A broad-spectrum antibacterial strain of Lactobacillus plantarum YN-63 and its application

Abstract

This invention belongs to the field of microbiology and oral care technology, and discloses a strain of Lactobacillus plantarum ( Lactiplantibacillus plants Lactobacillus plantarum YN-63 and its applications. The Lactobacillus plantarum YN-63 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 29, 2026, with accession number CGMCC No. 39525. Microbial preparations containing this strain, cell-free fermentation supernatant of this strain, oral care products, functional foods, or probiotic products containing this strain or its cell-free fermentation supernatant are also disclosed. The Lactobacillus plantarum YN-63 and its cell-free fermentation supernatant can be used for non-therapeutic purposes to inhibit oral pathogenic bacteria, inhibit oral biofilm formation, reduce Streptococcus mutans colonization, and / or reduce the risk of tooth surface demineralization, and are suitable for the development of oral care products and functional probiotic products.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and oral care technology, specifically relating to a strain of Lactobacillus plantarum (… Lactiplantibacillus plantarum YN-63, microbial preparations containing this strain or its fermentation products, cell-free fermentation supernatants, oral care products, functional foods or probiotic products, and their use for non-therapeutic purposes in inhibiting oral pathogens, inhibiting oral biofilm formation, reducing Streptococcus mutans colonization and / or reducing the risk of tooth surface demineralization. Background Technology

[0002] Dental caries is closely related to oral microecological imbalance, and Streptococcus mutans (S. mutagenesis) Streptococcus mutans Candida albicans is a common caries-causing pathogen that produces and is resistant to acid, forming a biofilm containing extracellular polysaccharides, thereby promoting tooth demineralization and caries development. Besides Streptococcus mutans, Candida albicans (…) is another common caries-causing pathogen. Candida albicans ) can work with Streptococcus mutans to promote biofilm stability, Porphyromonas gingivalis ( Porphyromonas gingivalis Oral pathogens, such as bacteria, can also affect the local microecological environment of the oral cavity. Therefore, ecological regulation targeting various oral pathogens and their biofilm formation processes is an important direction for the development of oral care products and functional probiotic products.

[0003] Traditional oral care and caries prevention strategies typically include mechanical removal, fluoride preparations, chemical antimicrobials, and behavioral management. These methods can reduce plaque load on tooth surfaces or promote remineralization to some extent. However, the effectiveness of mechanical removal depends on user compliance, and long-term use of chemical antimicrobials may affect the balance of oral flora, with limited ability to sustainably regulate biofilms in hidden areas. In recent years, lactic acid bacteria and their fermentation products have been increasingly used in the development of oral probiotics and oral care products due to their relatively safe origin and ability to regulate the microecology through metabolites or competitive colonization.

[0004] Previous studies have shown that some *Lactobacillus plantarum* strains can inhibit oral microorganisms such as *Streptococcus mutans* or *Candida albicans*, and affect biofilm formation under certain conditions. However, significant differences exist in the functional characteristics among different strains, and strains capable of simultaneously targeting *Streptococcus mutans*, *Candida albicans*, and *Porphyromonas gingivalis* still require further screening. For strains suitable for the oral environment, it is also necessary to comprehensively consider their tolerance to oral environmental factors such as lysozyme, their interaction with oral pathogens, the composition of fermentation products, their biofilm regulation capabilities, and their safety for in vivo use.

[0005] Therefore, there is an urgent need to develop a strain of Lactobacillus plantarum with a clear strain source that can be used in oral care products or functional foods, as well as related preparations, so that it can inhibit a variety of oral pathogens and interfere with the formation of caries-related biofilms in non-therapeutic use scenarios, and provide new strain resources and productization basis for oral microecological regulation. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Lactobacillus plantarum YN-63 and its applications, in order to solve the problems in existing oral care products, such as the limited number of candidate strains that can be used for the ecological regulation of various oral pathogens, the narrow antibacterial spectrum of some strains, and the lack of evidence on the active ingredients of fermentation products and biofilm regulation.

[0007] The core of this invention lies in: screening and obtaining a strain of *Lactobacillus plantarum* YN-63, and using this strain, microbial preparations containing this strain, its cell-free fermentation supernatant, and oral care products, functional foods, or probiotic products containing the above-mentioned components for non-therapeutic purposes of oral microecological regulation. This strain and its fermentation products can inhibit a variety of oral pathogenic bacteria and have the basis for application as active ingredients in oral care products or functional foods.

[0008] To achieve the above objectives, the present invention adopts the following technical solution.

[0009] In a first aspect, the present invention provides a strain of *Lactobacillus plantarum* (… Lactiplantibacillus plantarum Lactobacillus plantarum YN-63, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39525 and is classified as Lactobacillus plantarum (YN-63). Lactiplantibacillus plantarum ).

[0010] Secondly, the present invention provides a microbial preparation comprising the aforementioned *Lactobacillus plantarum* YN-63.

[0011] In some embodiments, the *Lactobacillus plantarum* YN-63 exists in the form of live bacteria, inactivated cells, cultures, lyophilized powder, or a combination thereof.

[0012] In some embodiments, the microbial preparation further comprises orally acceptable, food-acceptable, or pharmaceutically acceptable excipients. The excipients may be selected from one or more of the following, depending on the product form: carrier, diluent, excipient, sweetener, humectant, thickener, stabilizer, buffer, flavoring agent, flavoring agent, preservative, disintegrant, filler, embedding material, or lyophilization protectant.

[0013] Thirdly, the present invention provides a cell-free fermentation supernatant of Lactobacillus plantarum YN-63, which is obtained by fermenting and culturing Lactobacillus plantarum YN-63 and removing the bacterial cells.

[0014] In some embodiments, the cell-free fermentation supernatant contains organic acids. Preferably, the organic acids include lactic acid and phenyllactic acid.

[0015] In some embodiments, the cell-free fermentation supernatant also contains protein components that can be degraded by proteinase K. These protein components can be characterized by a decrease in antibacterial activity after proteinase K treatment; however, this characterization does not preclude the presence of organic acids, short-chain fatty acids, phenylalanine metabolism-related products, or other fermentation metabolites in the cell-free fermentation supernatant.

[0016] Fourthly, the present invention provides an oral care product comprising one or more of the above-mentioned Lactobacillus plantarum YN-63, the above-mentioned microbial preparation, the above-mentioned cell-free fermentation supernatant, and oral-acceptable excipients.

[0017] In some embodiments, the oral care product is toothpaste, mouthwash, oral spray, oral gel, lozenges, chewing tablets, chewing gum, or oral probiotic powder.

