Lactobacillus salivarius LT017 and application thereof in preventing dental caries and / or removing dental plaque

Abstract

The application provides a lactobacillus salivarius LT017 and application thereof in preventing dental caries and / or removing dental plaque, and belongs to the technical field of microorganisms.The application provides the lactobacillus salivarius LT017, and the preservation number is CGMCC No.30724.The application further provides a preparation method of the lactobacillus salivarius LT017 bacterial powder, and application of the lactobacillus salivarius LT017 bacterial powder in preventing dental caries and / or removing dental plaque.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of Lactobacillus salivae LT017 and its application in preventing dental caries and / or removing dental plaque. Background Technology

[0002] Dental caries, a prevalent chronic oral disease, is closely related to the imbalance of the oral microecology and has become a significant burden on public health. The core of dental caries lies in the formation and metabolic activities of dental plaque biofilm: Streptococcus mutans, as the main cariogenic bacteria, forms plaque by adhering to the tooth surface. It then metabolizes carbohydrates to produce a large amount of lactic acid, causing the local pH value of the tooth surface to drop below 4.5. This value is far below the critical pH value (5.5) for demineralization of tooth hard tissues, directly disrupting the balance between enamel demineralization and remineralization, ultimately leading to the formation of cavities.

[0003] Lactobacilli play a dual role in the development of dental caries, acting as both "co-cariogenic agents" and "potential probiotics." The complexity of their mechanisms of action presents challenges for oral health interventions. On the one hand, strains such as *Lactobacillus salivarius* and *Lactobacillus rhamnosus* exhibit strong acid production and resistance, significantly reducing plaque pH in the presence of sucrose and even surviving in extreme environments with pH levels as low as 2.2, further exacerbating acidification damage to tooth tissues. On the other hand, some lactobacillus strains have been shown to exert caries-preventing potential by inhibiting *Streptococcus mutans* proliferation and regulating oral flora structure. This strain-specific difference leads to significant uncertainties in traditional lactobacillus application protocols. Therefore, screening for lactobacillus strains with high antibacterial activity, strong oral colonization ability, and non-pathogenicity can provide innovative solutions for the prevention and treatment of oral diseases that combine safety and effectiveness, possessing significant clinical value and market potential. Summary of the Invention

[0004] To address the problems existing in the prior art, the primary objective of this invention is to provide a *Lactobacillus saliva-associated* (Saliva-associated lactobacillus) Ligilactobacillus salivarius LT017, deposited by China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No.30724, deposit date: May 22, 2024.

[0005] A second objective of this invention is to provide the above-mentioned Lactobacillus saliva-associated powder LT017 and its preparation method.

[0006] A third objective of this invention is to provide the use of the above-mentioned Lactobacillus salivae LT017 or the above-mentioned Lactobacillus salivae LT017 powder in the preparation of products for preventing dental caries and / or removing dental plaque.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a *Lactobacillus salivarius* LT017, wherein *Lactobacillus salivarius* ( Ligilactobacillus salivarius The accession number of LT017 is CGMCC No.30724.

[0009] The present invention also provides the application of the above-mentioned Lactobacillus saliva-associated LT017 in the preparation of products for preventing dental caries.

[0010] The present invention also provides the application of the above-mentioned Lactobacillus saliva-associated LT017 in the preparation of products for removing dental plaque.

[0011] The present invention also provides a method for preparing Lactobacillus salivarius LT017 bacterial powder, comprising the following steps: inoculating the above-mentioned Lactobacillus salivarius LT017 into a culture medium for fermentation to obtain a fermentation broth; centrifuging the fermentation broth to obtain bacterial cells, adding a freeze-drying protectant and then freeze-drying to obtain Lactobacillus salivarius LT017 bacterial powder.

