Pediococcus pentosaceus MG-T267 capable of resisting inflammation and improving oral health and application of Pediococcus pentosaceus MG-T267

Abstract

The invention relates to the field of microorganisms, in particular to anti-inflammatory pediococcus pentosaceus MG-T267 capable of improving oral health and application of the pediococcus pentosaceus MG-T267. The pediococcus pentosaceus MG-T267 disclosed by the invention has the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.35097, the preservation time of the pediococcus pentosaceus MG-T267 is 2025-07-03, and the pediococcus pentosaceus MG-T267 is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms. According to the invention, pediococcus pentosaceus MG-T267 can inhibit harmful microorganisms, such as streptococcus mutans, fusobacterium nucleatum, porphyromonas gingivalis and candida albicans; the microbial agent containing the pediococcus pentosaceus MG-T267 can improve oral problems such as halitosis, gingival bleeding, tooth sensitivity, dental plaque and oral ulcer.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to an anti-inflammatory, oral health-improving Pediococcus pentosaccharide MG-T267 and its uses. Background Technology

[0002] The oral cavity is a complex micro-ecosystem, and the dynamic balance of its internal microbiome is crucial for maintaining oral health. However, when certain pathogenic microorganisms proliferate excessively and form pathogenic biofilms, this balance is disrupted, leading to a series of oral diseases. Among the many oral pathogens, *Streptococcus mutans*, *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, and *Candida albicans* are four of the most significant and damaging pathogens, which respectively dominate or synergistically participate in the occurrence and development of dental caries, periodontal disease, and oral mucosal infections.

[0003] First, *Streptococcus mutans* is considered the most important pathogenic bacterium causing dental caries. Its pathogenic mechanism lies in its ability to efficiently ferment dietary sugars to produce large amounts of organic acids, leading to demineralization of the hard tissues of teeth. At the same time, it can use sucrose to synthesize viscous dextran, which adheres firmly to the tooth surface, forming the basic structure of dental plaque biofilm and providing conditions for the colonization of other pathogens.

[0004] Secondly, *Porphyromonas gingivalis* and *Fusobacterium nucleatum* are key pathogens of periodontitis. *Porphyromonas gingivalis* secretes virulence factors such as gingival protease, directly damaging periodontal epithelium and connective tissue, and stimulating excessive inflammatory responses in the host, leading to alveolar bone resorption. *Fusobacterium nucleatum* plays a "bridging" role in the maturation of dental plaque biofilm, promoting adhesion between early-establishing bacteria and late-establishing highly pathogenic bacteria (such as *Porphyromonas gingivalis*) through copolymerization, accelerating the structural complexity and pathogenicity escalation of the biofilm community. The synergistic effect of both is a core factor leading to gingival bleeding, periodontal pocket formation, and tooth loosening.

[0005] Furthermore, as a common oral fungus, Candida albicans overgrowth can lead to oral mucosal diseases such as oral thrush and denture stomatitis. More importantly, Candida albicans can physically aggregate and signal with Streptococcus mutans to form a denser, more drug-resistant bacterial-fungal mixed biofilm, significantly exacerbating the severity of dental caries, especially in the development of rampant caries in young children.

[0006] Currently, conventional control methods for the aforementioned oral pathogenic microorganisms mainly include mechanical removal (such as brushing and scaling) and chemical antibacterial agents (such as mouthwashes containing chlorhexidine, triclosan, etc.). However, mechanical removal is difficult to be thorough and requires high compliance; while broad-spectrum chemical antibacterial agents, while inhibiting pathogenic bacteria, also indiscriminately kill beneficial symbiotic bacteria in the oral cavity, and long-term use may lead to oral flora imbalance, increased microbial resistance, and side effects such as tooth discoloration and altered taste. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an anti-inflammatory, oral health-improving Pediococcus pentosaccharide MG-T267 and its uses, in order to solve the problems in the prior art.

[0008] To achieve the above and other related objectives, this invention provides a Pediococcus pentosaceus MG-T267, with accession number CGMCC No.35097, accession date 2025-07-03, deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beijing.

[0009] Preferably, the Pediococcus pentosaceus MG-T267 comprises a DNA fragment with a nucleotide sequence as shown in SEQ ID No. 1.

[0010] The present invention also provides a microbial agent containing the aforementioned Pediococcus pentosaceus MG-T267.

[0011] The present invention also provides the use of the aforementioned Pediococcus pentosaceus MG-T267 or the aforementioned bacterial agent in the preparation of anti-inflammatory products or products for improving oral health.

