Antibacterial polypeptide compound and application thereof in preparation of anti-caries drugs

Abstract

The invention provides an antibacterial polypeptide compound and application thereof in preparation of anti-caries drugs, and belongs to the technical field of biochemistry, the antibacterial polypeptide compound comprises a polypeptide Ap-3 and / or a polypeptide Ap-4, the amino acid sequence of the polypeptide Ap-3 is as shown in SEQ ID NO.1, and the amino acid sequence of the polypeptide Ap-4 is as shown in SEQ ID NO.2. The invention further provides a preparation method of the antibacterial polypeptide compound. The compound is screened from wheat structural protein, has excellent anti-streptococcus mutans activity and anti-caries performance, and provides a new choice for prevention and treatment of early caries clinically.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and particularly relates to antibacterial polypeptide compounds and their application in the preparation of anti-caries drugs. Background Art

[0002] Antibacterial peptides are a large class of bioactive polypeptides with antibacterial functions against harmful living organisms (or pathogens) such as bacteria, fungi, parasites, and viruses. Because they generally carry a sufficient amount of positive charge and are often accompanied by hydrophobicity, they can bind to negatively charged biological membranes under electrostatic action, penetrate and destroy the membrane structure, resulting in cell death. Different from the single-target bactericidal principle of traditional antibiotics, antibacterial peptides can carry out multi-target destruction on pathogens, can greatly reduce the generation of drug-resistant bacteria, and have broad-spectrum antibacterial properties, making them one of the best choices to replace antibiotics in the future.

[0003] In the past few decades, although there have been some studies on the application of natural and synthetic antibacterial peptides in the prevention and treatment of dental caries and some promising results have been obtained. However, the prevention and treatment of dental caries is a long-term process, and long-term use requires not only considering drug resistance but also economic efficiency and population accessibility. Therefore, there is an urgent need to expand the new anti-caries peptide library for the prevention and treatment of dental caries. Summary of the Invention

[0004] To solve the problem of insufficient antibacterial peptides in the anti-caries peptide library, the present invention provides antibacterial polypeptide compounds, which are screened from wheat structural proteins, have excellent Streptococcus mutans inhibitory activity and anti-caries performance, and provide a new choice for the prevention and treatment of early dental caries clinically.

[0005] The present invention also provides the application of the antibacterial polypeptide compound in the preparation of anti-caries drugs.

[0006] The present invention is achieved by the following technical solutions:

[0007] The present invention provides an antibacterial polypeptide compound, which includes polypeptide Ap-3 and / or polypeptide Ap-4. The amino acid sequence of polypeptide Ap-3 is shown in SEQ ID NO.1, and the amino acid sequence of polypeptide Ap-4 is shown in SEQ ID NO.2.

[0008] Based on the same inventive concept, the present invention provides the application of the antibacterial polypeptide compound in the preparation of anti-caries drugs.

[0009] Based on the same inventive concept, the present invention provides the application of the antibacterial polypeptide compound in the preparation of anti-caries oral care products, and the oral care products include any one of toothpaste, mouthwash, oral spray, and chewing gum.

[0010] Based on the same inventive concept, the present invention provides the application of the antibacterial polypeptide compound in being or preparing a Streptococcus mutans inhibitor.

[0011] Based on the same inventive concept, the present invention provides an anti-caries drug, and the active ingredient of the drug comprises the above-mentioned antibacterial polypeptide compound.

[0012] Furthermore, the dosage form of the drug comprises any one of tablets, injections, sprays, solutions, ointments and gels.

[0013] Based on the same inventive concept, the present invention also provides an anti-caries oral cleaning product, and the active ingredient of the anti-caries oral cleaning product comprises the above-mentioned antibacterial polypeptide compound, and the oral cleaning product comprises any one of toothpaste, mouthwash, oral spray and chewing gum.

[0014] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0015] 1. The antibacterial polypeptide compound of the present invention comprises two food-derived polypeptides, polypeptide Ap-3 and polypeptide Ap-4, which are screened from wheat structural proteins, and has the advantages of safety, low toxicity and few side effects, and has excellent Streptococcus mutans inhibitory activity and anti-caries performance, providing a new option for the prevention and treatment of early caries in clinic.

