Polypeptides smug15, smuv13 inhibiting streptococcus mutans biofilm formation and applications thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to peptides SmuG15 and SmuV13 that inhibit biofilm formation in Streptococcus mutans and their applications. The amino acid sequence of SmuG15 is shown in SEQ ID NO: 1, and the amino acid sequence of SmuV13 is shown in SEQ ID NO: 2. The peptides of this invention do not exert their anti-caries effect by inhibiting the planktonic growth of Streptococcus mutans or by directly killing bacteria, but rather by targeting and interfering with the cariogenic virulence-related processes of this strain. Specifically, this includes inhibiting biofilm formation, dissociating the three-dimensional biofilm structure, and reducing the synthesis and accumulation of extracellular polysaccharides, thereby achieving selective intervention in the cariogenic process. This "targeting virulence rather than survival" characteristic avoids the drug resistance selection pressure and non-specific interference with the oral microecology caused by traditional antibacterial drugs, and also differs from existing peptide models such as C16G2 that rely on bactericidal action, providing a new technical path and candidate molecules for the precise ecological prevention and treatment of dental caries.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to polypeptides SmuG15 and SmuV13 that inhibit the formation of biofilms in Streptococcus mutans and their applications. Background Technology

[0002] Dental caries is one of the most common chronic infectious diseases of the oral cavity. According to the Fourth National Oral Health Epidemiological Survey, the prevalence of dental caries in deciduous teeth among 5-year-old children in my country is 70.9%, in permanent teeth among 12-year-old children it is 34.5%, and in the elderly aged 65-74 it reaches as high as 98.0%. Dental caries can lead to progressive destruction of the hard tissues of the teeth, causing complications such as pulpitis and periapical periodontitis. In severe cases, it can cause crown defects or tooth loss, affecting chewing function. With changes in dietary structure, the prevention and control of dental caries is becoming increasingly serious, and developing safe and effective caries prevention strategies has become an important direction in the field of oral medicine.

[0003] The occurrence and development of dental caries are closely related to the imbalance of the oral microecology. Among the many cariogenic microorganisms, *Streptococcus mutans* is recognized as one of the core pathogens of dental caries. The cariogenic virulence of *Streptococcus mutans* is mainly reflected in the following three aspects: Firstly, this bacterium secretes glucosyltransferase to synthesize a large amount of insoluble extracellular polysaccharides from sucrose. These extracellular polysaccharides form the biofilm skeleton, promoting bacterial adhesion and aggregation on the tooth surface. Secondly, *Streptococcus mutans* can form a three-dimensional biofilm, constructing a highly organized bacterial community on the tooth surface. This structure can effectively inhibit the penetration of antimicrobial drugs and weaken the clearance efficiency of host immune cells, thereby enhancing the survival advantage and pathogenic persistence of the bacterial community in the oral environment. Third, Streptococcus mutans can efficiently metabolize carbohydrates to produce organic acids, lowering the local pH and leading to enamel demineralization.

[0004] Among the aforementioned virulence factors, extracellular polysaccharide synthesis and biofilm formation are upstream in the cariogenic cascade. Without the "scaffolding" provided by extracellular polysaccharides, bacteria cannot stably colonize the tooth surface, nor can they form mature plaque biofilms with cariogenic activity. Therefore, targeted intervention in the synthesis of extracellular polysaccharides and the structural assembly of biofilms in Streptococcus mutans has become an important research direction in current cariogenic prevention strategies.

[0005] Strategies for preventing dental caries targeting Streptococcus mutans biofilms currently face two main technical bottlenecks: First, while traditional chemical antibacterial drugs (such as chlorhexidine) can effectively inhibit cariogenic bacteria, long-term use can easily induce bacterial resistance, and their broad-spectrum bactericidal effects can non-specifically kill resident oral flora, disrupting the oral microecological balance. Second, although novel bioactive molecules, represented by antimicrobial peptides, have shown application potential in the field of dental caries prevention, the reported anti-caries peptides vary in their targets and modes of action. For example, C16G2 is a specific targeted antimicrobial peptide composed of a targeting peptide coupled with a broad-spectrum antimicrobial peptide domain (G2), which can selectively kill bacteria by recognizing specific signaling molecules of Streptococcus mutans. However, the mechanism of action of C16G2 depends on the direct killing of target bacteria, and its G2 domain is derived from a broad-spectrum antimicrobial peptide, which carries a certain risk of cytotoxicity. In addition, most of the currently reported anti-caries peptides lack systematic comparisons in terms of their concentration range, biocompatibility, and impact on the oral microecology. Therefore, there is an urgent need to develop novel peptide molecules that are highly efficient and can precisely target the cariogenic virulence process. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide polypeptides SmuG15 and SmuV13 involved in inhibiting the formation of biofilms in Streptococcus mutans and their applications.

