Postbiotic composition for effectively inhibiting dental caries in oral cavity and preparation method thereof

Abstract

The invention belongs to the technical field of biomedical treatment and biological fermentation, discloses a metagen composition for effectively inhibiting oral dental caries and application thereof, and aims to solve the problems of great side effect, weak effect and unknown mechanism of the existing dental caries prevention and treatment means. The composition is prepared by mixing a metagen 1 which is prepared by fermenting lactobacillus salivarius Postbio-YY with a metagen 2 which is prepared by fermenting lactococcus lactis Postbio-F3 and lactobacillus paracasei Postbio-P6 step by step. In the preparation process, the active ingredients are reserved by accurately controlling the fermentation pH, time and pasteurization parameters. The composition can be prepared into multiple dosage forms such as toothpaste and the like, can remarkably inhibit the growth of streptococcus mutans and destroy the thallus structure, inhibits biofilm and exopolysaccharide synthesis by down-regulating fimI, wcaJ and wcae genes, is safe and free of side effects, and provides an efficient scheme for decayed tooth prevention and treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine and biological fermentation, and particularly discloses a postbiotic composition for effectively inhibiting oral caries bacteria and a preparation method thereof. BACKGROUND

[0002] As a globally high-incidence oral bacterial disease, caries has a high prevalence rate and harm, which has become an important challenge in the field of public health. According to statistics, the global prevalence rate of caries is about 44%, directly affecting 3.1 billion people, and the prevalence rate of caries in China is as high as 75%, among which the prevalence risk of children, the elderly and other groups is significantly rising. The pathological process of caries has a chronic and progressive characteristic, and in the early stage, it is characterized by demineralization of hard tissue of teeth, and with the development of the disease, it can cause pulpitis, periapical periodontitis, and even alveolar bone and jaw inflammation, which not only destroys the masticatory function of the oral cavity, but also may cause systemic health risks through oral local infection, and poses multiple threats to the life quality and health of patients.

[0003] Streptococcus mutans in the oral cavity is a recognized core cariogenic microorganism, and its pathogenic ability depends on the synergistic mechanism of "biofilm formation-acid production-acid tolerance-extracellular polysaccharide synthesis". The strain efficiently binds to the dental hydroxyapatite through surface adhesin, gradually aggregates to form dental plaque biofilm; in the microenvironment of the biofilm, Streptococcus mutans metabolizes carbohydrates (such as sucrose) to synthesize extracellular polysaccharides such as dextran and fructan, which not only provide mechanical stability and adhesion for the biofilm, but also build a "protective barrier" against host immune cell clearance and antibacterial drug penetration. At the same time, the acid substances such as lactic acid produced by the continuous metabolism of Streptococcus mutans will break the balance of tooth mineralization and demineralization, and accelerate the demineralization and dissolution of enamel; more importantly, the strain maintains intracellular pH homeostasis through the ATPase-driven proton efflux system, so that it can survive and reproduce in an extremely acidic environment, further aggravating the progression of caries. In addition, recent studies have confirmed that Streptococcus mutans may also participate in the occurrence of systemic diseases such as subacute endocarditis and atherosclerosis through the blood circulation, and its harm has exceeded the scope of oral local.

[0004] Current caries prevention and treatment methods have significant technical limitations, which are difficult to meet the "safe, efficient and long-acting" clinical and daily care needs: Chemical agents (such as fluoride): as a traditional anti-caries means, fluoride can play a role by promoting enamel remineralization, but has clear side effects - excessive intake (such as children swallowing fluoride toothpaste by mistake) can easily cause fluorosis, fluorosis; long-term use may also induce drug resistance of Streptococcus mutans, leading to decreased anti-caries effect, and non-target inhibition of beneficial oral bacteria (such as bifidobacteria and lactobacilli), disrupting the balance of oral microecology.

[0005] Natural extracts (such as matrine, propolis flavones): existing studies show that most natural extracts have weak antibacterial activity (the inhibition zone diameter of Streptococcus mutans is mostly <12 mm), and the mechanism is unclear, only the phenomenon of "inhibiting growth" can be observed, and the regulation target of biofilm and exopolysaccharide synthesis cannot be determined, which leads to difficulties in optimizing the preparation formula during industrialization, and unstable actual application effect.

[0006] Physical intervention (such as post and crown repair, tooth filling): such means belong to "symptomatic treatment", which is only suitable for middle and late stages of dental caries, has the problems of high cost (the cost of single treatment is hundreds to thousands of yuan), long time (multiple rechecks are required), and irreversible damage to tooth structure during treatment, and cannot meet the early prevention needs.

[0007] Existing research on postbiotics: in recent years, there has been preliminary exploration of postbiotics against dental caries, but most of them focus on a single strain (such as fermentation products of a single lactobacillus), lack of synergistic design among strains; the preparation process is rough (such as single fermentation process with fixed pH and temperature), resulting in low content of active ingredients (such as bacteriocin and short-chain fatty acid); and the mechanism is not clear, only the surface research of "inhibition zone" and "growth curve" is carried out, which cannot provide theoretical support for formulation optimization and targeted synergistic effect.

[0008] As a "preparation of microorganisms and / or components beneficial to the human body", the core advantage of postbiotics lies in: containing peptidoglycan, intracellular polysaccharide and other bacterial components, as well as organic acids, short-chain fatty acids, bacteriocins and other active metabolites, which have the potential to inhibit the growth of pathogens and destroy the formation of biofilm; at the same time, postbiotics does not need to rely on the survival of live bacteria, is not strictly limited by temperature and storage conditions, has fast action speed and strong targeting, and can also reduce the risk of drug-resistant gene production of pathogenic bacteria, which is an ideal food-grade anti-caries raw material. Based on the defects of existing technologies and the unique advantages of postbiotics, developing a natural and safe postbiotic product with clear mechanism and high efficiency in inhibiting Streptococcus mutans becomes a key direction to solve the problem of dental caries prevention and treatment. SUMMARY

[0009] Therefore, the present application provides a postbiotic composition for effectively inhibiting oral dental caries bacteria.

