Pediococcus pentosaceus with efficacy of preventing or treating dental caries and periodontal disease, preparation and application thereof

Abstract

The present application relates to the technical field of probiotics, in particular to a Pediococcus pentosaceus with the effect of preventing or treating dental caries and periodontal disease, and a preparation and application thereof. The Pediococcus pentosaceus has been preserved in the China General Microbiological Culture Collection Center on May 15, 2023, and the address of the preservation center is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 27356. The Pediococcus pentosaceus provided by the present application can significantly inhibit the growth and reproduction of oral pathogenic bacteria such as Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum, Actinomyces viscosus and Aggregatibacter actinomycetemcomitans, is conducive to the balance of oral flora, and can be widely used for preventing or treating oral diseases such as dental caries and periodontitis.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, specifically to a strain of Pediococcus pentosaceus that has the effect of preventing or treating dental caries and periodontal disease, its preparations, and applications. Background Technology

[0002] Dental caries is a chronic, progressive disease that damages the hard tissues of the teeth, primarily caused by bacteria and other factors. The formation of caries mainly involves pathogenic bacteria in the oral cavity metabolizing carbohydrates in food debris, producing acidic substances that, over time, act on the hard tissues of the teeth, leading to demineralization of the enamel and dentin, and ultimately forming cavities. If left untreated, the disease will continue to progress, causing not only tooth pain and impaired chewing function, but also complications such as pulpitis and periapical periodontitis, and even affecting overall health. Periodontal disease, on the other hand, is a group of diseases characterized by inflammation and destruction of periodontal tissues, mainly including gingivitis and periodontitis. Common clinical manifestations include swollen and bleeding gums, periodontal pocket formation, and alveolar bone resorption. In later stages, it can also lead to tooth loosening, displacement, and even loss, severely affecting the patient's oral and maxillofacial system function and aesthetics. When the oral microecology is imbalanced, opportunistic pathogens become dominant pathogens, leading to periodontal disease through mechanisms such as invading periodontal tissues and triggering inflammatory responses, causing numerous inconveniences to the patient's daily life.

[0003] The pathogens closely related to dental caries and periodontal disease mainly include *Streptococcus mutans*, *Fusobacterium nucleatum*, *Porphyromonas gingivalis*, *Actinomyces villosa*, and *Actinomyces acicularis*. *Streptococcus mutans* is a recognized major cariogenic bacterium, producing large amounts of acidic metabolites and possessing strong tooth surface adhesion, forming biofilms on the tooth surface and continuously damaging the hard tissues of the tooth. It is a core pathogen causing and developing dental caries. *Porphyromonas gingivalis* is one of the key pathogens of periodontitis. This strain can produce various toxins and enzymes, damaging the structure and function of periodontal tissue cells, inhibiting the host's immune defense response, accelerating periodontal pocket formation and alveolar bone resorption, posing a serious threat to periodontal health. *Fusobacterium nucleatum*, as an important periodontal disease-associated pathogen, is often found in deep periodontal pockets. It not only has its own pathogenic effects but also promotes bacterial colonization and inflammatory spread in periodontal tissues, exacerbating periodontal tissue damage. Mucilaginous actinomycetes can participate in the formation of dental plaque biofilm, and the acidic substances produced by their metabolism can also damage the hard tissues of teeth and trigger inflammatory responses in the gingival tissues. They are common pathogens associated with both dental caries and gingivitis. Aggregates actinomycetes are the main pathogens of invasive periodontitis. They have a strong invasive ability, can directly invade periodontal tissues, leading to rapid inflammation progression, severe alveolar bone resorption, and a relatively younger age of onset, posing a greater threat to the oral health of adolescents and young adults. These pathogens interact and synergistically cause disease in the oral cavity, jointly promoting the occurrence and development of dental caries and periodontal disease.

[0004] Traditional treatments for oral diseases mainly include mechanical cleaning and drug therapy. Mechanical cleaning, such as brushing and scaling, can reduce plaque and tartar in the short term, but it's difficult to completely remove bacteria from hidden areas like periodontal pockets and cannot fundamentally regulate the oral microecological balance. Drug therapy, such as the use of antibiotics, can temporarily inhibit the growth of pathogenic bacteria, but long-term use can easily lead to increased bacterial resistance and disrupt the normal oral flora structure, further exacerbating the oral microecological imbalance. Therefore, finding safer, gentler, and more effective intervention methods is of great significance. Probiotics, as a class of live microorganisms beneficial to host health, have been studied extensively for their beneficial effects through regulating the intestinal microecological balance, and their application in oral health has received widespread attention in recent years. Probiotics can inhibit the growth and reproduction of oral pathogenic bacteria through mechanisms such as secreting antibacterial substances, competitively colonizing with pathogenic bacteria, and regulating the host's immune response, thereby improving the oral microecological environment and providing new ideas for the prevention and treatment of dental caries and periodontal disease.

[0005] Chinese invention patent application CN 118146972 A discloses a probiotic composition for improving oral health. This composition contains *Lactobacillus helveticus* K6, as well as *Lactobacillus acidophilus*, *Lactobacillus curvatureii*, *Pediococcus pentosaceus*, and other probiotics. It effectively aggregates harmful bacteria in the oral cavity, improves the oral microecological environment, prevents tooth decay, alleviates gingival swelling and pain, and reduces bad breath. According to its description, *Pediococcus pentosaceus* inhibits foodborne pathogens, helps improve immunity, lowers cholesterol, and regulates metabolism. However, its functional targeting is not related to the inhibition of pathogens associated with tooth decay and periodontal disease, and it fails to exert a direct antagonistic effect on the core pathogens of tooth decay and periodontal disease, thus limiting its effectiveness in targeted prevention and treatment of oral diseases.

[0006] Chinese invention patent application CN 120888446 A discloses a probiotic composition for preventing and / or inhibiting oral pathogens. This probiotic composition contains multiple strains, including *Lactobacillus rhamnosus* strain A21149, *Lactobacillus plantarum* strain A21241, and *Pediococcus pentosus* strain A21358. Its metabolites can effectively inhibit various pathogens such as *Streptococcus mutans*, *Porphyromonas gingivalis*, and *Helicobacter pylori*, showing promise in preventing and alleviating oral problems such as dental caries, periodontitis, and halitosis. Although the probiotic composition provided by this technical solution contains *Pediococcus pentosus* strain A21358, its specification clearly states that the core efficacy of this strain is to effectively inhibit the growth and infection of *Helicobacter pylori* and reduce the inflammatory response it causes through multiple mechanisms, such as enhanced aggregation, reduced urease activity, and regulation of inflammatory factors. It does not primarily inhibit the core pathogens causing dental caries and periodontal disease. More importantly, this composition requires the use of multiple strains in specific mass ratios to achieve a good inhibitory effect on common oral pathogens. The effect of a single strain is limited, and the strain combination is relatively complex, which increases the difficulty of product preparation and quality control.

[0007] Chinese invention patent application CN 116445367 A discloses a *Pediococcus pentosaceus* strain JYPR 9330 and its agent for improving oral health. However, the function of this *Pediococcus pentosaceus* strain JYPR 9330 is limited to inhibiting *Streptococcus mutans*, a caries-causing bacterium. This strain does not show inhibitory effects on key periodontal disease-related pathogens such as *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, and *Aggregobacter actinomycetes*, thus failing to meet the needs of periodontal disease prevention and treatment. Its application scope is relatively narrow, making it difficult to comprehensively address the various oral health problems caused by oral microecological imbalance.