[0018] Fifthly, the present invention provides a functional food or probiotic product, the functional food or probiotic product comprising one or more of the above-mentioned Lactobacillus plantarum YN-63, the above-mentioned microbial preparation, the above-mentioned cell-free fermentation supernatant, and food science acceptable excipients.

[0019] In a sixth aspect, the present invention provides the use of the above-mentioned Lactobacillus plantarum YN-63, the above-mentioned microbial preparation, or the above-mentioned cell-free fermentation supernatant in the preparation of products for non-therapeutic purposes to inhibit oral pathogenic bacteria, inhibit oral biofilm formation, reduce Streptococcus mutans colonization, and / or reduce the risk of tooth surface demineralization.

[0020] In some embodiments, the oral pathogens include at least one of Streptococcus mutans, Candida albicans, and Porphyromonas gingivalis. Preferably, the oral pathogens include Streptococcus mutans, Candida albicans, and Porphyromonas gingivalis.

[0021] In some embodiments, the product is an oral care product, a functional food, or a probiotic product.

[0022] The technical solution of the present invention can achieve the following beneficial effects:

[0023] 1. This invention provides a definitive strain of *Lactobacillus plantarum* YN-63. This strain was obtained through screening and confirmed as *Lactobacillus plantarum* by 16S rRNA gene sequence analysis. Lactiplantibacillus plantarum ( ), which can be used as strains for oral care products, functional foods or probiotic products.

[0024] 2. The *Lactobacillus plantarum* YN-63 provided by this invention has inhibitory effects against a variety of oral pathogens. Examples show that this strain has significant inhibitory effects on *Streptococcus mutans* and *Candida albicans*, and its cell-free fermentation supernatant also has an inhibitory effect on *Porphyromonas gingivalis*, making it suitable for oral microecological regulation scenarios that require simultaneous monitoring of bacteria, fungi, and anaerobic oral pathogens.

[0025] 3. The *Lactobacillus plantarum* YN-63 provided by this invention possesses tolerance and colonization-related characteristics suitable for the oral environment. Examples show that this strain can still grow under 20 mg / mL lysozyme conditions and exhibits high cell surface hydrophobicity and co-aggregation ability with *Streptococcus mutans* and *Candida albicans*, making it a suitable candidate strain for oral probiotics.

[0026] 4. The cell-free fermentation supernatant provided by this invention contains organic acids and protein-based antibacterial components. Examples show that the cell-free fermentation supernatant of YN-63 retains some antibacterial activity after pH adjustment to neutral, but the antibacterial activity decreases after treatment with proteinase K. Simultaneously, metabolomics and organic acid quantification results show that it contains lactic acid and phenyllactic acid. These results indicate that the effect of this supernatant is not solely dependent on a single acidic condition, but can be achieved through the combined participation of multiple fermentation products.

[0027] 5. The *Lactobacillus plantarum* YN-63 and its fermentation products provided by this invention can inhibit the formation of *Streptococcus mutans* biofilm. Examples show that the cell-free fermentation supernatant of YN-63 can reduce the biomass of *Streptococcus mutans* biofilm, inhibit the formation of water-insoluble extracellular polysaccharides, and disrupt the three-dimensional structure of the biofilm.

[0028] 6. The *Lactobacillus plantarum* YN-63 provided by this invention has an application basis for the development of oral care products for non-therapeutic purposes. Animal model results show that topical application of YN-63 bacterial suspension can reduce the level of *Streptococcus mutans* colonization in the oral cavity and alleviate tooth surface demineralization and caries-related manifestations; safety evaluation shows that it did not have adverse effects on rat body weight gain and major organ coefficients.

[0029] Biological Preservation

[0030] The Lactobacillus plantarum provided by this invention ( Lactiplantibacillus plantarum Lactobacillus plantarum YN-63 was deposited on June 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 39525. The depository address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (CGMCC). Its classification name is Lactobacillus plantarum. Lactiplantibacillus plantarum ). Attached Figure Description

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0032] Figure 1 The inhibitory effect of Lactobacillus plantarum YN-63 on oral pathogens is shown; where A is the inhibition zone against Candida albicans and Streptococcus mutans, and B is the concentration-dependent inhibition rate against Porphyromonas gingivalis.

[0033] Figure 2 A phylogenetic tree of Lactobacillus plantarum YN-63 constructed based on the 16S rRNA gene sequence is shown.

[0034] Figure 3 The physiological tolerance of Lactobacillus plantarum YN-63 is shown; where A represents tolerance to lysozyme, B represents survival rate in simulated gastric juice, and C represents survival rate in simulated intestinal juice.

[0035] Figure 4 The surface characteristics of Lactobacillus plantarum YN-63 are shown; where A represents the surface hydrophobicity and acid-base charge characteristics, and B represents the co-aggregation rate with Streptococcus mutans and Candida albicans.

[0036] Figure 5 The antioxidant capacity of cell-free fermentation supernatant and intact cell suspension of Lactobacillus plantarum YN-63 was demonstrated.

[0037] Figure 6 The analysis of the active components against Streptococcus mutans in cell-free fermentation supernatant of Lactobacillus plantarum YN-63 is shown; where A is the result of MIC and MBC determination, B is the antibacterial activity under different pH conditions, and C is the antibacterial activity after treatment with different concentrations of proteinase K.

[0038] Figure 7 The composition of organic acids and their antibacterial activity in the cell-free fermentation supernatant of Lactobacillus plantarum YN-63 are shown; where A is the absolute quantitative result of the major organic acids, and B is the minimum inhibitory concentration of representative organic acids against Streptococcus mutans.

[0039] Figure 8 The inhibitory effect of cell-free fermentation supernatant of Lactobacillus plantarum YN-63 on the biofilm of Streptococcus mutans is shown; where A is the crystal violet staining result, B is the biofilm inhibition rate, and C is the inhibitory effect of different concentrations of cell-free fermentation supernatant on water-insoluble extracellular polysaccharides.

[0040] Figure 9 The effects of treating cell-free fermentation supernatant of Lactobacillus plantarum YN-63 on the microscopic morphology of Streptococcus mutans and its biofilm are shown.

[0041] Figure 10 The results of real-time quantitative PCR detection of biofilm and virulence-related gene expression in Streptococcus mutans are shown.

[0042] Figure 11 The changes in the number of Streptococcus mutans in the oral cavity of rats in different groups of a rat caries model are shown.

[0043] Figure 12 The effect of Lactobacillus plantarum YN-63 on the morphology of dental caries in rats is shown; where A is the result of stereomicroscopic observation and B is the result of Micro-CT imaging.

