Polypeptides that inhibit streptococcus mutans biofilm formation and exopolysaccharide synthesis and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a polypeptide for inhibiting the formation of Streptococcus mutans biofilm and the synthesis of exopolysaccharide and application thereof. The polypeptide is polypeptide 98-12, and the amino acid sequence is shown as SEQ ID NO. 1. The polypeptide 98-12 can effectively inhibit the formation of Streptococcus mutans biofilm and the synthesis of exopolysaccharide, significantly change the three-dimensional structure characteristics of the biofilm, and has good biological safety. Compared with the prior art, the application has obvious advantages in the effect of preventing dental caries, provides a new safe solution for the prevention and treatment of dental caries, and widens the polypeptide resource library for inhibiting the virulence of Streptococcus mutans and preventing and treating dental caries.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide that inhibits biofilm formation and extracellular polysaccharide synthesis in Streptococcus mutans and its applications. Background Technology

[0002] Dental caries is one of the most common oral diseases worldwide. According to the World Health Organization, approximately 2.5 billion people globally suffer from permanent tooth caries, and nearly 3.5 billion people are affected by various oral diseases. The results of the Fourth National Oral Health Epidemiological Survey show that the caries rate among 12-year-old children in my country is approximately 34.5%, and the rate among people aged 35 to 44 is as high as 89%, making dental caries one of the most common diseases endangering oral health. Dental caries not only damages tooth enamel and dentin but can also cause a series of serious consequences such as pain, chewing dysfunction, and premature tooth loss, severely impacting patients' quality of life. More seriously, dental caries is closely related to systemic diseases, increasing the risk of cardiovascular, digestive, and respiratory diseases. Therefore, effective prevention and control of dental caries has significant clinical importance.

[0003] Dental caries occurs when an imbalance in the oral microecology leads to the demineralization of tooth hard tissues by bacterial metabolic products. Streptococcus mutans (Streptococcus mutans) Streptococcus mutans Streptococcus mutans is widely considered the core microorganism in the development of dental caries. It can efficiently adhere to the tooth surface, synthesizing extracellular polysaccharides from sucrose to form a dense biofilm structure. The extracellular polysaccharides synthesized by glucosyltransferase are important structural components of the biofilm and key virulence factors in the caries-causing process.

[0004] The biofilm structure formed by *Streptococcus mutans* plays a crucial role in the development and progression of dental caries. Firstly, the dense structure of the biofilm restricts the diffusion of organic acids, resulting in a persistently low pH at the plaque-tooth interface and prolonged demineralization of the enamel. Secondly, the biofilm matrix provides adhesion sites and a protective microenvironment for other cariogenic microorganisms. Thirdly, the high content of extracellular polysaccharides enhances the stability and structural integrity of the biofilm, providing a physical barrier against host immune factors and exogenous antimicrobial drugs. Therefore, the ability of *Streptococcus mutans* to form biofilms and synthesize extracellular polysaccharides are its two most significant cariogenic virulence characteristics and key intervention targets for the development of anti-cariogenic drugs.

[0005] Currently, commonly used clinical anti-caries drugs such as chlorhexidine have inherent drawbacks that are difficult to overcome. Long-term use of chlorhexidine can inhibit the growth of Streptococcus mutans and other oral microorganisms, potentially disrupting the oral microecology. Therefore, developing novel and highly effective Streptococcus mutans extracellular polysaccharides and biofilm inhibitors has become a pressing technical challenge in the field of anti-caries drug research and development.

[0006] To address the aforementioned technical issues, existing technologies have explored relevant approaches. For example, Chinese Patent CN119638790B discloses a polypeptide and its application in the preparation of Streptococcus mutans inhibitors. The amino acid sequence of this polypeptide is GLDWWQL. Experiments have shown that, at different concentrations and durations of action, the inhibitory activity of this polypeptide against Streptococcus mutans is significantly superior to other control and comparative groups, and it is non-toxic to cells and exhibits good cell compatibility. Furthermore, Chinese Patent CN113336826B discloses an antimicrobial peptide and its application. The amino acid sequence of this antimicrobial peptide is KRLFKKLLFWLRKY (KR-1). Compared to its parent peptide, KR-1 exhibits higher anti-Streptococcus mutans activity and anti-Streptococcus mutans biofilm activity, more effectively inhibiting both planktonic and biofilm-bound Streptococcus mutans, while also exhibiting low toxicity and good biocompatibility, demonstrating certain advantages in preventing tooth decay.