[0007] This invention provides two functional peptides, SmuG15 and SmuV13, with regulatory effects on the biofilm formation process of *Streptococcus mutans*. These two peptides significantly inhibit the in vitro formation of *Streptococcus mutans* biofilms and effectively block the synthesis and accumulation of extracellular polysaccharides. They also exhibit significant remodeling and dissociation effects on the three-dimensional biofilm structure. Notably, the inhibition of biofilm formation and extracellular polysaccharide synthesis is not accompanied by a decrease in bacterial growth capacity, indicating good targeting. Compared to existing caries prevention strategies, the peptides provided in this invention demonstrate excellent efficacy in inhibiting biofilm formation and extracellular polysaccharide accumulation. This not only enriches the current peptide library for *Streptococcus mutans* biofilm intervention but also provides novel candidate molecules with independent intellectual property rights for caries prevention and treatment.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A polypeptide, namely SmuG15 or SmuV13, wherein the amino acid sequence of SmuG15 is shown in SEQ ID NO: 1 and the amino acid sequence of SmuV13 is shown in SEQ ID NO: 2.

[0010] An oral care product comprising the aforementioned polypeptide.

[0011] As a preferred technical solution, the concentration of the added peptide is 64–256 µg / mL.

[0012] As a preferred technical solution, the dosage form of the oral care product includes toothpaste, mouthwash, oral spray, tooth powder, gel, oral lozenges, or chewing gum. The oral care product also contains orally acceptable excipients, which are selected from one or more of thickeners, surfactants, humectants, sweeteners, flavorings, preservatives, or pH adjusters.

[0013] As a preferred technical solution, the oral care product is in the form of a liquid dosage form, a semi-solid dosage form, or a solid dosage form.

[0014] A biofilm inhibitory pharmaceutical composition comprising the aforementioned polypeptide.

[0015] As a preferred technical solution, the concentration of the added peptide is 64–256 µg / mL.

[0016] The above-mentioned peptides are used in the preparation of drugs for preventing and treating dental caries.

[0017] As a preferred technical solution, dental caries is prevented by inhibiting the formation of Streptococcus mutans biofilm and / or dissociating the three-dimensional biofilm structure of Streptococcus mutans and / or reducing the synthesis and accumulation of extracellular polysaccharides of Streptococcus mutans and / or not inhibiting the growth of planktonic strains of Streptococcus mutans.

[0018] It is worth noting that the dosage forms of the oral care products described in this invention include, but are not limited to, toothpaste, mouthwash, oral spray, tooth powder, gel, oral lozenges, or chewing gum.

[0019] The present invention has the following advantages: This invention provides two novel functional peptides, SmuG15 and SmuV13, derived from the culture supernatant of *Streptococcus mutans*, with amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Unlike existing anti-caries peptides, the peptides of this invention do not exert their anti-caries effect by inhibiting the planktonic growth of *Streptococcus mutans* or by directly killing bacteria. Instead, they work by targeting and interfering with cariogenic virulence-related processes of this strain. Specifically, this includes inhibiting biofilm formation, dissociating the three-dimensional biofilm structure, and reducing the synthesis and accumulation of extracellular polysaccharides, thereby achieving selective intervention in the cariogenic process. This "targeting virulence rather than survival" characteristic avoids the drug resistance selection pressure and non-specific interference with the oral microecology caused by traditional antibacterial drugs, and also differs from existing peptide models such as C16G2 that rely on bactericidal action. This provides a new technical pathway and candidate molecules for the precise ecological prevention and treatment of dental caries. Attached Figure Description

[0020] Figure 1This figure shows the effect of different concentrations of peptides SmuG15 and SmuV13 on biofilm formation in *Streptococcus mutans* in Example 3. In the figure, a: representative image of crystal violet staining in the SmuG15 treatment group; b: representative image of crystal violet staining in the SmuV13 treatment group, with peptide concentrations from left to right being 0, 64, 128, and 256 μg / mL. The staining depth reflects the amount of biofilm attached; darker staining indicates greater biofilm formation; c: quantitative analysis results of crystal violet staining in the SmuG15 treatment group; d: quantitative analysis results of crystal violet staining in the SmuV13 treatment group, with the ordinate representing the absorbance (OD) at 595 nm. 595 This reflects the relative biomass of the biofilm.