[0010] The technical scheme of the present application is realized as follows: the present application provides a postbiotic composition for effectively inhibiting oral dental caries bacteria, which is prepared by mixing "postbiotic 1" and "postbiotic 2" at a mass ratio of 1:1-3:1, and the core feature lies in that three specific functional lactobacillus strains are used for synergistic fermentation, and the strain information and preparation process are as follows: Lactobacillus salivarius Postbio-YY: from the Lactic Acid Bacteria Strain Library of the Probiotics and Postbiotics Basic Research and Development Application Innovation Laboratory of Qingdao Agricultural University, preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 25, 2022, with the preservation number CGMCC No. 26239.

[0011] Lactobacillus paracasei Postbio-P6: from the Lactic Acid Bacteria Strain Library of the Probiotics and Postbiotics Basic Research and Development Application Innovation Laboratory of Qingdao Agricultural University, preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 25, 2022, with the preservation number CGMCC No. 26237; Lactococcus lactis Postbio-F3: from the Lactic Acid Bacteria Strain Library of the Probiotics and Postbiotics Basic Research and Development Application Innovation Laboratory of Qingdao Agricultural University, preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 25, 2022, with the preservation number CGMCC No. 26236.

[0012] Preparation steps of Postbiotic 1: (1) Strain activation: Lactobacillus salivarius Postbio-YY was inoculated in MRS liquid medium (formula: peptone 10.0 g / L, beef infusion powder 8.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, potassium phosphate dibasic 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, pH 5.7±0.2), and incubated at 37°C for 18 h, and subcultured twice to make the viable count reach 5-10×10 6 CFU / mL; (2) Fermentation: the activated strain was inoculated into the fermentation medium (same as MRS liquid medium) at an inoculation amount of 2%, and fermented at 37°C, 150 rpm for 35 h. The pH of the system was controlled to be stable at 6.5 during the fermentation process by pH regulator; (3) Post-treatment: after fermentation, the fermentation broth was pasteurized at 95°C for 30 min (inactivated viable bacteria and retained active metabolites), and then clarified and removed impurities (centrifugation or membrane filtration to remove bacterial fragments), concentrated and desalted (reduced pressure concentration combined with ion exchange resin desalting) in sequence to obtain Postbiotic 1.

[0013] Preparation steps of Postbiotic 2: (1) First stage fermentation (Lactococcus lactis Postbio-F3): The strain was inoculated into MRS liquid medium at a 2% inoculum and fermented at 37℃ and 150rpm for 24h. The pH at the end of the fermentation was controlled to be 4.2. (2) Second stage fermentation (Lactobacillus paracasei Postbio-P6): The activated Lactobacillus paracasei Postbio-P6 (activated under the same conditions as Lactobacillus salivarius) was inoculated into the above fermentation system at an inoculation rate of 5%, and fermented for 15 hours at 37°C and 150 rpm, with the final pH controlled at 3.5. (3) Post-processing: After fermentation, the mixed fermentation broth was pasteurized at 95℃ for 40-100 min, and then clarified, purified, concentrated and desalted in sequence to obtain post-generic 2.

[0014] This invention also provides a formulation for preventing and treating Streptococcus mutans, using the above-mentioned metabiotic composition as the active ingredient, and further comprising a pharmaceutically acceptable carrier, solvent, diluent, or excipient; wherein, the selection of the carrier / excipient is adapted to the needs of different dosage forms, for example: lactose can be added as a filler in powder, potassium sorbate can be added as a stabilizer in oral liquid, and silica (abrasive), glycerin (humectant), sodium lauryl sulfate (surfactant), etc. can be added in toothpaste.

[0015] In some embodiments, the dosage form of the formulation can be selected according to the application scenario, including but not limited to: powder, granules, capsules, injections, oral liquids, tablets, and oral care-specific dosage forms (such as toothpaste, mouthwash, and oral spray); wherein, the mass concentration of the postbiotic composition in the toothpaste dosage form is 2%-6%, which is suitable for the differentiated needs of daily oral cleaning and high-risk groups of dental caries.

[0016] Mechanism of action and uses of the postbiotic composition in this invention: This invention, through transcriptomics analysis and qPCR verification, clarifies the core mechanism by which the metabiotic composition inhibits Streptococcus mutans: 1. Targeted downregulation of pathogenic genes: Significant downregulation of the fimI gene (expression level reduced by 6.07-fold), wcaJ gene (expression level reduced by 4.57-fold), and wcae gene (expression level reduced by 8.71-fold); Among them, the enzyme encoded by the fimI gene regulates the adhesion ability of Streptococcus mutans to the tooth surface, and the enzymes encoded by the wcaJ and wcae genes catalyze the synthesis of β-1,6-glucan (the core component of extracellular polysaccharides). Gene downregulation directly inhibits bacterial adhesion and biofilm construction. 2. Regulation of metabolic pathways: KEGG enrichment analysis confirmed that the composition can upregulate pathways such as ribosomes and bacterial secretion systems (interfering with bacterial protein synthesis), and downregulate pathways such as phenylalanine / tyrosine / tryptophan biosynthesis, propionic acid metabolism, and peptidoglycan biosynthesis (cutting off bacterial energy supply and cell wall synthesis), thereby inhibiting the survival and reproduction of the strain at the metabolic level.

[0017] Based on the above mechanism, this postbiotic composition can be used to prepare products that "inhibit Streptococcus mutans-related activities". Specific uses include: inhibiting the growth of Streptococcus mutans, disrupting the biofilm structure of Streptococcus mutans, reducing the synthesis of extracellular polysaccharides of Streptococcus mutans, thereby preventing or assisting in the treatment of dental caries.

