Streptococcus salivarius for producing bacteriocin and inhibiting oral pathogenic bacteria and postbiotic prepared from the same

Abstract

The application discloses a streptococcus salivarius for producing bacteriocin and inhibiting oral pathogenic bacteria and a prepared probiotic, and belongs to the technical field of microorganisms.The fermentation supernatant of the streptococcus salivarius provided by the application contains bacteriocin with an amino acid sequence of IMFKIVGLRYGMTFGILAGFLNMIP, which inhibits oral pathogenic bacteria through mechanisms such as destroying cell membrane integrity of the pathogenic bacteria and degrading intracellular DNA.Compared with traditional bacteriocin Nisin A, the bacteriocin produced by the strain can improve the antibacterial ability (minimum inhibitory concentration) on fusiform bacterium nucleatum and streptococcus mutans by 17-70 times, and has a wide application prospect in the fields of oral care, food health care, feed additives and the like.

Description

Technical Field

[0001] This invention relates to a bacteriocin-producing Streptococcus salivarius that inhibits oral pathogens and to a metabiotic prepared therefrom, belonging to the field of microbial technology. Background Technology

[0002] Streptococcus mutans is a major pathogen causing dental caries. It adheres to the tooth surface and forms a biofilm, then metabolizes and produces acid, leading to demineralization of the tooth structure and ultimately causing cavities. On the other hand, Fusobacterium nucleatum is a key pathogen causing periodontitis. It can damage periodontal supporting tissues through adhesion and colonization, tissue invasion, and the release of metabolic products, thus triggering periodontal inflammation. Furthermore, studies have shown that Fusobacterium nucleatum is also closely related to various oral diseases such as halitosis, periapical periodontitis, and oral cancer, posing a serious threat to overall oral health.

[0003] Common broad-spectrum antibiotics such as tinidazole and chlorhexidine are widely used in the prevention and treatment of oral diseases. Tinidazole is effective in inhibiting Fusobacterium nucleatum, while chlorhexidine has a strong bactericidal effect against Streptococcus mutans. However, long-term or widespread use of these drugs may disrupt the homeostasis of the oral microbiota and increase the risk of bacterial resistance. In contrast, probiotics that colonize the same ecological niche can directly inhibit the growth of pathogenic bacteria (such as Fusobacterium nucleatum and Streptococcus mutans) by secreting antagonistic substances such as bacteriocins, while also helping to maintain the balance of the oral microecology.

[0004] Patent CN113633666B reports the application of *Streptococcus salivarius* K12 as an oral microecological preparation. This bacterium can alleviate halitosis and inflammatory responses by reducing sulfide production, but it does not explicitly mention its direct inhibitory effect on *Fusobacterium nucleatum*. Another patent, CN115093989A, indicates that *Streptococcus salivarius* has inhibitory effects on various pathogenic bacteria (such as *Escherichia coli*, *Staphylococcus aureus*, *Salmonella*, and *Bacillus subtilis*), but its inhibitory scope does not cover *Fusobacterium nucleatum* and *Streptococcus mutans*. Therefore, screening probiotic strains that can target and inhibit *Fusobacterium nucleatum* and *Streptococcus mutans*, and identifying the specific antibacterial active substances they produce, is of great significance for developing oral health products targeting *Fusobacterium nucleatum* and *Streptococcus mutans*. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a bacteriocin-producing Streptococcus salivarius that inhibits the growth of oral pathogens, so as to solve the problem of the lack of oral symbiotic bacteria strains that can directly inhibit the growth of oral pathogens in the prior art.

[0006] This invention provides a strain of Streptococcus salivarius (Salivarius) Streptococcus salivarius CCFM1469 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 17, 2025, with accession number GDMCC NO: 65820.

[0007] In one embodiment, the *Streptococcus salivarius* CCFM1469 is isolated from the dorsum of the tongue mucosa of a healthy person and has the following characteristics: The colonies of *Streptococcus salivarius* CCFM1469, after inoculation on BHI solid medium and cultured for 48 h, are generally white, raised, round, with a smooth, moist surface and neat edges. *Streptococcus salivarius* CCFM1469 is a Gram-positive, facultative anaerobic bacterium with an optimal growth temperature of 35-40℃ and an optimal growth pH of 5.5-7.0.

[0008] The present invention also provides a microbial preparation containing the said Streptococcus salivarius CCFM1469 or its postgenetic agent.