[0021] Figure 2 The image shows the results of scanning electron microscopy (SEM) observation of the effects of different concentrations of peptides SmuG15 and SmuV13 on the microstructure of *Streptococcus mutans* biofilm in Example 4. In the image, a: SEM image of the SmuG15 treatment group; b: SEM image of the SmuV13 treatment group. The peptide concentrations from top to bottom are 0, 64, 128, and 256 µg / mL, respectively; the magnifications from left to right are 1000×, 5000×, and 20000×, respectively.

[0022] Figure 3 The image shown in Example 5 is a confocal laser scanning microscope image of the effects of different concentrations of peptides SmuG15 and SmuV13 on the synthesis and accumulation of extracellular polysaccharides in Streptococcus mutans. In the image, a: confocal image of the SmuG15 treatment group; b: confocal image of the SmuV13 treatment group; from top to bottom, each group is Bacteria (SYTO 9 labeled Streptococcus mutans, green fluorescence), EPS (Alexa Fluor 647 labeled dextran indicating extracellular polysaccharides, red fluorescence) and Merged (green and red fluorescence fused image). The scale bar is 25 μm, and the peptide concentrations from left to right are 0, 64, and 128 μg / mL.

[0023] Figure 4 This is a graph showing the effect of different concentrations of peptides SmuG15 and SmuV13 on the planktonic growth of *Streptococcus mutans* in Example 6. a: Growth curve of the SmuG15 treatment group; b: Growth curve of the SmuV13 treatment group. The horizontal axis represents the culture time (hours), and the vertical axis represents the absorbance at 600 nm (OD). 600 The concentrations of peptides in each treatment group were 0, 64, 128, and 256 μg / mL, respectively, reflecting the planktonic growth density of bacteria. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments. The scope of protection of the present invention is not limited to the following description:

[0025] The amino acid sequence of the polypeptide SmuG15 described in this invention is shown in SEQ ID NO.1, specifically: GVTLSSATTLSAVKA.

[0026] The amino acid sequence of the polypeptide SmuV13 described in this invention is shown in SEQ ID NO.2, specifically: VIFAFTFTSKAFA.

[0027] The polypeptides SmuG15 and SmuV13 described in this invention can be used as active ingredients in the formulation of oral care products to prevent or assist in the treatment of dental caries. In application, the polypeptides possess at least one of the following functions: inhibiting the formation of Streptococcus mutans biofilm, dissociating three-dimensional biofilm structures, reducing the synthesis and accumulation of extracellular polysaccharides, and not inhibiting the proliferation activity of planktonic bacterial strains.

[0028] In one specific embodiment of the present invention, SmuG15 and / or SmuV13 can be added to oral care products, including but not limited to toothpaste, mouthwash, oral spray, tooth powder, gel, oral lozenges, or chewing gum. Orally acceptable carriers or excipients may also be added to the above-mentioned oral care products, and the excipients may be selected from one or more of thickeners, surfactants, humectants, sweeteners, flavorings, preservatives, or pH adjusters. Depending on actual usage requirements, the dosage form of the oral care products can be flexibly adopted as a liquid dosage form, a semi-solid dosage form, or a solid dosage form.

[0029] The core contribution of this invention lies in providing two novel functional peptides, SmuG15 and SmuV13. These peptides can target and interfere with the formation of biofilms and the synthesis of extracellular polysaccharides in Streptococcus mutans, while not inhibiting the normal growth of the strain, thus ensuring both efficacy in preventing tooth decay and selectivity. Furthermore, these peptides are adaptable to various oral care product formulations, effectively compensating for the shortcomings of traditional anti-caries drugs in terms of functional modality and application scope, and providing a new technological approach to caries prevention and treatment that combines selectivity and flexibility.

[0030] Terminology Explanation:

[0031] "Inhibition of Streptococcus mutans biofilm formation" means that within the effective concentration range, SmuG15 or SmuV13 can significantly reduce the ability of Streptococcus mutans to form biofilms on tooth surfaces or other solid-phase interfaces.