[0012] As described above, the anti-inflammatory and oral health-improving Pediococcus pentosaccharide MG-T267 of the present invention and its uses have the following beneficial effects: In this invention, Pediococcus pentosaceus MG-T267 can inhibit the activity of harmful microorganisms such as Streptococcus mutans, Fusobacterium nucleatum, Porphyromonas gingivalis, and Candida albicans, and can efficiently adhere to simulated oral tooth surfaces, exhibiting strong self-aggregation and co-aggregation capabilities with pathogenic bacteria. Bacterial agents containing Pediococcus pentosaceus MG-T267 can improve the oral microecology, alleviate oral problems such as halitosis, gingival bleeding, tooth sensitivity, dental plaque, and oral ulcers, overcoming the shortcomings of existing oral probiotics, such as narrow antibacterial spectrum, weak colonization, and single function. Attached Figure Description

[0013] Figure 1 The results show the statistical results of the inhibition zone diameter of *Pediococcus pentosus* MG-T267 of the present invention against *Streptococcus mutans*, *Fusobacterium nucleatum* subsp. *prolifera*, and *Candida albicans* (compared with the positive strain group, *p<0.05, ***p<0.001).

[0014] Figure 2 The results show the inhibition rate of biofilm formation of *Pediococcus pentosus* MG-T267 against *Streptococcus mutans* (**p<0.01 compared with the positive strain group).

[0015] Figure 3The results show the adhesion rate of Pediococcus pentosaceus MG-T267 of the present invention on saliva-coated hydroxyapatite surface (**p<0.01 compared with the positive strain group).

[0016] Figure 4 The results shown are comparative studies on the improvement of dental plaque by the bacterial agent containing Pediococcus pentosaceus MG-T267 in the colorimetric test of this invention. Detailed Implementation

[0017] This application provides a Pediococcus pentosaceus MG-T267, with accession number CGMCC No.35097, accession date 2025-07-03, deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beijing.

[0018] In some specific embodiments, the Pediococcus pentosaceus MG-T267 comprises a DNA fragment with a nucleotide sequence as shown in SEQ ID NO.1.

[0019] In some specific embodiments, the *Pediococcus pentosaceus* MG-T267 contains a DNA fragment with a nucleotide sequence similarity of more than 50% to that shown in SEQ ID NO. 1. Specifically, the *Pediococcus pentosaceus* MG-T267 comprises a DNA fragment with a nucleotide sequence similarity of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more to that of SEQ ID NO. 1. Preferably, the *Pediococcus pentosaceus* MG-T267 comprises a DNA fragment with a nucleotide sequence similarity of more than 90% to that of SEQ ID NO. 1; more preferably, the *Pediococcus pentosaceus* MG-T267 comprises a DNA fragment with a nucleotide sequence similarity of more than 99% to that of SEQ ID NO. 1. Specifically, the DNA fragment contained in the Pediococcus pentosaceus MG-T267 can be obtained by nucleotide substitution, deletion, insertion, or homologous recombination of nucleic acid fragments on the DNA strand with the nucleotide sequence shown in SEQ ID No. 1.

[0020] In some specific embodiments, the Pediococcus pentosaceus MG-T267 is isolated from the oral cavity of a healthy human.

[0021] In some specific embodiments, the *Pediococcus pentosaceus* MG-T267 exhibits inhibitory activity against one or more of the following microorganisms: *Listeria*, *Streptococcus*, *Helicobacter*, *Staphylococcus*, *Porphyromonas*, *Clostridium*, *Fusobacterium*, *Bacillus*, *Candida*, or *Escherichia coli*; preferably, the microorganism is one or more of *Streptococcus mutans*, *Fusobacterium nucleatum* subsp. *proteus*, *Porphyromonas gingivalis*, or *Candida albicans*. The inhibitory effect can be expressed as the size of the inhibition zone; that is, the better the inhibitory effect, the larger the area of ​​the inhibition zone.

[0022] The present invention also provides a microbial agent containing the aforementioned Pediococcus pentosaceus MG-T267.

[0023] In some specific embodiments, the aforementioned Pediococcus pentosaceus MG-T267 in the bacterial agent exists in one or more of the following forms: live bacteria, inactivated bacteria, fermentation products, or fermentation extracts.

[0024] In some specific embodiments, the microbial agent contains one or more of the following: Lactobacillus paracasei, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus salivarius, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium adolescentis, or Bifidobacterium lactis; preferably, the microbial agent contains Lactobacillus paracasei.