[0007] In summary, existing technologies have provided effective components such as GLDWWQL peptides and KR-1 antimicrobial peptides that can inhibit Streptococcus mutans and its biofilm. These components are characterized by low toxicity and good biocompatibility, providing valuable candidate molecules for the development of anti-caries drugs. However, different peptides or antimicrobial peptides differ in their specific mechanisms of action, inhibitory activity intensity, applicable concentration range, and duration of action, each possessing different functional characteristics and potential application scenarios. To enrich the market supply of anti-caries products, meet diverse clinical prevention and treatment needs, and provide more selectable active ingredients for oral care, it remains necessary to continue developing novel, highly effective, and safe anti-caries drugs based on existing technologies. Therefore, this application proposes a new solution different from the aforementioned existing technologies, aiming to further expand the types and selectivity of anti-caries drugs. Summary of the Invention

[0008] The purpose of this invention is to provide a polypeptide (polypeptide 98-12) that inhibits biofilm formation and extracellular polysaccharide synthesis in *Streptococcus mutans* and its applications. Polypeptide 98-12 effectively inhibits the production of biofilms and extracellular polysaccharides in *Streptococcus mutans*, significantly altering the three-dimensional structural characteristics of its biofilm without affecting the growth of *Streptococcus mutans*, and exhibits good biocompatibility. Compared with existing technologies, this invention has significant advantages in caries prevention, providing a novel and safe solution for caries prevention and treatment, while also expanding the existing polypeptide resource library for inhibiting the virulence of *Streptococcus mutans* and preventing caries.

[0009] The present invention is achieved through the following technical solution: a polypeptide that inhibits the formation of biofilm and the synthesis of extracellular polysaccharides in Streptococcus mutans, wherein the polypeptide is polypeptide 98-12 and its amino acid sequence is shown in SEQ ID NO.1.

[0010] Another technical solution of the present invention is to provide the application of the above-mentioned polypeptide in the preparation of oral care products, wherein the polypeptide 98-12 achieves the effect of oral care products in preventing and treating dental caries through at least one of the following functions: (1) Inhibits the formation of biofilms in Streptococcus mutans; (2) Disrupts the biofilm structure of Streptococcus mutans; (3) Inhibits the ability of Streptococcus mutans to produce extracellular polysaccharides; (4) It does not affect the growth of Streptococcus mutans and maintains oral microbial homeostasis.

[0011] The concentration of peptide 98-12 in the oral care product is 32–128 μmol / L.

[0012] The concentration of peptide 98-12 in the oral care product is 128 μmol / L.

[0013] The oral care products include at least one of toothpaste, mouthwash, oral spray, tooth powder, gel, oral lozenges or chewing gum.

[0014] The oral care product also contains pharmaceutically acceptable excipients selected from at least one of thickeners, surfactants, humectants, sweeteners, flavorings, preservatives, or pH adjusters.

[0015] The oral care products are available in liquid, semi-solid, or solid dosage forms.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention provides a novel polypeptide—polypeptide 98-12 (amino acid sequence as shown in SEQ ID NO.1). This polypeptide has a small molecular weight, good solubility, is easy to prepare, and has good biocompatibility.

[0017] (2) Experiments have confirmed that the polypeptide 98-12 of the present invention significantly inhibits the formation of biofilm and the synthesis of extracellular polysaccharides in Streptococcus mutans and can effectively destroy the biofilm structure, especially at 128 μmol / L.

[0018] (3) The polypeptide 98-12 described in this invention has extremely low hemolytic activity and no obvious cytotoxicity to normal cells. It has high safety and is suitable for the development of oral care products, which helps to meet the diverse clinical prevention and daily oral care needs.