[0012] Preferably, the components of the culture medium include: 30 g / L glucose, 5 g / L yeast extract FM902, 5 g / L yeast extract FM502, 10 g / L bovine bone peptone, 10 g / L beef extract, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 2 g / L disodium citrate, 0.5 g / L calcium carbonate, 0.58 g / L magnesium sulfate, 0.25 g / L manganese sulfate, and 1 g / L Tween 80.

[0013] Preferably, the fermentation temperature is 36~38℃ and the fermentation time is 10~14h.

[0014] Preferably, the centrifugation speed is 4000~8000 rpm and the time is 10~40 min.

[0015] Preferably, the components of the freeze-drying protectant include: 20g / 100ml skim milk, 20g / 100ml trehalose, 5g / 100ml sucrose, 1.5g / 100ml glycerol and 0.1g / 100ml vitamin E.

[0016] The present invention also provides Lactobacillus saliva-associated LT017 bacterial powder prepared by the above preparation method.

[0017] The present invention also provides the application of the above-mentioned Lactobacillus saliva-associated powder LT017 in the preparation of products for preventing dental caries and / or removing dental plaque.

[0018] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0019] This invention provides the first strain of *Lactobacillus saliva* LT017, which significantly inhibits *Streptococcus mutans*, *Streptococcus grosvenorii*, and *Porphyromonas gingivalis*. It exhibits high tolerance to oral lysozyme, high antibiotic sensitivity, strong colonization ability in the oral cavity, and can effectively inhibit and remove dental plaque biofilm. It also has low corrosiveness to teeth, demonstrating good efficacy in preventing tooth decay and removing dental plaque, and high safety.

[0020] Biological Preservation Instructions

[0021] Lactobacillus salivarius LT017, classified as Lactobacillus salivarius ( Ligilactobacillus salivarius (), depositary institution: China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, accession number: CGMCCNo. 30724, deposit date: May 22, 2024. Attached Figure Description

[0022] Figure 1 The corrosive effect of lactic acid bacteria on teeth;

[0023] Figure 2 : The self-aggregation ability of the strain;

[0024] Figure 3 Hydrophobicity of the strain;

[0025] Figure 4 Colony morphology of Lactobacillus salivae LT017;

[0026] Figure 5 Microstructure of Lactobacillus saliva-associated LT017. Detailed Implementation

[0027] This invention provides a *Lactobacillus salivarius* LT017, wherein *Lactobacillus salivarius* ( Ligilactobacillus salivarius The accession number for LT017 is CGMCC No. 30724. The *Lactobacillus salivarius* LT017 described in this invention was isolated and purified from oral scraping samples from healthy individuals, and identified as belonging to *Lactobacillus salivarius* (…). Ligilactobacillus salivarius It was deposited on May 22, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0028] The *Lactobacillus saliva-associated* LT017 described in this invention can significantly inhibit *Streptococcus mutans*, *Streptococcus Gordonii*, and *Porphyromonas gingivalis*. It exhibits high tolerance to oral lysozyme, high antibiotic sensitivity, and strong colonization ability in the oral cavity. It effectively inhibits and removes dental plaque biofilm, and has low corrosiveness to teeth, demonstrating good efficacy in preventing tooth decay and removing dental plaque, with high safety. Based on this, this invention provides the application of *Lactobacillus saliva-associated* LT017 in the preparation of products for preventing tooth decay and / or removing dental plaque, preferably including oral hygiene products, oral care products, or oral medicines.

[0029] The present invention also provides a method for preparing Lactobacillus salivarius LT017 bacterial powder, comprising the following steps: inoculating the above-mentioned Lactobacillus salivarius LT017 into a culture medium for fermentation to obtain a fermentation broth; centrifuging the fermentation broth to obtain bacterial cells, adding a freeze-drying protectant and then freeze-drying to obtain Lactobacillus salivarius LT017 bacterial powder.