[0008] Furthermore, the three technical solutions mentioned above share common shortcomings. Firstly, none of these solutions systematically evaluate the potential hazards of the strains involved. Oral probiotics act directly on the oral environment, coming into direct contact with the hard tissues of teeth and the oral mucosa. Their acid-producing capacity and corrosiveness to teeth directly affect their safety. If the strain's acid-producing capacity is too strong, it may cause additional damage to the hard tissues of teeth, thus increasing the risk of tooth decay. However, existing technologies have not assessed this crucial safety indicator. Secondly, these solutions lack evaluation of the level of dental inflammation, fail to clarify the specific effects of the strains in alleviating oral inflammation, and do not involve research on the immunomodulatory function of gingival epithelial cells. The occurrence and development of oral diseases are closely related to the host's immune response. The inflammatory response caused by pathogenic bacteria is an important mechanism leading to periodontal tissue damage. As an important component of the oral mucosa, the balance of the immune regulation function of gingival epithelial cells is crucial for maintaining oral health. Current technologies have failed to focus on the regulatory role of bacterial strains on the immune response of gingival epithelial cells, and cannot effectively intervene in oral diseases from the perspective of inflammation regulation. As a result, their efficacy in maintaining oral health is not comprehensive enough, and it is difficult to meet people's multifunctional and in-depth needs for oral care products.

[0009] Therefore, developing a probiotic strain and related products that can specifically inhibit caries and periodontal disease pathogens, have good biosafety, and can regulate oral immune inflammatory responses is of great practical significance and has broad application prospects. Summary of the Invention

[0010] To address the current lack of a suitable Pediococcus pentosaceus strain that is safe for oral health, can regulate inflammatory and immune responses caused by oral pathogens, and can inhibit various oral pathogens such as Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum, Actinomyces colistii, and Aggregates actinomycetes, this invention provides a Pediococcus pentosaceus strain with preventative or therapeutic effects on dental caries and periodontal disease, along with its formulation and applications. This Pediococcus pentosaceus strain can significantly inhibit the growth and reproduction of oral pathogens such as Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum, Actinomyces colistii, and Aggregates actinomycetes, thus promoting oral flora balance and can be widely used for the prevention or treatment of dental caries, periodontitis, and other oral diseases.

[0011] The technical solution of this invention is as follows:

[0012] In a first aspect, the present invention provides a strain of *Pediococcus pentosaceus* that has the effect of preventing or treating dental caries and periodontal disease. Pediococcus pentosaceus HC3368, this strain was deposited on May 15, 2023 at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27356.

[0013] Secondly, the present invention provides a probiotic preparation made from the above-mentioned Pediococcus pentosaceus HC3368.

[0014] Furthermore, the probiotic preparation contains Pediococcus pentosaceus HC3368 and / or the fermentation products of Pediococcus pentosaceus HC3368.

[0015] Furthermore, the preparation method of probiotic preparations is as follows:

[0016] Pediococcus pentosaceus HC3368 was inoculated into MRS liquid medium at a volume percentage of 1% and cultured at 37°C for 24 h. The fresh bacterial culture was then centrifuged at 8000 r / min for 10 min, and the supernatant was discarded to obtain the bacterial cells. The bacterial cells were washed and resuspended to obtain the final product.

[0017] Furthermore, the preparation method of probiotic preparations is as follows:

[0018] Pediococcus pentosaceus HC3368 was inoculated into MRS liquid medium at a volume percentage of 1% and cultured at 37°C for 24 h. The fresh bacterial culture was then centrifuged at 8000 r / min for 10 min at 4°C to obtain the fermentation supernatant.

[0019] Furthermore, probiotic preparations can be health supplements, functional foods, pharmaceuticals, or oral care products. Preferably, probiotic preparations are oral care products, which are limited to localized action within the oral cavity. Specifically, they refer to products that can exert their effects through application, rinsing, or cleaning on the oral mucosa, tooth surfaces, and other areas within the oral cavity. Common categories include toothpaste, polishing powder, tooth powder, dental floss, dental cleaning solutions, mouthwash, oral sprays, or dental foam.

[0020] Thirdly, the present invention provides the application of the above-mentioned Pediococcus pentosaceus HC3368 in the preparation of probiotic preparations with the efficacy of preventing or treating dental caries and periodontal disease.

[0021] Furthermore, the probiotic preparation made from Pediococcus pentosaceus HC3368 has an inhibitory effect on oral pathogens, including Streptococcus mutans, Fusobacterium nucleatum, Porphyromonas gingivalis, Actinomyces coliformis, and Aggregates actinomycetes.

[0022] Furthermore, probiotic preparations made from Pediococcus pentosaceus HC3368 can regulate the inflammatory immune response caused by oral pathogens and reduce the levels of pro-inflammatory cytokines IL-1β, IL-6, and IL-8.

[0023] The beneficial effects of this invention are as follows:

[0024] The *Pediococcus pentosaceus* HC3368 strain provided by this invention exhibits strong tolerance to artificial gastrointestinal fluid and can germinate in it. This strain does not produce hemolysin and cannot lyse blood cells, demonstrating good biocompatibility. This strain can scavenge DPPH and HRS free radicals, inhibit lipid peroxidation, possesses certain antioxidant activity, and can degrade cholesterol, exhibiting probiotic properties that lower serum cholesterol.

[0025] The *Pediococcus pentosaceus* HC3368 provided by this invention exhibits weaker acid-producing ability and less corrosiveness to teeth than oral pathogens, posing no potential harm to teeth. *Pediococcus pentosaceus* HC3368 can produce certain extracellular polysaccharides, exhibiting good adhesion properties to both tooth and gingival epithelial cells, which is beneficial for the colonization of this strain in the oral cavity. *Pediococcus pentosaceus* HC3368 can also inhibit oral pathogens such as *Streptococcus mutans*, *Fusobacterium nucleatum*, *Porphyromonas gingivalis*, *Actinomyces myxoides*, and *Actinomyces actinomyces* by producing bacteriocins and other proteinaceous substances; both bacterial suspensions and fermentation supernatants can exert this effect. Its inhibitory mechanism includes a co-aggregation effect with oral pathogens, reducing the biofilm formed by oral pathogens on the tooth surface, thereby reducing the risk of oral diseases such as dental caries and periodontal disease. Furthermore, *Pediococcus pentosaceus* HC3368 can also regulate the inflammatory immune response induced by oral pathogens, reducing the levels of pro-inflammatory cytokines IL-1β, IL-6, and IL-8.

[0026] The Pentosacchariphyte HC3368 provided by this invention can protect oral health without being combined with prebiotics and / or other probiotics, and there is no risk of side effects or overdose with long-term use. Attached Figure Description

[0027] 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, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a colony morphology diagram of Pediococcus pentosaceus HC3368 from Example 1.

[0029] Figure 2 This is a crystal violet-stained microscopic image of Pediococcus pentosaceus HC3368 from Example 1.

[0030] Figure 3 This is the API 50 CHL carbon source metabolism identification map of Pediococcus pentosaceus HC3368 in Example 2.

[0031] Figure 4This is the RAPD fingerprint of Pediococcus pentosaceus HC3368 in Example 2.

[0032] Figure 5 This is the rep-PCR fingerprint of Pediococcus pentosaceus HC3368 in Example 2.

[0033] Figure 6 This is a diagram showing the tooth-corrosion effect of Pediococcus pentosaceus HC3368 in Example 5.

[0034] Figure 7 This is a graph showing the sugar production results of Pediococcus pentosacchari HC3368 in Experiment 1 of Example 6.

[0035] Figure 8 This is a diagram showing the simulated tooth adhesion results of Pediococcus pentosaceus HC3368 in Experiment 2 of Example 6.

[0036] Figure 9 This is a diagram showing the self-agglutination effect of Pediococcus pentosaceus HC3368 in Experiment 1 of Example 9. Figure 9 (a) shows the effect of self-agglomeration after 2 hours, (b) shows the effect after 4 hours, and (c) shows the effect after 6 hours.

[0037] Figure 10 This is a diagram showing the coagulation effect of *Pediococcus pentosaceus* HC3368 with pathogenic bacteria in Experiment 2 of Example 9. Figure 10 (a) shows the coagulation effect of Pediococcus pentosaceus HC3368 with Streptococcus mutans; (b) shows the coagulation effect of Pediococcus pentosaceus HC3368 with pathogenic bacteria; (c) shows the coagulation effect of Pediococcus pentosaceus HC3368 with Fusobacterium nucleatum; (d) shows the coagulation effect of Pediococcus pentosaceus HC3368 with Actinomyces viscous; and (e) shows the coagulation effect of Pediococcus pentosaceus HC3368 with Aggregates actinomycetes.