[0019] (4) The polypeptide 98-12 described in this invention has no obvious hemolytic activity and no obvious toxicity to human cells. It has a high safety profile and is suitable for the development of oral care products, which helps to meet diverse clinical prevention and daily oral care needs. This polypeptide can be added as an active ingredient to a variety of oral care products such as toothpaste, mouthwash, oral spray, tooth powder, gel, oral lozenges or chewing gum, and its application is flexible. Attached Figure Description

[0020] Figure 1 The figure shows the effect of different concentrations of polypeptide on the formation of Streptococcus mutans biofilm in Example 3.

[0021] Figure 2 The image shows the results of scanning electron microscopy observation of the effects of the polypeptide-treated group and the control group without polypeptide on the biofilm structure of Streptococcus mutans in Example 4.

[0022] Figure 3 The figure shows the effect of different concentrations of polypeptide on the production of extracellular polysaccharides by Streptococcus mutans in Example 5.

[0023] Figure 4 This is a graph showing the effects of co-focused laser scanning microscopy on the extracellular polysaccharides of Streptococcus mutans in Example 6, comparing the peptide-treated group and the control group without added peptides.

[0024] Figure 5 The figure shows the effect of different concentrations of polypeptide on the growth of Streptococcus mutans in Example 7.

[0025] Figure 6 This is a graph showing the hemolytic activity test results of different concentrations of peptides in Example 8.

[0026] Figure 7 This is a graph showing the cytotoxicity test results of different concentrations of peptides at different incubation times in Example 9. Detailed Implementation

[0027] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0028] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the claimed invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] This invention aims to address the technical problem that long-term use of existing anti-caries drugs (such as chlorhexidine) leads to drug resistance in Streptococcus mutans and disrupts the oral microecology. It also overcomes the current situation where existing peptides (such as GLDWWQL and KR-1) differ in their mechanisms of action, inhibitory activity, and applicable concentration ranges, making it difficult to meet diverse application needs. Therefore, this invention provides a novel, highly efficient, and safe peptide, namely peptide 98-12, that inhibits the synthesis of Streptococcus mutans biofilms and extracellular polysaccharides. This peptide can be used to prepare oral care products, further enriching the variety of anti-caries products and providing a better solution for caries prevention and treatment.

[0030] The technical solution of the present invention can be summarized in detail as follows: The amino acid sequence of polypeptide 98-12 of the present invention is shown in SEQ ID NO.1, specifically: LKVGWSLYLRGTKITKLPKKLKVKGEIIW.

[0031] The polypeptide 98-12 described in this invention can be used as an effective active ingredient in the preparation of oral care products to achieve the effect of preventing and treating dental caries. Its active functions include at least one of the following: (1) Inhibits the formation of biofilms in Streptococcus mutans; (2) Disrupts the biofilm structure of Streptococcus mutans; (3) Inhibits the ability of Streptococcus mutans to produce extracellular polysaccharides; (4) It does not affect the growth of Streptococcus mutans and maintains the oral microecology.

[0032] In one specific embodiment of the present invention, the polypeptide 98-12 can be prepared into an oral care product, such as at least one of toothpaste, mouthwash, oral spray, tooth powder, gel, oral lozenges, or chewing gum. The oral care product may also contain at least one pharmaceutically acceptable excipient, such as a thickener, surfactant, humectant, sweetener, flavoring agent, preservative, or pH adjuster. The dosage form of the oral care product may be a liquid, semi-solid, or solid dosage form.

[0033] The core innovation of this invention lies in proposing a novel polypeptide 98-12, which can inhibit the formation of biofilms in Streptococcus mutans, disrupt the biofilm structure, and inhibit the synthesis of extracellular polysaccharides, without affecting the growth of Streptococcus mutans, thus achieving a highly efficient and safe anti-caries effect. This polypeptide can be flexibly applied to a variety of oral care products, overcoming the limitations of traditional drugs in terms of functional diversity and application breadth, and providing a new solution for the prevention and treatment of dental caries that is both safe and practical.

[0034]

Terminology Explanation

[0035] "Disrupting the biofilm structure of Streptococcus mutans" means that an effective concentration of polypeptide 98-12 can loosen the three-dimensional structure of the Streptococcus mutans biofilm and reduce its thickness.

[0036] "The ability to inhibit the production of extracellular polysaccharides by Streptococcus mutans" means that an effective concentration of polypeptide 98-12 can significantly reduce the ability of Streptococcus mutans to synthesize water-insoluble extracellular polysaccharides. "Does not affect the growth of Streptococcus mutans" means that the effective concentration of polypeptide 98-12 does not produce statistically significant inhibition on the growth of Streptococcus mutans in the planktonic state.