[0018] The present invention has the following advantages over the prior art: Prepared using food-grade lactic acid bacteria fermentation, and pasteurized to leave no live bacteria residue, avoiding the risk of intestinal discomfort that may be caused by live bacteria preparations; at the same time, the composition targets and inhibits Streptococcus mutans, without inhibiting beneficial bacteria such as oral Bifidobacteria and harmless Lactobacillus, and will not disrupt the oral microecological balance, solving the pain points of fluoride fluorosis bacterial flora imbalance and the side effects of chemical agents, making it suitable for long-term use by sensitive groups such as children and pregnant women.

[0019] Compared to natural extracts, the postbiotic composition of this invention has better antibacterial activity against Streptococcus mutans, a higher inhibition rate of extracellular polysaccharides, and can completely inhibit the growth of Streptococcus mutans. Its effects include inhibiting growth, destroying structure, and blocking metabolism, which solves the problem of limited antibacterial activity in existing technologies.

[0020] The three strains were designed with stepwise fermentation and mixed compatibility. Lactococcus lactis regulated the microenvironment during the early fermentation stage, creating conditions for Lactobacillus paracasei to produce anti-polysaccharide components efficiently. Lactobacillus salivarius supplemented the basic antibacterial activity. The three strains worked synergistically to achieve a "1+1+1>3" effect. The fermentation process parameters were precise and controllable, and the post-processing was standardized to ensure the consistency of activity of different batches of products and meet the needs of large-scale industrial production. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The inhibition zone diagrams of different metabiotics against Streptococcus mutans in Example 1 are shown. Figure 2 The graph shows the inhibitory effects of different concentrations of postbiotic D3 on the growth of Streptococcus mutans in Example 2. Figure 3 This is a scanning electron microscope image showing the effect of metabiotic D3 on the morphology and structure of Streptococcus mutans in Example 3. Figure 4 This is a differential gene volcano plot of the transcriptome analysis of *Streptococcus mutans* by metagenic D3 in Example 6; Figure 5 This is a KEGG enrichment analysis diagram of differentially expressed genes in the transcriptome analysis of Streptococcus mutans by metagenic D3 in Example 6. Figure 6 This is a qPCR verification diagram of the expression levels of key differentially expressed genes in Streptococcus mutans by metagenic D3 in Example 6. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] The bacterial strains used in this invention are: Lactobacillus salivarius Postbio-YY, Lactobacillus paracasei Postbio-P6, and Lactococcus lactis Postbio-F3. All of them are from the Lactic Acid Bacteria Strain Bank of the Laboratory for Basic Research and Development of Probiotics and Metabiotics Innovation, Qingdao Agricultural University, and were deposited at the China General Microbiological Culture Collection Center on December 25, 2022, with accession numbers CGMCC No. 26239, CGMCC No. 26237, and CGMCC No. 26236, respectively.

[0025] Culture medium: MRS liquid medium (formulation: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, pH 5.7 ± 0.2); Other reagents and equipment: pH adjuster (hydrochloric acid / sodium hydroxide), high-speed centrifuge, pasteurization equipment, membrane filtration device, vacuum concentrator, ion exchange resin column.

[0026] Preparation of post-genetic agent 1: Strain activation: Take *Lactobacillus salivarius* Postbio-YY strain, inoculate it into 100 ml MRS liquid medium, and incubate statically at 37℃ for 18 h; after the first culture, transfer it to fresh MRS liquid medium at a 1% inoculum rate, and repeat the culture once (passage twice) to ensure sufficient strain activation. After activation, the viable count should reach 5-10 × 10⁻⁶. 6 CFU / mL; Fermentation culture: The activated Lactobacillus salivarius Postbio-YY bacterial culture was inoculated into 1 LMRS liquid fermentation medium at an inoculation rate of 2% and placed in a shaker at 37℃ and 150 rpm for fermentation. During the fermentation process, the pH of the fermentation system was controlled to be stable at 6.5 in real time by adding 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution, and the fermentation was continued for 35 hours. Pasteurization: After fermentation, the fermentation broth is transferred to a pasteurization tank and kept at 95°C for 30 minutes to inactivate live bacteria while retaining active metabolites. Clarification and impurity removal: The sterilized fermentation broth was cooled to room temperature and centrifuged at 8000 rpm for 15 min to remove cell fragments; then filtered through a 0.22 μm organic membrane to further remove fine impurities, resulting in a clarified fermentation broth. Concentration and desalting: The clarified fermentation broth was passed into a vacuum concentrator and concentrated to 1 / 5 of its original volume at 50℃ and -0.08MPa. The concentrate was then passed through a 732 cation exchange resin column and a D301 anion exchange resin column to remove salt from the system, finally obtaining the post-generic 1 concentrate.

[0027] Preparation of post-genetic agent 2: Strain activation: Lactococcus lactis Postbio-F3 and Lactobacillus paracasei Postbio-P6 were inoculated separately into 100 ml MRS liquid medium and incubated at 37°C for 18 h. The cultures were then transferred to fresh MRS medium at a 1% inoculum and passaged twice. After activation, the viable counts of both strains reached 5-10 × 10⁻⁶. 6 CFU / mL; First stage fermentation (Postbio-F3 lactococcus): The activated Postbio-F3 lactococcus bacterial culture was inoculated into 1 LMRS liquid fermentation medium at a 2% inoculation rate and fermented at 37°C and 150 rpm on a shaker. The pH of the system was controlled during the fermentation process. The first stage fermentation was ended when the pH dropped to 4.2 after 24 hours of fermentation. Second stage fermentation (Lactobacillus paracasei Postbio-P6): Activated Lactobacillus paracasei Postbio-P6 bacterial culture was inoculated into the first stage fermentation broth at a 5% inoculum rate. Fermentation was continued at 37°C and 150 rpm. The pH of the system was controlled to drop to 3.5 by a pH adjuster. After fermentation continued for 15 hours, the overall fermentation was terminated. Pasteurization: Transfer the mixed fermentation broth to a pasteurization tank and keep it at 95°C for 40-100 minutes (adjust according to the volume of the batch fermentation broth; keep it at 100 minutes if the volume is >1L, and keep it at 40 minutes if the volume is <1L). Clarification, impurity removal, concentration and desalting: Same as steps 4-5 of post-genetic agent 1, namely centrifugation at 8000 rpm for 15 min, filtration through a 0.22 μm membrane, concentration under reduced pressure at 50 °C to 1 / 5 volume, and desalting with anion and cation exchange resin to obtain post-genetic agent 2 concentrate; Mixing ratio: Mix postbiotic 1 and postbiotic 2 concentrates at a mass ratio of 1:1-3:1 to obtain basic postbiotic raw materials; then add excipients to prepare postbiotic samples with concentrations of 2% (B3), 4% (C3), and 6% (D3) (such as adding the corresponding concentration of postbiotic raw materials to toothpaste base) for subsequent experiments.

