A strain of Lactobacillus rhamnosus and its application in preventing or treating dental caries and periodontal disease

Abstract

The present invention relates to the technical field of probiotic screening and application, and specifically to a strain of Lactobacillus rhamnosus and its application in preventing or treating dental caries and periodontal disease. The Lactobacillus rhamnosus has a deposit number of CCTCC NO: M2022171, and has a significant inhibitory effect on pathogenic bacteria such as Streptococcus mutans and Actinomyces viscosus that cause dental caries, and Aggregatibacterium semiactinomycetemcomitans, Porphyromonas gingivalis, and Fusobacterium nucleatum that cause periodontal disease, and can be used as an oral probiotic to effectively prevent and improve oral diseases. The lysate of Lactobacillus rhamnosus can also effectively remove the biofilm formed by Streptococcus mutans and inhibit the growth and reproduction of Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum, Actinomyces viscosus, and Aggregatibacterium semiactinomycetemcomitans, and can be added to medicines and tooth care products for use.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic screening and application, and particularly relates to a Lactobacillus rhamnosus strain and application thereof in preventing or treating dental caries and periodontal disease. Background Art

[0002] The oral cavity is a complex ecosystem with a diverse microbiome, with an estimated surface population of more than 700 bacterial species. These oral microbes are distributed in different oral habitats, such as the tongue, teeth, subgingival sulcus, buccal mucosa, and tonsils. The load of specific microbial communities in these habitats varies due to factors such as metabolic activity, pH, nutrient availability, shedding / non-shedding, and saliva and sulcular fluid. Oral microbes maintain checks and balances to maintain health, but once pathogenic microbes multiply in large numbers, they can lead to various oral diseases such as caries, periodontitis, and halitosis.

[0003] In oral care products, people are increasingly interested in probiotics. By taking the right amount of probiotics, it can play an effective role in the health of the consumer. Bifidobacterium and Lactobacillus are the most common probiotics, and other species such as Bacillus, Enterococcus and Streptococcus, as well as yeast, are also classified as probiotics. Lactobacillus is a natural oral microbiome, mainly including Lactobacillus casei, Lactobacillus pseudocasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus acidophilus and Lactobacillus salivarius. Compared with Streptococcus mutans, lactic acid bacteria do not adhere to teeth and cause caries. Lactobacillus also has the effect of antagonizing periodontal pathogens, such as inhibiting the growth of Actinomycetes, Prevotella intermedia and Porphyromonas gingivalis. It can be seen that lactic acid bacteria play an important role in maintaining oral health and help maintain the microecological balance of the oral cavity. Summary of the invention

[0004] The present invention solves the problems of the prior art and provides a strain of Lactobacillus rhamnosus and its application in preventing or treating dental caries and periodontitis. The strain is screened from fermented cheese, can significantly inhibit the growth and reproduction of oral pathogens such as Streptococcus mutans, Porphyromonas gingivalis, and Aggregatibacterium semiactinomycetemcomitans, and can effectively colonize in the oral cavity, which is beneficial to restoring the balance of the flora, and can be widely used in preventing or treating oral diseases such as dental caries and periodontitis.

[0005] The Lactobacillus rhamnosus provided by the present invention is Lactobacillus rhamnosus VHProbi M14

[0006] (Lacticaseibacillus rhamnosus VHProbi M14) strain, which was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on March 1, 2022, and its deposit number is CCTCC NO: M2022171.

[0007] The 16s rDNA sequence of the Lactobacillus rhamnosus VHProbi M14 strain is SEQ ID NO: 1; its Riboprinter fingerprint is as follows Figure 1 As shown, the protein spectrum is Figure 2 shown.

[0008] The invention discloses an application of the Lactobacillus rhamnosus VHProbi M14 strain in preparing a product having the function of removing DPPH free radicals or removing hydroxyl free radicals.