[0037] "Caries prevention and treatment" refers to the ability to prevent and alleviate dental caries, encompassing a range of beneficial effects related to the occurrence and development of dental caries, from preventing early demineralization and inhibiting the virulence of cariogenic bacteria to removing mature dental plaque and promoting remineralization.

[0038] The specific implementation of the present invention will be described below with reference to the embodiments. Of course, the scope of protection of the present invention is not limited to the following embodiments.

[0039] Example 1: Synthesis of polypeptides In this embodiment, peptide 98-12 was synthesized using the Fmoc solid-phase synthesis method (prepared by Sangon Biotech (Shanghai) Co., Ltd.). The purity of peptide 98-12 is greater than 95%, and its amino acid sequence is shown in SEQ ID NO. 1.

[0040] Example 2: Cultivation of Streptococcus mutans and preparation of polypeptide solutions This embodiment obtains a Streptococcus mutans suspension in the logarithmic growth phase and liquid culture media containing different concentrations of peptides, providing an experimental basis for subsequent determination of the inhibitory activity of peptide 98-12 on the growth, biofilm formation, extracellular polysaccharide synthesis, and biofilm structure of Streptococcus mutans.

[0041] 1. Preparation of test strain: The test strain Streptococcus mutans in this example was obtained from the National Key Laboratory for Prevention and Treatment of Oral Diseases, strain number: ATCC 700610.

[0042] 2. Components and preparation of the culture medium: (1) Components and preparation method of bovine heart and brain extract (hereinafter referred to as BHI) liquid culture medium: 37 g of commercially available BHI powder was added to 1000 mL of distilled water and sterilized by high temperature and high pressure (121.3℃, 103.4 kPa) steam sterilization for 15 minutes. After cooling, it was ready for use.

[0043] (2) Components and preparation method of BHI solid culture medium: Add 37 g of commercially available BHI powder and 15 g of agar powder to 1000 mL of distilled water, sterilize by high temperature and high pressure (121.3℃, 103.4 kPa) steam sterilization for 15 minutes, cool to about 60℃ and pour an appropriate amount into bacterial culture dishes, and let it cool and solidify before use.

[0044] (3) Components and preparation method of BHI sucrose liquid culture medium: 37 g of commercially available BHI powder and 10 g of sucrose were added to 1000 mL of distilled water and sterilized by high temperature and high pressure (121.3℃, 103.4 kPa) steam sterilization for 15 minutes. After cooling, it was ready for use.

[0045] 3. Preparation of the peptide: In this embodiment, peptide 98-12 is used in solution form. Specific preparation method: First, dissolve 5 mg of peptide 98-12 powder in 147.18 μL of commercially available dimethyl sulfoxide (DMSO) to obtain a peptide stock solution with an initial concentration of 10 mmol / L, which is then stored for later use. In subsequent experiments, the prepared peptide stock solution is serially diluted using a two-fold dilution method with liquid culture medium to obtain peptide-containing liquid culture media with final concentrations of 32 µmol / L, 64 µmol / L, and 128 µmol / L, respectively.

[0046] 4. Inoculation and Culture Methods: *Streptococcus mutans* culture was inoculated onto BHI solid medium and cultured at 37°C and 5% CO2 for 24 hours. A single colony was picked using an inoculation loop and inoculated into 5 mL of BHI liquid medium, and cultured at 37°C and 5% CO2 for 24 hours. The absorbance at 600 nm (Optical Density 600nm, abbreviated as OD) was measured using a UV spectrophotometer. 600 ), will be cultured to the logarithmic growth phase (OD) 600 The bacterial suspension (approximately 0.5 g / L) was diluted 1:100 in BHI liquid medium (for culturing planktonic Streptococcus mutans) or BHI sucrose liquid medium (for culturing Streptococcus mutans biofilms) and then cultured. The entire procedure was performed under aseptic conditions.