[0016] 2. In the application of the antibacterial polypeptide compound of the present invention in preparing an anti-caries drug, polypeptide Ap-3 and polypeptide Ap-4 have excellent Streptococcus mutans inhibitory activity and good specificity. Applying the polypeptide Ap-3 and / or polypeptide Ap-4 derived from wheat structural proteins to prepare an anti-caries drug can not only effectively prevent and treat dental caries, but also has the advantages of safety, low toxicity and low side effects compared with existing antibacterial peptides, and is not easy to destroy the oral flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without creative efforts.

[0018] Figure 1 HPLC results and mass spectrometry results of compounds Ap-3 and Ap-4: (A) HPLC result of Ap-3; (B) HPLC result of Ap-4; (C) mass spectrometry result of Ap-3; (D) mass spectrometry result of Ap-4.

[0019] Figure 2 Results of hemolytic activity experiment of compounds Ap-3 and Ap-4.

[0020] Figure 3 1h cytotoxicity results of compound Ap-4.

[0021] Figure 4 It is the bactericidal kinetic curve of compound Ap-4.

[0022] Figure 5 It is the observation result of compound Ap-4 by scanning electron microscope (SEM).

[0023] Figure 6 It is the result of the propidium iodide (PI) uptake experiment of compound Ap-4.

[0024] Figure 7 It is the result of the experiment for inhibiting biofilm formation of compound Ap-4.

[0025] Figure 8 It is the result of the effect of Ap-4 on preventing dental caries formation, organs and oral flora in rats: (A) Intraoral photos of the maxilla of rats in each group; (B) Results under a stereomicroscope of the mandible of rats in each group; (C) Results of 16S rDNA sequencing of rat saliva; (D) Results of HE staining of the heart, liver, spleen, lung, kidney, stomach, and gingival tissues of rats in each group.

[0026] Figure 9 It is the result of the saliva stability experiment of Ap-4: (A) Line graph of the retention rate of Ap-4 in saliva; (B) HPLC result graph of Ap-4. Detailed implementation manners

[0027] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than to limit the present invention.

[0028] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. In case of contradiction, this specification shall prevail.

[0029] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0030] Unless otherwise specifically stated, the experimental methods used in the following examples are all conventional methods.

[0031] The overall idea of the present invention is as follows:

[0032] In the past few decades, although there have been some studies on the application of natural and synthetic antimicrobial peptides in the prevention and treatment of dental caries, and some promising results have been achieved. However, the prevention and treatment of dental caries is a long-term process. Long-term use requires not only considering drug resistance but also economic efficiency and population accessibility. Therefore, there is an urgent need to expand the library of novel anti-caries peptides for the prevention and treatment of dental caries.

[0033] In recent years, functional peptides derived from plants, especially food sources, have received extensive attention because these bioactive peptides are generally considered safe, low-toxic, and have few side effects, especially when used in the prevention and treatment of chronic diseases. However, the research and development cost of conventional chemical modification methods for antimicrobial peptides is relatively high and time-consuming. By using artificial intelligence, the characteristics of antimicrobial peptides can be deeply learned, useful information can be obtained from a large number of polypeptide sequences, so as to predict or generate bioactive peptides. This step simplifies the research process of traditional methods, reduces production costs, and can also ensure the safety and effectiveness of drugs. To solve the problem of identifying anti-caries peptides from food source proteins, here we use a new anti-caries peptide classification model to screen out bioactive peptides with potential application value from wheat structural proteins.

[0034] Based on this, the present invention provides an antimicrobial polypeptide compound and its application in the preparation of anti-caries drugs. By capturing the antibacterial characteristics of antimicrobial peptides by computer, artificial intelligence learns these characteristics, establishes a screening model, and finally screens out polypeptides with high activity against Streptococcus mutans from wheat structural proteins. Their structural formulas are FPVTWRWWKWWKG (theoretical molecular weight: 1860.9279) and FPVTWRWWKWW (theoretical molecular weight: 1675.8565), respectively, labeled as compound Ap-3 and Ap-4.

[0035] The following will combine examples and experimental data to elaborate in detail on the antimicrobial polypeptide compound of the present invention and its application in the preparation of anti-caries drugs.

[0036] Example 1

[0037] This example provides a method for synthesizing and purifying an antimicrobial polypeptide compound.

[0038] The antimicrobial polypeptide compound is synthesized by the classical solid-phase peptide synthesis method, and the specific steps are as follows:

[0039] 1) Swell the resin: Weigh a certain amount of resin and add it to a peptide synthesizer. Then add an appropriate amount of redistilled DCM and stir for 30 min to completely swell the resin. After suction drying, wash the resin 4 times with redistilled DMF, 3 min each time, and then suction dry.