[0028] Example 1 Evaluation of the antibacterial effects of various metabiotics on oral Streptococcus mutans The antibacterial activity of different types and concentrations of postbiotic samples (B3, C3, D3) against oral mucosa streptococci was evaluated, and the sample with the best antibacterial effect was screened for subsequent experiments.

[0029] Test materials 1. Bacterial strain: Streptococcus mutans (purchased from Beijing Baocang Biotechnology Co., Ltd., model ATCC 25175); 2. Culture media: Brain and heart extract liquid culture medium (formula: tryptone 10.0 g / L, disodium hydrogen phosphate 2.5 g / L, ox heart extract powder 17.5 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, pH 7.4±0.2), brain and heart extract solid culture medium (brain and heart extract liquid culture medium with 1.5%-2.0% agar added); 3. Test samples: 25% B3, 50% B3, 25% C3, 50% C3, 25% D3, 50% D3 (all are preparations containing the corresponding concentration of postbiotic raw materials); 4. Other reagents and equipment: physiological saline, Oxford cup (6mm in diameter), petri dishes, pipettes, 37℃ incubator, -4℃ refrigerator.

[0030] Test Procedure 1. Activation of the strain: Take 1 mL of Streptococcus mutans bacterial suspension, inoculate it into 100 mL of brain heart extract liquid culture medium, and incubate it in an aerobic incubator at 37℃ for 24 h to complete the activation of the strain; 2. Diluting the bacterial suspension: Take 0.5 mL of the activated Streptococcus mutans bacterial suspension and add it to 4.5 mL of physiological saline, diluting to a -2 gradient (i.e., 10⁻²). -2 (Dilution) 3. Pouring and Adding Samples: Inoculate the diluted Streptococcus mutans bacterial suspension into the still-solidified brain heart extract solid medium at a volume ratio of 0.7%. After gently mixing, pour the bacterial medium evenly into the petri dishes using the pouring method. After the medium solidifies, punch holes in the surface of the medium using an Oxford cup (3-4 holes per petri dish, with a hole spacing of ≥2cm). Then, use a pipette to add 150μL of different concentrations of post-biotic samples to each well, with 3 parallel wells for each sample. 4. Cultivation and observation: Place the culture dishes with the sample added in a -4℃ refrigerator and let them stand for 4-8 hours to allow the sample to fully penetrate into the culture medium; then transfer the culture dishes to a 37℃ incubator and incubate aerobically for 24 hours; after cultivation, measure the diameter of the inhibition zone around each well with calipers (accurate to 0.1 mm), record the data and analyze the antibacterial activity.

[0031] Test results The antibacterial effects of different metabiotic samples against *Streptococcus mutans* are shown in the table below. "-" indicates no antibacterial activity, and an inhibition zone diameter ≥10 mm is considered to have antibacterial activity. Larger diameters indicate stronger antibacterial effects. (Specific details are as follows...) Figure 1 As shown in the figure, from left to right, they are B3, C3, and D3.

[0032]

[0033] As shown in the table above, 25%B3 and 25%C3 have no antibacterial activity against Streptococcus mutans; although 50%B3 and 50%C3 have antibacterial activity, the diameter of the inhibition zone is small; 25%D3 and 50%D3 both have antibacterial activity against Streptococcus mutans, and 50%D3 has the largest inhibition zone diameter (25.00 mm), showing the most significant antibacterial effect. Therefore, sample D3 was selected for subsequent tests.

[0034] Example 2 The study investigated the inhibitory effect of sample D3 on the growth of oral pathogen Streptococcus mutans, explored the dynamic inhibitory effect of different concentrations of postbiotic D3 on the growth process of Streptococcus mutans, and clarified the degree of influence of D3 on the growth of Streptococcus mutans.

[0035] Test materials 1. Bacterial strain: Same as the *Streptococcus mutans* strain in Example 1; 2. Culture medium: The same liquid culture medium for brain and heart extract as in Example 1; 3. Test samples: 25% D3, 50% D3; 4. Other reagents and equipment: 48-well cell culture plates, microbial growth analyzer, pipettes, OD value detector (wavelength 600nm).

[0036] Test Procedure 1. Activation of the strain: Take 1 mL of Streptococcus mutans bacterial suspension and inoculate it into 100 mL of brain heart extract liquid culture medium. Incubate aerobically at 37℃ for 24 h to complete the activation. 2. Diluting the bacterial suspension: Take 0.5 mL of activated Streptococcus mutans bacterial suspension and add it to 4.5 mL of brain heart extract liquid culture medium. Adjust the OD value of the bacterial suspension using an OD value detector (600 nm). 600 Set the value to around 0.2 to ensure a consistent initial bacterial count; 3. Sample addition and treatment: Add 0.5 mL of the above-adjusted OD value Streptococcus mutans bacterial suspension to each well of the 48-well culture plate, and then add 0.5 mL of the corresponding concentration of D3 sample (25% D3 or 50% D3), and mix gently; at the same time, set up a sterile control group, add 0.5 mL of bacterial suspension and 0.5 mL of sterile brain heart extract culture medium to the control group wells, and set up 3 parallel wells for each concentration group and control group; 4. Growth monitoring: The 48-well culture plate was placed in a microbial growth analyzer, the internal temperature was set to 37℃, the shaking speed was set to 800 rpm, the measurement interval was 5 min, and continuous monitoring was performed for 24 h; after 24 h, the OD of each well at different time points was counted. 600 Values, plot growth curves, such as Figure 2 As shown, the inhibitory effect of D3 on the growth of Streptococcus mutans was analyzed.