[0047] Example 3: Effect of polypeptides on biofilm formation in Streptococcus mutans In this embodiment, the effect of different concentrations of polypeptide 98-12 on the amount of biofilm formation in Streptococcus mutans was quantitatively analyzed by crystal violet staining to determine its inhibitory effect on biofilm.

[0048] Following the aseptic procedure of Example 2, the *Streptococcus mutans* suspension from Example 2 was inoculated into BHI sucrose liquid medium containing different concentrations of polypeptides to determine the effect of the polypeptides on *Streptococcus mutans* biofilm formation. The specific method is as follows: The cultures from Example 2, grown to the logarithmic growth phase (OD), were prepared at a ratio of 1:100. 600 ≈0.5) Streptococcus mutans bacterial suspensions were diluted in BHI sucrose liquid medium containing different concentrations (32 µmol / L, 64 µmol / L, 128 µmol / L) of peptides and cultured at 37 ℃ and 5% CO2 for 24 hours. Planktonic bacteria and supernatant were aspirated, and the biofilm at the bottom was washed three times with PBS. After fixation with 200 µL of paraformaldehyde for 45 minutes, the paraformaldehyde was aspirated, and the mixture was washed three times with PBS. 200 µL of 0.01% crystal violet staining solution was added, and the mixture was allowed to stand at room temperature for 5 minutes. The crystal violet staining solution was aspirated, and the mixture was washed three times with PBS and dried at 37 ℃.

[0049] Experimental results are as follows Figure 1 As shown.

[0050] from Figure 1 It can be seen that, compared with the control group without peptide, the treatment groups with added peptide (concentrations of 32 µmol / L, 64 µmol / L, and 128 µmol / L) showed a significant reduction in Streptococcus mutans biofilm, and this reduction was concentration-dependent, meaning that the higher the concentration of peptide, the more obvious the inhibitory effect. This indicates that peptide 98-12 can effectively inhibit the formation of Streptococcus mutans biofilm.

[0051] Example 4: Scanning electron microscopy observation of the effect of peptides on the biofilm structure of Streptococcus mutans In this embodiment, scanning electron microscopy was used to observe the microstructural changes of Streptococcus mutans biofilm after treatment with different concentrations of polypeptide 98-12, in order to evaluate its destructive effect on biofilm structure.

[0052] Following the aseptic procedure of Example 2, the *Streptococcus mutans* suspension from Example 2 was inoculated into BHI sucrose liquid medium containing different concentrations of polypeptides to determine the effect of the polypeptides on the biofilm structure of *Streptococcus mutans*. The specific method is as follows: The cultures from Example 2, grown to the logarithmic growth phase (OD), were prepared at a ratio of 1:100. 600≈0.5) of *Streptococcus mutans* bacterial suspension was diluted in BHI sucrose liquid medium containing different concentrations (32 µmol / L, 64 µmol / L, 128 µmol / L) of peptides and cultured at 37°C and 5% CO2 for 24 hours. Planktonic bacteria and supernatant were aspirated, and the biofilm at the bottom was washed three times with PBS. 1 mL of 2.5% glutaraldehyde was added and the mixture was fixed overnight at 4°C. The supernatant was aspirated, and the mixture was washed three times with PBS. Gradual dehydration was performed by adding 1 mL of ethanol solutions of different concentrations (30%, 40%, 50%, 60%, 70%, 80%, 90%) for 15 minutes at each gradient. The samples were stored in anhydrous ethanol solution, and images of the biofilm structure were acquired under a scanning electron microscope at different magnifications.

[0053] Experimental results are as follows Figure 2 As shown.

[0054] from Figure 2 It can be seen that, compared with the control group without peptide, the treatment groups with added peptides (concentrations of 32 µmol / L, 64 µmol / L, and 128 µmol / L) showed reduced biofilm formation in *Streptococcus mutans*. When the peptide concentration reached 128 µmol / L, *Streptococcus mutans* hardly formed a biofilm, indicating that peptide 98-12 significantly affected the biofilm structure of *Streptococcus mutans* in a concentration-dependent manner; that is, the higher the peptide concentration, the more obvious the structural changes.

[0055] Example 5: Effect of polypeptides on extracellular polysaccharide production by Streptococcus mutans This embodiment evaluates the effect of different concentrations of peptide 98-12 on the ability of Streptococcus mutans to produce extracellular polysaccharides, thus verifying its inhibitory effect on extracellular polysaccharide synthesis.