[0009] In one embodiment, the metabiotic includes, but is not limited to, one or more of the fermentation supernatant of *Streptococcus salivarius* CCFM1469, inactivated bacterial cells, or bacterial lysate species.

[0010] In one embodiment, the fermentation supernatant contains bacteriocins that inhibit Fusobacterium nucleatum and / or Streptococcus mutans.

[0011] In one embodiment, the bacteriocin contains the antimicrobial peptide IMFKIVGLRYGMTFGILAGFLNMIP (shown in SEQ ID NO.1).

[0012] In one embodiment, the content of *Streptococcus salivarius* CCFM1469 in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL.

[0013] The present invention also provides an antimicrobial peptide having the amino acid sequence IMFKIVGLRYGMTFGILAGFLNMIP (as shown in SEQ ID NO.1).

[0014] The present invention also provides a product containing the aforementioned Streptococcus salivarius CCFM1469, the aforementioned bacteriocin and / or the aforementioned antimicrobial peptide IMFKIVGLRYGMTFGILAGFLNMIP.

[0015] In one embodiment, the product is a drug, health product, daily chemical product, or feed additive.

[0016] In one embodiment, the product comprises the saliva streptococcus CCFM1469 and conventional excipients.

[0017] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0018] In one embodiment, the drug comprises the salicylic streptococcus CCFM1469 and a pharmaceutically permissible carrier.

[0019] In one embodiment, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0020] In one embodiment, the personal care products include toothpaste, mouthwash, and oral spray, and the personal care products do not contain live cells.

[0021] The present invention also provides the use of the aforementioned Streptococcus salivarius CCFM1469, or the fermentation supernatant, or the antimicrobial peptide in the preparation of a drug for inhibiting the growth of oral pathogenic bacteria.

[0022] In one embodiment, inhibiting the growth of oral pathogens includes disrupting cell membranes and / or disrupting pathogen DNA.

[0023] In one embodiment, the oral pathogens include Fusobacterium nucleatum and Streptococcus mutans.

[0024] In one embodiment, the site of infection includes the oral cavity.

[0025] Beneficial effects: This invention provides a strain of Streptococcus salivarius (Salivarius) Streptococcus salivarius The fermentation supernatant of CCFM1469 containing bacteriocins, isolated from the dorsum of the tongue mucosa of a healthy human oral cavity, exhibits antibacterial activity against oral pathogens, specifically manifested in: (1) Advantages in antibacterial effect: For Fusobacterium nucleatum, the MIC value of salivarius bacteriocin is 6 μg / mL, which is 98.6% lower than that of Nisin A (418.875 μg / mL); while for Streptococcus mutans, the MIC value of salivarius bacteriocin is 24 μg / mL, which is 94.2% higher than that of Nisin A (418.875 μg / mL); (2) It disrupts the cell membrane integrity of Fusobacterium nucleatum and Streptococcus mutans, thus inhibiting the growth of pathogenic bacteria; (3) It causes leakage of nucleic acid and protein from Fusobacterium nucleatum, damages the cell membrane, and inhibits the growth of Fusobacterium nucleatum; (4) It causes depolarization of the cell membrane of Streptococcus mutans and inhibits the growth of Streptococcus mutans; (5) It destroys the DNA of Fusobacterium nucleatum and Streptococcus mutans, inhibiting the growth of pathogenic bacteria.

[0026] This invention also screened and obtained the active antibacterial polypeptide IMFKIVGLRYGMTFGILAGFLNMIP from the postbiotic of Streptococcus salivarius CCFM1469, which has an inhibitory effect on both Fusobacterium nucleatum and Streptococcus mutans, and can be used to prepare pharmaceuticals or oral care products.

[0027] Therefore, Streptococcus salivarius ( Streptococcus salivarius CCFM1469, containing bacteriocin fermentation supernatant, achieves highly efficient inhibition of Fusobacterium nucleatum and Streptococcus mutans through a multi-target mechanism involving membrane damage and DNA intervention, and has a wide range of applications.

[0028] Preservation of biological materials Salinomyces ( Streptococcus salivarius (CCFM1469, categorized as follows) Streptococcus salivarius It was deposited on January 17, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65820, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0029] Figure 1 The liquid chromatogram for the preparation of bacteriocin semi-preparative salivarius from Streptococcus salivarius.