[0044] Figure 13 The Keyes histopathological score of rat molar caries lesions is shown for Lactobacillus plantarum YN-63. Detailed Implementation

[0045] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0046] It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0047] In this invention, the term "Lactobacillus plantarum YN-63" refers to the Lactobacillus plantarum strain screened and obtained in this invention and intended to be deposited at the China General Microbiological Culture Collection Center. When formally submitting the application, the placeholder information in this document should be replaced with the actual deposit date and accession number.

[0048] In this invention, the term "cell-free fermentation supernatant" refers to the liquid fermentation product obtained from Lactobacillus plantarum YN-63 after fermentation culture, through centrifugation, filtration, or other methods capable of removing bacterial cells. The cell-free fermentation supernatant may contain organic acids, proteins, and other metabolites produced or released by the strain during fermentation.

[0049] In this invention, the term "oral pathogens" includes, but is not limited to, Streptococcus mutans, Candida albicans, and Porphyromonas gingivalis. Depending on the specific application, the oral pathogens may also include other microorganisms related to dental caries, periodontal disease, oral biofilms, or oral microecological imbalance.

[0050] In this invention, the term "non-therapeutic purpose" refers to oral care, oral cleaning, oral microecological maintenance, reducing the risk of tooth demineralization, reducing the oral pathogen load, or regulating oral biofilm formation, which are not directly aimed at disease diagnosis or treatment. The use for non-therapeutic purposes does not exclude the product's auxiliary role in maintaining oral health.

[0051] In some embodiments, *Lactobacillus plantarum* YN-63 can be used as a live bacterium in the product. The live bacterium can be prepared using conventional methods such as culturing, centrifugation, washing, resuspension, freeze-drying, or encapsulation.

[0052] In some embodiments, *Lactobacillus plantarum* YN-63 can be used as an inactivated bacterial cell in the product. The inactivated bacterial cell can be obtained through heat treatment, pressure treatment, drying treatment, or other methods that do not alter its basic cellular structure.

[0053] In some embodiments, *Lactobacillus plantarum* YN-63 can be used in the product as a culture or as a lyophilized powder. The culture may contain bacterial cells and residual culture medium components; the lyophilized powder may contain a lyophilization protectant, a carrier, or an excipient.

[0054] In some embodiments, the cell-free fermentation supernatant can be used alone as the active component, or in combination with live Lactobacillus plantarum YN-63, inactivated cells, lyophilized powder, or culture.

[0055] In some embodiments, the oral care product may be toothpaste, mouthwash, oral spray, oral gel, lozenges, chewing tablets, chewing gum, or oral probiotic powder. These product forms can be prepared using conventional processes in the art, and appropriate oral-acceptable excipients can be selected based on the product form.

[0056] In some embodiments, the functional food or probiotic product may be a solid beverage, compressed candy, probiotic powder, lozenges, chewable tablets, fermented food, or other food forms suitable for oral administration or oral retention. The functional food or probiotic product may contain *Lactobacillus plantarum* YN-63, a microbial preparation containing *Lactobacillus plantarum* YN-63, or a cell-free fermentation supernatant of *Lactobacillus plantarum* YN-63.

[0057] Example

[0058] Example 1: Screening and identification of Lactobacillus plantarum YN-63, which is resistant to oral pathogens.

[0059] In this embodiment, Candida albicans and Streptococcus mutans were used as indicator bacteria to screen lactic acid bacteria with anti-oral pathogenic bacteria activity from 178 lactic acid bacteria strains preserved in the laboratory.

[0060] The screening method employed the double-layer streak plate method: MRS medium containing 1.5% agar was poured into a petri dish as the bottom layer. After solidification, activated lactic acid bacteria strains were inoculated along the marked lines and incubated at 37°C for 48 h. Subsequently, YPD medium or BHI medium containing 0.7% agar was mixed with *Candida albicans* or *Streptococcus mutans*, respectively, and poured onto the surface of the bottom culture. Incubation was continued at 37°C for 12–24 h, and the diameter of the transparent inhibition zone was measured to evaluate the antibacterial effect of the lactic acid bacteria strains.

[0061] Through the above screening, strain YN-63 was obtained. The results showed that the inhibition zone diameter of this strain against Candida albicans was 32.19 mm, and the inhibition zone diameter against Streptococcus mutans was 46.58 mm. Figure 1 A).

[0062] The inhibitory activity of YN-63 cell-free fermentation supernatant against *Porphyromonas gingivalis* was further evaluated using the micro-broth dilution method. The results showed that the inhibitory effect of YN-63 cell-free fermentation supernatant on the growth of *Porphyromonas gingivalis* was concentration-dependent, with an inhibition rate of 17.88% in the 5% (v / v) treatment group and 30.51% in the 10% (v / v) treatment group. Figure 1 B).

[0063] The 16S rRNA gene of strain YN-63 was amplified and sequenced, and a phylogenetic tree was constructed. Figure 2 The results showed that this strain belongs to *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum ).

[0064] The proposed Lactobacillus plantarum YN-63 is to be deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 29, 2026, with accession number CGMCC No. 39525.

[0065] This embodiment illustrates that Lactobacillus plantarum YN-63 is a strain of Lactobacillus plantarum that has inhibitory effects on a variety of oral pathogens, and can be used as the strain basis for the microbial preparations, oral care products, functional foods or probiotic products of the present invention.

[0066] Example 2: Evaluation of the tolerance, surface characteristics and antioxidant capacity of Lactobacillus plantarum YN-63

[0067] (1) Lysozyme tolerance evaluation

[0068] The tolerance of *Lactobacillus plantarum* YN-63 to lysozyme was determined using the agar diffusion method, with appropriate adjustments made to the method established by Schillinger et al. Specifically, YN-63 bacterial culture in the logarithmic growth phase (OD...) was... 600= 1.0 ± 0.05) was inoculated at a v / v ratio and thoroughly mixed with MRS medium containing 0.7% agar at approximately 50°C. The mixture was then poured into sterile petri dishes to prepare bacterial plates. After the plates had completely solidified, five equidistant wells were punched using a sterile Oxford cup (8 mm outer diameter). 25 μL of different concentrations of lysozyme working solution (5, 10, 15, and 20 mg / mL, prepared with PBS) sterilized through a 0.22 μm filter was added to each well. An equal volume of sterile distilled water was added to the control well. All plates were incubated at 37°C for 24 h to observe the formation of inhibition zones under lysozyme action. The laboratory-preserved strain of *Lactobacillus MY-71* (lysozyme-resistant, positive control) was used as a reference and was measured simultaneously. Each experiment was independently repeated three times.