[0032] "Disintegration of the three-dimensional biofilm structure" refers to the fact that effective concentrations of SmuG15 or SmuV13 can loosen the three-dimensional structure of Streptococcus mutans biofilms, accompanied by a significant reduction in biofilm thickness.

[0033] "Reducing the synthesis and accumulation of extracellular polysaccharides" means that effective concentrations of SmuG15 or SmuV13 can significantly reduce the amount of extracellular polysaccharides produced by Streptococcus mutans.

[0034] "Does not exert inhibitory pressure on the normal growth of the strain" means that, at effective concentrations, the effect of SmuG15 or SmuV13 on the growth rate of Streptococcus mutans in the planktonic state is not statistically significant.

[0035] "Dental caries prevention and control" refers to the ability to prevent, alleviate, or improve the occurrence and development of dental caries. Its scope covers a series of beneficial biological effects related to the dental caries process, such as inhibiting early enamel demineralization, weakening the virulence factors of cariogenic bacteria, removing mature dental plaque, and promoting tooth surface remineralization.

[0036] Example 1: Preparation of polypeptides

[0037] The polypeptides SmuG15 and SmuV13 involved in this embodiment were derived from peptidomics analysis of the culture supernatant of *Streptococcus mutans* and were artificially prepared using the Fmoc solid-phase synthesis strategy. The synthesis was commissioned to Sangon Biotech (Shanghai) Co., Ltd. After synthesis, the chromatographic purity of the obtained products was higher than 95%, and their corresponding amino acid sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0038] GVTLSSATTLSAVKA SEQ ID NO. 1;

[0039] VIFAFTFTSKAFA SEQ ID NO.2.

[0040] Example 2: Preparation of polypeptide solution and culture conditions of Streptococcus mutans

[0041] To facilitate subsequent evaluation of the activities of SmuG15 and SmuV13 in inhibiting the proliferation of Streptococcus mutans, biofilm formation, extracellular polysaccharide accumulation, and biofilm spatial structure, this embodiment first prepared a bacterial suspension of the strain in the logarithmic growth phase and configured a liquid culture system containing gradient concentrations of peptides.

[0042] 1. Preparation and preservation of polypeptide stock solutions

[0043] The lyophilized powders of SmuG15 and SmuV13 peptides obtained by Fmoc solid-phase synthesis (prepared by Sangon Biotech (Shanghai) Co., Ltd., purity >95%) were respectively prepared into stock solutions according to the following methods:

[0044] (1) SmuG15 stock solution: Accurately weigh 5 mg of SmuG15 peptide powder, dissolve it in 1 mL of sterile deionized water, shake thoroughly until completely dissolved, and prepare a stock solution with a concentration of 5 mg / mL.

[0045] (2) SmuV13 stock solution: Accurately weigh 5 mg of SmuV13 peptide powder, add 200 μL of dimethyl sulfoxide (DMSO), and repeatedly blow until completely dissolved to prepare a stock solution with a concentration of 25 mg / mL.

[0046] The above stock solution was aliquoted into sterile microcentrifuge tubes and stored at -80 °C for later use. When needed, take out the required amount, thaw it, and dilute it with the appropriate culture medium to the target working concentration. Prepare and use immediately, avoiding repeated freeze-thaw cycles.

[0047] 2. Source of the experimental strain: The test strain used in this example is *Streptococcus mutans*, whose standard strain number is ATCC 700610. This strain was provided by the National Key Laboratory for Prevention and Treatment of Oral Diseases.

[0048] 3. Components and preparation process of various culture media:

[0049] (1) Components and preparation process of bovine heart and brain extract (hereinafter referred to as BHI) liquid culture medium: Weigh 14.8 g of commercial BHI dry powder, dissolve it in 400 mL of deionized water, mix thoroughly, and then sterilize it by high pressure steam at 121.3℃ and 103.4 kPa for 15 min. After it cools down naturally, it is ready for use.

[0050] (2) Components and preparation process of BHI solid culture medium: Add 14.8 g of BHI dry powder and 6 g of agar powder to 400 mL of distilled water and stir to disperse evenly. After autoclaving under the same conditions (121.3℃, 103.4 kPa, 15 min), lower the temperature of the culture medium to about 65℃, and then dispense it into sterilized bacterial culture dishes under aseptic conditions. Let it stand until the agar is completely solidified before use.