[0030] Figure 2 This is a mass spectrometry diagram of peptides from Streptococcus salivarius bacteriocins.

[0031] Figure 3 The effect of salivarius bacteriocins on the growth kinetics of pathogenic bacteria is shown; where a-indicator bacteria: Fusobacterium nucleatum; b-indicator bacteria: Streptococcus mutans.

[0032] Figure 4 The effects of salivarius bacteriocins on the cell membrane integrity of pathogenic bacteria are shown in the following diagrams: a - the microscopic state of *Fusobacterium nucleatum* without salivarius bacteriocin intervention; b - the microscopic state of *Fusobacterium nucleatum* without salivarius bacteriocin intervention; c - the microscopic state of *Streptococcus mutans* without salivarius bacteriocin intervention; and d - the microscopic state of *Streptococcus mutans* without salivarius bacteriocin intervention.

[0033] Figure 5 The effects of salivarius bacteriocins on the membrane potential of pathogenic bacterial cells were investigated; specifically, the effect of α-salivarius bacteriocins on the membrane potential of Fusobacterium nucleatum and the effect of β-salivarius bacteriocins on the membrane potential of Streptococcus mutans.

[0034] Figure 6The effects of salivarius bacteriocins on the in vitro nucleic acid and protein leakage of pathogenic bacteria were investigated; specifically, in vitro nucleic acid leakage assays were performed on b-Fusobacterium nucleatum, c-Fusobacterium nucleatum, and d-Fusobacterium mutagenesis.

[0035] Figure 7 The effect of bacteriocin intervention on DNA content of *Fusobacterium nucleatum* and *Streptococcus mutans* was investigated; where α-indicator bacteria: *Fusobacterium nucleatum*; β-indicator bacteria: *Streptococcus mutans*.

[0036] In this context, "*" indicates a statistically significant difference compared to the Model group; the difference levels of p < 0.05, p < 0.01, and p < 0.001 are represented by "*", "**", and "***", respectively. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The strains involved in the following embodiments are as follows: *Streptococcus salivarius* CCFM1469 is a strain obtained through screening in this invention; *Fusobacterium nucleatum* ATCC 23726 and *Streptococcus mutans* ATCC 25175 were purchased from ATCC.

[0038] The culture media involved in the following examples are as follows: Heme chloride and vitamin K1 stock solution: 0.5 g / L heme chloride, 1% (v / v) vitamin K1.

[0039] BHI liquid culture medium: tryptone 10.0 g / L, ox heart extract 17.5 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate 2.5 g / L, pH 7.2~7.4.

[0040] BHI solid medium: tryptone 10.0 g / L, ox heart extract 17.5 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate 2.5 g / L, agar 20.0 g / L, pH 7.2~7.4.

[0041] BHIL liquid medium (BHI with added heme chloride and VK): tryptone 10.0 g / L, ox heart extract 17.5 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate 2.5 g / L, 1% heme chloride, vitamin K1 stock solution (v / v), pH 7.2~7.4.

[0042] BHIL solid medium (BHI with added heme chloride and VK): tryptone 10.0 g / L, ox heart extract 17.5 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate 2.5 g / L, 1% heme chloride, vitamin K1 stock solution (v / v), agar 20.0 g / L, pH 7.2~7.4.

[0043] The sample preparation methods involved in the following examples are as follows: (1) Preparation of Fusobacterium nucleatum bacterial culture: A small amount of *Fusobacterium nucleatum* ATCC23726 bacterial suspension was taken with a sterile inoculation loop and activated in BHIL solid medium supplemented with 5% sheep blood. The culture was then anaerobic at 37°C (85% N2, 10% H2, 5% CO2) for 48 h. Subsequently, a single colony was picked and inoculated into BHIL liquid medium, and anaerobically cultured at 37°C for 24 h to prepare a seed culture. The prepared seed culture was then inoculated into BHIL liquid medium at a 4% (v / v) inoculation rate, anaerobically cultured at 37°C for 24 h, and the bacterial cells were collected by centrifugation and adjusted to a colony count of 1 × 10⁻⁶ using BHIL. 8 CFU / mL available for use.