[0038] Figure 11 This is a diagram showing the results of Pediococcus pentosaceus HC3368 eliminating oral pathogenic bacteria biofilm in Example 10.

[0039] Figure 12 This is a bar chart showing the concentrations of pro-inflammatory cytokines in different treatment groups of Experiment 1 in Example 11. Figure 12 (a) is a bar chart of IL-1β concentration, (b) is a bar chart of IL-6 concentration, and (c) is a bar chart of IL-8 concentration.

[0040] Figure 13 This is a bar chart showing the concentrations of pro-inflammatory cytokines in different treatment groups of Experiment 2 in Example 11. Figure 13 (a) is a bar chart of IL-1β concentration, (b) is a bar chart of IL-6 concentration, and (c) is a bar chart of IL-8 concentration. Detailed Implementation

[0041] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0042] Unless otherwise specified, the inoculum solutions referred to in the specific embodiments of the present invention are obtained according to the following method:

[0043] Under aseptic conditions, take an appropriate amount of fresh Pediococcus pentosaceus HC3368 bacterial suspension, centrifuge at 5000 r / min for 5 min, wash twice with PBS buffer, resuspend the bacterial cells with an equal volume of PBS buffer, and then dilute 50 times as the inoculum.

[0044] Unless otherwise specified, the *Pediococcus pentosaceus* HC3368 bacterial suspension referred to in the specific embodiments of the present invention is obtained according to the following method:

[0045] The bacterial strain was inoculated into MRS liquid medium at a volume percentage of 1% and cultured at 37°C for 24 h. The fresh bacterial suspension was then centrifuged at 8000 r / min for 10 min, and the supernatant was discarded to obtain the bacterial cells. The cells were washed twice with PBS buffer (pH=7.0) and resuspended until the absorbance OD value reached a certain level. 600 nm Once the concentration reaches between 0.5 and 0.6, a bacterial suspension is obtained for later use.

[0046] Unless otherwise specified, the fermentation supernatant of Pediococcus pentosaceus HC3368 referred to in the specific embodiments of the present invention is obtained according to the following method:

[0047] The bacterial strain was inoculated into MRS liquid medium at a volume percentage of 1% and cultured at 37°C for 24 h. Then, the fresh bacterial culture was centrifuged at 8000 r / min for 10 min at 4°C to obtain the fermentation supernatant.

[0048] Example 1: Isolation and screening of Pediococcus pentosaceus HC3368

[0049] 1. Initial screening

[0050] In December 2022, fresh fermented soybean samples were collected in Yangjiang City, Guangdong Province. After dilution with 100 mL of sterile physiological saline, the samples were placed in sterile sample bags and homogenized using a homogenizer. 100 μL of the homogenate was serially diluted, spread onto MRS agar medium, and anaerobically incubated at 37°C for 48 h. Single colonies were observed under a microscope after growth. Based on the microscopic examination results, 35 potential lactobacillus strains were screened and named HC3350, HC3351, ..., HC3383, and HC3384, respectively.

[0051] 2. Secondary screening

[0052] Prepare 1 L of MRS liquid culture medium, autoclave at 121℃ for 15 min, and after cooling, add 3.2 g of porcine mucosal pepsin, shake well to dissolve, and incubate in a 37℃ water bath for 1 h to prepare an acid-resistant culture medium. Inoculate the 35 strains of lactobacillus obtained from the initial screening into the above acid-resistant culture medium at a 6% inoculum, and incubate anaerobically at 37℃ for 48 h. Collect the fermentation broth for bacterial count.

[0053] The results showed that, after rescreening on acid-resistant culture medium, strain HC3368 among the 35 Lactobacillus strains had the highest viable count, with a logarithmic value as high as 11.03 Log. 10 The CFU / mL indicates that strain HC3368 has the highest acid resistance.

[0054] The HC3368 strain was inoculated onto MRS agar medium and anaerobically cultured at 37°C for 24 h. Figure 1 As shown, single colonies of HC3368 are off-white, with a diameter of approximately 1.5-2.5 mm. The surface is moist and smooth, with neat, opaque edges and a raised surface. After crystal violet staining, under an optical microscope, HC3368 strains appear spherical, smooth, arranged singly, in clusters, or in groups. They are Gram-positive and do not form spores (e.g., ...). Figure 2 (As shown).

[0055] Example 2 Identification of Pediococcus pentosaceus HC3368

[0056] 1. API 50 CHL carbon source metabolism experiment

[0057] The carbon source metabolism capacity of strain HC3368 was verified using API 50 CHL reagent strips. For experimental methods and result analysis, please refer to the API 50 CHL kit instructions. API test results are shown below. Figure 3 The HC3368 strain had an ID value of 99.9% and a T value of 0.98 with Pediococcus pentosaceus, and its carbohydrate metabolic activity was basically the same, indicating an excellent identification result.

[0058] Therefore, based on the carbon source metabolism results, strain HC3368 can be preliminarily identified as Pediococcus pentosaceus (Pentose-60). Pediococcus pentosaceus ).

[0059] 2. Molecular biological identification

[0060] Single colonies of strain HC3368 were picked from the plate and placed in MRS liquid medium. The culture was carried out at 37°C for 24 h. Then, 500 μL of fermentation broth was taken and the genome of the strain was obtained by following the procedure of Tiangen Bacterial Genomic DNA Extraction Kit (DP302). The genome was used for subsequent molecular biological identification.

[0061] 2.1 Identification of 16S rDNA gene sequence

[0062] Primers 27F (AGAGTTTGATCCTGGCTCA, SEQ ID NO.1) and 1492R (GGTTACCTTGTTACGACTT, SEQ ID NO.2) were used. The total PCR amplification system was 50 μL, containing 5 μL of 10×PCR amplification buffer, 4 μL of deoxyribonucleotides (dNTPs), 2 μL of the 27F upstream primer, 2 μL of the 1492R downstream primer, 2.5 μL of DNA template, 0.5 μL of recombinant Taq DNA polymerase (rTaq), and 34 μL of double-distilled water (ddH2O). Electrophoresis was used to verify that the PCR product nucleic acid electrophoresis result was approximately 1500 bp, which met the requirements.

[0063] Sequencing results showed that the 16S rDNA sequence (SEQ ID NO.3) of strain HC3368 is as follows:

[0064]

[0065] 2.2 RAPD fingerprint identification

[0066] M13 (GAGGGTGGCGGTTCT, SEQ ID NO.4) was used as the primer. The RAPD reaction system consisted of 20 μL of RAPD, containing 0.2 μL of Taq DNA polymerase (5 U / μL) and 10× buffer (containing Mg). 2+ 2 μL of deoxyribonucleic acid (dNTPs, 2.5 mM), 0.8 μL of M13 primer (10 μM), 1 μL of DNA template, and 2 μL of double-distilled water (ddH2O) were added to prepare a 1.5% agarose gel plate. Using a DL2000 DNA Marker as a control, electrophoresis was performed at a constant voltage of 100 V for 80 min. The electrophoresis results were then analyzed using a gel imaging system. The RAPD fingerprint of strain HC3368 is shown below. Figure 4 As shown.

[0067] 2.3 Rep-PCR fingerprint identification

[0068] The primer sequence for rep-PCR is GTGGTGGTGGTGGTG (SEQ ID NO. 5). The total reaction volume for rep-PCR is 20 μL, containing 0.2 μL of recombinant Taq DNA polymerase (rTaq) and 10×Ex Taq DNA buffer (containing Mg). 2+ 2 μL of deoxyribonucleic acid (dNTPs, 2.5 mM), 2 μL of primers (10 μM), 1 μL of DNA template, and 12.8 μL of double-distilled water (ddH2O) were added. A 1.5% agarose gel plate was prepared, and an DL2000 DNA Marker was used as a control. Electrophoresis was performed at a constant voltage of 100 V for 80 min. The electrophoresis results were then analyzed using a gel imaging system. The rep-PCR fingerprint of strain HC3368 is shown below. Figure 5 As shown.