[0069] The results showed that no transparent inhibition zone appeared around strain YN-63 under conditions of 0-20 mg / mL lysozyme. Figure 3 A). The concentration of lysozyme in human saliva is usually 0.1-0.3 mg / mL, while YN-63 can still grow normally at a lysozyme concentration as high as 20 mg / mL, indicating that it can maintain strong growth activity in a high concentration of lysozyme environment, showing good tolerance and having the potential to resist the host's innate immune factors in the oral environment.

[0070] (2) Evaluation of tolerance to simulated gastrointestinal fluid

[0071] Activated *Lactobacillus plantarum* YN-63 bacterial suspension was collected by centrifugation at 4 ℃ and 6000×g for 5 min, and the cells were resuspended in sterile physiological saline. Simulated gastric fluid was prepared using PBS buffer, with the pH adjusted to 2.5, 3.0, and 4.0 respectively using HCl. Pepsin, sterilized by 0.22 μm filtration, was then added to achieve a final concentration of 3 g / L. The bacterial suspension was inoculated into simulated gastric fluids at different pH values, and the initial OD was adjusted. 600 The culture was adjusted to 1.0 ± 0.05 and incubated at 37°C with constant temperature and shaking. 100 μL samples were taken at 0 h and 3 h, serially diluted with sterile PBS buffer, plated on MRS solid medium, and incubated at 37°C for 48 h before viable cell counting. Three h after gastric fluid treatment, 100 μL of bacterial culture was transferred to 900 μL of simulated intestinal fluid (based on pH 8.0 PBS containing 1 g / L sterile filtered trypsin) and incubated at 37°C for another 4 h. 100 μL samples were taken at 0 h and 4 h after intestinal fluid treatment, appropriately diluted, plated on MRS plates, and incubated at 37°C for 48 h before colony counting. The survival rate of the strains under each treatment condition was calculated using the following formula:

[0072] Strain survival rate (%) = Log CFU N1 / Log CFU N0 × 100%

[0073] Where N0 is the total number of viable bacteria before treatment, and N1 is the total number of viable bacteria after treatment with simulated gastrointestinal fluid.

[0074] The results showed that after 3 hours of treatment in simulated gastric fluid, the survival rates of the bacterial strains were pH-dependent, at 85.63%, 88.12%, and 90.49%, respectively, with the viable bacterial concentration in each treatment group remaining at 10. 9 The concentration of CFU / mL was above 100%, indicating that the strain maintained high metabolic activity and survival ability even under strongly acidic conditions. After being treated with gastric juice, the bacterial cells were transferred to simulated intestinal fluid (pH 8.0) containing 1 g / L trypsin and cultured for another 4 h. The survival rate of the strain reached 91.58%-94.87%, and the viable count remained stable at 10⁻⁶. 8 Effective concentration of CFU / mL ( Figure 3 (BC), demonstrating that YN-63 can withstand trypsin hydrolysis and weak alkaline pressure in the intestinal environment, maintaining a sufficient number of live bacteria throughout the digestive process.

[0075] The above results indicate that YN-63 has good gastrointestinal tolerance and can adapt to the complete physiological transition from the gastric acid environment to the intestinal fluid environment. It maintains a high survival level in all stages of digestion, providing reliable experimental evidence for its oral application and in-depth research on the "gut-mouth axis" related mechanisms.

[0076] (3) Hydrophobicity and surface acid-base charge

[0077] The hydrophobicity of bacterial cell surfaces was quantitatively assessed using the microbial adhesion hydrocarbon method (MATH). The specific procedure was as follows: *Lactobacillus plantarum* YN-63 bacterial suspension in the logarithmic growth phase was centrifuged at 4 °C and 6000 × g for 5 min to collect the bacterial pellet. The pellet was washed twice with PBS and resuspended. The OD value of the bacterial cells was adjusted. 600 = 1.0 ± 0.05 A1). Measure 3 mL of bacterial suspension and mix with 1.0 mL of analytical grade xylene. Incubate at 37 ℃ for 10 min, then vortex vigorously for 2 min to ensure thorough mixing. Allow to stand at room temperature for 15 min to complete phase separation. Carefully aspirate the lower aqueous phase, avoiding disturbance of the organic phase, and measure its absorbance at 600 nm using a microplate reader (denoted as A2). The hydrophobicity index (HPHBI) is calculated using the following formula. Three biological replicates are required for each sample:

[0078] HPBI (%) = [A1- A2] / A1 × 100%

[0079] Microbial Adhesion to Solvents (MATS) was used to analyze the amphoteric properties of bacterial cell surfaces. This method indirectly reflects the surface charge distribution by measuring the affinity of bacterial cells for electron donor and acceptor solvents. Bacterial cultures in the logarithmic growth phase were centrifuged at 4 °C and 6000 × g for 5 min. The bacterial precipitate was collected, washed twice with PBS, and resuspended. The OD of the bacterial suspension was adjusted. 600 = 0.60 ± 0.05. Take 3 mL of bacterial suspension and mix it with 1 mL of chloroform (electron acceptor solvent) or ethyl acetate (electron donor solvent) in sterile test tubes. Vortex for 2 min, then let stand at room temperature for 15 min until the two phases are completely separated. Carefully aspirate the aqueous phase and measure the absorbance at 600 nm (denoted as A1). By comparing the difference in adhesion rates between the two solvents, the relative contributions of electron donors (basic) and electron acceptors (acidic) on the bacterial surface are determined. The adhesion rate is calculated using the following formula, and the experiment is independently repeated three times:

[0080] Adhesion rate (%) = [A0 – A1] / A0 × 100%

[0081] The results showed that YN-63 had an affinity of 83.15% for xylene, an adhesion rate of 93.78% for chloroform, and an adhesion rate of 70.44% for ethyl acetate. Figure 4 A) The high xylene affinity indicates that the cell surface of this strain is strongly hydrophobic. According to MATS results, YN-63 showed high adhesion rates to both chloroform and ethyl acetate, suggesting the presence of both acidic and basic sites on its cell surface, with a higher affinity for chloroform, indicating that acidic sites are relatively dominant. Overall, YN-63 possesses strong hydrophobicity and specific acid-base charge characteristics, exhibiting certain amphoteric surface features and strong initial adhesion potential. These surface characteristics contribute to its colonization potential during initial contact with the host mucosa and acquired membranes on the tooth surface.