[0044] (2) Preparation of Streptococcus mutans bacterial suspension: A small amount of *Streptococcus mutans* ATCC25175 bacterial suspension was taken with a sterile inoculation loop and activated in BHI solid medium, then anaerobically cultured at 37°C for 48 h. A single colony was then picked and inoculated into BHI liquid medium, and anaerobically cultured at 37°C for 18 h to prepare a seed culture. The prepared seed culture was then inoculated into BHI liquid medium at a 2% (v / v) inoculation rate and anaerobically cultured at 37°C for 18 h. The cells were then collected by centrifugation and adjusted to a colony count of 1 × 10⁻⁶ with BHI. 7 CFU / mL.

[0045] (3) Culture of Streptococcus salivarius CCFM1469 and preparation of its fermentation supernatant: For bacterial activation, a small amount of *Streptococcus salivarius* CCFM1469 culture was taken with a sterile inoculation loop and activated on BHI solid medium, and cultured at 37°C for 48 h. Then, a single colony was picked and inoculated into BHI liquid medium, and anaerobically cultured at 37°C for 18 h to prepare a seed culture. The prepared seed culture was then inoculated into BHI liquid medium at a 4% (v / v) inoculation rate, and anaerobically cultured at 37°C for 18 h, adjusting the colony count to 5 × 10⁻⁶. 9 The solution was centrifuged at 8000 r / min for 5 min to obtain the supernatant, which was then filtered through a 0.22 μm microporous membrane and stored at 4℃ for later use. Example 1: Screening and identification of Streptococcus salivarius CCFM1469 Strain screening: A swab was used to scrape the dorsum of the tongue from a healthy human oral cavity and placed in an EP tube containing 0.5 mL of sterile PBS for immediate bacterial isolation and culture. 0.2 mL of the spores was then added to 1.8 mL of sterile physiological saline to obtain 10... -1 Diluent, then take another 0.5 mL of 10 -1 The diluent was diluted in 4.5 mL of physiological saline to obtain 10 -2 Diluent, follow these steps to obtain 10 -3 10 -4 10 -5 10 -6 Serial dilution buffer. Take 10... -4 10 -5 10 -6 Add 1 mL of each diluent to a petri dish, pour in BHI solid medium, mix gently, and incubate at 37°C upside down for 48 h after the medium has solidified.

[0046] Strain identification: Single colonies were picked using a sterile inoculation loop and streaked onto BHI plates for purification. The purified single colonies were then inoculated into 10 mL of liquid culture medium and incubated at 37 ℃ for 18 h. 1.0 mL of the cultured bacterial suspension was centrifuged at 5000 r / min for 3 min, the supernatant was discarded, and the suspension was washed three times with 1.0 mL of sterile physiological saline and resuspended in 1.0 mL of sterile water. A 20 μL PCR system was prepared, containing 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), 10 μL of 2xTaq Mixture, 0.5 μL of bacterial suspension, and 8.5 μL of double-distilled water. Primer information is shown in Table 1. The obtained 16S sequence was analyzed using NCBI BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST) for species confirmation. The strain was identified as *Streptococcus salivarius* and named *Streptococcus salivarius* CCFM1469.

[0047] Table 1 Primer Information Table

[0048] Preservation of bacterial strains: For identified strains, take 1.0 mL of bacterial culture into a 2 mL preservation tube, centrifuge at 6000 r / min for 3 min, remove the supernatant in a clean bench, add 1 mL of 30% sterile glycerol, mix thoroughly with a vortex mixer, and store at -80℃.

[0049] Example 2: Isolation and purification of bacteriocin from Streptococcus salivarius CCFM1469 1. Crude isolation and antibacterial titer determination of bacteriocins Bacteriocins are proteins or polypeptides that have antibacterial or bactericidal activity, synthesized by bacterial strains through ribosomes during metabolism.

[0050] Bacteriocin was crudely separated from the fermentation supernatant of Streptococcus salivarius CCFM1469 by ultrafiltration, pH adsorption and solid phase extraction in sequence.

[0051] Ultrafiltration: First, rinse with 0.1 M NaOH, add 5 mL of fermentation broth supernatant and centrifuge at 4℃, 8000 r / min for 30 min. Check the concentration volume every 15 min until concentrated to 100 μL. Collect the <3 kDa concentrate and the >3 kDa concentrate, adjust to equal volume with sterile water, and then perform antibacterial experiments.