[0051] (3) Components and preparation process of BHI sucrose liquid culture medium: Dissolve 14.8 g BHI powder and 4 g sucrose together in 400 mL distilled water. After complete dissolution, autoclave the medium for 15 min using the same parameters (121.3℃, 103.4 kPa). After sterilization, cool the medium for later use.

[0052] Example 3: Effects of peptides SmuG15 and SmuV13 on biofilm formation in Streptococcus mutans

[0053] In this embodiment, crystal violet staining combined with quantitative analysis was used to evaluate the effects of different concentrations of peptides SmuG15 and SmuV13 on the amount of biofilm formation in Streptococcus mutans, so as to determine their inhibitory effect on biofilm formation.

[0054] Following the aseptic technique described in Example 2, the *Streptococcus mutans* suspension cultured to the logarithmic growth phase in Example 2 was diluted 1:100 by volume in BHI sucrose liquid medium containing different concentrations (64, 128, 256 μg / mL) of peptide SmuG15 or SmuV13. A peptide-free medium was used as a blank control group. The cultures were incubated statically at 37 °C and 5% CO2 for 24 hours.

[0055] After incubation, carefully aspirate the airborne bacteria and culture supernatant, and gently wash the biofilm adhering to the bottom of the wells three times with PBS buffer. Then, add 200 μL of methanol to each well and fix at room temperature for 15 minutes. After fixation, discard the methanol and, after the residual methanol has evaporated, add 200 μL of 0.1% crystal violet staining solution to each well and stain at room temperature for 15 minutes. After staining, discard the staining solution, rinse three times with PBS, and dry in a 37 ℃ oven. After drying, photograph the 96-well plate; it is visibly less stained than the control group by the naked eye.

[0056] Subsequently, 200 μL of 33% acetic acid solution was added to each well, and the plate was shaken at 80 rpm for 30 minutes to ensure complete dissolution of the crystal violet dye bound to the biomembrane. After extraction, 100 μL of supernatant was transferred from each well to a new 96-well plate, and the absorbance (OD) at 595 nm was measured using a microplate reader. 595 Using the control group without added peptides as a reference, the relative inhibition rate of biofilm formation in each concentration treatment group was calculated. Each experiment was set up with 3 replicates, and each experiment was independently repeated 3 times. Experimental data are expressed as mean ± standard deviation. One-way ANOVA was used for multiple comparisons to analyze the differences between each peptide treatment group and the control group.

[0057] Experimental results are as follows Figure 1 As shown.

[0058] Depend on Figure 1 a and Figure 1 As shown in Figure b, the direct images of the 96-well plate show that the amount of crystal violet staining after treatment with different concentrations of SmuG15 or SmuV13 was significantly less than that in the control group, and the amount of staining decreased in a gradient manner as the peptide concentration increased. Figure 1 c and Figure 1 Quantitative analysis results showed that, compared with the control group without added peptides, the OD values ​​of each peptide-treated group were significantly higher. 595The values ​​all decreased gradually with increasing concentration, with the OD values ​​of the 256 μg / mL treatment group showing the lowest values. 595 The value was the lowest, while the OD value of the control group was the lowest. 595 The highest value indicates a clear concentration-dependent inhibitory characteristic. These results demonstrate that peptides SmuG15 and SmuV13 can effectively inhibit the formation of *Streptococcus mutans* biofilm, and their inhibitory effect is positively correlated with peptide concentration.

[0059] Example 4: Scanning electron microscopy observation of the effects of peptides SmuG15 and SmuV13 on the biofilm structure of Streptococcus mutans

[0060] In this embodiment, scanning electron microscopy was used to observe the changes in the microstructure of Streptococcus mutans biofilm after treatment with different concentrations of peptides SmuG15 and SmuV13, in order to evaluate the intervention effect of the two on the spatial structure of the biofilm.

[0061] Following the aseptic procedure described in Example 2, the *Streptococcus mutans* suspension cultured to the logarithmic growth phase in Example 2 was diluted at a volume ratio of 1:100 in BHI sucrose liquid medium containing different concentrations (64, 128, 256 μg / mL) of peptide SmuG15 or SmuV13. A peptide-free medium was used as a blank control group. The cultures were incubated statically at 37 °C and 5% CO2 for 24 hours.