[0009] The invention discloses an application of the Lactobacillus rhamnosus VHProbi M14 strain in preparing a product having the function of inhibiting oral pathogenic bacteria.

[0010] The oral pathogenic bacteria include Streptococcus mutans, Porphyromonas gingivalis, Actinomyces viscosus, Fusobacterium nucleatum or Aggregatibacter actinomycetemcomitans.

[0011] Another aspect of the present invention is to provide a product for inhibiting oral pathogens, wherein the product contains live bacteria of the above-mentioned Lactobacillus rhamnosus VHProbi M14 strain;

[0012] Furthermore, the product for inhibiting oral pathogens may also contain a lysate of Lactobacillus rhamnosus VHProbi M14 strain.

[0013] The Lactobacillus rhamnosus VHProbi M14 provided by the present invention is a strain screened from fermented cheese and has the most significant inhibitory effect on Streptococcus mutans. The diameter of the inhibition zone produced by its fermented bacterial liquid reaches 23 mm. The strain can also effectively inhibit oral pathogens such as Porphyromonas gingivalis, Actinomyces viscosus, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans, and has a wide antibacterial spectrum.

[0014] Lactobacillus rhamnosus VHProbi M14 can effectively colonize in the oral cavity, and effectively bind to oral pathogens to prevent them from adhering to teeth, significantly reducing the number of oral pathogens, thereby reducing the load of pathogens in the oral cavity. Among them, the self-agglutination rate of this strain reached 43.05% at 6 hours, and the co-agglutination effect on Streptococcus mutans and Semiactinomycetemcomitans was the best, with a co-agglutination rate of 42.27%-59.11% at 6 hours.

[0015] Lactobacillus rhamnosus VHProbi M14 can effectively remove biofilms formed by Streptococcus mutans and effectively prevent and treat dental caries. Among them, the bacterial suspension, that is, the removal rate of pure bacteria on biofilm is as high as 99.42%, while the removal rate of fermentation liquid and lysate is 100%, achieving unexpected technical results.

[0016] Lactobacillus rhamnosus VHProbi M14 can effectively inhibit the growth and reproduction of Streptococcus mutans. In the control group, Streptococcus mutans began to grow rapidly 10 hours after inoculation and reached a stable period around 20 hours; while in the experimental group added with the supernatant of the lysate of Lactobacillus rhamnosus VHProbi M14, Streptococcus mutans hardly grew within 40 hours.

[0017] Lactobacillus rhamnosus VHProbi M14 is non-toxic to normal human gingival epithelial cells and can adhere to the cells, achieving effective colonization in the oral cavity, which helps to balance the oral flora. Lactobacillus rhamnosus VHProbi M14 can significantly inhibit the adhesion and growth of periodontal pathogens (Semiactinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum) on normal human gingival epithelial cells, effectively preventing and improving symptoms such as periodontitis and gingival bleeding caused by the pathogens.

[0018] In addition, Lactobacillus rhamnosus VHProbi M14 also has strong gastrointestinal fluid tolerance and antioxidant properties, with scavenging rates of hydroxyl radicals and DPPH radicals of 18.1% and 16.8%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The Riboprinter fingerprint of Lactobacillus rhamnosus VHProbi M14;

[0020] Figure 2 The protein spectrum of Lactobacillus rhamnosus VHProbi M14;

[0021] Figure 3 This is an agglutination test of Lactobacillus rhamnosus VHProbi M14 and four pathogenic bacteria; A is a control of Lactobacillus rhamnosus, B is Lactobacillus rhamnosus + Streptococcus mutans; C is Lactobacillus rhamnosus + Porphyromonas gingivalis; D is Lactobacillus rhamnosus + Fusobacterium nucleatum; E is Lactobacillus rhamnosus + Aggregatibacterium semiactinomycetemcomitans;

[0022] Figure 4 This is a test of the inhibition of adhesion of Lactobacillus rhamnosus VHProbi M14 to Streptococcus mutans; A is the control group and B is the experimental group;