[0052] pH adsorption method: Take 1.6 L of fermentation broth, centrifuge to collect bacterial cells and fermentation supernatant separately. The bacterial cells were resuspended in 320 mL of sodium phosphate solution (one-fifth volume) and heated at 70 °C for 30 min to obtain inactivated cells. The inactivated cells were mixed with 800 mL of supernatant (2:1), the pH was adjusted to 6.0, and the culture was stirred at 4 °C for 24 h to allow bacteriocin adsorption onto the bacterial cells. The bacterial cells were collected by centrifugation and washed with 320 mL of 5.0 mM sodium phosphate buffer (pH 6.0) to remove impurities. After centrifugation at 8000 r / min for 30 min at 4 °C, the bacterial cells were resuspended in 160 mL of 5.0 mM sodium phosphate buffer (pH 6.0) containing 100 mM NaCl, and the pH was adjusted to 2.0. The bacterial suspension was stirred in a refrigerator (4 °C) for 12 h, after which the bacteriocin was desorbed and released into the supernatant. After centrifugation and filtration through a 0.22 µm filter membrane, 120 mL of the supernatant was collected, concentrated using a rotary evaporator, reconstituted with PBS to 24 mL, pH adjusted back to 6.0, filtered through a 0.22 μm filter membrane, and stored at -20 °C for later use.

[0053] Solid-phase extraction (SPE): A C18 solid-phase extraction column was used for desalting and enrichment. The specific steps are as follows: (1) Activation: Rinse with 3-5 mL of methanol and balance the column with 3-5 mL of water until no more liquid drips down; (2) Sample loading: Pass 1 mL of fermentation supernatant containing bacteriocin obtained by pH adsorption through the SPE column at a slow flow rate of 1 mL / min; (3) Washing: Wash away impurities with 3-5 mL of water; (4) Drying: Vacuum dry the SPE column (about 10 min); (5) Elution: Elute with 10 mL of methanol in multiple fractions to improve the recovery rate. Collect the concentrated solution, filter it through a 0.22 μm filter membrane, and store it at -20℃ for later use.

[0054] The crudely separated bacteriocin solution was diluted using a two-fold dilution method, and antibacterial experiments were conducted for each dilution gradient. The specific steps were as follows: The bacteriocin solution was diluted twofold with sterile water in a 96-well plate, with 100 μL of dilution buffer remaining in each well. 100 μL of *Fusobacterium nucleatum* (containing 1×10⁻⁶) was added to each well of the two-fold dilution buffer. 6 CFU) and Streptococcus mutans (containing 1×10) 5 CFU suspension was gently shaken to mix, and then incubated in a 37°C anaerobic incubator for 24 h and 18 h, respectively. The highest dilution that forms an inhibition zone is defined as one unit of activity (1 AU), and its reciprocal is the potency value (AU / mL) of the bacteriocin in each isolation and purification step. The highest dilution with sterile growth was observed, and the potency per milliliter (AU / mL) was calculated using the formula: Where n is the minimum inhibitory dilution number and x is the sample volume (μL).

[0055] Table 2. Effects of salivarius bacteriocins on the antibacterial titer of Fusobacterium nucleatum.

[0056] Table 3. Effects of bacteriocin from salivarius on the antibacterial potency of Streptococcus mutans.

[0057] The results are shown in Tables 2 and 3. After three-step purification treatment—ultrafiltration, pH adsorption, and solid-phase extraction—the activity of bacteriocins was significantly enhanced. Using *Fusobacterium nucleatum* as the indicator bacterium, the final specific activity after the final solid-phase extraction reached 1333.33 AU / mg, which was 76.67 times the initial value. Using *Streptococcus mutans* as the indicator bacterium, the final specific activity after solid-phase extraction reached 666.67 AU / mg, which was 76.63 times the initial value. These results indicate that this three-step purification method can effectively enrich the active components of bacteriocins.

[0058] 2. Chromatographic separation and identification of bacteriocins A C18 reversed-phase high-performance liquid chromatography column (5 µm, 10 × 10 mm) was used. The mobile phase consisted of water (phase A) and acetonitrile (phase B). From 0 min to 10 min, phase B linearly increased from 0% to 10%; from 10 min to 40 min, phase B linearly increased from 10% to 30%; and from 40 min to 50 min, phase B remained constant at 30%. The flow rate was set at 2.0 mL / min, the detection wavelength was 280 nm, and the injection volume was 500 μL. After collecting each chromatographic peak component, it was lyophilized and concentrated, reconstituted with PBS, and its antibacterial activity was determined by the Oxford cup method.