[0062] After incubation, the airborne bacteria and culture supernatant were carefully aspirated, and the biofilm adhering to the bottom of the wells was gently washed three times with PBS buffer. Then, 1 mL of 2.5% glutaraldehyde fixative was added to each well, and the solution was fixed overnight at 4 °C. After fixation, the supernatant was discarded, and the samples were washed three times with PBS. Subsequently, different volume fractions (30%, 40%, 50%, 60%, 70%, 80%, 90%) of ethanol solution were added sequentially for gradient dehydration, with each concentration gradient treated for 15 minutes. Finally, the samples were stored in anhydrous ethanol, critically dried, and sputter-coated with gold. Images of the biofilm surface structure were then observed and acquired under a scanning electron microscope at different magnifications.

[0063] Experimental results are as follows Figure 2 As shown.

[0064] Depend on Figure 2 a and Figure 2As shown in Figure b, compared with the control group without added peptides, the experimental groups treated with different concentrations of SmuG15 or SmuV13 showed a significant reduction in the amount of *Streptococcus mutans* biofilm formed, and the biofilm structure tended to be loose and scattered. With increasing peptide concentration, the degree of biofilm aggregation gradually decreased. When the concentration reached 256 μg / mL, only a few scattered bacteria were visible on the material surface, and it was almost impossible to form a complete biofilm structure. These results indicate that peptides SmuG15 and SmuV13 can significantly dissociate the spatial structure of *Streptococcus mutans* biofilm, and this effect is concentration-dependent; that is, the higher the peptide concentration, the more significant the degree of biofilm dissociation.

[0065] Example 5: Evaluation of the effects of peptides SmuG15 and SmuV13 on extracellular polysaccharide synthesis in Streptococcus mutans using confocal laser scanning microscopy

[0066] This embodiment uses confocal laser scanning microscopy combined with fluorescence staining technology to evaluate the effects of different concentrations of peptides SmuG15 and SmuV13 on the synthesis of extracellular polysaccharides in Streptococcus mutans, in order to verify the intervention effect of the two on the accumulation of extracellular polysaccharides.

[0067] Following the aseptic procedure described in Example 2, the *Streptococcus mutans* suspension cultured to the logarithmic growth phase in Example 2 was diluted 1:100 by volume in BHI sucrose liquid medium containing different concentrations (64, 128 μg / mL) of peptide SmuG15 or SmuV13, with the peptide-free medium serving as a blank control. Simultaneously, Alexa Fluor® 647-labeled dextran was added to each well to indicate the distribution of extracellular polysaccharides. The bacterial suspensions were then added to confocal culture dishes, wrapped in aluminum foil, and incubated statically at 37 °C and 5% CO2 for 24 hours in the dark.

[0068] After incubation, airborne bacteria and culture supernatant were carefully aspirated under light-protected conditions. The biofilm adhering to the bottom of the dish was gently washed three times with physiological saline and air-dried at room temperature. Then, SYTO 9 green fluorescent nucleic acid dye to a final concentration of 2.5 μmol / L was added to each confocal culture dish, and staining was performed for 15 minutes in the dark to label viable Streptococcus mutans in the biofilm. After staining, the dishes were washed three times with physiological saline, and residual water was aspirated. An appropriate amount of anti-fluorescence quenching mounting medium was added, and the dishes were stored at 4 ℃ in the dark for later use. Observation and image acquisition were performed using a confocal laser scanning microscope (Olympus FV3000) in 60x oil immersion objective mode.

[0069] Experimental results are as follows Figure 3 As shown.

[0070] Depend on Figure 3 a and Figure 3As shown in Figure b, compared with the control group without added peptides, the fluorescence signals of extracellular polysaccharides labeled with Alexa Fluor® 647 were significantly weakened in the experimental groups treated with 64 μg / mL and 128 μg / mL peptides SmuG15 or SmuV13, with the fluorescence intensity reduction being more significant in the 128 μg / mL treatment group. These results indicate that peptides SmuG15 and SmuV13 can effectively inhibit the synthesis and accumulation of extracellular polysaccharides in Streptococcus mutans, and their inhibitory effect is positively correlated with concentration.

[0071] Example 6: Effects of peptides SmuG15 and SmuV13 on the planktonic growth of Streptococcus mutans

[0072] This embodiment was used to detect the effects of different concentrations of peptides SmuG15 and SmuV13 on the planktonic growth state of Streptococcus mutans.