[0040] 2) Ninhydrin test: Take a small amount of resin and place it in a test tube. Add ninhydrin reagent (ninhydrin: pyridine: phenol = 1:2:1), and heat it in boiling water for 3 min. If the color of the resin does not change, the next step can be carried out.

[0041] 3) Deprotection: Add the deprotection reagent (piperidine: DMF = 1:4) into the synthesizer, stir, repeat 3 times, 5 min each time, to completely remove the Fmoc protecting group. Wash the resin with redistilled DMF 4 times, 3 min each time. Conduct ninhydrin test again. If the resin turns blue-violet, it proves that the deprotection is successful.

[0042] 4) Condensation reaction: Weigh 3-fold excess of Fmoc-protected amino acid, condensing agents HOBT and HBTU, dissolve them with redistilled DMF, add 6-fold excess of initiator DIEA, stir and mix evenly, then add them into the synthesizer. Stir for 1 h under the protection of inert gas, drain, wash the resin with redistilled DMF 4 times, 3 min each time, drain, conduct ninhydrin test. If the color of the resin does not change, it proves that the condensation reaction is successful. Repeat the steps of removing the Fmoc protecting group and condensation reaction until the whole peptide chain is completely synthesized.

[0043] 5) Cleavage of polypeptide: After the condensation of the last amino acid is completed, remove the Fmoc protecting group, wash the resin with redistilled DMF 4 times, 3 min each time, then wash the resin with DCM and anhydrous methanol alternately 3 times each, 3 min each time, and put the resin into a vacuum drying oven. After the resin is completely dried, add the cleavage reagent (TFA:Tis:H2O = 95%:2.5%:2.5%), stir slowly for 3 h, and collect the cleavage solution.

[0044] 6) Extraction of polypeptide: Rotate the cleavage solution to dryness under reduced pressure, store it in a -20 °C refrigerator for 15 min, take it out and immediately add an appropriate amount of ice ether for precipitation, extract with water, collect the aqueous phase in a beaker, and obtain the crude peptide powder after freeze-drying.

[0045] 7) Purification of polypeptide: Purify the polypeptide with a reverse-phase high performance liquid chromatograph. Dissolve the crude peptide powder, filter and load the sample. The elution system is an aqueous solution of 15%-95% acetonitrile, the flow rate is 10 mL / min, elute for 80 min, observe the change of the peak shape of the chromatogram at 220 nm and 254 nm, collect the eluate at the main peak and label it, and obtain the pure peptide after freeze-drying. Conduct purity analysis with reverse-phase high performance liquid chromatography. Take a small amount of pure peptide to dissolve, load the sample, the elution system is an aqueous solution of 5%-95% acetonitrile, the flow rate is 1 mL / min, elute for 30 min, integrate the chromatogram, calculate the purity, and detect the actual molecular weight of the polypeptide with mass spectrometry analysis. The results are shown in Figure 1 A - D, the purity of Ap-3 and Ap-4 is > 95%, and the mass spectrometry results are correct.

[0046] Example 2

[0047] In this example, the minimum inhibitory concentration of compounds Ap-3 and Ap-4 is determined.

[0048] Standard strains used for the antibacterial activity assay of compounds Ap-3 and Ap-4: Streptococcus mutans (S. mutans, ATCC 25175), Escherichia coli (E. coli, ATCC 25922), Staphylococcus aureus (S. aureus, ATCC 25923), Enterococcus faecalis (E. faecalis, ATCC 29212), and Candida albicans (C. albicans, ATCC 10231) were purchased from the American Type Culture Collection (ATCC). Lactobacillus acidophilus (L. acidophilus, BNCC 185342) and Lactobacillus casei (L. casei, BNCC 364409) were purchased from BeNa Culture Collection (BNCC). When performing the drug susceptibility test, Mueller-Hinton Agar was used for Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus; Brain Heart Infusion Agar was used for Streptococcus mutans; and Sabouraud Dextrose Agar was used for Candida albicans. Lactobacillus acidophilus and Lactobacillus casei were cultured using MRS. The minimum inhibitory concentration (MIC) values of compounds Ap-3 and Ap-4 against bacteria were slightly modified according to the Clinical and Laboratory Standards Institute (CLSI) microdilution method.