[0037] Test results The inhibitory effects of different concentrations of D3 on the growth of Streptococcus mutans are shown in the table below. 600 The value reflects the concentration of bacteria in the bacterial solution; the higher the value, the more vigorous the bacterial growth.

[0038]

[0039] As shown in the table above, the growth of *Streptococcus mutans* in the blank control group was not inhibited, exhibiting a typical "lag phase-log phase-stationary phase" growth curve; 25% D3 significantly inhibited the growth of *Streptococcus mutans* before 16 hours (OD200). 600 (When the value is below 0.35), the bacteria gradually resume growth after 16 hours; 50% D3 can completely inhibit the growth of Streptococcus mutans, and the OD value within 24 hours is significantly lower. 600 The value remained basically at the initial level (around 0.20), indicating that the inhibitory effect of D3 on the growth of Streptococcus mutans is concentration-dependent, and the inhibitory effect is more thorough at higher concentrations of D3.

[0040] Example 3 The inhibitory effect of D3 on the cell morphology of oral pathogen Streptococcus mutans was investigated by scanning electron microscopy to explore the disruptive effect of the metabiotic D3 on the cell morphology and structure of Streptococcus mutans and to clarify the direct effect of D3 on the cells.

[0041] Test materials 1. Bacterial strain: Same as the *Streptococcus mutans* strain in Example 1; 2. Culture medium: The same liquid culture medium for brain and heart extract as in Example 1; 3. Test sample: 50% D3; 4. Other reagents and equipment: PBS buffer (pH 7.4), 2.5% glutaraldehyde solution, centrifuge tubes, high-speed centrifuge (10,000 rpm), scanning electron microscope (SEM).

[0042] Test Procedure 1. Activation of the strain: Take 1 mL of Streptococcus mutans bacterial suspension and inoculate it into 100 mL of brain heart extract liquid culture medium. Incubate aerobically at 37°C for 24 h to complete the activation. 2. Bacterial cell collection and washing: Take 1 mL of activated Streptococcus mutans bacterial solution and add it to a 2 mL centrifuge tube. Centrifuge at 10,000 rpm for 10 min. After centrifugation, carefully aspirate the supernatant and retain the bacterial sludge at the bottom of the tube. Add 1 mL of PBS buffer to the centrifuge tube, gently pipette to resuspend the bacterial sludge, and centrifuge again at 10,000 rpm for 10 min. Repeat this washing step 3 times to thoroughly remove culture medium residue. 3. D3 treatment and fixation: The washed bacterial sludge was reconstituted with 1 mL of PBS buffer and dispersed by blowing. It was divided into two groups: one group was added with 0.5 mL of 50% D3 (experimental group), and the other group was added with 0.5 mL of PBS buffer (control group). Both groups were incubated at 37℃ for 24 h. After treatment, both groups were centrifuged at 10000 rpm for 10 min, the supernatant was aspirated, and the bacterial sludge was washed 3 times with PBS buffer. Then, 1 mL of 2.5% glutaraldehyde solution was added to each centrifuge tube, the bacterial sludge was gently dispersed, and fixed under light-protected conditions for 24 h. 4. Electron Microscopy Observation: The fixed bacterial sludge samples were sent to a scanning electron microscope (SEM) platform. After dehydration, drying, and gold sputtering, the morphology and structure of the bacteria were observed under an SEM (magnification approximately 50.0 kx). The morphological differences between the control group and the experimental group were recorded. Figure 3 As shown, from left to right, the bacterial morphology diagrams are of the blank control group (PBS treatment) and the D3 experimental group (50% D3 treatment).

[0043] Test results There were significant differences in the cell morphology of *Streptococcus mutans* between the control group and the D3 treatment group, as shown in the table below:

[0044] As shown in the table above, the *Streptococcus mutans* in the control group treated with PBS exhibited typical morphological characteristics, with smooth surfaces, intact structures, and good growth. However, the *Streptococcus mutans* treated with 50% D3 showed significant damage to both the cell wall and cell membrane, resulting in cell deformation, dissolution, numerous wrinkles, depressions, and ruptures on the cell wall surface, and overall structural collapse. This indicates that D3 can directly destroy the cell structure of *Streptococcus mutans*, leading to cell inactivation.

[0045] Example 4 The inhibitory effect of D3 on the biofilm growth of oral pathogen Streptococcus mutans was investigated by using crystal violet staining to quantitatively determine the inhibition rate of different concentrations of D3 on the formation of Streptococcus mutans biofilm, thus clarifying the inhibitory effect of D3 on biofilm.

[0046] Test materials 1. Bacterial strain: Same as the *Streptococcus mutans* strain in Example 1; 2. Culture medium: The same liquid culture medium for brain and heart extract as in Example 1; 3. Test samples: 25% D3, 50% D3; 4. Other reagents and equipment: 96-well flat-bottom culture plate, sterile PBS buffer, methanol, 1% crystal violet solution, 30% acetic acid solution, microplate reader (wavelength 600nm), pipette.