[0059] The results are as follows Figure 1As shown, two components, T1 and T2, were successfully isolated. Only component T1 showed inhibitory effects on Fusobacterium nucleatum and Streptococcus mutans, while T2 had no antibacterial activity.

[0060] The purified component T1 with antibacterial effect was sent to Shanghai Sangon Biotech Co., Ltd., and its amino acid sequence was identified using LC-MS / MS technology.

[0061] The results are as follows Figure 2 As shown, two peptides were detected in fraction T1 purified by high-performance liquid chromatography (HPLC), with amino acid sequences of TGFLMKAT and IMFKIVGLRYGMTFGILAGFLNMIP, respectively. A search of the Uniprot database revealed no known proteins matching these sequences. Further analysis showed that the molecular weights of these two peptides were 867.4524 Da and 2818.4988 Da, respectively. Based on these results, it is speculated that these two peptides may be novel bacteriocins.

[0062] Example 3: Determination of minimum inhibitory concentration (MIC) and inhibition curve MIC determination: MIC refers to the lowest drug concentration that can inhibit the growth of pathogenic bacteria after 18 to 24 hours of in vitro bacterial culture. It is an important indicator for measuring the antibacterial activity of antimicrobial drugs. The bacteriocin purified by solid-phase extraction was serially diluted with PBS and added to a suspension of *Fusobacterium nucleatum* (v / v = 1:1). After 24 hours of culture, the OD was measured using a microplate reader. 600 The concentration of bacteriocins at 1×MIC was determined using a BCA protein quantification kit. The blank group used PBS, and the control group used Nisin A, with concentrations of 3.344, 1.672, 0.836, 0.418, and 0.209 mg / mL, respectively. For *Streptococcus mutans*, the experimental procedures were the same as for *Fusobacterium nucleatum*, except for the 18-hour incubation period.

[0063] Table 4. MIC (μg / mL) of bacteriocinogens from salivary streptococci against pathogenic bacteria.

[0064] The results are shown in Table 4. Under the minimum inhibitory concentration (MIC) condition, salinomycin bacteriocin exhibited a lower inhibitory concentration than Nisin A. Against *Fusobacterium nucleatum*, the MIC of salinomycin bacteriocin was 6 μg / mL, 98.6% lower than that of Nisin A (418.875 μg / mL); while against *Streptococcus mutans*, the MIC of salinomycin bacteriocin was 24 μg / mL, 94.2% lower than that of Nisin A (418.875 μg / mL). These results indicate that, compared to Nisin A, salinomycin bacteriocin is more effective in inhibiting the growth of *Fusobacterium nucleatum* and *Streptococcus mutans*.

[0065] Antibacterial curve determination: After determining the MIC, bacteriocins (final concentrations of 1×MIC and 2×MIC) were added to a solution containing 10... 6 CFU contains Fusobacterium nucleatum or contains 10 5 CFU-containing Streptococcus mutans suspension was used as a blank control and anaerobic culture was performed at 37°C for 24 h, with OD recorded every 2 h. 600 Values, plot time-growth curves.

[0066] The results are as follows Figure 3 As shown, for *Fusobacterium nucleatum*, in the control group, it entered the logarithmic growth phase at 4 h and reached the stationary phase at 12 h; however, at concentrations of 1×MIC and 2×MIC, the absorbance values ​​of *Fusobacterium nucleatum* remained the same as the initial absorbance values, suggesting that bacteriocin can achieve antibacterial effects in a short time. For *Streptococcus mutans*, in the control group, it entered the logarithmic growth phase at 4 h and reached the stationary phase at 8 h; however, at concentrations of 1×MIC and 2×MIC, the absorbance values ​​of *Streptococcus mutans* also remained unchanged, suggesting that bacteriocin can also inhibit its growth in a short time. The experimental results show that a concentration of 1×MIC is sufficient to completely inhibit the growth of both *Fusobacterium nucleatum* and *Streptococcus mutans*.