[0082] (4) Co-aggregation ability

[0083] The interaction between lactic acid bacteria and Streptococcus mutans and Candida albicans was evaluated using an in vitro co-aggregation assay: the three strains were cultured to the logarithmic growth phase, and the bacterial cells were collected by centrifugation at 4 ℃ and 6000×g for 5 min. The cells were washed twice with PBS and resuspended to adjust the concentration to OD. 600= 0.50 ± 0.05. Subsequently, 1.5 mL of lactic acid bacteria suspension and an equal volume of pathogenic bacteria suspension were placed in a sterile EP tube and gently mixed. At the same time, a self-aggregation control group for each strain was set up (1.5 mL of bacterial suspension mixed with an equal volume of PBS). All systems were incubated at 37°C for 24 h to promote aggregation. After the reaction, the mixture was allowed to stand at room temperature for 10 min to allow the aggregates to settle completely. The supernatant was carefully aspirated and the absorbance at 600 nm (Am) was measured. The co-aggregation rate was calculated according to the following formula, where Ax and Ay are the initial OD values ​​of lactic acid bacteria and pathogenic bacteria, respectively. The experiment was performed in three biological replicates and each sample was measured three times. At the same time, the self-aggregation rate of all tested strains was required to be less than 15% to ensure the accuracy of the results.

[0084] Copolymerization rate (%) = [(Ax + Ay) - 2Am] / [Ax + Ay] × 100%

[0085] Experimental results showed that the co-aggregation rate of YN-63 with Streptococcus mutans was 34.32%, and the co-aggregation rate with Candida albicans was 43.38%. Figure 4 (B) These results indicate that YN-63 can co-aggregate with both pathogens to some extent, and its co-aggregation ability with Candida albicans is higher than that with Streptococcus mutans. Overall, YN-63 showed a moderate level of co-aggregation ability with both Streptococcus mutans and Candida albicans in the co-aggregation experiment, suggesting that it may inhibit the colonization and spread of these two types of pathogens through physical interactions.

[0086] (5) Evaluation of antioxidant capacity

[0087] To systematically evaluate the antioxidant activity of lactic acid bacteria, this study employed three in vitro free radical scavenging systems with complementary chemical properties: DPPH free radical scavenging, ABTS free radical scavenging, and ABTS free radical scavenging. + Free radical and hydroxyl radical scavenging assays were performed on 48-h CFS and intact bacterial cell suspensions (OD). 600 Parallel determinations were performed (n = 1.0 ± 0.05). All experiments were set up with three biological replicates (n = 3), and each replicate was performed in three technical parallel determinations. Data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS 26.0 software. Tukey's HSD test was used for multiple comparisons between groups, and the significance level was set at p < 0.05.

[0088] ① DPPH free radical scavenging activity assay

[0089] Take 1 mL of the test sample and 1 mL of DPPH stock solution and vortex to mix in a sterile centrifuge tube. Incubate at 25°C in the dark for 30 min, then centrifuge at 4°C and 10,000×g for 5 min to obtain the supernatant. Measure the absorbance at 517 nm using an ELISA reader. Three control groups were set up: blank group (with anhydrous ethanol instead of the sample), sample background group (with ethanol instead of DPPH solution), and negative control group (DPPH solution + ethanol).

[0090] DPPH clearance rate (%) = [1 - [Sample A – Sample A background] / Blank group] × 100%

[0091] ② ABTS + Free radical scavenging capacity determination

[0092] Mix 7 mmol / L ABTS with 2.45 mmol / L potassium persulfate aqueous solution at a 1:1 (v / v) ratio and incubate at room temperature in the dark for 12–16 h to prepare ABTS⁺ stock solution. Before use, dilute with distilled water to a working solution with an absorbance of 0.70 ± 0.02 at 734 nm. Take 80 μL of sample and 1.6 mL of working solution, vortex for 20 s to mix, react at room temperature in the dark for 6 min, and then measure the absorbance at 734 nm. Use sterile water as a blank control.

[0093] ABTS + Clearance rate (%) = [1 – A sample / A blank] × 100%

[0094] ③ Hydroxyl radical scavenging activity assay

[0095] To initiate the reaction, add 0.5 mL of 0.75 mmol / L 1,10-o-phenanthroline, 1 mL of 0.15 mol / L PBS, 0.5 mL of sample, 0.5 mL of 0.75 mmol / L FeSO4, and 0.5 mL of 0.01% H2O2 sequentially to a 5 mL EP tube. After incubation at 37°C for 60 min, measure the absorbance at 536 nm. For the blank group, distilled water was used instead of H2O2, and for the control group, distilled water was used instead of the sample.

[0096] Hydroxyl radical scavenging rate (%) = [Sample A – Control A] / [Blank A – Control A] × 100%

[0097] The results showed that ( Figure 5YN-63 CFS exhibited significant scavenging capabilities in all three free radical systems. Specifically, CFS scavenged 49.93% of DPPH radicals, 51.61% of hydroxyl radicals, and 71.82% of ABTS⁺ radicals. These data indicate that the extracellular metabolites of YN-63 possess good multi-target free radical scavenging properties. In contrast, the antioxidant activity of intact cell suspensions was significantly lower than that of CFS. Specifically, intact cell suspensions scavenged 11.85% of DPPH radicals and 21.77% of hydroxyl radicals, while scavenging ABTS⁺ radicals was significantly lower. + No significant free radical scavenging effect was observed. The results indicate that the antioxidant capacity of *Lactobacillus plantarum* YN-63 primarily derives from its extracellular metabolites, rather than from structural components of the cell itself. This strain demonstrates strong potential in secreting antioxidant substances, suggesting that it may exert its biological functions through the synthesis and secretion of secondary metabolites with antioxidant properties. This finding supports the potential application of YN-63 as an oral probiotic and provides experimental evidence for further research on its antioxidant mechanisms.

[0098] Example 3: Analysis of cell-free fermentation supernatant of Lactobacillus plantarum YN-63 and its active components against Streptococcus mutans

[0099] (1) The CFS of lactic acid bacteria against the MIC and MBC of Streptococcus mutans after 48 h of culture was determined by the microbroth dilution method. The experiment was independently repeated three times, with two technical parallels for each experiment. The specific procedure was as follows: the fermentation broth of Lactobacillus plantarum YN-63 cultured for 48 h was centrifuged (8000×g for 10 min) and filtered (0.22 μm aqueous filter membrane) to obtain CFS. The CFS was serially diluted with BHI broth to a final concentration of 0%, 10%, 15%, 20%, 25%, and 30% (v / v) in a sterile 96-well polystyrene microplate to 200 μL per well. Then, Streptococcus mutans in the logarithmic growth phase (OD) was added to the well. 600 = 1.0 ± 0.05) was added at a 1% inoculum volume; no bacterial suspension was added to the control group. After incubation at 37 ℃ and 5% CO2 for 48 h, the lowest concentration at which the liquid was clear and transparent and no bacterial growth was observed at the bottom of the well was taken as the MIC value. Subsequently, 10 μL of the mixture was taken from all wells with no growth above the MIC and inoculated onto BHI agar plates. The plates were then incubated at 37 ℃ and 5% CO2 for another 48 h to reduce the colony count by 99.9% (i.e., < 5 CFU or ≥ 3 log) compared to the initial inoculum volume. 10 The lowest CFS concentration is the MBC value.