[0056] Following the aseptic procedure of Example 2, the *Streptococcus mutans* suspension from Example 2 was inoculated into BHI sucrose liquid medium containing different concentrations of polypeptides to determine the effect of the polypeptides on the production of extracellular polysaccharides by *Streptococcus mutans*. The specific method is as follows: The cultures from Example 2, grown to the logarithmic growth phase (OD), were prepared at a ratio of 1:100. 600≈0.5 g of *Streptococcus mutans* bacterial suspension was diluted in BHI sucrose liquid medium containing different concentrations (32 µmol / L, 64 µmol / L, 128 µmol / L) of peptides and cultured at 37°C and 5% CO2 for 24 hours. Planktonic bacteria and supernatant were aspirated, and the biofilm at the bottom was washed three times with PBS. 200 µL of PBS was added, and all the biofilm at the bottom was scraped off and transferred to a 1.5 mL EP tube. The tube was centrifuged at 10,000 r / min for 10 minutes at 4°C, the supernatant was discarded, and the precipitate was resuspended in PBS. The centrifugation and washing were repeated three times. The precipitate was resuspended in 200 µL of 0.4 mol / L NaOH solution and incubated at 37°C for 2 hours. Centrifuge the mixture from the previous step at 8000 r / min for 10 minutes at 4℃. Transfer 100 μL of the supernatant to another EP tube, and slowly add 300 μL of anthrone reagent (30 mg anthrone dissolved in 15 mL concentrated sulfuric acid) along the wall. Mix thoroughly by inverting the tube, and incubate at 95℃ for 6 minutes. After cooling to room temperature, take 100 μL of the mixture and measure its absorbance (OD) at 625 nm using a microplate reader. 625 ).

[0057] Experimental results are as follows Figure 3 As shown, Figure 3 In the above, **: P≤0.01; ***: P≤0.001.

[0058] from Figure 3 It can be seen that, compared with the control group without peptide, the treatment groups with added peptide (concentrations of 32 µmol / L, 64 µmol / L, and 128 µmol / L) produced less extracellular polysaccharide, indicating that peptide 98-12 has a significant inhibitory effect on extracellular polysaccharide production by Streptococcus mutans, and this effect is concentration-dependent, meaning that the higher the concentration of peptide, the more obvious the inhibitory effect.

[0059] Example 6: Evaluation of the effect of peptides on extracellular polysaccharide formation in Streptococcus mutans using confocal laser scanning microscopy In this embodiment, confocal laser scanning microscopy combined with fluorescence staining was used to evaluate the effect of peptide 98-12 (128 µmol / L) on the formation of extracellular polysaccharides in Streptococcus mutans, in order to verify its inhibitory effect on extracellular polysaccharide synthesis.

[0060] Following the aseptic procedure of Example 2, the *Streptococcus mutans* suspension from Example 2 was inoculated into BHI sucrose liquid medium containing different concentrations of polypeptides to determine the effect of the polypeptides on the production of extracellular polysaccharides by *Streptococcus mutans*. The specific method is as follows: The cultures from Example 2, grown to the logarithmic growth phase (OD), were prepared at a ratio of 1:100. 600≈0.5) of *Streptococcus mutans* bacterial suspension was diluted in BHI sucrose liquid medium containing 128 µmol / L polypeptide, and then Alexa Fluor® 647 dye was added to label the extracellular polysaccharide to a final concentration of 1 µmol / L. The culture was wrapped in aluminum foil and incubated at 37°C and 5% CO2 for 24 hours. Under light-protected conditions, the air-drying bacteria and supernatant were aspirated, and the biofilm at the bottom was washed three times with physiological saline. After drying, SYTO 9 fluorescent dye was added to a final concentration of 2.5 µmol / L and stained for 15 minutes to label *Streptococcus mutans*. The bacteria were washed three times with physiological saline, dried, and then an anti-fluorescence quencher was added. The culture was stored at 4°C in the dark and observed under a 60x oil immersion microscope using a confocal fluorescence microscope (Olympus FV3000).

[0061] Experimental results are as follows Figure 4 As shown.