[0069] The 16S rRNA sequence of strain HC3368 was uploaded to the EzBioCloud website for comparison. Based on the identification results of the combined physiological and biochemical characteristics, strain HC3368 was identified as a novel Pediococcus pentosaccharis strain and named Pediococcus pentosaccharis HC3368.

[0070] On May 15, 2023, *Pediococcus pentosaceus* HC3368 was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27356, and classified as *Pediococcus pentosaceus*. Pediococcus pentosaceus .

[0071] Example 3 Physicochemical properties of Pediococcus pentosaceus HC3368

[0072] Experiment 1: Salinity Tolerance Test

[0073] Add 190 μL of BSM liquid medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8% to each well of a 96-well plate, performing triplicate for each salt concentration. Then add 10 μL of inoculum solution to each well. The uninoculated wells serve as controls. Add 50 μL of autoclaved paraffin oil to each well to prevent moisture evaporation during culture. Incubate at 37°C and observe whether the medium becomes turbid.

[0074] Salinity tolerance test results showed that the maximum salt concentration that Pediococcus pentosaceus HC3368 could tolerate was 6%.

[0075] Experiment 2: Temperature Growth Range Experiment

[0076] The inoculum was inoculated into 10 mL of MRS liquid medium at a volume percentage of 10%, with 10 mL of uninoculated MRS liquid medium serving as a control. Both MRS liquid media were incubated at 15°C in a constant temperature shaking incubator for 7 days, and at 37°C, 45°C, and 60°C for 2 days, respectively. The turbidity of each MRS liquid medium was observed.

[0077] The results showed that after 7 days of constant temperature incubation at 15℃, the culture medium remained clear; after 2 days of constant temperature incubation at 37℃, the culture medium became turbid; and after 2 days of constant temperature incubation at 45℃ and 60℃, the culture medium became clear. Therefore, *Pediococcus pentosaceus* HC3368 cannot grow at 15℃ and below or 45℃ and above, but can grow normally at 37℃.

[0078] Experiment 3: Tolerance test to artificial gastrointestinal fluid

[0079] Accurately weigh 0.5144 g KCl, 0.1225 g KH2PO4, 2.75085 g NaCl, 2.1002 g NaHCO3, 0.0203 g MgCl2·6H2O, 0.0480 g (NH4)2CO3, and 0.0083 g CaCl2, and bring the volume to 1000 mL to obtain a gastric juice buffer. Dissolve 3 g pepsin in 1000 mL of the gastric juice buffer, adjust the pH to 3.0 with 1 M HCl, and filter through a 0.22 μm filter membrane for sterilization to obtain simulated gastric juice. The simulated gastric juice should be prepared fresh each time it is used.

[0080] Accurately weigh 0.5069 g KCl, 0.1089 g KH2PO4, 2.2442 g NaCl, 7.1408 g NaHCO3, 0.0067 g MgCl2·6H2O, and 0.0333 g CaCl2, and bring the volume to 1000 mL to obtain intestinal fluid buffer. Dissolve 1 g trypsin and 1 g bile salt in 1000 mL of intestinal fluid buffer, adjust the pH to 8.0 with 1 M NaOH, and filter through a 0.22 μm filter membrane for sterilization to obtain simulated intestinal fluid. The simulated intestinal fluid should be prepared fresh each time it is used.

[0081] Nine mL of artificial gastric fluid was incubated in a 37°C water bath shaker for 1 h to simulate human body temperature. One mL of inoculum was added to the nine mL of artificial gastric fluid and incubated in a 37°C water bath shaker (200 r / min). One mL samples were taken before inoculation and 2 h after incubation and added to 99 mL of PBS buffer. The mixture was homogenized using a homogenizer, and one mL of the homogenate was transferred to a sterile Petri dish. Approximately 15 mL of MRS agar medium cooled to 48°C was poured into the Petri dish, and the dish was anaerobically incubated at 37°C for 72 h for colony counting. Two parallel assays were performed.

[0082] 24 mL of artificial intestinal fluid was incubated in a 37°C water bath shaker for 1 h to simulate human body temperature. 1 mL of artificial gastric fluid, digested for 2 h, was added to the 24 mL of artificial intestinal fluid and incubated in a 37°C water bath shaker (200 r / min). At 3 h, 1 mL of the sample was added to 99 mL of PBS buffer and homogenized using a homogenizer. 1 mL of the homogenate was transferred to a sterile Petri dish, and approximately 15 mL of MRS agar medium cooled to 48°C was poured into the dish. The dish was then anaerobically incubated at 37°C for 72 h for colony counting, and two parallel assays were performed.

[0083] The viable count (Log) of Pediococcus pentosaceus HC3368 after digestion with artificial gastrointestinal fluid. 10 The CFU / mL values ​​are shown in Table 1 below.

[0084] Table 1. Viable bacterial count after digestion with artificial gastrointestinal fluid (unit: Log) 10 CFU / mL

[0085]

[0086] As shown in Table 1, the viable count of Pediococcus pentosaceus HC3368 decreased by 0.08 Log after exposure to artificial gastric fluid. 10 After digestion with artificial intestinal fluid, the viable bacterial count remained at 9.56 Log CFU / mL. 10 CFU / mL, total viable bacteria decreased by only 0.13 Log. 10The CFU / mL indicates that Pediococcus pentosaceus HC3368 has good resistance to gastric acid and bile salts, and can tolerate harsh environments in the gastrointestinal tract.

[0087] Experiment 4, Hemolytic Experiment

[0088] Weigh all components of the TBS basal medium, dissolve them, autoclave at 121°C for 15 min, and when the medium cools to 50°C, add 5% sterile defibrinated sheep blood, mix well, and pour into plates to obtain hemocytocyte agar plates. Streak *Pediococcus pentosaceus* HC3368 onto the hemocytocyte agar plates and incubate at 37°C. Observe whether *Pediococcus pentosaceus* HC3368 exhibits hemolysis after 24–48 h.

[0089] The results showed no changes in the blood cell plate, and Pediococcus pentosaceus HC3368 could not grow, indicating that Pediococcus pentosaceus HC3368 does not produce hemolysin and cannot lyse blood cells, thus demonstrating good biosafety.

[0090] Experiment 5: Antibiotic Tolerance Test

[0091] The minimum inhibitory concentration (MIC) of antibiotics against Pediococcus pentosaceus HC3368 was determined using the microbroth dilution method. The specific results are shown in Table 2 below.

[0092] Table 2. MIC values ​​of antibiotics against Pediococcus pentosaceus HC3368 (unit: μg / mL)

[0093]

[0094] As shown in Table 2, Pediococcus pentosaceus HC3368 is sensitive to common antibiotics such as erythromycin, streptomycin, ampicillin and clindamycin, and has good biosafety.

[0095] Experiment 6: Hydrophobic cell surface test

[0096] Purified Pediococcus pentosaceus HC3368 colonies were picked and inoculated into MRS liquid medium, and cultured at 40°C with shaking for 24–48 h. Then, 1% (v / v) of the inoculum was transferred to fresh MRS liquid medium and cultured at 40°C with shaking for another 24–48 h. The cells were then centrifuged at 6000 × g for 10 min, collected, and washed twice with sterile physiological saline. The cells were then resuspended in 1 mL of 0.1 M sterile KNO3 solution as the test solution.

[0097] Add 50 μL of the above-mentioned bacterial culture to 2450 μL of 0.1 M KNO3 solution and test the OD of the solution. 600 nmThe value is recorded as A0; 1.5 mL of the above-mentioned bacterial solution is mixed with 500 μL of xylene and allowed to stand at room temperature for 10 min. The resulting two-phase system is then vortexed for 2 min and allowed to stand for another 20 min to reform the aqueous and organic phases. The aqueous phase is carefully aspirated for OD testing. 600 nm The value is recorded as A1. Cell hydrophobicity is calculated as hydrophobicity% = (A0 - A1) / A1 × 100%. Three parallel experiments are conducted, and the average value of the obtained data is taken.

[0098] The results showed that the hydrophobicity of the cell surface of Pediococcus pentosaceus HC3368 was 70.96% ± 10.12%.