[0049] Briefly, a single bacterial colony was picked and incubated in 4 mL of the corresponding medium at 37 °C on a shaker at 180 rpm for 18 h. The bacterial suspension was diluted to 1×10 5 CFU / ml with the corresponding medium for standby. The minimum inhibitory concentration of the compound against the selected strains was detected by the microbroth dilution method: different concentrations of the compound solution after serial dilution were added to each well of a sterile 96-well plate, and the final concentration of the bacterial suspension in each well was 1×10 4 CFU / ml. First, 100 μl of the corresponding medium was added to each well. The drug solution at 2 mg / ml was diluted to 512 μM. 100 μl of the drug solution was added to each of the first 5 wells in row A of the first row to make the concentration 256 μM, and then serially diluted to wells B-H with concentrations of 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, and 2 μM, respectively. 100 μl of the medium was added to each well to make the volume of each well system 200 μl. 20 μl of the bacterial suspension with a concentration of 1×10 5 CFU / ml was added to each well to ensure that the final concentration of the bacterial suspension in each well was 1×10 4 CFU / ml. The cultures were incubated for 24 h under the culture conditions corresponding to each strain, and the lowest concentration at which no bacterial growth was detected at OD600nm was defined as the MIC. The above experiments were repeated effectively 3 times.

[0050] The experimental results are shown in Table 1. Ap-3 and Ap-4 have high selectivity for antibacterial activity, showing good antibacterial effects against Gram-positive bacteria (S. mutans, S. aureus, E. faecalis), especially better antibacterial activity against Streptococcus mutans. The MIC of both peptides is 4 μM. However, the antibacterial effects against Gram-negative bacteria (E. coli), C. albicans, and probiotics (L. casei, L. acidophilus) are poor.

[0051] Table 1 Minimum inhibitory concentrations of compounds against standard bacterial strains

[0052]

[0053] Example 3

[0054] In this example, a hemolytic activity experiment was carried out.

[0055] 100 μL of the peptide and an equal volume of 8% (v / v) chicken red blood cells were added to a 96-well plate. After incubation at 37 °C for 1 h, centrifuged at 800 g for 5 min, 100 μL of the supernatant was collected, and OD was measured 490nm . The red blood cells treated with PBS and 1% Triton X-100 were used as negative and positive controls, respectively. Three independent experiments were carried out, and each experiment was repeated three times.

[0056]

[0057] The detection results are shown in Figure 2 : Ap-4 showed extremely low hemolytic activity against red blood cells, and Ap-3 showed relatively high hemolytic activity at high concentrations.

[0058] Example 4

[0059] In this example, a cytotoxicity experiment of compound Ap-4 was carried out.

[0060] The cytotoxicity of compound Ap-4 was determined by measuring the proliferation effect on mouse epithelial-like fibroblasts L-929 by the CCK-8 method. The cell lines used in this experiment were all purchased from Pricella. The in vitro toxicity of the compound to L-929 cells was detected. The cell suspension was inoculated in a 96-well plate (the number of cells per well was 8×10 3Add 100 μl of high-glucose DMEM medium containing 10% fetal bovine serum to each well, place it in an incubator for culturing. After the cells adhere to the wall, aspirate and discard the old medium. Set up 4 drug groups, a negative group (only add DMEM) and a blank group (only add basal medium). The drug groups are respectively added with the drug solution diluted with DMEM (final concentrations are 1 / 2×MIC, 1×MIC, 2×MIC, 4×MIC), then incubated at 37°C. After culturing for 1 h, take out the 96-well plate, add 100 μL of CCK-8 solution to each well, and culture at 37°C for 1.5 h. Use a multifunctional microplate reader to measure the absorbance at 450 nm. Finally, calculate the cytotoxicity (%).

[0061]

[0062] The experimental results are as Figure 3 shown: After short-term contact of L-929 cells with Ap-4, no obvious cytotoxicity was shown at all tested concentrations. On the contrary, it even promoted the proliferation of cells, and the cell survival rate exceeded 100%, indicating its good biocompatibility in the short term.

[0063] Example 5

[0064] This example conducts a bactericidal kinetics test.