[0025] In some specific embodiments, the microbial agent further includes one or more of the following: prebiotics, vitamins, minerals, amino acids, polypeptides, emulsifiers, thickeners, sweeteners, flavorings, stabilizers, or preservatives.

[0026] Furthermore, the prebiotic may be selected from one or more of fructooligosaccharides, inulin, galactooligosaccharides, or polydextrose.

[0027] In some specific embodiments, the concentration of Pediococcus pentosaceus MG-T267 in the bacterial agent can be at least 1×10⁸ CFU / g, at least 2×10⁸ CFU / g, at least 3×10⁸ CFU / g, at least 4×10⁸ CFU / g, at least 5×10⁸ CFU / g, at least 6×10⁸ CFU / g, at least 7×10⁸ CFU / g, at least 8×10⁸ CFU / g, at least 9×10⁸ CFU / g, 1×10⁹ CFU / g, at least 2×10⁹ CFU / g, at least 3×10⁹ CFU / g, at least 4×10⁹ CFU / g, at least 5×10⁹ CFU / g, or at least 5×10⁹ CFU / g. FU / g, at least 6×10⁹ CFU / g, at least 7×10⁹ CFU / g, at least 8×10⁹ CFU / g, at least 9×10⁹ CFU / g, at least 1×10¹⁰ CFU / g, at least 2×10¹⁰ CFU / g, at least 3×10¹⁰ CFU / g, at least 4×10¹⁰ CFU / g, at least 5×10¹⁰ CFU / g, at least 6×10¹⁰ CFU / g, at least 7×10¹⁰ CFU / g, at least 8×10¹⁰ CFU / g, at least 9×10¹⁰ CFU / g, or at least 1×10¹¹ CFU / g or more.

[0028] In some specific embodiments, the microbial agent may be selected from one or more of the following forms: compressed candy, tablets, powders, granules, oral liquids, drops, gummies, capsules, jellies, solid beverages, or fermented dairy products.

[0029] The present invention also provides the use of the aforementioned Pediococcus pentosaceus MG-T267 or the aforementioned bacterial agent in the preparation of anti-inflammatory products or products for improving oral health.

[0030] In some specific embodiments, the oral health improvement product has one or more of the following functions: prevention, relief and / or treatment of dental caries; improvement of breath odor; regulation of the oral microecological environment; prevention, relief and / or treatment of periodontal disease; prevention, relief and / or treatment of gingival swelling and pain; reduction of dental plaque or tartar formation; prevention, relief and / or treatment of oral ulcers.

[0031] In some specific embodiments, the anti-inflammatory product or oral health improvement product is a medicine, health product, or food.

[0032] When the product is a pharmaceutical product, it contains pharmaceutically acceptable excipients. Specifically, the excipients include various excipients and diluents that are not essential active ingredients and do not cause excessive toxicity after administration. The excipients include sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), or binders. The excipients also contain other low molecular weight peptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol, or sorbitol. When the excipients are used in an aqueous solution for injection, they are selected from physiological saline, isotonic glucose solution, isotonic D-sorbitol solution, isotonic D-mannose solution, or isotonic D-mannitol or sugar alcohol solution. The aqueous solution for injection contains a solubilizer. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG) and / or nonionic surfactants (Tween 80 or HCO-50).

[0033] In this invention, the term "similarity" is defined as the percentage of identical nucleotide residues in a candidate nucleotide sequence to a reference nucleotide sequence after aligning nucleotide sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine the percentage nucleotide sequence similarity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0034] In this invention, the term "vitamin" refers to a class of trace organic substances that humans and animals must obtain from food to maintain normal physiological functions. Common examples may be selected from one or more of vitamin A, B vitamins, vitamin C, vitamin D, vitamin E, or vitamin K.

[0035] In this invention, the term "mineral" refers to an inorganic element essential to the human body, which often exists in the form of metal salts, such as calcium salts, phosphates, potassium salts, sodium salts, magnesium salts, iron salts, zinc salts, copper salts, iodates, or selenates.

[0036] In this invention, the term "probiotics" refers to live microorganisms that, when administered in adequate amounts, confer health benefits to the host, such as restoring or improving the composition of the gut microbiota. They are often provided as dietary supplements containing potentially beneficial bacteria or yeasts and are widely used in foods, including dairy products and probiotic-fortified foods.