[0067] Example 4: Scanning electron microscopy detection of pathogenic bacterial cell membrane integrity Salinomycin bacteriocin (final concentration 1×MIC) and 1×10 8 CFU contains Fusobacterium nucleatum and contains 1×10 7 CFU-mutant Streptococcus was incubated anaerobicly at 37°C for 1 h, with PBS as a blank control. Subsequently, the bacterial cells were washed twice with PBS and fixed with 2.5% glutaraldehyde at 4°C for 12 h. Finally, the cells were observed by scanning electron microscopy.

[0068] The results are as follows Figure 4As shown, in the blank control group, *Fusobacterium nucleatum* exhibited a typical rod-shaped structure with an intact cell membrane. After treatment with 1×MIC bacteriocin, *Fusobacterium nucleatum* cells showed significant shrinkage, with pores forming on the membrane surface and a large amount of intracellular material leaking out. *Streptococcus mutans* in the control group was a regular spherical shape with a smooth surface, but in the bacteriocin-treated group at the same concentration, its cell morphology changed significantly, with an incomplete membrane structure and pores. These results indicate that bacteriocin at this concentration exerts its bactericidal effect by disrupting the cell membrane integrity of *Fusobacterium nucleatum* and *Streptococcus mutans*.

[0069] Example 5: Determination of cell membrane permeability of pathogenic bacteria The effect of bacteriocins on the membrane potential of pathogenic bacteria was assessed using a DiSC3(5) fluorescent probe. A sample containing 10... 6 CFU contains Fusobacterium nucleatum or contains 10 5 CFU-mutant streptococcal suspension was incubated with 20 μM DiSC3(5) in the dark for 30 min. Bacteriocin solutions with final concentrations of 1×MIC and 2×MIC were then added, with sterile water and melitrix venom (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number 20449-79-0, 32 μg / mL, positive control) used as control groups. A fluorescence microplate reader (λ) was used. ex =622 nm, λ em =670 nm) Dynamic monitoring for 20 min (reading once every 2 min).

[0070] The results are as follows Figure 5 As shown, in the 1×MIC bacteriocin treatment group, the degree of membrane depolarization of *Fusobacterium nucleatum* was not significantly different from that of the blank control group, suggesting that its membrane damage mechanism may not depend on changes in membrane potential. In contrast, *Streptococcus mutans* showed significant membrane depolarization under both 1×MIC and 2×MIC bacteriocin treatment (p<0.05). However, the magnitude of this MIC change did not show a concentration-dependent relationship, indicating that the antibacterial effect of bacteriocin on *Streptococcus mutans* may be achieved through transient disruption of the cell membrane rather than continuous alteration of membrane potential.

[0071] Example 6: Determination of leakage of nucleic acid and protein from pathogenic bacteria in vitro Membrane damage was assessed by detecting extracellular macromolecule leakage. The bacterial cell concentration was 1×10⁻⁶. 8 CFU / mL of Fusobacterium nucleatum or a bacterial cell concentration of 1×10⁻⁶ 7 CFU / mL Streptococcus mutans suspension was incubated with salivarius bacteriocin at a final concentration of 1×MIC for 0, 2, 4, 8, 16, and 24 h under anaerobic conditions at 37°C. At each time point, the in vitro nucleic acid (OD) of the pathogenic bacteria was detected using Nanodrop. 260 ) and protein (OD) 280The absorbance change was observed, with PBS serving as the control group.

[0072] The results are as follows Figure 6 The results showed that in the 1×MIC bacteriocin treatment group, the extracellular nucleic acid and protein content of *Fusobacterium nucleatum* increased over time. From 0 h to 2 h, the extracellular nucleic acid and protein levels increased by 2031 μg / mL and 38 μg / mL, respectively, which were significantly higher than those in the control group at the same time point (p<0.05), suggesting that impaired cell membrane integrity led to leakage of intracellular substances. After 2 h, the extracellular nucleic acid and protein levels tended to stabilize. However, under the same concentration treatment, the extracellular nucleic acid (OD) of *Streptococcus mutans* was significantly higher. 260 ) and protein (OD) 280 The levels of salivarius bacteriocins were not significantly different from those in the blank control group. These results further validate that salivarius bacteriocins exert their antibacterial effect by disrupting the cell membrane structure of Fusobacterium nucleatum, while their antibacterial mechanism against Streptococcus mutans may depend on non-membrane damage pathways.