[0047] Test Procedure 1. Preparation of bacterial suspension: Take Streptococcus mutans bacterial suspension, activate it, and then adjust the bacterial suspension concentration to 10⁻⁶ without dilution. 7 CFU / mL; 2. Sample addition and incubation: Add 100 μL of the above-mentioned 10 to each well of a 96-well flat-bottomed culture plate. 7 CFU / mL Streptococcus mutans suspension was added to the test wells, followed by 100 μL of the corresponding concentration of D3 sample (25% D3 or 50% D3) and 100 μL of sterile PBS buffer was added to the control group. The mixture was gently mixed. Three parallel wells were set for each concentration group and control group. The culture plate was placed in a 37°C incubator for 24 h to allow the biofilm to fully form. 3. Biofilm washing and fixation: After 24 hours of culture, carefully aspirate the culture medium from each well using a pipette, add 200 μL of sterile PBS buffer to each well, gently shake the culture plate, and then aspirate the PBS. Repeat the washing process three times to remove any unattached airborne bacteria. Then, add 100 μL of methanol to each well and incubate at room temperature for 15 minutes to fix the biofilm. After fixation, aspirate the methanol and allow the culture plate to air dry at room temperature. 4. Staining, Destaining, and Detection: Add 100 μL of 1% crystal violet solution to each well after air-drying and stain at room temperature for 15 min. After staining, remove the crystal violet solution, wash three times with sterile water to remove excess color, and air-dry again at room temperature. Add 100 μL of 30% acetic acid solution to each well and let stand at room temperature for 10 min to allow the crystal violet to decolorize and dissolve from the biofilm. Finally, measure the OD of each well at 600 nm using a microplate reader. 600 value; 5. Inhibition rate calculation: The inhibition rate of D3 on Streptococcus mutans biofilm formation was calculated according to the following formula, where ODc is the average OD of the control group. 600 Value, ODt is the average OD of the experimental group 600 value: Inhibition rate (%) = [(ODc-ODt) / ODc] × 100%.

[0048] Test results The inhibitory effects of different concentrations of D3 on the formation of Streptococcus mutans biofilm are shown in the table below. All data are the average values ​​of three parallel experiments, and P<0.05 indicates that the difference is statistically significant.

[0049]

[0050] As shown in the table above, compared with the PBS control group, both 25% D3 and 50% D3 significantly inhibited the formation of Streptococcus mutans biofilm (P<0.05), and the inhibition rate increased with increasing D3 concentration: the inhibition rate of 25% D3 was 60.30%, and the inhibition rate of 50% D3 reached 71.55%, indicating that D3 has a significant and concentration-dependent inhibitory effect on the formation of Streptococcus mutans biofilm.

[0051] Example 5 The inhibitory effect of D3 on the extracellular polysaccharide of Streptococcus mutans, an oral pathogen, was investigated. The inhibition rate of different concentrations of D3 on the synthesis of extracellular polysaccharides of Streptococcus mutans was quantitatively determined by phenol-sulfuric acid method to clarify the regulatory role of D3 on extracellular polysaccharides.

[0052] Test materials 1. Bacterial strain: Same as the *Streptococcus mutans* strain in Example 1; 2. Culture medium: The same liquid culture medium for brain and heart extract as in Example 1; 3. Test samples: 25% D3, 50% D3; 4. Other reagents and equipment: sterile PBS buffer, 0.22μm filter membrane, ice ethanol, deionized water, phenol-sulfuric acid chromogenic solution, centrifuge tubes, high-speed centrifuges (11300×g, 12857×g), and microplate reader (wavelength 490nm).

[0053] Test Procedure 1. Preparation of bacterial suspension: Take Streptococcus mutans bacterial suspension, activate it, and adjust the concentration to 1×10⁻⁶. 7 CFU / mL; 2. D3 treatment and incubation: Add the above-mentioned 1×10 to the culture medium containing different concentrations of D3 (25%, 50%). 7 The bacterial suspension of CFU / mL was placed in a constant temperature incubator at 37℃ and incubated for 24h. At the same time, a control group was set up, in which an equal amount of bacterial suspension and sterile PBS buffer were added to the culture medium and incubated under the same conditions. 3. Extraction of extracellular polysaccharides: After incubation for 24 h, take 1 mL of each group mixture and centrifuge at 11300×g for 10 min at 4 °C. After centrifugation, filter the supernatant through a 0.22 μm filter membrane to remove bacterial residue. Add 3 mL of ice-cold ethanol to the filtered supernatant, mix gently, and place in a 4 °C refrigerator for 24 h to allow the extracellular polysaccharides to precipitate fully. Then centrifuge at 12857×g for 10 min at 4 °C and collect the extracellular polysaccharide precipitate at the bottom of the tube. 4. Quantitative detection and inhibition rate calculation: The collected extracellular polysaccharide precipitate was dissolved in 1 mL of deionized water. Phenol-sulfuric acid chromogenic solution was added to the solution, and the reaction was carried out in a boiling water bath for 15 min. After cooling, the absorbance was measured at a wavelength of 490 nm using an ELISA reader (DL). 490 (Value); calculate the inhibition rate of D3 on extracellular polysaccharides according to the following formula, where D 490 (Control group) refers to the absorbance of the control group, D. 490 (Experimental group) refers to the absorbance of the experimental group: Inhibition rate (%) = [(D 490 (Control group) - D 490 (Experimental group) / D 490 (Control group) × 100%.

[0054] Test results The inhibitory effects of different concentrations of D3 on the synthesis of extracellular polysaccharides in Streptococcus mutans are shown in the table below. All data are the average values ​​of three parallel experiments, and P<0.05 indicates that the difference is statistically significant.

[0055]

[0056] As shown in the table above, compared with the PBS control group, both 25% D3 and 50% D3 significantly inhibited the synthesis of extracellular polysaccharides in Streptococcus mutans (P<0.05), and the inhibition rate increased with increasing D3 concentration: the inhibition rate of 25% D3 was 23.42%, and the inhibition rate of 50% D3 reached 38.74%. This indicates that D3 can reduce the synthesis of extracellular polysaccharides, weaken the stability of the Streptococcus mutans biofilm, and thus inhibit its pathogenicity.