[0037] In this invention, the term "anti-inflammatory" refers to the ability of probiotics to restore and maintain a healthy, balanced immune state by regulating the host's (such as human) immune system and microbial community, characterized by effectively controlling unnecessary, excessive, or harmful inflammatory responses.

[0038] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0039] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0040] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0041] The nucleotide sequence described in this application is as follows: SEQ ID No.1 Example 1: Inhibitory effect of Pediococcus pentosaceus MG-T267 on pathogenic bacteria 1. Experimental strain Target bacterium: Pediococcus pentosaceus MG-T267 (identified by 16S rDNA, the nucleotide sequence of which is shown in SEQ ID No. 1), isolated from the oral cavity of a healthy Chinese woman, was cryopreserved in glycerol tubes at -80°C. Under normal circumstances, colonies are obtained by inoculating the bacteria onto the surface of MRS solid agar plates and incubating them upside down at 37°C for 24-48 hours. Single colonies are then picked and inoculated into MRS liquid agar and incubated at 37°C for 18-24 hours to obtain the activated strain.

[0042] Pathogenic bacteria culture: Streptococcus mutans GDMCC1.518 / Fusobacterium nucleatum subsp. polymorphum GDMCC1.386: Take glycerol cryovials of the two strains stored at -80℃, and aseptically inoculate 200 μL of the cryopreserved bacterial solution into sterile test tubes containing 10 mL of TSB medium. Incubate anaerobically at 37℃ for 16-20 h. Transfer the enriched cultures of the two bacteria to sterile centrifuge tubes and centrifuge at 4000 r / min for 10 min. Adjust the viable count of the two bacteria to 1.0 × 10⁶ CFU / mL with sterile physiological saline, seal and store temporarily at 4℃ for later use. Candida albicans ATCC18804: Take a glycerol cryopreservation tube stored at -80℃, and inoculate 200µL of the cryopreserved bacterial culture into a test tube containing 10 mL of SD medium. Place the inoculated SD medium test tube in a 37℃ constant temperature shaker, set the rotation speed to 180 r / min, and incubate in an aerobic environment for 16-20 h. Transfer the enriched culture to a sterile centrifuge tube, centrifuge at 4000 r / min for 10 min, adjust the viable count to 1.0×106 CFU / mL with sterile physiological saline, seal and store at 4℃ for later use. Porphyromonas gingivalis bio-53046: Glycerol cryovials stored at -80℃ were plated on TSB blood agar plates for activation. The culture was then strictly anaerobic at 37℃ for 4-7 days. Single colonies were inoculated onto TSB medium and strictly anaerobic at 37℃ for 48-72 h. The enriched culture was transferred to sterile centrifuge tubes and centrifuged at 4000 r / min for 10 min. The viable count was adjusted to 1.0 × 10⁶ CFU / mL with sterile physiological saline. The tubes were then sealed and stored at 4℃ for later use.

[0043] Culture medium preparation: Preparation of MRS medium: Weigh 10.0 g tryptone, 10.0 g beef extract, 5.0 g yeast extract, 20.0 g glucose, 2.0 g diammonium citrate, 5.0 g sodium acetate, 0.58 g magnesium sulfate (7H2O), 0.25 g manganese sulfate (4H2O), 1.0 mL Tween-80, and 0.5 g L-cysteine. Dissolve in 1 L of distilled water, adjust the pH to 5.7 ± 0.2, and sterilize at 121℃ for 15 min. For MRS solid medium, an additional 20 g agar needs to be added.

[0044] SD medium: Weigh 10.0 g of an equal mixture of animal tissue pepsin hydrolysate and tryptone, and 20.0 g of glucose, dissolve in 1 L of distilled water, adjust the pH to 5.6±0.2, and sterilize at 121℃ for 15 min. SD solid medium requires the addition of 20 g of agar.

[0045] TSB medium: Weigh 17.0 g tryptone, 3.0 g soybean papain hydrolysate, 5.0 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, 2.5 g glucose, 1.0 g yeast extract, 5.0 mg heme chloride, and 0.5 mg L-cysteine ​​hydrochloride, and bring the volume to 1 L. Adjust the pH to 7.3±0.1 and sterilize at 121℃ for 15-20 min. After sterilization and cooling, add 1 mg vitamin K1. For TSB solid medium, an additional 20 g agar needs to be added.

[0046] TSB blood agar plates: Based on 1 L of modified TSB medium with 15 g agar added, mix well and sterilize at 121℃ for 15-20 min. After sterilization, wait until the temperature is about 55℃, add 1 mg vitamin K1 and 53 mL of defibrinated sheep blood, mix well and pour into plates, each plate containing 20-25 mL of medium.