[0099] Experiment 7. Determination of aflatoxin B1 scavenging ability

[0100] Aflatoxins are secondary metabolites produced by various fungi, including Aspergillus flavus and Aspergillus parasiticus. Eighteen types of aflatoxins have been isolated, among which aflatoxin B1 (AFB1) is classified as a Group 1 carcinogen by the International Agency for Research on Cancer due to its extremely strong carcinogenic, mutagenic, and teratogenic properties. Even trace amounts of aflatoxin can have harmful effects on humans and animals. Prepare the required solution according to the instructions of the aflatoxin B1 assay kit and perform the detection procedure.

[0101] The results showed that the aflatoxin B1 clearance capacity of Pediococcus pentosaceus HC3368 was 68.43% ± 6.55%.

[0102] Experiment 8: In vitro cholesterol degradation test

[0103] Accurately weigh 1 g of cholesterol, dissolve it in anhydrous ethanol, and bring the volume to 100 mL. Filter the solution under sterile conditions using a 0.22 µm microporous membrane to obtain a cholesterol solution.

[0104] Weigh out 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 2.0 g of diammonium hydrogen citrate, 20.0 g of glucose, 1.0 mL of Tween 80, 5.0 g of sodium acetate, 0.1 g of magnesium sulfate, 0.05 g of manganese sulfate, 2.0 g of dipotassium hydrogen phosphate, and 1000 mL of distilled water. After dissolving, adjust the pH to 7.3 and sterilize at 115℃ for 30 min. Then add cholesterol solution to make the final cholesterol concentration 0.1% to obtain a cholesterol-containing liquid culture medium.

[0105] Inoculate the inoculum into a cholesterol-containing liquid culture medium at a volume percentage of 0.1%, and incubate statically at 37°C for 48 h. Then, take 0.2 mL of bacterial culture, add 1.8 mL of anhydrous ethanol, mix well, let stand for 10 min, centrifuge at 3000 r / min for 5 min, take the supernatant, and determine the cholesterol content according to the method specified in GB 5009.128-2016 "Determination of Cholesterol in Food", and calculate the cholesterol degradation rate.

[0106] The results showed that the in vitro cholesterol degradation rate of Pediococcus pentosaceus HC3368 was 73.76% ± 2.57%.

[0107] Example 4: Determination of the antioxidant function of Pediococcus pentosaceus HC3368

[0108] Experiment 1. Determination of the ability of Pediococcus pentosaceus HC3368 to scavenge 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH)

[0109] Take 1 mL of Pediococcus pentosaceus HC3368 bacterial suspension and 1 mL of Pediococcus pentosaceus HC3368 fermentation supernatant, add 1 mL of freshly prepared 0.4 mM DPPH free radical solution to each, mix well, and incubate at room temperature in the dark for 30 min. Then measure the absorbance A of the sample at a wavelength of 517 nm. 样品 Three parallel experiments were conducted, and the average value of the obtained data was taken.

[0110] During the experiment, an equal volume of PBS buffer and ethanol mixture was used as a blank for instrument zeroing; the absorbance was recorded as A. 空白 A mixture of equal volumes of PBS buffer and DPPH ethanol solution was used as a control, and the absorbance was recorded as A. 对照 DPPH free radical scavenging rate is calculated as scavenging rate % = [1 - (A...]]. 样品 -A 空白 ) / A 对照 The result of the calculation is shown in Table 3 below.

[0111] Table 3. Scavenging rate of DPPH free radicals by Pediococcus pentosaceus HC3368 (unit: %)

[0112]

[0113] Experiment 2. Determination of the ability of Pediococcus pentosaceus HC3368 to scavenge hydroxyl radicals (HRS).

[0114] Take 200 μL of *Pediococcus pentosaceus* HC3368 bacterial suspension and 200 μL of *Pediococcus pentosaceus* HC3368 fermentation supernatant, and mix them separately with 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate solution, and 500 μL of deionized water. Then add 100 μL of 3 mM hydrogen peroxide solution to each. After incubating in a water bath at 37℃ for 15 min, measure the absorbance of the sample at a wavelength of 510 nm and record it as A. 样品 Replace the bacterial suspension or fermentation supernatant with an equal volume of deionized water, and mix with 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate solution, and 500 μL of deionized water. Then add 100 μL of 3 mM hydrogen peroxide solution to each. After incubating in a 37°C water bath for 15 min, measure the absorbance of the sample at a wavelength of 510 nm and record it as A. 控制 Replace the bacterial suspension or fermentation supernatant and hydrogen peroxide solution with an equal volume of deionized water, and mix with 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate solution, and 500 μL of deionized water. After incubating in a water bath at 37°C for 15 min, measure the absorbance of the sample at a wavelength of 510 nm, and record it as A. 空白 HRS free radical scavenging rate is calculated as scavenging rate % = (A 样品 -A 控制 ) / (A 空白 -A 控制 The result of the calculation (100%) is shown in Table 4 below.

[0115] Table 4. Scavenging rate of HRS free radicals by Pediococcus pentosaceus HC3368 (unit: %)

[0116]

[0117] Experiment 3: Anti-lipid peroxidation experiment of Pediococcus pentosaceus HC3368

[0118] Measure 0.1 mL of linoleic acid, 0.2 mL of Tween 20, 19.7 mL of deionized water, and 0.5 mL of PBS buffer (pH=7.4). Mix the above components thoroughly and stir until homogeneous to obtain a linoleic acid emulsion for later use.

[0119] Take 1 mL of the prepared linoleic acid emulsion, add 1 mL of FeSO4 solution (1%), and then add 0.5 mL of the sample to be tested (a suspension or fermentation supernatant of *Pediococcus pentosaceus* HC3368). Gently shake to homogenize the mixture. Place the mixture in a 37℃ water bath and incubate for 1.5 h. After the reaction, add 0.2 mL of TCA solution (4%) and 2 mL of TBA solution (0.8%) to the mixture, mix well, and heat in a 100℃ water bath for 30 min. After color development, rapidly cool the mixture, then centrifuge at 4000 r / min for 15 min to remove precipitated impurities. Collect the supernatant after centrifugation and measure the absorbance at 532 nm, denoted as A. Simultaneously, repeat the above operation using 0.5 mL of distilled water instead of the sample to be tested, and record the measured absorbance as A0. The lipid peroxidation inhibition rate was calculated as inhibition rate % = (A0-A) / A0 × 100%, and the results are shown in Table 5 below.

[0120] Table 5. Lipid peroxidation inhibition rate of Pediococcus pentosaceus HC3368 (unit: %)

[0121]

[0122] Example 5: Potential Hazard Assessment of Pediococcus pentosaceus HC3368

[0123] Experiment 1: Evaluation of Acid Production Capacity

[0124] The acid-producing capacity of *Pediococcus pentosaceus* HC3368 was evaluated using the endpoint pH method. *Pediococcus pentosaceus* HC3368 was cultured in MRS liquid medium for 24 h, and the acidity of the culture medium was measured using a pH meter. *Pediococcus pentosaceus* CECT8330 and *Porphyromonas gingivalis* BNCC353909, both commercially available products, were used as controls. The results are shown in Table 6 below. The endpoint pH value of *Pediococcus pentosaceus* HC3368 was higher than that of the control strain, indicating that its acid-producing capacity was weaker than that of the control strain. Therefore, the use of *Pediococcus pentosaceus* HC3368 can reduce the risk of dental caries caused by acid production by this strain.

[0125] Table 6. Fermentation endpoint pH values ​​of the strains

[0126]

[0127] Experiment 2, Corrosion Evaluation

[0128] The corrosiveness of *Pediococcus pentosaceus* HC3368 to teeth was measured using the iso-element method. The fermentation supernatant was mixed with hydroxyapatite, stirred thoroughly, and incubated at 37°C. Equal volumes of fermentation broth were centrifuged at 30 min, 1 h, 2 h, and 3 h to obtain the supernatant. The OD of the supernatant was determined using acid-molybdate spectrophotometry.620 nm Value, OD 620 nm The value reflects the change in phosphorus content in the supernatant, thus characterizing the corrosion of hydroxyapatite. Meanwhile, the fermentation supernatant of *Porphyromonas gingivalis* BNCC353909 was used as a control; the method for obtaining the fermentation supernatant of *Porphyromonas gingivalis* BNCC353909 was the same as that for *Pediococcus pentosaceus* HC3368. The fermentation supernatant at 0 min was used as a blank to correct for the endogenous phosphorus content in the samples.