[0065] Mix equal volumes of the peptide (final concentrations are 1×MIC, 2×MIC, and 4×MIC) with the pre-activated Streptococcus mutans solution (about 1×10 6 CFU / mL) and incubate at 37°C. At the same time, set up a blank group (medium + bacterial solution). Then, at different time points (0 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h), take 100 μL of the co-incubation solution for appropriate dilution, and spread it on BHI agar plates. After incubating at 37°C for 24 h, count the number of bacterial colonies by CFU (colony forming unit). Plot the bactericidal kinetics curve. The results are shown in Figure 4 : Ap-4 has a significant bactericidal effect, and the bactericidal speed is concentration-dependent.

[0066] Example 6

[0067] This example conducts a scanning electron microscope (SEM) observation experiment.

[0068] Take the bacteria (1×10 6CFU / mL) were treated with peptide Ap-4 at a concentration of 2×MIC at 37°C for 40 min, and then fixed overnight at 4°C with 2.5% glutaraldehyde. Subsequently, dehydration was carried out with gradient ethanol of 30%, 50%, 60%, 70%, 80%, 90% and 100%, and the bacteria were diluted to an appropriate concentration with 100% ethanol. Finally, 10 μL of the sample was dropped on a silicon wafer. After air drying at room temperature, the sample was gold-plated and observed under a scanning electron microscope. The results are shown in Figure 5 : Compared with Streptococcus mutans treated with PBS (control group), after treatment with Ap-4 at 2-fold MIC for 40 min, the surface of the bacteria became rough, deformed, with unclear outlines, obvious irregular cracks and fragments, indicating that bacterial lysis occurred.

[0069] Example 7

[0070] In this example, a propidium iodide (PI) uptake experiment was carried out.

[0071] Single colonies of Streptococcus mutans were picked and inoculated into 4 ml of liquid medium, and cultured in a constant temperature shaker at 37°C for 24 h. After activation, the bacterial solution (concentration of 1×10 6 CFU / mL) was centrifuged at 4000 rpm for 10 min and washed twice with PBS. Set up blank control group (PBS), positive control group (Triton 4%), negative control group (PBS), experimental groups (final concentration of Ap-4 was 4×MIC, 8×MIC, 16×MIC), 500 μL of each drug was mixed evenly with an equal volume of the washed bacterial solution, and incubated in a shaker at 37°C at 180 rpm for 2 hours. 200 uL of PI was added to the co-incubates, sampled, and detected by flow cytometry. The results are as Figure 6 shown: It indicates that Ap-4 may play an antibacterial role through a membrane disruption mechanism, which is similar to the mechanism of action of most antibacterial peptides.

[0072] Example 8

[0073] In this example, a biofilm formation inhibition experiment was carried out.

[0074] 100 μL of different concentrations of polypeptide Ap-4 (1 / 2×MIC, 1×MIC, 2×MIC, 4×MIC) was mixed evenly with an equal volume of Streptococcus mutans bacterial solution (1×10 6 CFU / mL) in a 96-well plate and incubated at 37°C for 24 h. Static culture was carried out at 37°C for 48 hours. The culture medium was aspirated, and washed 3 times with PBS to remove planktonic bacteria. 200 μL of methanol was added to each well to fix the biofilm for 15 minutes, and after discarding the methanol, it was air dried. The material was washed 3 times with PBS to remove excess dye and air dried. 95% alcohol was added to dissolve the bound dye, and the OD was measured with an enzyme-labeled instrument 590nm . The results are as Figure 7Shown: It shows that the target peptide with a concentration of more than 1×MIC of Ap-4 can significantly inhibit the formation of Streptococcus mutans biofilm.

[0075] Example 9

[0076] In this example, an in-vivo animal experiment on the anti-caries effect of antibacterial peptides was carried out.

[0077] Twenty 21-day-old (about 70 g) male Sprague-Dawley (SD) rats were selected. The SD rats used in this example were provided by the Medical Experimental Animal Center of Lanzhou University (Gansu Province, China). All animals were housed in a constant-temperature room (22±1°C) with sufficient water and feed. In this example, we closely monitored the health status of the mice and tried our best to reduce their suffering. All experiments were carried out in accordance with the Council Directive of the European Communities (86 / 609 / EEC) of 24 November 1986.