[0030] In this invention, the components of the culture medium include: 30 g / L glucose, 5 g / L yeast extract FM902, 5 g / L yeast extract FM502, 10 g / L bovine bone peptone, 10 g / L beef extract, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 2 g / L disodium citrate, 0.5 g / L calcium carbonate, 0.58 g / L magnesium sulfate, 0.25 g / L manganese sulfate, and 1 g / L Tween 80. The fermentation temperature is 36~38℃, preferably 37℃, and the fermentation time is 10~14 hours, preferably 12 hours; the centrifugation speed is 4000~8000 rpm, preferably 6000 rpm, and the centrifugation time is 10~40 minutes, preferably 20 minutes. The centrifugation is preferably carried out at a temperature below 10℃, more preferably at 4℃; the components of the freeze-drying protectant include: 20g / 100ml skim milk, 20g / 100ml trehalose, 5g / 100ml sucrose, 1.5g / 100ml glycerol, and 0.1g / 100ml vitamin E.

[0031] As an optional implementation, the present invention inoculates *Lactobacillus saliva-associated* LT017 into a culture medium and incubates it at 37°C for 12 hours until OD... 600 The concentration was 5.0 to obtain activated Lactobacillus salivae LT017. Then, the activated Lactobacillus salivae LT017 inoculum was inoculated into the culture medium at 3% (v / v) for three-stage expansion culture. The fermentation broth obtained from the culture was centrifuged at 4℃, 6000rpm for 20min. The centrifuged precipitate was added with a freeze-drying protectant and then freeze-dried to prepare Lactobacillus salivae LT017 bacterial powder (probiotic powder).

[0032] The present invention also provides Lactobacillus saliva-associated LT017 bacterial powder prepared by the above preparation method, wherein the viable bacterial count of the bacterial powder prepared by the present invention is 100 billion CFU / g.

[0033] The present invention also provides the application of the above-mentioned Lactobacillus saliva-associated powder LT017 in the preparation of products for preventing tooth decay and / or removing dental plaque. The products are preferably oral cleaning products, oral health care products or oral medicines, which can effectively inhibit and remove dental plaque biofilm, and have low corrosiveness to teeth, have good tooth decay prevention and dental plaque removal effects, and have high safety.

[0034] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0035] In a specific embodiment of the present invention, *Streptococcus mutans* ATCC 25175 was purchased from the China Industrial Microbial Culture Collection Center; *Streptococcus Gordonii* ATCC 10558 was purchased from Baosai Biotechnology; *Porphyromonas gingivalis* ATCC 33277 was purchased from the China Industrial Microbial Culture Collection Center; *Streptococcus salivarius* K12 was purchased from Shanghai Xuanya Biotechnology Co., Ltd.; and oral probiotic LS97 was purchased from Jiangsu Weikang Biotechnology Co., Ltd.

[0036] Unless otherwise specified, the following embodiments are all conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] Example 1

[0039] Isolation and identification of Lactobacillus saliva-associated with Salivary Lactobacillus LT017:

[0040] 1. Strains Isolation

[0041] Ten oral scraping samples were collected from healthy individuals and sent to the laboratory for isolation and screening experiments. After amplification of the samples on MRS liquid medium, they were spread onto the surface of MRS solid medium containing 2% calcium carbonate. After 48 hours of incubation, single colonies with a calcium dissolution zone were picked and streaked for purification. After three generations of purification, the strains were preserved in glycerol tubes and stored at -80°C for later use. Forty strains were screened based on morphology and the presence of a calcium dissolution zone.