[0057] Example 6 Based on transcriptomics research on the antibacterial mechanism of D3 against Streptococcus mutans and qPCR verification, differentially expressed genes of Streptococcus mutans after D3 treatment were screened through transcriptomics analysis to clarify the regulatory pathway; at the same time, the expression changes of key differentially expressed genes were verified by qPCR to reveal the molecular mechanism by which D3 inhibits Streptococcus mutans.

[0058] Test materials 1. Bacterial strain: Same as the *Streptococcus mutans* strain in Example 1; 2. Culture medium: The same liquid culture medium for brain and heart extract as in Example 1; 3. Test sample: 50% D3; 4. Other reagents and equipment: sterile PBS buffer, lysis buffer, chloroform, anhydrous ethanol, RNase-free ddH2O, reverse transcription kit, qPCR kit, large-capacity refrigerated high-speed centrifuge, nucleic acid and protein detector, Agilent 2100 BioAlyzer, Illumina sequencing platform, real-time quantitative PCR instrument.

[0059] Test Procedure (1) Sample preparation 1. Preparation of bacterial suspension: Take Streptococcus mutans bacterial suspension, activate it, and adjust the concentration to 10⁻⁶ without dilution. 7 CFU / mL; 2. D3 treatment and cell collection: Take 1 mL of the above bacterial culture and centrifuge at 10,000 rpm for 10 min using a large-capacity refrigerated high-speed centrifuge. Remove the supernatant and wash the bacterial sludge three times with sterile PBS buffer. After the last wash, reconstitute the sludge with PBS. Add 500 μL of 50% D3 (experimental group) to the centrifuge tube and 500 μL of sterile PBS buffer to the control group. Mix gently and place in a 37°C water bath for 30 min. After the reaction, collect the bacterial precipitate by centrifuging at 10,000 rpm for 10 min. Immediately freeze the precipitate with liquid nitrogen and then store it in an ultra-low temperature freezer at -80°C for later use.

[0060] (2) RNA extraction and quality testing 1. RNA extraction: Add 1 mL of lysis buffer to the bacterial cell pellet, mix by pipetting, and incubate at room temperature for 5 min to allow complete separation of the nucleic acid-protein complex; add 200 μL of chloroform, shake vigorously for 15 s until the solution turns milky white, and incubate at room temperature for 3-5 min; centrifuge at 12000 rpm for 10 min at 2-8℃, and carefully transfer the upper colorless aqueous phase (containing RNA) to a new centrifuge tube; add 200 μL of anhydrous ethanol to the aqueous phase, mix well, transfer to an adsorption column, incubate for 2 min, centrifuge at 12000 rpm for 2 min at 2-8℃, and discard the waste liquid; Add 500 μL of wash buffer to the adsorption column, incubate at room temperature for 2 min, then centrifuge at 12000 rpm for 2 min at 2-8℃ and discard the waste liquid; add 600 μL of rinse buffer (add anhydrous ethanol before use), centrifuge at 12000 rpm for 2 min at 2-8℃, and repeat the rinse buffer step once; centrifuge the empty column at 12000 rpm for 2 min to remove residual rinse buffer; add 50-100 μL of Nase-free ddH2O to the center of the adsorption column membrane, incubate at room temperature for 5 min, then centrifuge at 12000 rpm at room temperature for 2 min to collect the RNA sample; 2. Quality control: RNA concentration was measured using a nucleic acid protein analyzer, and RNA integrity (RIN value) was detected using an Agilent 2100 BioAlyzer. Qualified RNA samples (RIN≥7.0) were stored at -80℃.

[0061] (3) Library construction and sequencing 1. Library Construction: Ribosomal RNA (rRNA) was removed from Total RNA using a probe to enrich mRNA; mRNA was randomly fragmented into short fragments using a fragmentation buffer; using the short mRNA fragments as templates, the first strand of cDNA was synthesized, followed by degradation of the RNA strand and synthesis of the second strand of cDNA containing dUTP; the purified cDNA underwent end repair, A-tailing, and ligation of sequencing adapters, and cDNAs of 370-420 bp were screened using AMPureXPbeads; the second strand containing U was degraded using USER enzyme, amplified by PCR, and purified again using AMPureXPbeads to obtain the final library; 2. Library quality control and sequencing: The library was initially quantified using a Qubit 2.0 Fluorometer (diluted to 1.5 ng / μL), and the library insert size was detected using an Agilent 2100 Bioanalyzer; the effective concentration of the library was accurately quantified by qRT-PCR (≥1.5 nM); after pooling the qualified libraries in proportion, sequencing was performed on the Illumina platform using sequencing by synthesis (SBS) technology.

[0062] (4) Data Analysis 1. Data filtering: Remove adapter sequences, trim terminal bases below Q20, and remove reads containing ≥10% N from the Raw Data (fastq format) obtained from sequencing to obtain Clean Data; 2. Gene alignment and differential expression analysis: The CleanData genome was aligned with the *Streptococcus mutans* reference genome using Bowtie2 software; differentially expressed genes were screened using DESeq2 software, with the screening criteria being |log2FoldChange| > 0 and padj < 0.05. The differential gene volcano plot is shown below. Figure 4 As shown; 3. KEGG enrichment analysis: KEGG pathway enrichment analysis was performed on differentially expressed genes to identify the core regulatory pathways. The results of the KEGG enrichment analysis are as follows: Figure 5 As shown, 1422 upregulated genes were mainly enriched in pathways such as ribosomes, bacterial secretion systems, and protein export, while 1031 downregulated genes were mainly enriched in pathways such as phenylalanine, tyrosine and tryptophan biosynthesis, propionic acid metabolism, and peptidoglycan biosynthesis.