[0062] from Figure 4 It can be seen that the fluorescence intensity of extracellular polysaccharides was significantly reduced in the treatment group with 128 µmol / L peptide compared with the control group without peptide, indicating that peptide 98-12 has a significant inhibitory effect on the production of extracellular polysaccharides by Streptococcus mutans.

[0063] Example 7: Effect of polypeptides on the growth of Streptococcus mutans This embodiment is used to detect the effect of different concentrations of polypeptide 98-12 on the planktonic growth state of Streptococcus mutans.

[0064] Following the aseptic procedure of Example 2, the *Streptococcus mutans* suspension from Example 2 was inoculated into BHI liquid culture medium containing different concentrations of peptides to determine the effect of the peptides on the growth status of *Streptococcus mutans*. The specific method is as follows: The cultures from Example 2, grown to the logarithmic growth phase (OD), were prepared at a ratio of 1:100. 600 ≈0.5) Streptococcus mutans bacterial suspensions were diluted in BHI liquid medium containing different concentrations (32 µmol / L, 64 µmol / L, 128 µmol / L) of peptides, with BHI liquid medium without peptides serving as the control group. After incubation at 37°C and 5% CO2 for 24 hours, the suspensions were serially diluted 10-fold with commercially available sterile phosphate-buffered saline (PBS) to a final concentration of 10. -6 The different dilutions were then spotted onto BHI solid medium to compare and determine whether the growth of Streptococcus mutans was affected.

[0065] Experimental results are as follows Figure 5 As shown.

[0066] from Figure 5It can be seen that the growth of the treatment groups with added peptides (concentrations of 32 µmol / L, 64 µmol / L, and 128 µmol / L, respectively) was not significantly different from that of the control group without added peptides, indicating that peptide 98-12 did not have a significant inhibitory effect on the growth of Streptococcus mutans.

[0067] Example 8: Detection of hemolytic activity of peptides This embodiment assesses the biocompatibility and safety of peptide 98-12 by detecting its hemolytic activity at different concentrations.

[0068] 400 µL of sterile defibrinated sheep blood was diluted to 20 mL with PBS, centrifuged at 3000 rpm for 10 minutes at 4°C, and the supernatant was discarded. The red blood cells were washed three times with PBS until the supernatant no longer showed a red color. The obtained red blood cells were then prepared into a 5% suspension with PBS for experimental use. In the experimental group, peptide 98-12 was dissolved in PBS and diluted to 32 µmol / L, 64 µmol / L, and 128 µmol / L, respectively. PBS and Triton X-100 were used as negative and positive controls, respectively. 100 µL of 5% red blood cell suspension was added to each of the experimental group, negative control, and positive control group, with a final reaction volume of 200 µL. Each sample was incubated at 37°C for 1 hour. After incubation, the samples were centrifuged at 3000 rpm for 10 minutes at 4°C, and 100 µL of the supernatant was collected. The absorbance (OD) at 540 nm was measured using a microplate reader. 540 ).

[0069] Hemolysis rate = (OD value of experimental sample - OD value of negative control group) ÷ (OD value of positive control group - OD value of negative control group) × 100%.

[0070] Experimental results are as follows Figure 6 As shown.

[0071] from Figure 6 It can be seen that the hemolysis rate of peptide 98-12 at different concentrations (32 µmol / L, 64 µmol / L, 128 µmol / L) is less than 5%, indicating that peptide 98-12 has a high safety profile.

[0072] Example 9: Cytotoxicity detection of peptides This embodiment assesses the biocompatibility and safety of peptide 98-12 by detecting its cytotoxicity on human gingival epithelial cells at different concentrations.

[0073] Human gingival epithelial cells (HGE) were used as the test cell line. The peptides prepared in Example 1 were serially diluted using a two-fold dilution method with DMEM medium to obtain peptide concentrations of 32 µmol / L, 64 µmol / L, and 128 µmol / L. Cells with different concentrations of peptides were used as experimental groups, cells without peptides were used as control groups, and cells with only DMEM medium were used as blank groups.