[0129] The results are as follows Figure 6 As shown, the corrosive ability of *Pediococcus pentosaceus* HC3368 reached its maximum at 1 hour, and almost no further corrosion occurred after 1 hour. In contrast, *Porphyromonas gingivalis* BNCC353909 showed a continuously increasing corrosive effect on hydroxyapatite, indicating that *Pediococcus pentosaceus* HC3368 has a weaker corrosive ability, and its destructive effect on teeth is far less than that of oral pathogens such as *Porphyromonas gingivalis*.

[0130] Example 6: Evaluation of the adhesion properties of Pediococcus pentosaceus HC3368

[0131] Experiment 1: Evaluation of Glycoproductivity

[0132] Probiotics can enhance their adhesion to oral mucosa, tooth surfaces, and other sites by producing extracellular polysaccharides. Stable adhesion is a key prerequisite for probiotics to successfully colonize the host and further exert their beneficial effects. Therefore, the adhesion performance and colonization potential of probiotics in the oral environment can be indirectly assessed by measuring the content of extracellular polysaccharides in the probiotic fermentation broth.

[0133] Take 10 mL of fermentation supernatant, add 2 mL of 80% trichloroacetic acid, mix, and stir on ice for 30 min. Centrifuge to remove residual bacterial cells and proteins from the supernatant. Collect the centrifuged supernatant and add three volumes of pre-cooled anhydrous ethanol at 0°C. Then, refrigerate the mixture at 4°C for 24 h and observe the precipitation of extracellular polysaccharides (EPS). Centrifuge the mixture at 10,000 r / min for 15 min at 4°C to obtain polysaccharide precipitate. Then, redissolve the precipitate in 10 mL of distilled water to form a polysaccharide solution. The polysaccharide content in the polysaccharide solution is determined using the phenol-sulfuric acid method. Simultaneously, Lactobacillus rhamnosus LGG from a commercially available product is used as a control.

[0134] The results are as follows Figure 7 As shown, the extracellular polysaccharide yield of *Pediococcus pentosaceus* HC3368 was 106 mg / L, while the extracellular polysaccharide yield of the control strain *Lactobacillus rhamnosaceus* LGG was 84 mg / L, significantly lower than that of *Pediococcus pentosaceus* HC3368 provided in this invention (*). P <0.05).

[0135] Experiment 2: Evaluation of tooth adhesion

[0136] The absorbance of a suspension of *Pediococcus pentosaceus* HC3368 at a wavelength of 600 nm was measured and denoted as OD. 600前 10 mg of sterile hydroxyapatite powder (d=82 μm) was added to a suspension of *Pediococcus pentosaceus* HC3368, mixed well, and incubated in a 37°C water bath for 1 h. After incubation, the suspension was filtered through a 0.45 μm pinhole membrane, and the filtrate was collected. The pinhole membrane was then washed twice with PBS buffer, and the washed liquid was combined with the previously collected filtrate. The combined filtrate was then centrifuged, and the bacterial pellet at the bottom of the centrifuge tube was collected. 1 mL of PBS buffer was added to the bacterial pellet for resuspending, and the absorbance at 600 nm was measured and recorded as OD. 600后 Simultaneously, *Lactobacillus rhamnosus* LGG was used as a control. The simulated tooth adhesion rate was calculated using the following formula:

[0137] .

[0138] The results are as follows Figure 8 As shown, both *Pediococcus pentosaceus* HC3368 and *Lactobacillus rhamnosaceus* LGG exhibit certain adhesive properties. The simulated tooth adhesion rate of *Lactobacillus rhamnosaceus* LGG was 36.18%, significantly lower than the 87.86% of *Pediococcus pentosaceus* HC3368 (**). P <0.01). The high adhesion rate of Pediococcus pentosaceus HC3368 allows it to adhere to the tooth surface and effectively reduces the risk of the strain being carried into the digestive tract by saliva and swallowing, thus enhancing its residence time.

[0139] Experiment 3: Evaluation of Cell Adhesion

[0140] Human gingival epithelial cells C1052, after resuscitation, culture, and counting, were cultured at a concentration of 2 × 10⁻⁶. 6 Inoculation was performed at a rate of 1000 cells / well in 6-well plates and incubated in a CO2 incubator for 24 h. The experimental group was treated by resuspending *Pediococcus pentosaceus* HC3368 in logarithmic growth phase in MRS liquid medium to a concentration of 5 × 10⁻⁶. 7CFU / mL, 1 mL of this suspension was added to a 6-well plate containing pre-adhered cells and incubated in a CO2 incubator for 2 h. After incubation, the cells were washed three times with PBS buffer to remove unadhered bacteria. 500 μL of trypsin was added for 3 min of digestion, followed by 1.5 mL of cell culture medium to terminate the digestion and repeated pipetting. The resulting solution was collected in sterile EP tubes and serially diluted 10-fold, 100-fold, 1000-fold, and 10000-fold, then plated and counted. A 6-well plate without the *Pediococcus pentosaceus* HC3368 suspension was used as a control group, and cell counting was performed using the same procedure as the experimental group. The cell adhesion ability of *Pediococcus pentosaceus* HC3368 was calculated using the following formula:

[0141] Adhesion capacity (CFU / cell) = Total number of bacteria adhering in each culture well / Total number of cells in each culture well.

[0142] The results showed that the adhesion ability of Pediococcus pentosaceus HC3368 was 52.05±9.39 CFU / cell, indicating that this strain has a strong adhesion ability to oral epithelial cells, which is conducive to its colonization in the oral environment.

[0143] Example 7: Antibacterial test of Pediococcus pentosaceus HC3368 against oral pathogens

[0144] Commercially available Streptococcus mutans ATCC25175, Fusobacterium nucleatum BNCC336949, Actinomyces viscous ATCC27044, Porphyromonas gingivalis BNCC353909, and Aggregates actinomycetii BNCC336945 were cultured separately. The bacterial cultures were inoculated at a volume percentage of 1% into BHI broth medium (supplemented with 5% bovine serum). After anaerobic incubation at 37°C for 48 h, the bacterial cells were collected by centrifugation at 8000 r / min for 10 min. The bacterial cells were washed twice with PBS buffer (pH=7.0), and then resuspended in PBS buffer (pH=7.0) to adjust the initial absorbance (OD) of the bacterial suspension. 600 nm Reserve 0.5~0.6.

[0145] The inhibitory effects of Pediococcus pentosaceus HC3368 bacterial suspension and fermentation supernatant on the aforementioned oral pathogens were determined using the Oxford cup double-layer plate method. BHI medium with a 0.7% agar content was prepared and sterilized. After the medium temperature dropped below 47℃, 0.2% of the mixed pathogenic bacterial solution was added and shaken well. 7 mL of medium was poured onto the bottom agar plate. After the medium solidified, Oxford cups were placed on top, and 150 μL of Pediococcus pentosaceus HC3368 bacterial suspension or fermentation supernatant was added to each well. After incubation at 37℃ for 48 h, the diameter of the inhibition zone was measured. The results are shown in Table 7 below.

[0146] Table 7. Antibacterial effect of Pediococcus pentosaceus HC3368 against oral pathogens (unit: mm)

[0147]

[0148] As shown in Table 7, *Pediococcus pentosaceus* HC3368 exhibits significant inhibitory effects against five oral pathogens: *Streptococcus mutans*, *Fusobacterium nucleatum*, *Porphyromonas gingivalis*, *Actinomyces myxobolus*, and *Aggregobacter gingivalis*, demonstrating broad-spectrum antibacterial activity. Among these, the bacterial suspension of *Pediococcus pentosaceus* HC3368 showed better antibacterial effects than the fermentation supernatant, and its inhibitory effects against *Streptococcus mutans*, *Fusobacterium nucleatum*, and *Porphyromonas gingivalis* were most pronounced, with inhibition zones exceeding 20 mm in diameter for each.