[0100] The results showed that after 48 h of culture, when the CFS concentration reached 25% or higher, the OD of each treatment well was [data missing]. 600 The value decreased significantly and stabilized, the liquid in the wells became clear, and no obvious bacterial deposition was observed, indicating that 25% was the MIC. Further, the culture medium from the 30% CFS treatment group was plated on BHI agar plates, and after 48 h of incubation, no colony growth was observed, indicating that 30% was the MBC (microbial culture). Figure 6 A). The above results indicate that the CFS of YN-63 has a concentration-dependent inhibitory effect on Streptococcus mutans, effectively inhibiting its growth at a concentration of 25% and achieving a bactericidal effect at a concentration of 30%.

[0101] (2) pH sensitivity experiment

[0102] The 48-hour CFS of YN-63 was prepared using the same method as described above. The CFS was titrated to pH 4.0, 5.0, 6.0, and 7.0 using 1 mol / L NaOH solution, with two control groups: ① activity control: unadjusted original CFS; ② substrate control: sterilized MRS medium adjusted to the same pH. Each sample was serially diluted in BHI broth at 0%, 5%, 10%, 20%, and 30% (v / v), and inoculated with 1% (v / v) of *Streptococcus mutans* (OD) in the logarithmic growth phase. 600 = 1.0 ± 0.05), and incubated in 96-well plates at 37℃ and 5% CO2 for 24 h. After incubation, the absorbance at 600 nm was measured using a microplate reader, and the inhibition rate of each treatment group against the growth of Streptococcus mutans was calculated.

[0103] Inhibition rate (%) = [1 - (experimental group OD)] 600 / Corresponding pH matrix control group OD 600 )] × 100%

[0104] Under acidic conditions (pH 3.8, CFS stock solution pH) and pH 4.0, the antibacterial activity of CFS showed a clear concentration-dependent effect with increasing concentration. In the 30% CFS treatment group, the inhibition rate was close to 100%. When the pH increased to 5.0, 6.0, and 7.0, the antibacterial activity of CFS decreased significantly, with the inhibition rate at the 30% concentration not exceeding 30%, and no obvious concentration-dependent trend was observed. This result indicates that under acidic conditions, the organic acids in CFS mainly exist in an undissociated form, which can effectively penetrate the bacterial cell membrane, thus playing a dominant antibacterial role. However, under higher pH conditions, the degree of dissociation of organic acids increases, membrane penetration decreases, leading to a weakening of antibacterial efficacy. Nevertheless, CFS still retains approximately 20% residual antibacterial activity under neutral conditions. Figure 6 B) suggests that it may contain non-acidic antibacterial ingredients that are not sensitive to pH.

[0105] (3) Proteinase K sensitivity test

[0106] To assess the protein properties of the active ingredient, CFS was co-incubated with different concentrations of proteinase K. The final concentrations of proteinase K in each treatment group were set at 0.5, 1.0, and 2.0 mg / mL, respectively. All samples were incubated at 37°C for 2 h, followed by treatment in a boiling water bath for 10 min to terminate the enzyme reaction. All samples were added to BHI medium at a final concentration of 10% (v / v), and three control groups were set up simultaneously: blank control group (BHI + bacterial suspension), nutrient control group (BHI + 10% sterile MRS + bacterial suspension), and activity control group (BHI + 10% untreated CFS + bacterial suspension). Each group was inoculated with 1% (v / v) of Streptococcus mutans (OD) in the logarithmic growth phase. 600 = 1.0 ± 0.05) and then incubated at 37℃ and 5% CO2 for 24 h, and the OD was measured. 600 The study aimed to quantify bacterial growth and calculate the retention rate of protease activity in samples after treatment, thereby evaluating the contribution of protein components to the overall antibacterial activity. All experiments were performed in triplicate, and data are presented as mean ± standard deviation.

[0107] The results showed that the antibacterial activity of the cell-free fermentation supernatant decreased with increasing proteinase K concentration, and the highest enzyme concentration treatment resulted in a loss of approximately 45% of the antibacterial activity. Figure 6 C).

[0108] (4) Metabolomics analysis

[0109] Untargeted metabolomics analysis was performed on the cell-free fermentation supernatant of Lactobacillus plantarum YN-63. The results showed that the cell-free fermentation supernatant of YN-63 was significantly different from the culture medium control group. A total of 787 differential ion peaks were screened, of which 312 differential metabolites were successfully annotated; organic acids and their derivatives accounted for a high proportion of the differential metabolites.

[0110] (5) Absolute quantitative analysis of organic acids

[0111] The main organic acids in the cell-free fermentation supernatant were analyzed by absolute quantification using gas chromatography-mass spectrometry. The results showed that the lactic acid content was 12,400 ± 850 μmol / L, and the phenyllactic acid content was 418 ± 32 μmol / L. Figure 7 A). The results of the antibacterial activity assay for representative organic acids showed that the MIC of phenyllactic acid against *Streptococcus mutans* was 1.25 mg / mL, while the MICs of lactic acid and hexanoic acid against *Streptococcus mutans* were 2.5 mg / mL. Figure 7 B).

[0112] (6) Whole genome sequencing and analysis

[0113] Whole-genome sequencing of *Lactobacillus plantarum* YN-63 was performed, and bacteriocinogen synthesis genes were predicted using the BAGEL4 database. The results showed that gene clusters related to bacteriocinogen synthesis were predicted in the YN-63 genome. Combined with the results of proteinase K sensitivity experiments, this suggests the possibility of the presence of protein-based antibacterial components in the cell-free fermentation supernatant of YN-63.

[0114] This embodiment illustrates that the cell-free fermentation supernatant of Lactobacillus plantarum YN-63 contains organic acids such as lactic acid and phenyllactic acid, and has proteinase K-sensitive antibacterial active ingredients, which can be used as active fermentation product components in oral care products, functional foods or probiotic products of the present invention.