[0042] 2. Screening of antibacterial strains

[0043] The Oxford cup method was used to screen the antibacterial effects of the selected strains. Indicator bacteria were *Streptococcus mutans* ATCC25175, *Streptococcus Gordonii* ATCC 10558, and *Porphyromonas gingivalis* ATCC 33277. After the indicator bacteria reached the logarithmic growth phase, the final bacterial concentration was adjusted to 1.0 × 10⁻⁶. 6 Prepare plates by mixing CFU / mL into solid culture medium. Place Oxford cups on the plates, then add 100 μL of lactic acid bacteria suspension (OD200). 600 The sample (5.0) was applied to Oxford cups, pre-diffused at 4°C for 12 hours, and then incubated at 37°C for 24 hours. The diameter of the inhibition zone was measured. Chlorhexidine acetate at 2.5 mg / L was used as a positive control, and MRS broth as a negative control. The results are as follows:

[0044] Table 1. Size of inhibition zones for each strain

[0045]

[0046] Based on the size of the inhibition zone, the results showed that a total of 28 strains of bacteria had antibacterial effects, of which 25 strains had inhibitory effects on all three types of oral harmful bacteria.

[0047] 3. Biomembrane inhibition and clearance experiments

[0048] The activated indicator bacteria (Streptococcus mutans ATCC 25175, Streptococcus Gordonii ATCC 10558, and Porphyromonas gingivalis ATCC 33277) were adjusted to a bacterial concentration of 1.0 × 10⁻⁶ using BHI broth medium supplemented with 2.5% sucrose. 8 Add 200 μL of indicator bacterial suspension (CFU / mL) to a 96-well plate, incubate at 37°C for 48 hours, then discard the supernatant. Add 100 μL of lactic acid bacteria suspension (OD200). 600 In the control group (using physiological saline instead of MRS), the culture medium was incubated at 4°C for 12 hours, and the supernatant was discarded. Airborne bacteria were removed by washing with PBS (0.01 mol / L, pH 7.2). After air drying, 200 μL of 0.1% crystal violet solution was added for staining for 30 min. Excess dye was aspirated, and the surface stain was washed away with PBS. After air drying, 95% ethanol solution was added, and the OD was measured using a microplate reader. 600 The biofilm removal rate of the strain is calculated using the following formula:

[0049]

[0050] In the formula: OD0 is the OD of the control group bacterial solution. 600 OD1 represents the OD of the bacterial culture in the treatment group. 600 .

[0051] The results are as follows:

[0052] Table 2. Scavenging rate results for each strain

[0053]

[0054] Oral pathogens exert their pathogenic effects by forming dental plaque biofilms; therefore, investigating the inhibitory effects on oral pathogenic bacteria plaque biofilms is crucial. The biofilm clearance rate of the strains was determined using crystal violet-ultraviolet spectrophotometry. The results showed that 19 strains exhibited biofilm clearance activity, with 13 strains achieving clearance rates exceeding 50%. Based on the inhibition zone results, these 13 strains were selected for further testing.

[0055] 4. Safety evaluation of the strain

[0056] (1) Acid-producing:

[0057] Each type of lactic acid bacteria was inoculated into MRS liquid medium at an inoculum size of 3% (v / v) and cultured at 37°C for 12 h. The pH value at the end of fermentation was measured using a pH meter to analyze the acid production of the lactic acid bacteria. *Streptococcus salivarius* K12 was used as the control group. The results are as follows:

[0058] Table 3 pH of fermentation broth for each strain

[0059]

[0060] The results showed that the pH of the fermentation broth of commercially available Streptococcus salivarius K12 was 3.65. Among the screened strains, 5 strains had a pH below 3.65 and 10 strains had a pH above 3.65.

[0061] (2) Corrosiveness to teeth:

[0062] Using *Streptococcus salivarius* K12 as a control strain, each lactic acid bacteria was inoculated into MRS liquid medium at an inoculum size of 3% (v / v) and cultured at 37°C for 36 h. 3 mL of the lactic acid bacteria fermentation supernatant (OD200) was then collected. 600 After mixing 0.05 g of hydroxyapatite with 0.003 g of hydroxyapatite and stirring thoroughly, the mixture was incubated together to simulate a tooth environment. The supernatant of Streptococcus mutans ATCC 25175 fermentation was used as the OD value. 600 A positive control was defined as 0.05 μmol / L. Equal volumes of incubation solution were centrifuged at 4700 rpm for 10 min at 4°C after 4 h. A blank control was defined as 0 h. The supernatant (0.1 mL) was collected in a test tube, 2 mL of ferrous sulfate-ammonium molybdate solution was added, and the volume was adjusted to 4 mL with ultrapure water. The mixture was centrifuged at 4700 rpm for 10 min, and the OD of the supernatant was measured. 600 The phosphorus concentration in the supernatant was calculated based on the standard curve equation plotted using phosphorus standard solutions, and the corrosiveness of lactic acid bacteria to teeth was analyzed. The results are as follows: Figure 1 As shown.

[0063] Figure 1 The results of the tooth-corrosiveness test on the strains were consistent with the results of the acid-producing test. Compared with other strains, M3-2, M5-4, M7-2, M9-1, and M9-2 were more corrosive to teeth and were not suitable as oral probiotics. The remaining 8 strains had lower phosphorus concentrations and weaker tooth-corrosiveness compared with Streptococcus mutans, and could be used for further experiments.

[0064] (3) Lysozyme tolerance:

[0065] The bacterial cell concentration was prepared to be 1.0 × 10⁻⁶. 8 A CFU / mL suspension of logarithmic-phase lactic acid bacteria was mixed with MRS agar medium at 50–55°C and poured into petri dishes. After the medium solidified, Oxford cups were placed in the petri dishes. 100 μL of lysozyme solutions at different concentrations (0 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.6 mg / mL, 2.0 mg / mL) were added to the Oxford cups, and the mixture was incubated overnight at 37°C. The diameter of the inhibition zone was measured, and the concentration of lysozyme without an inhibition zone was considered the highest tolerance level of the lactic acid bacteria to lysozyme. The results are as follows:

[0066] Table 4 Lysozyme tolerance of each strain

[0067]

[0068] For oral probiotics to colonize the human oral cavity and exert their antibacterial effect, they must be able to tolerate the oral environment. The human oral cavity contains lysozyme, a natural bactericidal substance; therefore, it is necessary to investigate the tolerance of lactic acid bacteria to lysozyme. The known reported concentrations of lysozyme in the human oral cavity range from 0 to 57 μg / mL. Eight strains of bacteria exhibited lysozyme tolerance levels above 1 mg / mL, indicating their ability to tolerate the oral environment and serve as oral probiotics.

[0069] (4) Antibiotic sensitivity:

[0070] The prepared concentration was 1.0 × 10⁻⁶. 8 A suspension of logarithmic-phase lactic acid bacteria (CFU / mL) was added at a 3% concentration to MRS agar medium at 50–55°C, mixed thoroughly, and poured into petri dishes. After the medium solidified, 22 antimicrobial susceptibility test discs were placed on plates and incubated at 37°C for 12 hours. The diameter of the clear zone was then measured. The results are as follows:

[0071] Table 5. Antibiotic susceptibility of each strain

[0072]

[0073] Note: R is resistant; I is moderately sensitive; S is sensitive.

[0074] Currently, the international standard used is CLSI to determine the antibiotic susceptibility of lactic acid bacteria. This experiment was conducted according to international standards. The results showed that all 8 strains were not sensitive to streptomycin, norfloxacin, gentamicin, ciprofloxacin, enoxacin, and polymyxin B, but were sensitive to amoxicillin. Specifically, M1-4, M7-1, and M8-2 were sensitive to 4 antibiotics, moderately sensitive to 9 antibiotics, and insensitive to 9 antibiotics. M2-2 was sensitive to 6 antibiotics, moderately sensitive to 5 antibiotics, and insensitive to 11 antibiotics. M2-3 was sensitive to 5 antibiotics, moderately sensitive to 9 antibiotics, and insensitive to 8 antibiotics. M3-3 was sensitive to 2 antibiotics, moderately sensitive to 12 antibiotics, and insensitive to 8 antibiotics. M4-4 was sensitive to 2 antibiotics, moderately sensitive to 8 antibiotics, and insensitive to 12 antibiotics. M6-2 was sensitive to 10 antibiotics, moderately sensitive to 7 antibiotics, and insensitive to 5 antibiotics. Higher sensitivity to antibiotics indicates higher safety of the strain; among the eight strains, M6-2 showed higher safety.