[0063] (5) qPCR verification 1. Primer design: Specific primers were designed for three key differentially expressed genes (fimI, wcaJ, wcae), with 18S rRNA as the internal reference gene; 2. Reverse transcription: RNA was reverse transcribed into cDNA using a reverse transcription kit; 3. qPCR reaction: Using a qPCR kit, the reaction conditions were set to 95℃ for 15s, 60℃ for 20s, and 72℃ for 20s, for 40 cycles; each sample was set up with 3 parallel wells; 4. Data Analysis: The relative expression levels of the target gene were calculated using the 2⁻△△Ct method. The differences between the experimental group and the control group were compared, and the verification figure is shown below. Figure 6 As shown.

[0064] Test results Sequencing quality results The sequencing quality of all six groups of samples (3 experimental groups and 3 control groups) was satisfactory, as shown in the table below:

[0065] As shown in the table above, all samples had Q20 > 95%, Q30 > 90%, GC content was stable at around 55%, RNA integrity was good (RIN ≥ 7.5), and sequencing data accuracy was high, which can be used for subsequent analysis.

[0066] Differentially expressed gene results The differentially expressed genes in the experimental group (D3 treatment) compared to the control group are shown in the table below:

[0067] As shown in the table above, D3 treatment produced a total of 2453 differentially expressed genes in Streptococcus mutans, of which 1422 were upregulated and 1031 were downregulated. The downregulated genes were mainly enriched in pathogenic pathways such as extracellular polysaccharide biosynthesis and amino acid metabolism, indicating that D3 can inhibit the pathogenicity of Streptococcus mutans by regulating these pathways.

[0068] Key differentially expressed genes qPCR validation results The qPCR validation results for the three core pathogenic genes (fimI, wcaJ, wcae) are shown in the table below. p<0.01 indicates extremely significant differences.

[0069]

[0070] As shown in the table above, the qPCR validation results are completely consistent with the trends of the transcriptome analysis: the fimI gene was downregulated by 6.07-fold (affecting bacterial adhesion), the wcaJ gene by 4.57-fold, and the wcae gene by 8.71-fold (all affecting extracellular polysaccharide synthesis), and the differences were extremely significant (p<0.01). This confirms that D3 can inhibit the adhesion ability and extracellular polysaccharide synthesis of Streptococcus mutans by targeting and downregulating these three key genes, thereby reducing biofilm formation and exerting an anti-caries effect.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A metabiotic composition that effectively inhibits oral cariogenic bacteria, characterized in that, The postbiotic composition is composed of postbiotic 1 and postbiotic 2. Postbiotic 1 is prepared by Lactobacillus salivarius Postbio-YY through activation, fermentation, pasteurization, clarification, impurity removal, concentration, and desalting. Postbiotic 2 is prepared by first fermenting Lactococcus lactis Postbio-F3, then inoculating Lactobacillus paracasei Postbio-P6 for further fermentation, followed by pasteurization, clarification, impurity removal, concentration, and desalting. Lactobacillus salivarius Postbio-YY, Lactobacillus paracasei Postbio-P6, and Lactococcus lactis Postbio-F3 are all derived from the lactic acid bacteria strain bank of the Probiotics and Postbiotics Basic Research and Development Application Innovation Laboratory of Qingdao Agricultural University.

2. The post-genetic composition as described in claim 1, characterized in that, The preservation number of *Lactobacillus salivarius* Postbio-YY is CGMCC No. 26239, the preservation number of *Lactobacillus paracasei* Postbio-P6 is CGMCC No. 26237, and the preservation number of *Lactococcus lactis* Postbio-F3 is CGMCC No. 26236.

3. The post-genetic composition as described in claim 1, characterized in that, The mixing mass ratio of postgenetic 1 to postgenetic 2 is (1-3):

1.

4. The post-genetic composition as described in claim 1, characterized in that, In the preparation of the post-generic 1, the fermentation conditions are: inoculum amount 2%, temperature 37℃, rotation speed 150rpm, pH 6.5, and fermentation time 35h; the pasteurization conditions are: sterilization at 95℃ for 30min.

5. A method for preparing a metabiotic composition that effectively inhibits oral caries bacteria as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Preparation of postbiotic 1: After activation, Lactobacillus salivarius Postbio-YY was inoculated into MRS liquid medium. The fermentation conditions were: inoculum size 2%, temperature 37℃, rotation speed 150rpm, pH 6.5, fermentation time 35h. After fermentation, the postbiotic 1 was obtained by pasteurization, clarification and impurity removal, concentration and desalting. (2) Preparation of postbiotic 2: Lactococcus lactis Postbio-F3 was activated and inoculated into MRS liquid medium. It was fermented at 37℃ and 150 rpm until pH 4.

2. Then, it was inoculated with activated Lactobacillus paracasei Postbio-P6 and fermented at 37℃ and 150 rpm until pH 3.

5. After fermentation, it was sterilized at 95℃ for 40-100 min, clarified to remove impurities, concentrated and desalted to obtain postbiotic 2. (3) Mixing: Mix postgene 1 and postgene 2 to obtain the postgene composition.

6. The preparation method according to claim 5, characterized in that, In steps (1) and (2), the strain was activated under the following conditions: cultured in MRS liquid medium at 37°C for 18 hours, passaged twice, and the viable count after activation was 5-10 × 10⁻⁶. 6 CFU / mL.

7. A preparation for preventing oral caries bacteria, characterized in that, The formulation comprises the postbiotic composition according to any one of claims 1-4, and a pharmaceutically acceptable carrier, solvent, diluent, or excipient.

8. The formulation as described in claim 7, characterized in that, The dosage form of the preparation is powder, granules, capsules, oral liquid, tablets or toothpaste.

9. The formulation as described in claim 8, characterized in that, When the dosage form is toothpaste, the mass concentration of the postbiotic composition in the toothpaste is 2%-6%.

10. The use of the metabiotic composition according to any one of claims 1-4 in the preparation of an oral care product that inhibits Streptococcus mutans biofilm or extracellular polysaccharide, wherein the oral care product is toothpaste, mouthwash or oral spray.

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