[0073] Following the aseptic procedure described in Example 2, the *Streptococcus mutans* suspension cultured to the logarithmic growth phase in Example 2 was diluted 1:100 by volume in BHI liquid medium containing different concentrations (64, 128, 256 μg / mL) of peptide SmuG15 or SmuV13. BHI liquid medium without peptides served as a blank control group. The bacterial suspensions were inoculated into 96-well plates, with 200 μL of a mixed culture medium containing the bacterial suspension and the corresponding peptide concentration added to each well. 50 μL of mineral oil was then added to each well to prevent water evaporation and cross-contamination during culture. The plates were incubated statically at 37 °C and 5% CO2 for 12 hours. The absorbance (OD) of each well was measured every 2 hours using a microplate reader at 600 nm. 600 Record the growth density of Streptococcus mutans at different time points, and plot the growth density of Streptococcus mutans on the x-axis and OD on the y-axis. 600 The values ​​are plotted on the ordinate to show the growth curves of the different concentrations of peptide treatment groups and the control group.

[0074] Experimental results are as follows Figure 4 As shown.

[0075] Depend on Figure 4 It can be seen that different concentrations (64, 128, 256 μg / mL) of peptide SmuG15 ( Figure 4 a) or SmuV13 ( Figure 4 OD at various time points of Streptococcus mutans after treatment (b) 600 The values ​​and overall growth curve trends were basically consistent with those of the control group without added peptides, and there were no statistically significant differences between the treatment groups and the control group at each culture time point. These results indicate that peptides SmuG15 and SmuV13 did not significantly inhibit the planktonic growth of *Streptococcus mutans* within the concentration range of 64–256 μg / mL.

[0076] In summary, this invention provides a technical solution consisting of two novel functional peptides, SmuG15 and SmuV13, whose amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The core feature of this solution is that SmuG15 and SmuV13 do not exert their anti-caries effect by inhibiting the planktonic growth of *Streptococcus mutans*, but rather by targeting and interfering with the cariogenic virulence-related processes of this strain. Specifically, this includes: inhibiting biofilm formation (see Example 3), dissociating the three-dimensional spatial structure of the biofilm (see Example 4), and reducing the synthesis and accumulation of extracellular polysaccharides (see Example 5), while not inhibiting the normal proliferation of planktonic strains (see Example 6). Compared to previously reported functional peptides such as C16G2, the SmuG15 and SmuV13 provided by this invention exhibit significant differences in their mechanism of action and effective working concentration range (64–256 μg / mL), making them suitable for various oral care product formulations such as toothpaste, mouthwash, and oral sprays. This approach effectively avoids the non-selective interference of traditional antibacterial drugs with the oral microecology, expands the resource reserve of anti-caries active molecules, and provides a new technical path for clinical intervention of dental caries that is both selective and operable.

[0077] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. A polypeptide, namely SmuG15 or SmuV13, wherein the amino acid sequence of SmuG15 is shown in SEQ ID NO: 1 and the amino acid sequence of SmuV13 is shown in SEQ ID NO:

2.

2. An oral care product, characterized in that, It contains the polypeptide as described in claim 1.

3. The oral care product according to claim 2, characterized in that, The concentration of the added peptide was 64–256 µg / mL.

4. The oral care product according to claim 2, characterized in that, The dosage forms of the oral care products include toothpaste, mouthwash, oral spray, tooth powder, gel, oral lozenges or chewing gum.

5. The oral care product according to claim 2, characterized in that, The oral care products are formulated in liquid, semi-solid, or solid forms.

6. A biofilm inhibitory drug composition, characterized in that, It contains the polypeptide as described in claim 1.

7. The biofilm inhibitory pharmaceutical composition according to claim 6, characterized in that, The concentration of the added peptide was 64–256 µg / mL.

8. The use of the polypeptide according to claim 1 in the preparation of a caries prevention and treatment drug.

9. The application according to claim 8, characterized in that, Dental caries can be prevented by inhibiting the formation of Streptococcus mutans biofilm and / or dissociating the three-dimensional biofilm structure of Streptococcus mutans and / or reducing the synthesis and accumulation of extracellular polysaccharides of Streptococcus mutans and / or not inhibiting the growth of planktonic strains of Streptococcus mutans.

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