[0075] 5. Colonization ability of the strain

[0076] (1) Self-cohesive ability:

[0077] Using oral probiotic LS97 as a control strain, the OD of lactic acid bacteria suspension was prepared using PBS. 600 Adjust the pH to 0.6, take 1 mL of bacterial suspension, and let it stand at 37℃ for 2, 4, 19, and 24 hours. Then, slowly take 2 mL of the upper bacterial suspension to measure the OD. 600 The self-polymerization capacity of lactic acid bacteria is calculated using the following formula:

[0078]

[0079] In the formula: OD0 is the initial OD of the bacterial suspension. 600 OD1 is the OD of the upper bacterial solution after standing. 600 .

[0080] The results are as follows Figure 2 As shown, the self-aggregation ability of a bacterial strain is one of the important indicators for evaluating its colonization in the oral cavity. Strains with poor self-aggregation ability have poor colonization ability, while excessively high self-aggregation ability can easily form biofilms and cause oral diseases. Therefore, strains with strong self-aggregation ability in the early stage and poor self-aggregation ability in the later stage are excellent oral probiotics. Figure 2 The results showed that M1-4 and the commercially available oral probiotic LS97 had relatively poor self-aggregation ability compared to other strains, making them difficult to colonize in the oral cavity. M2-2, M2-3, M4-4, M7-1, and M8-2 showed gradually increasing self-aggregation ability over time, but the acid production in the later stages could be corrosive to teeth. M6-2, compared to other strains, exhibited strong initial self-aggregation ability but weaker later self-aggregation ability, making it suitable as an oral probiotic.

[0081] (2) Surface hydrophobicity of the strain:

[0082] Using oral probiotic LS97 as a control strain, the OD of lactic acid bacteria suspension was prepared using PBS. 600 Adjust the nm to 0.6, take 3 mL of bacterial suspension, add 1 mL of xylene as a hydrophobic organic solvent, let stand at room temperature for 10 min, then shake to mix and let stand for another 20 min before measuring the OD of the upper aqueous phase. 600 The hydrophobicity of lactic acid bacteria surfaces is calculated using the following formula:

[0083]

[0084] In the formula: OD0 is the initial OD of the bacterial suspension. 600 OD1 is the OD of the upper aqueous phase after settling. 600 .

[0085] The results are as follows Figure 3 As shown in the figure, 'af' is used to distinguish differences between groups. The same letter indicates no significant difference between groups (P>0.05), while different letters indicate significant differences between groups (P<0.05). Hydrophobicity is a non-specific adhesion mechanism related to the adhesion and colonization ability of bacterial strains. The MATH standard classifies bacterial hydrophobicity into three levels: less than 20% is low hydrophobicity, 20%–50% is moderate hydrophobicity, and greater than 50% is high hydrophobicity. Figure 3 The results show that strain M6-2 has a hydrophobicity of 41.85%, significantly higher than other strains (P<0.05), followed by M2-2, M7-1, and M2-3, while the other strains are of low hydrophobicity. Therefore, strain M6-2 has a stronger oral colonization ability than the commercially available strain LS097. Based on previous experiments, strain M6-2 was selected as an oral probiotic for further research.

[0086] 6. Identification of strains

[0087] The selected bacterial strain M6-2, which exhibits high safety by inhibiting caries pathogens, clearing biofilms, and causing no corrosive damage to teeth, was submitted for preservation. After four generations of purification on MRS agar plates, M6-2 was sent to Shanghai Sangon Biotech Co., Ltd. for 16S rDNA sequencing identification (sequence shown in SEQ ID No. 1). The identification results were compared with the NCBI database for homology.