[0023] Figure 5 This is the inhibitory growth curve of Lactobacillus rhamnosus VHProbi M14 against Streptococcus mutans;

[0024] Figure 6 This is a picture of Lactobacillus rhamnosus VHProbi M14 inhibiting the adhesion of Porphyromonas gingivalis to normal human gingival epithelial cells; A is the control group and B is the experimental group;

[0025] Figure 7 This is a picture of Lactobacillus rhamnosus VHProbi M14 inhibiting the adhesion of Fusobacterium nucleatum to normal human gingival epithelial cells; A is the control group and B is the experimental group;

[0026] Figure 8 This is a picture of Lactobacillus rhamnosus VHProbi M14 inhibiting the adhesion of Semiactinomycetemcomitans to gingival epithelial cells; A is the control group and B is the experimental group. DETAILED DESCRIPTION

[0027] The Lactobacillus rhamnosus VHProbi M14 provided by the present invention is identified as a new type of lactobacillus by polyphasic taxonomy, meets the food safety regulations, and can be taken as a food raw material for a long time without side effects. The Lactobacillus rhamnosus VHProbi M14 provided by the present invention can effectively inhibit common oral pathogens and has important application value.

[0028] On March 1, 2022, the applicant deposited the screened Lactobacillus rhamnosus VHProbi M14 strain with the China Type Culture Collection of Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M2022171.

[0029] The present invention is described in detail below in conjunction with embodiments and drawings.

[0030] Example 1: Strain Isolation and Screening

[0031] 1. Initial screening:

[0032] Fermented cheese was purchased from the market, and then 10 g of cheese was added to a sample homogenizer bag, 90 mL of saline was added, and the mixture was beaten on a homogenizer for 10 min. After a series of dilutions, 10 g of cheese was taken. -1 , 10 -2 , 10 -3 100 μL of three dilution gradients were spread on MRS selective medium and cultured in a 37°C incubator under anaerobic conditions for 48 h. After single colonies grew on the plates, rod-shaped lactic acid bacteria were selected through microscopic examination. There were 12 strains in total and were named ZY1, ZY2, ..., ZY12.

[0033] 2. Rescreening against Streptococcus mutans strains

[0034] (1) Prepare the bottom agar plate

[0035] Prepare the bottom 1.5% agar plate in advance and let it dry.

[0036] (2) Preparation of Streptococcus mutans liquid

[0037] Streak and activate mutans streptococci ATCC 25175, CCTCC AB 99010, and BNCC700610 on BHI plates respectively, then pick a single colony into BHI broth medium, culture it aerobically at 37°C for 24 hours, then transfer it to new BHI broth medium at a ratio of 1%, culture it aerobically at 37°C for 24 hours to obtain a fresh bacterial solution, and mix the fresh bacterial solution at equal volumes of 1:1:1 to obtain a mutans streptococcus bacterial solution.

[0038] The mutans streptococcus bacterial liquid described in the following examples is the same as that in this example.

[0039] Using three different mutans Streptococcus as indicator strains can better reflect the antibacterial ability of the screened lactic acid bacteria against mutans Streptococcus.

[0040] (3) Oxford cup antibacterial experiment

[0041] Prepare BHI medium with 0.7% agar content and sterilize it; when the temperature drops below 47°C, add 0.3% (volume ratio) mixed mutans streptococcus liquid and shake well; pour 7mL onto the bottom agar plate, wait for solidification, put an Oxford cup on it, add 150μL of the fermentation liquid of the initially screened lactobacillus into each well, culture at 37°C for 48h, and observe whether there is an inhibition zone.

[0042] The results showed that among the 12 lactobacilli obtained by the initial screening of the present invention, only one strain had an inhibitory effect on Streptococcus mutans, with an obvious inhibition zone. Among them, the ZY7 strain had the most significant inhibitory effect on Streptococcus mutans, and the diameter of the inhibition zone produced by its fermentation liquid reached 23 mm.