[0047] 2 Experimental Methods Preparation of bacterial suspension: Take the activated bacterial suspension of Pediococcus pentosaceus to be tested, first determine the viable count by plate counting method, and adjust the bacterial suspension to 10. 8 CFU / mL; Sterilize the SD / TSB solid culture medium and then cool it to about 55°C for later use. Sterilize disposable sterile culture dishes and Oxford cups, and place the sterilized Oxford cups into the disposable sterile culture dishes under aseptic conditions.

[0048] Add the pathogenic bacterial suspension to the SD / TSB solid medium cooled to about 55°C at an inoculation rate of 1%, mix quickly and gently (avoiding the formation of air bubbles), and then quickly pour the bacterial medium into sterile petri dishes with Oxford cups placed in them. The amount poured into each dish should be enough to cover the bottom of the petri dish with a uniform thickness (about 15-20 mL). Allow the medium to cool and solidify naturally.

[0049] Gently rotate the Oxford cup with sterile forceps to remove it, creating uniform wells. Use a sterile pipette to draw 100 μL of the bacterial solution to be tested and accurately inject it into the wells. Simultaneously, a positive control group is set up, with 100 μL of the positive strain *Pediococcus pentosaceus* JYPR9330 bacterial solution injected into each well. Avoid spilling liquid into the wells during the procedure.

[0050] After adding the samples, gently cover the culture dishes. Place *Streptococcus mutans*, *Fusobacterium nucleatum* subsp. *mucinosa*, and *Porphyromonas gingivalis* in the anaerobic chamber, and place *Candida albicans* in the ordinary culture medium. Incubate at 37°C for the appropriate time.

[0051] Data recording: After the culture is completed, remove the petri dish and use a vernier caliper to measure the diameter of the inhibition zone around each well (referring to the entire diameter of the circular transparent zone). Each sample should be measured at least 3 times. Record the measurement data and calculate the average value (retain 2 decimal places).

[0052] 3. Experimental Results and Explanation The results are as follows Figure 1 As shown, *Pediococcus pentosaceus* MG-T267 exhibits antibacterial activity against *Streptococcus mutans*, *Fusobacterium nucleatum* subsp. *proliferate*, *Candida albicans*, and *Porphyromonas gingivalis* (forming a clear zone of inhibition). Compared with the positive control *Pediococcus pentosaceus* JYPR9330, MG-T267 showed significantly larger zones of inhibition against all four pathogens (* for *Streptococcus mutans* group and *** for *Fusobacterium nucleatum* subsp. *proliferate* group). This indicates that *Pediococcus pentosaceus* MG-T267 has stronger antibacterial activity against *Streptococcus mutans*, *Fusobacterium nucleatum* subsp. *proliferate*, *Candida albicans*, and *Porphyromonas gingivalis* than *Pediococcus pentosaceus* JYPR9330.

[0053] Example 2: Inhibitory effect of Pediococcus pentosaceus MG-T267 on biofilm formation of Streptococcus mutans 1. Experimental Objective In this embodiment, the inhibitory activity of the test strain *Pediococcus pentosaceus* MG-T267 against *Streptococcus mutans* (Sm) biofilm formation was evaluated to clarify its intervention effect on the biofilm of this pathogenic bacterium. *Pediococcus pentosaceus* JYPR9330 was used as a positive control, and the difference in inhibitory ability of MG-T267 and JYPR9330 against *Streptococcus mutans* biofilm was compared to determine the relative antibacterial activity of MG-T267.

[0054] 2 Experimental Methods Treatment with Streptococcus mutans: Take a suspension of activated Streptococcus mutans (concentration 1.0 × 10⁻⁶). 8 The concentration of *Streptococcus mutans* (CFU / mL) was serially diluted 10-fold using TSB medium, for a total of three dilutions. The final concentration of *Streptococcus mutans* after dilution was 1 × 10⁻⁶. 5 CFU / mL, for later use.

[0055] Lactic acid bacteria treatment: Activated Pediococcus pentosaceus suspensions MG-T267 and JYPR9330 were serially diluted using MRS medium to a final concentration of 1×10⁻⁶ for both types of Pediococcus pentosaceus. 6 CFU / mL, for later use.

[0056] Co-culture: In 96-well plates, a blank control group, a Streptococcus mutans culture group, and a pathogenic bacteria + lactic acid bacteria experimental group were set up. Each group consisted of 100 μL of bacterial suspension + 100 μL of bacterial suspension (or culture medium). The 96-well plates were placed in an anaerobic incubator and co-cultured at 37℃ for 24 h.