[0078] After recording the body weights, the rats were randomly divided into 4 groups: PBS group, sodium fluoride group, low-concentration peptide group, and high-concentration peptide group, with 5 rats in each group. The body weights were recorded every 2 days. After 2 days of feeding with conventional feed for adaptation, the rats in each group started the experiment and were fed with cariogenic feed (Keyes 2000#) throughout the process. At the same time, ampicillin (1 g / L) was used for drinking water on days 1-4 to make it easier for Streptococcus mutans to colonize and grow in the oral cavity, so as to better study the effect of Streptococcus mutans on caries formation. From days 5-9, the rats were given Streptococcus mutans drinking water (concentration 1×10 6 CFU / mL) and 5% sucrose drinking water alternately every day. From day 10 until the end of the experiment, 5% sucrose drinking water was used. Group administration started on day 5 of the experiment. Among them, 0.05% sodium fluoride was used in the sodium fluoride group; antibacterial peptide Ap-4 with 1× and 2×MIC was used in the low-concentration and high-concentration peptide groups respectively; PBS was used as a negative control. Before administration, the tooth surfaces and oral cavities of the rats were wiped with sterile cotton swabs. When administering the drugs, a brush was used to smear and 0.2 mL of the drug solution was dropped into the mouth onto the tooth surfaces, 2 times a day. The rats were fasted within 1 hour after smearing the bacterial solution. The experiment was terminated after obvious early caries symptoms appeared in the negative control group on day 28. Saliva samples of the rats were taken with oral swabs and sent to Zhongke New Life for 16S rDNA sequencing. Intraoral photos of the rats were taken. The rats were anesthetized and sacrificed with an animal anesthetic (isoflurane). The hearts, livers, spleens, lungs, kidneys, stomachs, and gingival tissues of the animals were weighed to calculate the organ indices. After fixation with 4%-paraformaldehyde and HE staining, the mandibles were removed and the surrounding soft tissues were dissected. Stereomicroscope photos of the mandibles were taken, and the formation of dental caries plaques at the first molar site of the mandible was observed mainly.

[0079] The results are as Figure 8 shown in Table 2: Figure 8A - D indicate that Ap - 4 can effectively prevent the occurrence of dental caries lesions, cause no damage to major organs, and has high biocompatibility. When preventing dental caries, it also has no obvious effect on the oral flora and does not disrupt the balance of the oral flora.

[0080] Table 2. Comparison of organ coefficients of mice in different groups (x ± s, %)

[0081]

[0082]

[0083] Example 10

[0084] In this example, a saliva stability experiment was conducted.

[0085] Take about 5 ml of morning brushing - before saliva from healthy volunteers. Aliquot it into EP tubes and centrifuge at 12000 rpm for 20 min. Aspirate the supernatant and filter it. Mix 1600 μL of Ap - 4 polypeptide solution (250 μmol / L) with 1600 μL of filtered saliva and incubate it in a shaker at 37 °C at 180 rpm. Take 300 μL at 0, 0.5 h, 1 h, 2 h, 4 h, and 6 h for HPLC sample injection and detection. The results are as Figure 9 shown in A and B: Within 1 h, the target peptide degrades very little in saliva, showing a certain stability.

[0086] Finally, it should also be noted that the term "comprises", "comprising", or any other variation thereof is intended to cover a non - exclusive inclusion, such that a process, method, article, or apparatus that comprises a series of elements includes not only those elements but also other elements not expressly listed, or elements that are inherent to such process, method, article, or apparatus.

[0087] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0088] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. An antimicrobial polypeptide compound, characterized in that: The antibacterial polypeptide compound comprises polypeptide Ap-3 and / or polypeptide Ap-4, the amino acid sequence of polypeptide Ap-3 is shown as SEQ ID NO.1, and the amino acid sequence of polypeptide Ap-4 is shown as SEQ ID NO.

2.

2. Use of the antibacterial polypeptide compound as claimed in claim 1 in the preparation of anti-caries drugs.

3. The use of the antibacterial polypeptide compound according to claim 1 in the preparation of an anti-caries oral cleaning product, characterized in that: The oral cleaning product includes any one of toothpaste, mouthwash, oral spray and chewing gum.

4. Use of the antimicrobial polypeptide compound as claimed in claim 1 as or in the preparation of a mutans Streptococcus inhibitor.

5. An anti-caries drug, characterized in that: The active ingredient of the medicine includes the antibacterial polypeptide compound according to claim 1.

6. An anti-caries drug according to claim 1, characterized in that: The dosage form of the drug includes any one of tablets, injections, sprays, solutions, ointments and gels.

7. An anti-caries oral cleaning product, characterized in that: The effective ingredients of the anti-caries oral cleaning product include the antibacterial polypeptide compound according to claim 1, and the oral cleaning product includes any one of toothpaste, mouthwash, oral spray and chewing gum.

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