[0088] The strain M6-2 was identified as *Lactobacillus salivarius* (…). Ligilactobacillus salivarius ), and named it LT017.

[0089] Macroscopic morphology of Lactobacillus saliva-associated LT017 colonies in Petri dishes as follows: Figure 4The image shows that Lactobacillus salivae LT017 grows well on MRS, is milky white, has neat circular edges, and has a glossy surface that is raised above the culture medium surface.

[0090] The microstructure of Lactobacillus salivae LT017 is as follows: Figure 5 The image shows that Lactobacillus salivae LT017 is Gram-positive and short rod-shaped.

[0091] Lactobacillus saliva-associated LT017 was deposited on May 22, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30724.

[0092] Example 2

[0093] A method for preparing Lactobacillus saliva-associated LT017 bacterial powder:

[0094] Activation: Take the Lactobacillus saliva-associated LT017 glycerol cryovial stored at -80℃, thaw it at room temperature, then use an inoculation loop to pick up the bacteria under aseptic conditions and streak it into MRS liquid medium. Incubate at 37℃ for 12 hours until OD reaches 100%. 600 The concentration was 5.0, resulting in activated Lactobacillus salivans LT017.

[0095] Preparation of bacterial culture: The activated inoculum of *Lactobacillus saliva-associated* LT017 was inoculated at 3% (v / v) into *Lactobacillus saliva-associated* enrichment medium (medium components: glucose 30 g / L, yeast extract FM902 5 g / L, yeast extract FM502 5 g / L, bovine bone peptone 10 g / L, beef extract 10 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, disodium citrate 2 g / L, calcium carbonate 0.5 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, Tween 80 1 g / L). Three-stage expansion was performed. The resulting fermentation broth was centrifuged (4℃, 6000 rpm for 20 min). A lyophilization protectant (lyophilization protectant components: skim milk 20 g / 100 ml, trehalose 20 g / 100 ml, sucrose 5 g / 100 ml, glycerol 1.5 g / L) was added to the precipitate. After freeze-drying (pre-freezing at -40℃ for 4 hours, primary drying at -40℃ for 1 hour, -35℃ for 2 hours, -30℃ for 4 hours, -25℃ for 4 hours, -20℃ for 4 hours, -15℃ for 4 hours, -10℃ for 4 hours, -5℃ for 4 hours, 0℃ for 3 hours; desorption drying at 6℃ for 4 hours, 12℃ for 3 hours, 24℃ for 12 hours), the probiotic powder was prepared with a live bacteria count of 100 billion CFU / g.

[0096] Experimental Example 1

[0097] The activity of the Lactobacillus LT017 bacterial powder prepared in Example 2 was tested.

[0098] 1. Prevention of dental caries

[0099] One hundred and twenty Wistar rats aged 22-23 days were randomly divided into six groups: high-dose, medium-dose, low-dose, modeling, positive control, and blank control, with twenty rats in each group. The blank control group was fed a normal diet. The experimental and modeling groups were inoculated with Streptococcus mutans to verify its colonization and survival in the oral cavity, and then fed a cariogenic diet (Diet). The high-, medium-, and low-dose groups in the experimental group applied 0.05 g, 0.1 g, and 0.2 g of probiotic powder to the tooth surface daily using cotton swabs, respectively. The positive control group received 1 ml of 0.02% chlorhexidine solution daily. The modeling group received no treatment. After two months, the rats were sacrificed, and the upper and lower jawbones were carefully dissected, with all attached muscle and other soft tissues thoroughly scraped away. The bones were immediately fixed in formalin for at least 24 hours. After fixation, the jawbones were decalcified, stained with thionine, decolorized with 70% ethanol, and then soaked in glycerol. The Keyes method was used to score the caries. The results are as follows.