[0057] Biofilm detection: After co-culturing, remove the 96-well plate, discard the supernatant from each well, and gently wash each well twice with 300 μL PBS buffer to remove any unattached airborne bacteria. Add 200 μL 99% methanol to each well and fix at room temperature for 15 min to stabilize biofilm adhesion. Discard the methanol, add 200 μL 0.1% crystal violet solution to each well, and incubate at room temperature for 30 min to ensure thorough staining of the biofilm. Discard the crystal violet solution, gently wash each well twice with 300 μL physiological saline to remove excess staining solution, and air dry the plate in a fume hood for 5 min. Add 200 μL 33% acetic acid to each well and dissolve at room temperature for 30 min. Measure the absorbance (OD value) of each well using a microplate reader at 595 nm. If the absorbance exceeds the range, dilute with 33% acetic acid and remeasure; only OD values ​​within the range of 0.2-1.2 are retained as valid data.

[0058] Data processing: Actual OD to be tested = Detected OD to be tested - OD (Streptococcus mutans + Lactic acid bacteria culture medium) control Inhibition rate (%) = [1 - (OD value of actual sample / OD value of Streptococcus variants cultured alone)] × 100% 3. Experimental Results and Explanation like Figure 2 As shown, the inhibition rate of *Pediococcus pentosaceus* MG-T267 against *Streptococcus mutans* biofilm formation was significantly higher than that of the positive control strain *Pediococcus pentosaceus* JYPR9330, and the difference between the two was extremely significant (**, p<0.01), suggesting that MG-T267 has a stronger inhibitory ability against *Streptococcus mutans* biofilm and may have more prominent application potential in the prevention and control of *Streptococcus mutans* biofilm-related diseases (such as dental caries).

[0059] Example 3: Test of the adhesion ability of Pediococcus pentosaceus MG-T267 to saliva-coated hydroxyapatite 1. Experimental Objective In this embodiment, to evaluate the adhesion ability of Pediococcus pentosaceus MG-T267 to saliva-coated hydroxyapatite, the adhesion rate was calculated by detecting the OD600 value of the supernatant after the adhesion experiment, so as to reflect the adhesion characteristics of probiotics.

[0060] 2 Experimental Methods Lactic acid bacteria treatment: Take the activated Pediococcus pentosacchari suspensions MG-T267 and JYPR9330 and perform serial dilutions using MRS medium until the final concentration of the two Pediococcus pentosacchari strains is 1×10⁹ CFU / mL, and set aside for later use.

[0061] Preparation of saliva-coated hydroxyapatite: 50 mg of hydroxyapatite was suspended in 10 mL of 0.2 mol / L NaOH solution and allowed to stand for 10 min to remove surface-active particles. This process was repeated three times or more. The precipitate was then washed with pure water until the pH of the supernatant was close to 7.0. The hydroxyapatite was allowed to precipitate naturally, and the supernatant was discarded. The precipitate was dried overnight at 80°C. The precipitate was coated with saliva at a ratio of 1 mg hydroxyapatite to 200 μL saliva, incubated at 37°C for 60 min, and then centrifuged at 2000 r / min for 5 min. The supernatant was discarded, and the resulting precipitate was the fully coated hydroxyapatite. It was resuspended in 20 times its weight of physiological saline for later use.

[0062] Adhesion test: In a 24-well plate, add 1 mL of the bacterial suspension to be tested and 100 μL of saliva-coated hydroxyapatite to each well, mix thoroughly, and incubate at 37 ℃ on a shaker (180 r / min) for 60 min. After removing the plate and letting it stand for 5 min, collect the supernatant and measure its OD600 value.

[0063] Data Processing: Adhesion Rate Calculation Formula: Adhesion Rate % = (1 - Supernatant OD600 value / Initial OD600 value) × 100% 3. Experimental Results and Explanation Experimental results are as follows Figure 3 As shown, the adhesion rate of *Pediococcus pentosaceus* MG-T267 to saliva-coated hydroxyapatite was significantly higher than that of JYPR9330: the adhesion rate of *P. pentosaceus* JYPR9330 was approximately 58.16%, while the adhesion rate of *P. pentosaceus* MG-T267 was approximately 74.47%; there was a significant difference between the two groups (**, usually indicating p < 0.01). This indicates that the adhesion ability of *Pediococcus pentosaceus* MG-T267 is significantly better than that of JYPR9330, and it may be more likely to colonize in scenarios involving adhesion and colonization, such as the oral cavity, and thus may more fully exert the probiotic efficacy.