[0115] Example 4: Effects of cell-free fermentation supernatant of Lactobacillus plantarum YN-63 on Streptococcus mutans biofilm

[0116] (1) Determination of biofilm biomass

[0117] The inhibitory effect of CFS on *Streptococcus mutans* biofilm formation was quantitatively assessed using crystal violet staining. *Streptococcus mutans* culture in the logarithmic growth phase (OD200) was used. 600 = 1.0 ± 0.05) was inoculated v / v into BHI medium containing 1% sucrose and 20% v / v CFS, with an equal volume of sterile MRS medium used as a negative control instead of CFS. 200 μL of the mixture was added to a 96-well plate and statically cultured at 37°C and 5% CO2 for 24 h. After incubation, the culture medium was carefully aspirated from the wells, and the plates were gently washed three times with pre-cooled PBS to remove unadhered airborne bacteria. Then, 100 μL of methanol was added for fixation for 15 min, and the fixative was discarded. The plates were then stained with w / v) crystal violet solution for 20 min. After staining, the plates were repeatedly rinsed with deionized water until colorless, and 100 μL of v / v) glacial acetic acid was added to dissolve the bound dye. After shaking at room temperature for 10 min, the absorbance was measured at 600 nm using a microplate reader. All experiments were performed in triplicate, and the CFS of strains XM and MY-71 was simultaneously measured for parallel comparison.

[0118] Biomembrane inhibition rate (%) = [1 - (experimental group OD)] 600 / Control group OD 600 )] × 100%

[0119] The staining results showed that the biofilm area and staining depth of the YN-63 CFS treatment group were lower than those of the control group and other strain treatment groups. The biofilm inhibition rate of the YN-63 CFS treatment group was significantly higher than that of the XM group and the MY-71 group. Figure 8 The differences (AB) were statistically significant. The results suggest that the metabolites secreted by YN-63 can effectively interfere with the formation of *Streptococcus mutans* biofilm.

[0120] (2) Determination of water-insoluble extracellular polysaccharides in biomembranes

[0121] The inhibitory effect of CFS on WI-EPS synthesis in biofilms was determined using the anthrone-sulfuric acid method. Biofilms were cultured in BHI medium containing different concentrations of CFS (0%, 5%, 10%, 20%, v / v) for 24 h. The biofilm precipitate was collected, sonicated (200 W, 2 s working, 3 s rest, 5 min total), and then centrifuged at 8000×g for 10 min at 4 °C. The precipitate was resuspended in PBS and washed twice, then extracted with 1.0 M NaOH solution in a 37 °C water bath with shaking for 2 h. The extract was mixed with freshly prepared anthrone-sulfuric acid reagent at a 1:4 volume ratio, heated in a boiling water bath for 5 min, and immediately cooled in an ice bath. The absorbance was measured at 625 nm. The WI-EPS content was calculated using a glucose standard curve (0-100 μg / mL).

[0122] WI-EPS inhibition rate (%) = [1 - (treatment group OD)] 625 / Control group OD 625 )] × 100%

[0123] The results showed that YN-63 CFS had a significant concentration-dependent inhibitory effect on the synthesis of WI-EPS. When the CFS concentration reached 30% (equivalent to its MBC), the inhibition rate approached 90%. Figure 8 C). This result indicates that, at bactericidal concentrations, YN-63 CFS can significantly inhibit the formation of the main structural components of the Streptococcus mutans biofilm matrix.

[0124] (3) Scanning electron microscopy observation of the morphology of planktonic cells and biofilms

[0125] The effects of cell-free fermentation supernatant treatment on the ultrastructure of *Streptococcus mutans* and its biofilm were observed using scanning electron microscopy. The results showed that after 24 h of treatment with 10% (v / v) and 20% (v / v) cell-free fermentation supernatant, the morphology of planktonic cells did not change significantly; however, the biofilm structure showed concentration-dependent damage. The 20% (v / v) treatment group showed reduced biofilm thickness, looser extracellular polymeric matrix, and impaired three-dimensional network structure integrity. Figure 9 ).

[0126] (4) Detection of biofilm and virulence-related gene expression

[0127] The effects of CFS treatment on the ultrastructure of *Streptococcus mutans* biofilm were observed using scanning electron microscopy. Sterile circular coverslips (10 mm in diameter) were placed in 24-well plates, and BHI medium containing 10% or 20% CFS was added, followed by inoculation with v / v *Streptococcus mutans* OD cells in the logarithmic growth phase. 600 = 1.0 ± 0.05 Incubated at 37℃ and 5% CO2 for 24 h. Coverslips were then fixed overnight with 2.5% glutaraldehyde at 4℃, followed by sequential dehydration with 30%, 50%, 70%, 80%, 90%, and 100% ethanol, each concentration for 15 min. After tert-butanol replacement, samples were freeze-dried in a vacuum freeze dryer for 24 h, sputter-coated with gold, and the three-dimensional structure of the biofilm was observed under a field emission scanning electron microscope with the assistance of Xi'an Nami Microbial Technology Co., Ltd. Samples containing an equal volume of sterile MRS medium were used as controls. Three independent biological replicates were set up for each treatment group, and three fields of view were randomly selected for image acquisition and analysis for each sample.

[0128] The results showed that, except for the stringent response regulator gene relA, all other detected genes were significantly upregulated. Figure 10 Based on the results of biofilm biomass, water-insoluble extracellular polysaccharides, and scanning electron microscopy, it is suggested that the cell-free fermentation supernatant of YN-63 may affect the formation of *Streptococcus mutans* biofilm by interfering with biofilm structure maintenance and stress regulation processes.

[0129] This embodiment illustrates that the cell-free fermentation supernatant of Lactobacillus plantarum YN-63 can inhibit the formation of Streptococcus mutans biofilm and can be used as an important fermentation product for non-therapeutic oral care purposes in this invention.

[0130] Example 5: Evaluation of the efficacy and safety of Lactobacillus plantarum YN-63 in a rat caries model

[0131] (1) Experimental scheme

[0132] Twenty-four 3-week-old male Sprague-Dawley rats were given distilled water containing 0.1% ampicillin on days 1-3 of the experiment to clear the oral flora. On day 4, the antibiotic water was replaced with ordinary distilled water to remove antibiotic residues. On day 5, the rats were randomly divided into four groups (n=6 per group): blank control group, model group (smeared with Streptococcus mutans), lactic acid bacteria intervention group (smeared with Streptococcus mutans and Lactobacillus plantarum YN-63), and drug control group (smeared with Streptococcus mutans + 0.12% CHX). Except for the blank control group, which was given ordinary maintenance diet and distilled water, the other groups were fed cariogenic diet Dirt 2000 and 5% sucrose water.