[0073] Example 7: Effects of salivarius bacteriocins on pathogenic bacterial DNA Genomic DNA was extracted from two pathogenic bacteria using a kit, and its purity (OD) was determined by Nanodrop. 260 / OD 280 After reaching a concentration of 1.88, the DNA was diluted to 60 μg / mL using 0.01 mol / L Tris-HCl buffer (pH 7.2). Subsequently, the diluted DNA was mixed with bacteriocin at final concentrations of 0, 0.5×MIC, 1×MIC, and 2×MIC, respectively, and incubated at 37°C in the dark for 10 min. The DNA was then analyzed using a fluorescence spectrometer (λ). ex =280 nm, λ em The fluorescence intensity changes of each group were measured at 300-700 nm (scanning slit width 5 nm) to assess the damaging effect of bacteriocins on DNA.

[0074] The results are as follows Figure 7 As shown, the fluorescence intensity of intracellular DNA in *Fusobacterium nucleatum* decreased in a concentration-dependent manner with increasing bacteriocin concentration. Compared to no bacteriocin intervention, the DNA content of *Fusobacterium nucleatum* decreased by 81.45% under 2×MIC treatment. The fluorescence intensity of intracellular DNA in *Streptococcus mutans* also decreased in a concentration-dependent manner. Under 2×MIC treatment, the DNA of *Streptococcus mutans* decreased by 43.68%. These results suggest that, in addition to exerting antibacterial effects by disrupting cell membrane structure, bacteriocins may also synergistically inhibit the growth of pathogenic bacteria by inducing DNA damage.

[0075] Example 8: Identification of the effective antibacterial peptide To determine the activity of the two peptides TGFLMKAT and IMFKIVGLRYGMTFGILAGFLNMIP obtained in Example 2, Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the two peptides. The purity of both peptides was >98%, and solutions of each peptide were prepared at a concentration of 200 μg / mL for later use. 100 μL of the prepared peptide solution (100 μL of sterile water was added to the control group) was placed in 96 wells, and an equal volume of *Fusobacterium nucleatum* (bacterial concentration 1×10⁻⁶) was added to each well. 8 CFU / mL) and Streptococcus mutans (concentration of 1×10⁻⁶ CFU / mL) 7 (CFU / mL), after incubation for 24 h, OD was determined by absorbance method. 600 The inhibition rate is calculated as follows: Inhibition rate (%) = (OD of control group) / ... 600 -Intervention group OD 600 ) / Control group OD 600 ×100%.

[0076] Table 5. Inhibitory effects of the two peptide segments on Fusobacterium nucleatum and Streptococcus mutans.

[0077] The results are shown in Table 5. The peptide TGFLMKAT had no in vitro antibacterial activity against Fusobacterium nucleatum and Streptococcus mutans, while the peptide IMFKIVGLRYGMTFGILAGFLNMIP inhibited Fusobacterium nucleatum and Streptococcus mutans by 42% and 28%, respectively. This indicates that IMFKIVGLRYGMTFGILAGFLNMIP is the active peptide in the bacteriocin CCFM1469 of Streptococcus salivarius.

[0078] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Streptococcus salivarius ( Streptococcus salivarius CCFM1469 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 17, 2025, with accession number GDMCC NO: 65820.

2. The metagene of *Streptococcus salivarius* CCFM1469 as described in claim 1.

3. A microbial preparation, characterized in that, Contains the salivary streptococcus CCFM1469 or its postgenetic agent as described in claim 1.

4. The microbial preparation according to claim 3, characterized in that, The metabiotic contains bacteriocins that inhibit Fusobacterium nucleatum and / or Streptococcus mutans.

5. An antimicrobial peptide, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

6. A product containing the *Streptococcus salivarius* CCFM1469 of claim 1, a bacteriocin prepared by fermentation of *Streptococcus salivarius* CCFM1469, and / or the antimicrobial peptide of claim 5.

7. The product according to claim 5, characterized in that, The products mentioned are pharmaceuticals, health products, daily chemical products, or feed additives.

8. The product according to claim 7, characterized in that, The drug contains the salicylic streptococcus CCFM1469 and a pharmaceutically permissible carrier.

9. The product according to claim 7, characterized in that, The aforementioned daily chemical products include toothpaste, mouthwash, or oral spray.

10. The use of the *Streptococcus salivarius* CCFM1469 of claim 1 or its fermentation supernatant, or the antimicrobial peptide of claim 5, in the preparation of a medicament for inhibiting the growth of oral pathogenic bacteria.

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