[0100] Table 6. Caries prevention efficacy of each bacterial strain

[0101]

[0102] In the table, the P-exposure rate refers to the pulp exposure rate, which is the number of rats in the group with at least one pulp exposure lesion (P) / the total number of rats in the group × 100.

[0103] The results showed that, compared with the control group, the model group had a total caries score of 38.03, a dentin caries rate of 74.3%, a deep dentin caries rate of 32.54%, and a P-positive animal rate of 66.74%, proving the successful establishment of the model. In the positive control group, the total caries score decreased to 9.63, and the dentin caries rate, deep dentin caries rate, and P-positive animal rate all decreased significantly, indicating a certain preventive effect against caries formation. The high, medium, and low doses of the bacterial powder all had a certain effect on caries formation, with the high-dose group showing an effect close to the positive control group: a total caries score of 7.94, a dentin caries rate of 31.44%, a deep dentin caries rate of 4.38%, and a P-positive animal rate of 10.23%, demonstrating a significant preventive effect against caries and significantly reducing caries formation.

[0104] 2. Plaque Removal Test

[0105] Thirty patients (aged 18-65) with oral diseases and a plaque index (PI) between 2.5 and 2.8 were selected, ensuring they had not undergone oral treatment within the past two months. Ten patients received no treatment, while ten rinsed their mouths three times daily (morning, noon, and evening) with sterile water containing 0.1 g / ml of the prescribed plaque powder, keeping the rinse in their mouths for 5 minutes each time. The remaining ten rinsed their mouths with mouthwash containing 0.12% chlorhexidine. During the washout period, all patients used the designated toothbrush, toothpaste, and other oral hygiene products. Follow-up visits were conducted at weeks 2, 4, and 6 to observe dental conditions and record the plaque index (PI). The results are as follows.

[0106] Table 7. Results of plaque removal experiments for each bacterial strain

[0107]

[0108] The results showed that the PI value in the experimental group decreased significantly compared with the blank control group, especially from the fourth week, with a final decrease of 2.13 over 6 weeks. Compared with the PI value decrease of 1.61 in the positive control group, the experimental group had a significant function in eliminating dental plaque.

[0109] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Lactobacillus saliva-associated with salivary bacteria LT017, characterized in that, The salivary lactobacillus ( Ligilactobacillus salivarius The accession number of LT017 is CGMCC No.30724.

2. The use of the Lactobacillus salivae LT017 as described in claim 1 in the preparation of products for preventing dental caries.

3. The use of the Lactobacillus salivae-associated LT017 as described in claim 1 in the preparation of a product for removing dental plaque.

4. A method for preparing Lactobacillus saliva-associated powder LT017, characterized in that, The process includes the following steps: inoculating the Lactobacillus salivae LT017 of claim 1 into a culture medium for fermentation to obtain a fermentation broth; centrifuging the fermentation broth to obtain bacterial cells, adding a freeze-drying protectant, and then freeze-drying to obtain Lactobacillus salivae LT017 bacterial powder.

5. The preparation method according to claim 4, characterized in that, The fermentation temperature is 36~38℃, and the fermentation time is 10~14h.

6. The preparation method according to claim 4, characterized in that, The centrifugation speed is 4000~8000 rpm, and the time is 10~40 min.

7. The preparation method according to claim 4, characterized in that, The components of the freeze-drying protectant include: 20g / 100ml skim milk, 20g / 100ml trehalose, 5g / 100ml sucrose, 1.5g / 100ml glycerol and 0.1g / 100ml vitamin E.

8. The Lactobacillus saliva-associated LT017 bacterial powder prepared by the preparation method according to any one of claims 4 to 7.

9. The use of the Lactobacillus saliva-associated LT017 powder according to claim 8 in the preparation of products for preventing dental caries and / or removing dental plaque.

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