[0133] Starting from day 5, the lactic acid bacteria intervention group received daily topical application of YN-63 bacterial suspension (1×10⁻⁶) using sterile cotton swabs.9 200 μL of CFU / mL was applied to the surface of both maxillary teeth of rats for 15 seconds for 5 consecutive days; other groups received no treatment during the same period. On day 10, after sampling to verify successful YN-63 colonization, the model group, drug control group, and lactic acid bacteria intervention group were inoculated daily with Streptococcus mutans suspension (1×10⁻⁶ CFU / mL) from day 11 to day 18. 8 CFU / mL was applied to the surface of both maxillary teeth, 200 μL each time, for 15 seconds, for 8 consecutive days to induce caries formation. Oral microbial samples were collected periodically during the experiment for bacterial count.

[0134] From day 20, the lactic acid bacteria intervention group continued to receive daily application of YN-63 bacterial suspension, while the drug group received 0.12% CHX, with 200 μL evenly applied to both maxillary teeth each time. This continued for 5 days, then the maintenance treatment was switched to 3 times per week until the end of the experiment. On day 60, all animals were sacrificed, and maxillary bones and major organs were collected. The effects of each intervention on rat body weight, oral microbiota, and other factors were comprehensively evaluated using organ coefficients, microbial culture, Micro-CT analysis, and the Keyes histological scoring system.

[0135] Plaque samples were collected from the bilateral maxillary tooth surfaces of rats on days 19, 38, and 60 of the experiment. The samples were then spread on MSA medium for counting to evaluate the colonization level of Streptococcus mutans in the oral cavity.

[0136] (2) Monitoring of oral mutagenic streptococcal colonization level

[0137] Plaque samples were collected from the bilateral maxillary tooth surfaces of rats on days 19 (before intervention), 38 (mid-intervention), and 60 (end of experiment), and were smeared on MSA medium for counting. Results showed that on days 38 and 60, the bacterial load in both the YN-63 group and the 0.12% CHX group was significantly lower than that in the model group (p < 0.05), and there was no significant difference between the two groups. Figure 11 ).

[0138] (3) Stereomicroscopic observation, Micro-CT analysis and Keyes histopathological scoring

[0139] After the experiment, maxillary bones were collected, stained with 0.4% ammonium purpurate, and then sagittally incised along the mesiodistal direction of the molars. The extent and depth of caries in each molar section were observed under a stereomicroscope. The results showed that both the YN-63 group and the 0.12% CHX group showed some degree of brown deposition in the pit and fissure areas, but the degree of caries was significantly less than that in the model group, and there was no significant difference in morphology between the two groups. Figure 12 A).

[0140] Micro-CT three-dimensional reconstruction images and two-dimensional tomographic scans further confirmed the above observations: obvious demineralization was observed in the molar fissure area of ​​the model group, with a decrease in enamel density; while no obvious demineralization was observed in the YN-63 group and the 0.12% CHX group. Figure 12 B).

[0141] Keyes histopathological scoring showed that caries in the YN-63 group were mostly confined to the enamel or superficial dentin, with no lesions extending into the deep dentin; while in the model group, some samples showed caries that had invaded the deep dentin. Quantitative analysis showed that the YN-63 group had significantly lower scores than the model group in enamel (E), superficial dentin (Ds), deep dentin (Dx), and total score (p < 0.01), and no significant difference compared to the 0.12% CHX group. Figure 13 ).

[0142] (4) Safety analysis of Lactobacillus plantarum YN-63

[0143] The in vivo safety of *Lactobacillus plantarum* YN-63 was evaluated using an SD rat dental caries model. Throughout the experimental period, the rats in the YN-63 intervention group were in good condition, with no deaths or abnormalities observed. Weight monitoring and organ coefficient analysis showed no significant differences in weight and organ coefficient between the YN-63 group and the control group at both the pre- and post-experiment time points (p > 0.05), indicating that this strain did not adversely affect the growth and development of rats or their major organs.

[0144] Further genomic safety assessments revealed no predicted toxin-producing genes in the YN-63 genome, and the predicted resistance genes showed less than 75% similarity to known sequences. In conclusion, *Lactobacillus plantarum* YN-63 exhibits high biocompatibility.

[0145] The above description is only intended to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum YN-63, characterized in that, The Lactobacillus plantarum YN-63 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39525 and is classified as Lactobacillus plantarum.

2. A microbial preparation, characterized in that, The microbial preparation comprises Lactobacillus plantarum YN-63 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The Lactobacillus plantarum YN-63 exists in the form of live bacteria, inactivated bacterial cells, culture, lyophilized powder, or a combination thereof; Preferably, the microbial preparation further comprises orally acceptable excipients, food-acceptable excipients, or pharmaceutically acceptable excipients.

4. A cell-free fermentation supernatant of Lactobacillus plantarum YN-63, characterized in that, The cell-free fermentation supernatant was obtained by fermenting and culturing the *Lactobacillus plantarum* YN-63 as described in claim 1 and removing the bacterial cells.

5. The cell-free fermentation supernatant according to claim 4, characterized in that, The cell-free fermentation supernatant contains organic acids, including lactic acid and phenyllactic acid. Preferably, the cell-free fermentation supernatant also contains protein components that can be degraded by proteinase K.

6. An oral care product, characterized in that, The oral care product comprises one or more of the following: Lactobacillus plantarum YN-63 as described in claim 1, the microbial preparation as described in claim 2 or 3, the cell-free fermentation supernatant as described in claim 4 or 5, and oral-acceptable excipients.

7. The oral care product according to claim 6, characterized in that, The oral care products mentioned are toothpaste, mouthwash, oral spray, oral gel, lozenges, chewing tablets, chewing gum, or oral probiotic powder.

8. A functional food or probiotic product, characterized in that, The functional food or probiotic product comprises one or more of the following: Lactobacillus plantarum YN-63 as described in claim 1, the microbial preparation as described in claim 2 or 3, the cell-free fermentation supernatant as described in claim 4 or 5, and food-grade excipients.

9. The use of Lactobacillus plantarum YN-63 as described in claim 1, the microbial preparation as described in claim 2 or 3, or the cell-free fermentation supernatant as described in claim 4 or 5 in the preparation of products for non-therapeutic purposes that inhibit oral pathogens, inhibit oral biofilm formation, reduce Streptococcus mutans colonization, and / or reduce the risk of tooth surface demineralization.

10. The application according to claim 9, characterized in that, The oral pathogens include Streptococcus mutans (… Streptococcus mutans Candida albicans ( Candida albicans ) and Porphyromonas gingivalis ( Porphyromonas gingivalis At least one of the following; Preferably, the product is an oral care product, a functional food, or a probiotic product.

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