. The results are shown in Table 2.

[0059] Table 2 Total colony count before and after treatment

[0060] Group Before rinsing your mouth After rinsing your mouth Group A 9301.7±86.5 4061.2±52.1# Group B 9258.9±78.4 5105.4±68.9# Group C 9349.4±95.4 8153.5±84.7

[0061] As can be seen from Table 2, the mouthwashes of Group A and Group B had a significant effect of reducing the total bacterial count compared with Group C after use (P<0.05), and in particular, the antimicrobial peptide mouthwash of the present invention had a better antibacterial effect.

[0062] Example 5 Preparation and efficacy verification of antimicrobial peptide probiotic toothpaste

[0063] Lactobacillus plantarum with the deposit number of CGMCC No.20496

[0064] Lactobacillus plantarum N13 strain, Lactobacillus acidophilus LA85 strain with a deposit number of CGMCC No.1.12735, and Lactobacillus acidophilus LA85 strain with a deposit number of

[0065] The Lactobacillus paracasei LC86 strain of CGMCC No.1.12731 was cultured in MRS liquid culture medium, and then the bacterial sludge was collected by centrifugation, and the bacterial sludges were mixed in a weight ratio of 1:1:1 to obtain a probiotic mixture.

[0066] The toothpaste is prepared by using the following components in mass fractions: 3% probiotic mixture, 5% silicon dioxide, 30% sorbitol, 10% glycerol, 1.5% sodium lauryl sulfate, 0.5% sodium hydroxymethyl cellulose, 0.8% xanthan gum, 0.8% sodium saccharin, 0.03% preservative, 0.2% tetrasodium pyrophosphate, 0.1% sodium chloride, 2% sodium lauroyl sarcosinate, 0.5% flavor and 2% MS-N-17 antimicrobial peptide, and the balance is deionized water.

[0067] The control toothpaste was prepared in the same manner as described above, but the antimicrobial peptide was replaced with an equal amount of PL-3301 antimicrobial peptide.

[0068] MS-N-17 antimicrobial peptide toothpaste: The toothpaste is prepared by mixing the following components by mass fraction: 5% silicon dioxide, 30% sorbitol, 10% glycerol, 1.5% sodium lauryl sulfate, 0.5% sodium hydroxymethyl cellulose, 0.8% xanthan gum, 0.8% sodium saccharin, 0.03% preservative, 0.2% tetrasodium pyrophosphate, 0.1% sodium chloride, 2% sodium lauroyl sarcosinate, 0.5% flavor and 2% MS-N-17 antimicrobial peptide, and the balance is deionized water.

[0069] Probiotic toothpaste: probiotic mixture 3%, silicon dioxide 5%, sorbitol 30%, glycerin 10%, sodium lauryl sulfate 1.5%, sodium hydroxymethyl cellulose 0.5%, xanthan gum 0.8%, sodium saccharin 0.8%, preservative 0.03%, tetrasodium pyrophosphate 0.2%, sodium chloride 0.1%, sodium lauroyl sarcosinate 2%, flavor 0.5%, and the balance is deionized water.

[0070] The preparation methods of the above toothpaste groups include the following steps: S1, weighing the raw materials, weighing the raw materials according to the aforementioned weight ratio; S2, mixing: adding the components weighed in S1 except the probiotics into a blender and stirring evenly first, and finally adding the probiotics (not adding the probiotics to the group without probiotics), stirring evenly to obtain a mixture; S3, vacuum degassing treatment: vacuumizing the mixture in S2, and then degassing to obtain a paste, and grinding to obtain the toothpaste of each group.

[0071] Take the toothpaste prepared above; select 80 subjects, the inclusion criteria of the subjects: the subjects are in good general condition; accompanied by chronic gingivitis; have the habit of brushing teeth in the morning and evening before the test. The subjects were randomly divided into 4 groups, 20 in each group, and there was no significant difference in gender, age and general data among the subjects in each group. The subjects in each group brushed their teeth once in the morning and evening, 3 minutes / time, for 4 consecutive weeks. The subjects in each group could not change other oral cleaning products during the test period, and the effect was evaluated after 4 weeks of use.

[0072] Gum index score: (1) 0 points: healthy gums; (2) 1 point: mild inflammation of the gums, slight change in gum color and edema, no bleeding on probing; (3) 2 points: moderate inflammation of the gums, red gums, edema and shiny gums, bleeding on probing; (4) 3 points: severe inflammation of the gums, obvious redness, swelling and ulceration of the gums, with a tendency to spontaneous bleeding. The results are shown in Table 3.

[0073] Table 3 The therapeutic effect of toothpaste prepared by the present invention on gingivitis (score)

[0074] Group Score before use After use score Antimicrobial peptides and probiotic toothpaste 1.89±0.17 0.53±0.13## Control toothpaste 1.83±0.13 0.76±0.09# Probiotic toothpaste 1.84±0.11 0.94±0.12 MS-N-17 Antimicrobial Peptide Toothpaste 1.86±0.16 0.73±0.04#

[0075] As can be seen from Table 3, the MS-N-17 antimicrobial peptide toothpaste of the present invention can significantly increase the therapeutic effect after the antimicrobial peptide is used in combination with probiotics (the difference is extremely significant compared with the probiotic toothpaste P<0.01). As can be seen from the results of Table 3, the MS-N-17 antimicrobial peptide combined with probiotics of the present invention can inhibit the reproduction of harmful bacteria and maintain the balance of oral microecology, thereby reducing the possibility of periodontal disease.

[0076] It should be understood that the specific embodiments of the present invention are only used to illustrate the spirit and principles of the present invention, and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes, substitutions, deletions, corrections or adjustments to the technical solutions of the present invention, and these equivalent technical solutions also fall within the scope defined by the claims of the present invention.

Claims

1. An antimicrobial peptide, characterized in that It was named MS-N-17, and its sequence was Ac-KKKSKKKNKKKFKAA-NH2.

2. Use of the antimicrobial peptide according to claim 1 in the preparation of a drug for inhibiting oral pathogens.

3. Use of the antimicrobial peptide according to claim 1 in oral care products for regulating the oral microenvironment.

4. The method of claim 1 wherein the probiotics are prepared by culturing Lactobacillus plantarum N13 strain (CGMCC No. 20496), Lactobacillus acidophilus LA85 strain (CGMCC No. 1.12735), and Lactobacillus paracasei LC86 strain (CGMCC No. 1.12731) in an MRS liquid medium, collecting bacterial mud by centrifugation, and mixing the bacterial mud in a weight ratio of 1:1:1 to obtain the probiotics; and the final concentration of the probiotics is 3%, and the final concentration of the antimicrobial peptides is 2%.

5. The method of claim 1 wherein the probiotics are prepared by culturing Lactobacillus plantarum N13 strain (CGMCC No. 20496), Lactobacillus acidophilus LA85 strain (CGMCC No. 1.12735), and Lactobacillus paracasei LC86 strain (CGMCC No. 1.12731) in an MRS liquid medium, centrifuging and collecting bacterial mud, and mixing the bacterial mud in a weight ratio of 1:1:1 to obtain the probiotics; and the final concentration of the probiotics is 3%, and the final concentration of the antimicrobial peptides is 2%.

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