[0064] Example 4: Human trial results of a Pediococcus pentosaceus MG-T267 bacterial agent 1. Experimental Objective In this embodiment, the effects of the self-developed probiotic sample on oral diseases were verified in the human oral environment. The differences in its effects with those of commercially available products were compared. Specific evaluation indicators included: subjective indicators: halitosis, tooth sensitivity, gingival bleeding, soft plaque and tartar, and oral ulcers; objective indicators: dental plaque (observed visually in conjunction with a colorimetric experiment).

[0065] Duration of effect assessment: By designing a “15-day trial period + 15-day observation period”, the changes in the improvement effect after discontinuation of medication (observation period) are tracked to determine whether the self-developed lozenges can achieve “effectiveness during the medication period and maintenance of effect after discontinuation”, thus avoiding short-term effects that depend on continuous use.

[0066] 2. Implementation Methods Test sample information and formulation description

[0067] Experimental Design and Grouping Group Test sample Recommended human dose Dosage Dosage time Group 1 Commercially available lozenges <![CDATA[2.5*10 9 CFU / slice]]> Take one tablet once daily before bedtime. 15d Group 2 Self-developed bacterial strain tablets <![CDATA[2.5*10 9 CFU / slice]]> Take one tablet once daily before bedtime. 15d Test metrics This experiment employed a double-blind parallel experimental design. The experimental period lasted 30 days, including a 15-day trial period and a 15-day observation period. Questionnaires were collected from volunteers at the same times before and after the trial (days 0, 3, 6, 9, 12, 15, and 30), and the following indicators were measured. index Related diseases Physiological significance Testing instruments / tools Dental plaque index (PI) Gingivitis, periodontitis, dental caries Dental plaque is a microbial biofilm on the surface of teeth and a major cause of most common oral diseases, including dental caries, gingivitis, and destructive periodontitis. Plaque buildup leads to the erosion of tooth enamel by acidic metabolic products (lactic acid), which cause tooth decay, and irritates the gums, triggering inflammation. Plaque staining agent Dental plaque and tartar Gingivitis, periodontitis, dental caries The main symptoms of dental calculus include bad breath, red and swollen gums or bleeding gums, and sometimes bad breath, bleeding gums, and frequent gum recession. Self-evaluation Tooth sensitivity Periodontal diseases such as gingivitis and periodontitis People with periodontal disease experience this due to exposed dentin. Self-evaluation Bleeding gums Periodontal diseases such as gingivitis and periodontitis Gingival bleeding refers to the bleeding of the gum tissue in response to spontaneous or external stimuli. The main causes include local inflammation and plaque buildup. Self-evaluation oral ulcers Oral inflammation Diet and weakened immunity may trigger the development of oral ulcers. Self-evaluation bad breath Gingivitis, periodontitis, dental caries A visual indicator for evaluating oral health. Self-evaluation 3. Experimental Results and Explanation 3.1 The total number of participants in this experiment was 70, and 54 valid questionnaires were collected, with a miss rate of 22.8%. Among them, 40.7% (22 questionnaires) of the participants used the self-developed lozenges, and 59.3% (32 questionnaires) of the participants used commercially available lozenges.

[0068]

[0069] 3.2 Feedback from test subjects of self-developed lozenges Positive feedback: Focusing on the improvement of oral health issues, specifically including "lighter color in plaque developer tests", "significantly reduced morning breath", and "reduction of soft plaque on tooth surfaces".

[0070] Subjects evaluate Subject 1 The biggest difference is that the first time I took it orally, the lozenge felt very sour and irritated my dental nerves. But in the last couple of times, I haven't felt any irritation from the lozenge. Subject 2 After rinsing my mouth briefly and applying the developer, the area with noticeable color was significantly reduced, and no other issues were observed. Subject 3 I noticed my breath was softer when I woke up this morning, but otherwise I didn't notice anything. 3.3 Explanation of plaque staining results The results are as follows Figure 4As shown, on day 15 of product use, the self-developed lozenges demonstrated significant advantages in core efficacy, especially in meeting oral care needs: the overall improvement rate for the two key indicators of bad breath and bleeding gums both exceeded 54%, with more than half of the subjects experiencing significant or certain improvements; for soft plaque and tartar on the tooth surface, the improvement rate reached 50%, and the improvement rates for dental plaque and tooth sensitivity both exceeded 40%; a small number of subjects also reported improvement in oral ulcers. Overall, it covers many common oral problems with clear improvement effects.