[0149] Example 8: Determination of Antibacterial Substances in Pediococcus pentosaceus HC3368

[0150] Studies have shown that lactic acid bacteria can exert oral health protection effects by producing substances such as organic acids, hydrogen peroxide, bacteriocins, and adhesion inhibitors. Based on this, this embodiment was designed to identify the substances that play a major antibacterial role in Pediococcus pentosaceus HC3368.

[0151] The pH of the fermentation supernatant of Pediococcus pentosaceus HC3368 was adjusted to between 5.5 and 6 with 5 M NaOH. The neutralized fermentation supernatant was then filtered through a 0.22 μm filter membrane to obtain an acid-free fermentation supernatant, which was used as sample 1 to be tested.

[0152] The pH of the fermentation supernatant of Pediococcus pentosaceus HC3368 was adjusted to between 5.5 and 6 with 5 M NaOH. Catalase was added to the supernatant, and after mixing well, it was incubated at 37°C for 30 min to obtain a fermentation supernatant free of H2O2. This supernatant was used as sample 2 to be tested.

[0153] The fermentation supernatant of Pediococcus pentosaceus HC3368 was adjusted to pH 5.5-6 with 5 M NaOH. Catalase, pepsin and trypsin were added to the supernatant, and the mixture was incubated at 37°C for 30 min to obtain a fermentation supernatant with proteins removed. This supernatant was used as sample 3 to be tested.

[0154] Antibacterial tests were conducted on samples 1, 2, and 3 to observe their antibacterial effects against five oral pathogens. The antibacterial test method was the same as in Example 7. The results are shown in Table 8 below.

[0155] Table 8. Antibacterial effect of fermentation supernatant under different treatments on oral pathogens (unit: mm)

[0156]

[0157] Note: Different letters in each column of Table 8 indicate significant differences. P <0.05, the same letters indicate no significant difference ( P 0.05).

[0158] As shown in Table 8, the fermentation supernatant of *Pediococcus pentosaceus* HC3368 still exhibited significant inhibitory effects on oral pathogens after acid neutralization and H2O2 removal. The size of its inhibition zone was not significantly different from that of the untreated fermentation supernatant. However, the size of its inhibition zone decreased significantly after protease treatment, indicating that the inhibitory substance of *Pediococcus pentosaceus* HC3368 against oral pathogens is a protein-based substance. Therefore, it is speculated that strain HC3368 can produce protein bacteriocins to inhibit oral pathogens, thereby playing a role in protecting oral health.

[0159] Example 9: Determination of the agglutination rate of Pediococcus pentosaceus HC3368 against oral pathogens

[0160] The stronger the self-agglomeration ability of probiotics, the better their colonization effect in the oral cavity. The bacteria can be less easily washed away by saliva through self-agglomeration, and high self-agglomeration helps increase the concentration of probiotics in the oral cavity, which is beneficial to their survival and healthy function. Simultaneously, probiotics can also compete with pathogenic bacteria for binding sites on the biofilm surface and for nutrients through co-agglomeration reactions, thus preventing pathogenic bacteria from adhering to the tooth surface. Therefore, measuring the self-agglomeration rate of probiotics and their co-agglomeration rate with pathogenic bacteria can be used to characterize the colonization effect of the strains.

[0161] Experiment 1: Determination of self-agglomeration rate

[0162] Add 1 mL of Pediococcus pentosaceus HC3368 bacterial suspension to a 24-well plate and let it stand at room temperature. Take 100 μL of the bacterial suspension and measure its initial absorbance A0 at 600 nm. Then, every 2 hours for the next 6 hours, take the upper layer of the bacterial suspension and measure its absorbance A at 600 nm. t Calculate the self-coagulation rate R 自 =(1-A t / A0)×100%, with three replicates for each sample. The autoagglutination effect of *Pediococcus pentosaceus* HC3368 at different time points is shown below. Figure 9 As shown in Table 9, the self-agglomeration rate results are as follows.

[0163] Table 9 Autoaggregation rate of Pediococcus pentosaceus HC3368 (unit: %)

[0164]

[0165] As shown in Table 9, the autoagglutination rate of Pediococcus pentosaceus HC3368 reached 52.15% at 6 h, indicating that Pediococcus pentosaceus HC3368 can effectively colonize in the oral cavity.

[0166] Experiment 2: Determination of Coagulation Rate

[0167] Take 100 μL of Pediococcus pentosaceus HC3368 bacterial suspension and measure its initial absorbance value A0 at 600 nm; take 100 μL of pathogenic bacteria (Streptococcus mutans ATCC25175, Fusobacterium nucleatum BNCC336949, Actinomyces viscous ATCC27044, Porphyromonas gingivalis BNCC353909 or Aggregates actinomycetii BNCC336945) bacterial suspension and measure its initial absorbance value B0 at 600 nm.

[0168] Equal volumes of *Pediococcus pentosaceus* HC3368 suspension and pathogenic bacteria suspension were mixed, shaken well, and then added to a 24-well plate and allowed to stand at room temperature. Every 2 hours, 100 μL of the upper suspension was taken and its absorbance at 600 nm was measured. t Calculate the coagulation rate R 共 =[1-2A t [ / (A0+B0)]×100%, with three replicates for each sample. The co-agglutination effect of *Pediococcus pentosaceus* HC3368 with different pathogenic bacteria at 6 h is shown below. Figure 10 As shown in Table 10, the coagulation rate results are as follows.

[0169] Table 10 Coagulation rates of Pediococcus pentosaceus HC3368 with different pathogenic bacteria (unit: %)

[0170]

[0171] As shown in Table 10, *Pediococcus pentosaceus* HC3368 can co-aggregate with five oral pathogens: *Streptococcus mutans*, *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, *Actinomyces viscerata*, and *Aggregobacter actinomycetes*. The co-aggregation effect at 2 h, 4 h, and 6 h is higher than that of *Pediococcus pentosaceus* HC3368 itself. Specifically, the co-aggregation effect of *Pediococcus pentosaceus* HC3368 against *Streptococcus mutans*, *Fusobacterium nucleatum*, and *Aggregobacter actinomycetes* can reach over 59%.

[0172] It is evident that *Pediococcus pentosaceus* strain HC3368 exhibited good autoagglutination ability within 6 hours, which is beneficial for its colonization in the oral cavity. Simultaneously, *Pediococcus pentosaceus* HC3368 also showed coagulation effects with *Streptococcus mutans*, *Fusobacterium nucleatum*, and other pathogens, effectively reducing the relative content of oral pathogens.

[0173] Example 10: Elimination effect of Pediococcus pentosaceus HC3368 on oral pathogenic bacterial biofilms

[0174] Biofilms of *Streptococcus mutans* ATCC25175, *Porphyromonas gingivalis* BNCC353909, and *Fusobacterium nucleatum* BNCC336949 were cultured using a well plate method. 300 μL of BHI medium supplemented with 2% (m / v) sucrose was added to each well of a 24-well plate, followed by inoculation with activated oral pathogens at a volume percentage of 2%. The 24-well plates were incubated at 37°C under micro-anaerobic conditions for 48 h. After incubation, mature biofilms of the pathogens were visible on the inner walls and bottom of the wells. The culture medium and unadhered bacterial cells were discarded, and the plates were washed twice with PBS buffer (pH=7.0). For the experimental groups, 300 µL of *Pediococcus pentosacchari* HC3368 fermentation supernatant was added to each well for co-culturing for another 24 h. Each group was run in triplicate, with an equal volume of MRS liquid medium used instead of the *Pediococcus pentosacchari* HC3368 fermentation supernatant serving as a control group. After culture, the supernatant containing planktonic cells was removed while maintaining the integrity of the biofilm. The microplate was then washed three times with sterile PBS buffer, fixed with methanol for 15 min, and the wells were emptied and air-dried for 45 min. The biofilm was stained with 0.1% (m / v) crystal violet for 30 min, and excess staining was removed. 300 µL of anhydrous ethanol was added to each well to release the bound crystal violet back into the well. Finally, the solution was transferred to a new well plate, and the absorbance at 600 nm was measured.