[0074] The cells from each group were cultured in a 37°C, 5% CO2 incubator. Logarithmic growth phase cells were digested with trypsin to prepare cell suspensions, and the density was adjusted to 2 × 10⁶ cells / mL. 4 Cells / mL: 100 µL of cell suspension was added to each well of a 96-well plate and incubated overnight to allow cell adhesion. The supernatant was aspirated, and 100 µL of peptides at different concentration gradients were added to each well. Cells treated with each peptide were incubated for 3 hours, 6 hours, and 24 hours, respectively. After incubation, the supernatant was discarded, and the cells were washed three times with PBS. 100 µL of CCK-8 reagent solution (1 mL CCK-8 reagent diluted to 9 mL DMEM medium) was added to each well, and the cells were incubated for 1.5 hours. The absorbance (OD) at 450 nm was measured using a microplate reader. 450 ).

[0075] The formula for calculating cell viability is: Cell viability = (Absorbance of experimental wells - Absorbance of blank wells) ÷ (Absorbance of negative control wells - Absorbance of blank wells) × 100%.

[0076] Experimental results are as follows Figure 7 As shown in the figure, the cell survival rates under different concentrations of peptide 98-12 were displayed after incubation times of 3 hours, 6 hours, and 24 hours.

[0077] from Figure 7 As can be seen, when the concentration of peptide 98-12 reached 128 µmol / L and the incubation time was 24 hours, the cell viability still exceeded 80%, and it did not inhibit cell proliferation or activity. This indicates that within the experimental concentration range, the peptide has almost no toxic effect on cells. This result further demonstrates the good biocompatibility of peptide 98-12, providing reliable support for its safety in clinical applications.

[0078] In summary, this invention provides a novel polypeptide 98-12 (amino acid sequence shown in SEQ ID NO. 1), the core of which is that this polypeptide 98-12 does not directly inhibit the growth of *Streptococcus mutans*, but rather inhibits its cariogenic virulence by effectively inhibiting it, including: inhibiting the formation of *Streptococcus mutans* biofilm (see Example 3), disrupting the three-dimensional structure of the biofilm (see Example 4), and inhibiting the synthesis of *Streptococcus mutans* extracellular polysaccharides (see Examples 5 and 6), while not affecting the growth of *Streptococcus mutans* (see Example 7). Furthermore, this polypeptide did not exhibit significant hemolytic activity (see Example 8, hemolysis rate less than 5%) and good cell compatibility (see Example 9, it showed no significant toxicity to human gingival epithelial cells, and cell survival rate still exceeded 80% after 24 hours), demonstrating high biosafety. Compared with existing peptides such as GLDWWQL and KR-1, the peptide 98-12 of this invention has a unique mechanism of action and concentration range (32-128 μmol / L), which can be flexibly applied to various oral care products such as toothpaste and mouthwash. It overcomes the defects of traditional anti-caries drugs that disrupt the oral microecology, enriches the types and choices of anti-caries peptides, and provides a safe and efficient solution for the prevention and treatment of dental caries.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A polypeptide that inhibits biofilm formation and extracellular polysaccharide synthesis in Streptococcus mutans, characterized in that: The polypeptide is polypeptide 98-12, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The application of the polypeptide as described in claim 1 in the preparation of oral care products, characterized in that: The polypeptide 98-12 achieves the effect of oral care products in preventing and treating dental caries through at least one of the following functions: (1) Inhibits the formation of biofilms in Streptococcus mutans; (2) Disrupts the biofilm structure of Streptococcus mutans. (3) Inhibits the ability of Streptococcus mutans to produce extracellular polysaccharides; (4) It does not affect the growth of Streptococcus mutans and maintains oral microbial homeostasis.

3. The application according to claim 2, characterized in that: The concentration of peptide 98-12 in the oral care product is 32–128 μmol / L.

4. The application according to claim 3, characterized in that: The concentration of peptide 98-12 in the oral care product is 128 μmol / L.

5. The application according to claim 2, characterized in that: The oral care products include at least one of toothpaste, mouthwash, oral spray, tooth powder, gel, oral lozenges or chewing gum.

6. The application according to claim 2, characterized in that: The oral care product also contains pharmaceutically acceptable excipients selected from at least one of thickeners, surfactants, humectants, sweeteners, flavorings, preservatives, or pH adjusters.

7. The application according to claim 2, characterized in that: The oral care products are available in liquid, semi-solid, or solid dosage forms.

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