[0071] After 15 days of product use, a follow-up period of 15 days was maintained (during which the product was not used). Both products showed long-term maintenance and improvement effects. Among them, the self-developed lozenge showed the best long-term improvement in bad breath, bleeding gums, and oral ulcers.

[0072] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A type of Pediococcus pentosaceus ( Pediococcus pentosaceus MG-T267, characterized in that, The Pediococcus pentosaceus ( Pediococcus pentosaceus The accession number of MG-T267 is CGMCC No.35097.

2. The Pediococcus pentosaceus according to claim 1 ( Pediococcus pentosaceus MG-T267, characterized in that, The Pediococcus pentosaceus ( Pediococcus pentosaceus MG-T267 contains a DNA fragment with the nucleotide sequence shown in SEQ ID NO.

1.

3. The Pediococcus pentosaceus according to claim 1 ( Pediococcus pentosaceus MG-T267, characterized in that, The Pediococcus pentosaceus ( Pediococcus pentosaceus MG-T267 has inhibitory activity against one or more of the following microorganisms: Listeria, Streptococcus, Helicobacter, Staphylococcus, Porphyromonas, Clostridium, Fusobacterium, Bacillus, Candida, or Escherichia coli; preferably, the microorganism is one or more of Streptococcus mutans, Fusobacterium nucleatum subsp. Proteus, Porphyromonas gingivalis, or Candida albicans.

4. A microbial agent, characterized in that, The bacterial agent contains Pediococcus pentosaceus as described in any one of claims 1-3. Pediococcus pentosaceus MG-T267.

5. The microbial agent according to claim 4, characterized in that, The microbial agent contains one or more of the following: Lactobacillus paracasei, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus salivarius, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium adolescentis, or Bifidobacterium lactis; preferably, the microbial agent contains Lactobacillus paracasei.

6. The microbial agent according to claim 4, characterized in that, The microbial agent also contains one or more of the following: prebiotics, vitamins, minerals, amino acids, polypeptides, emulsifiers, thickeners, sweeteners, flavorings, stabilizers, or preservatives.

7. The microbial agent according to claim 4, characterized in that, The bacterial agent contains Pediococcus pentosaceus ( Pediococcus pentosaceus The concentration of MG-T267 is at least 1×10⁻⁶. 8 CFU / g, at least 2×10 8 CFU / g, at least 3×10 8 CFU / g, at least 4×10 8 CFU / g, at least 5×10 8 CFU / g, at least 6×10 8 CFU / g, at least 7×10 8 CFU / g, at least 8×10 8 CFU / g, at least 9×10 8 CFU / g, 1×10 9 CFU / g, at least 2×10 9 CFU / g, at least 3×10 9 CFU / g, at least 4×10 9 CFU / g, at least 5×10 9 CFU / g, at least 6×10 9 CFU / g, at least 7×10 9 CFU / g, at least 8×10 9 CFU / g, at least 9×10 9 CFU / g, at least 1×10 10 CFU / g, at least 2×10 10 CFU / g, at least 3×10 10 CFU / g, at least 4×10 10 CFU / g, at least 5×10 10 CFU / g, at least 6×10 10 CFU / g, at least 7×10 10 CFU / g, at least 8×10 10 CFU / g, at least 9×10 10 CFU / g, or at least 1×10 11 CFU / g or higher.

8. The microbial agent according to claim 4, characterized in that, The microbial agent is selected from one or more of the following: compressed candy, tablets, powders, granules, powders, oral liquids, drops, soft candy, capsules, jellies, solid beverages, or fermented dairy products.

9. The Pediococcus pentosaceus according to any one of claims 1-3 ( Pediococcus pentosaceus Use of MG-T267 or any of the bacterial agents according to claims 4-8 in the preparation of anti-inflammatory products or products for improving oral health.

10. The use according to claim 9, characterized in that, The oral health improvement product has one or more of the following functions: prevention, relief and / or treatment of dental caries; improvement of breath odor; regulation of the oral microecological environment; prevention, relief and / or treatment of periodontal disease; prevention, relief and / or treatment of gingival swelling and pain; reduction of dental plaque or tartar formation; prevention, relief and / or treatment of oral ulcers; preferably, the anti-inflammatory product or oral health improvement product is a food, health product or drug; preferably, the product also contains excipients acceptable in food, health product or drug.

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