[0175] The results are as follows Figure 11 As shown, for *Streptococcus mutans* biofilm, co-culturing it with the fermentation supernatant of *Pediococcus pentosus* HC3368 reduced the biofilm by 73.65%, a significant difference between the experimental group and the control group (**). P<0.01); Regarding the *Porphyromonas gingivalis* biofilm, co-culturing it with the fermentation supernatant of *Pediococcus pentosus* HC3368 reduced the biofilm by 70.00%, showing a significant difference between the experimental group and the control group (**). P <0.01); For Fusobacterium nucleatum biofilm, co-culturing with Pediococcus pentosaceus HC3368 fermentation supernatant reduced the biofilm by 57.83%, showing a significant difference between the experimental group and the control group (**). P <0.01).

[0176] Biofilms effectively help bacteria resist changes in the external environment, promoting their growth and reproduction. Oral pathogens exist in the oral cavity in the form of biofilms. When the bacterial homeostasis within the biofilm changes, oral problems such as tooth decay and periodontal disease can occur. Pediococcus pentosaceus HC3368 can significantly reduce the biofilm formation of Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis, thereby reducing the risk of tooth decay and periodontal disease and contributing to improved oral health.

[0177] Example 11 Immunomodulatory effect of Pediococcus pentosaceus HC3368 on gingival epithelial cells

[0178] Experiment 1: Immunomodulation of gingival epithelial cells infected with Streptococcus mutans by Pediococcus pentosus HC3368

[0179] Resuscitated and passaged human gingival epithelial cells C1052 were administered at a rate of 5 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and, after cell adhesion, were divided into three groups for subsequent infection experiments.

[0180] The first group served as the control group and was not subjected to bacterial infection. The second group used DMEM medium (containing 10% FBS) to resuspend a suspension of *Streptococcus mutans* ATCC25175, prepared using the same method as in Example 7. Human gingival epithelial cells C1052 were infected with this suspension at a multiplicity of infection (MOI) of 250. This second group was named the *Streptococcus mutans* group. The third group used DMEM medium (containing 10% FBS) to resuspend an equal volume of *Streptococcus mutans* ATCC25175 and *Pediococcus pentosaceus* HC3368 suspensions. Human gingival epithelial cells C1052 were infected based on the number of pathogenic bacteria, according to an MOI of 250. This third group was named the *Streptococcus mutans* + probiotic group. Each treatment group had three replicates. The three groups of cells were cultured at 37°C for 24 h in an incubator containing 5% CO2. After the culture was completed, the cells were centrifuged at 1000×g for 20 min, and the supernatant was collected for the detection of cellular inflammatory factors IL-1β, IL-6 and IL-8. The detection steps were performed in accordance with the instructions of the ELISA kit.

[0181] Experimental results are as follows Figure 12As shown, after infection with *Streptococcus mutans*, the concentrations of pro-inflammatory cytokines IL-1β, IL-6, and IL-8 in the supernatant of human gingival epithelial cells reached 85.16 pg / mL, 464.03 pg / mL, and 1835.67 pg / mL, respectively, which were significantly higher than the inflammatory levels in normal gingival epithelial cells of the control group (**). P <0.01); however, after intervention with Pediococcus pentosaceus HC3368, the concentrations of IL-1β, IL-6, and IL-8 decreased to 60.47 pg / mL, 169.54 pg / mL, and 1247.96 pg / mL, respectively, representing reductions of 28.99%, 63.46%, and 32.02%, which were significantly different from those in the Streptococcus mutans group (**). P <0.01).

[0182] Experiment 2. Immunomodulation of gingival epithelial cells infected with Fusobacterium nucleatum by Pediococcus pentosus HC3368

[0183] The steps of Experiment 2 are basically the same as those of Experiment 1, with the only difference being:

[0184] (1) Use Fusobacterium nucleatum BNCC336949 instead of Streptococcus mutans ATCC25175 to infect human gingival epithelial cells C1052 alone, and name this treatment group the Fusobacterium nucleatum group;

[0185] (2) Used Fusobacterium nucleatum BNCC336949 instead of Streptococcus mutans ATCC25175 and Pediococcus pentosus HC3368 to co-infect human gingival epithelial cells C1052, and named this treatment group as nucleatum + probiotic group.

[0186] Experimental results are as follows Figure 13 As shown, the concentrations of pro-inflammatory cytokines IL-1β, IL-6, and IL-8 in the supernatant of human gingival epithelial cells infected with *Fusobacterium nucleatum* reached 108.49 pg / mL, 557.36 pg / mL, and 1249.00 pg / mL, respectively, which were significantly higher than the inflammatory levels in normal gingival epithelial cells of the control group (**). P <0.01); After intervention with Pediococcus pentosaceus HC3368, the concentrations of IL-1β, IL-6, and IL-8 decreased to 55.14 pg / mL, 226.21 pg / mL, and 643.96 pg / mL, respectively, representing reductions of 49.18%, 59.41%, and 48.44%, which were significantly different from those in the Fusobacterium nucleatum group (**). P <0.01).

[0187] The above experimental results indicate that Pediococcus pentosaceus HC3368 can effectively improve oral inflammation caused by oral pathogens such as Streptococcus mutans and Fusobacterium nucleatum, and is beneficial to the recovery of oral health.

[0188] Example 12: Preparation of probiotic formulation using Pediococcus pentosaceus HC3368

[0189] (1) Inoculate Pediococcus pentosaceus HC3368 into MRS liquid medium at an inoculation rate of 1% by volume and incubate at 37°C for 24 h;

[0190] (2) Centrifuge the fresh bacterial culture at 8000 r / min for 10 min to separate the supernatant and the bottom bacterial cells;

[0191] (3) Collect the supernatant as a probiotic preparation;

[0192] (4) Collect the bottom bacterial cells, wash twice with PBS buffer (pH=7.0), and resuspend the bacterial cells until the absorbance OD value is reached. 600 nm Reaching a concentration between 0.5 and 0.6 yields another probiotic preparation.

[0193] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A strain of Pediococcus pentosaceus that has the effect of preventing or treating dental caries and periodontal disease ( Pediococcus pentosaceus HC3368, characterized in that, Pediococcus pentosaceus HC3368 was deposited on May 15, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27356.

2. A probiotic preparation made from Pediococcus pentosaceus HC3368 as described in claim 1, characterized in that, The probiotic preparation contains Pediococcus pentosaceus HC3368 and / or the fermentation supernatant of Pediococcus pentosaceus HC3368.

3. The probiotic preparation as described in claim 2, characterized in that, The preparation method of probiotic preparations is as follows: Pediococcus pentosaceus HC3368 was inoculated into MRS liquid medium at a volume percentage of 1% and cultured at 37°C for 24 h. The fresh bacterial culture was then centrifuged at 8000 r / min for 10 min, and the supernatant was discarded to obtain the bacterial cells. The bacterial cells were washed and resuspended to obtain the final product.

4. The probiotic preparation as described in claim 2, characterized in that, The preparation method of probiotic preparations is as follows: Pediococcus pentosaceus HC3368 was inoculated into MRS liquid medium at a volume percentage of 1% and cultured at 37°C for 24 h. The fresh bacterial culture was then centrifuged at 8000 r / min for 10 min at 4°C to obtain the fermentation supernatant.

5. The probiotic preparation as described in claim 2, characterized in that, Probiotic preparations are oral care products.

6. The probiotic preparation as described in claim 5, characterized in that, Oral care products can be selected from any one of the following: dental gel, tooth powder, toothpaste, dental floss, dental cleaning solution, and dental cleaning foam.

7. The probiotic preparation as described in claim 5, characterized in that, The oral care products mentioned are mouthwashes or mouth sprays.

8. The use of Pediococcus pentosaceus HC3368 as described in claim 1 in the preparation of a probiotic preparation having the effect of preventing or treating dental caries and periodontal disease.

9. The application as described in claim 8, characterized in that, Probiotic preparations have an inhibitory effect on oral pathogens, including Streptococcus mutans, Fusobacterium nucleatum, Porphyromonas gingivalis, Actinomyces colistii, and / or Aggregates actinomycetes.

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