[0043] Example 2: Identification of ZY7 strain

[0044] 1. Colony morphology identification

[0045] The ZY7 strain was inoculated on MRS agar medium and cultured anaerobically at 37°C for 24 hours. Single colonies were observed to be milky white and shiny, with a smooth and moist surface, neat edges, and a colony diameter of 1-2 mm. Under a microscope, they were in the shape of curved rods or short rods.

[0046] 2. Carbon source metabolism test identification

[0047] API 50CHL reagent strips were used to determine the metabolism of 49 carbon sources by strain ZY7.

[0048] Under sterile conditions, take an appropriate amount of fresh bacterial liquid, centrifuge at 5000rpm for 5min, wash twice with pH7.0 phosphate buffer, and resuspend with the same volume of buffer to obtain a bacterial suspension. Add the fresh bacterial suspension to the culture medium of the API kit at a 10% addition rate, and then add the culture medium to the hole of the test strip according to the operation of the kit, seal it with paraffin, and then put the test strip into a box, add about 10ml of sterile deionized water to the box substrate, cover it with a lid, and place it in a 37℃ incubator for 24-48h. Observe the color change. If the bacteria grow, the color will change from blue to yellow, and if there is no growth, the color will remain unchanged. Record the test results and upload them to the identification software API web.

[0049] The results showed that the ZY7 strain could utilize glycerol, ribose, galactose, glucose, fructose, mannose, mannitol, sorbitol, α-methyl-D-glucoside, N-acetylglucosamine, amygdalin, phellodendron, esculin, salicin, rhamnose, dulcitol, inositol, cellobiose, maltose, lactose, sucrose, trehalose, melezitose, gentiobiose, D-tagatose and gluconate; but could not utilize erythritol, hyritol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-adunculose, β-methyl-D-xyloside, α-methyl-mannoside, melibiose, toulose, inulin, raffinose, starch, glycogen, xylitol, D-lyxose, D-fucose, L-fucose, D-arabinitol, L-arabinitol, 2-ketogluconate and 5-ketogluconate.

[0050] The API identification result showed that the strain was Lactobacillus rhamnosus, with ID=99.4% and T value=0.69.

[0051] 3. Molecular Biology Identification

[0052] 3.1 16S rDNA gene sequence analysis

[0053] 3.1.1 Genomic DNA extraction

[0054] A single colony of the ZY6 strain on the plate was picked up and placed in MRS medium, cultured at 37° C. for 24 h, and then 500 μL of the fermentation solution was taken and operated according to the Tiangen Bacteria Genomic DNA Extraction Kit (Catalog No.: DP302) to obtain the genome of the strain.

[0055] 3.1.2 16S rDNA gene amplification

[0056] (1) Primer sequence:

[0057] 27F:AGAGTTTGATCCTGGCTCA;

[0058] 1492R: GGTTACCTTGTTACGACTT.

[0059] (2) Reaction system (50 μL)

[0060] Table 1: 16s rDNA PCR amplification system

[0061]

[0062] (3) Electrophoresis verification: The PCR product meets the requirements when the nucleic acid electrophoresis result is about 1500 bp.

[0063] (4) PCR product sequencing

[0064] The 16s rDNA sequence SEQ ID NO: 1 of the ZY7 strain was obtained by sequencing, and the sequence was compared in the NCBI database, and it was preliminarily determined that the ZY7 strain was Lactobacillus rhamnosus.

[0065] 3.2Riboprinter fingerprint

[0066] Use a bacterial stick to pick up a purified single colony of the ZY7 strain from the agar medium plate, put it into a sample tube of buffer, and use a handheld stirring rod to suspend it in the buffer. Then put the sample into the heater to inactivate it and put it into the Riboprinter system. After DNA preparation, mold transfer, imaging detection and data processing, the bacterial identification results are obtained.

[0067] The identification results showed that strain ZY7 was Lactobacillus rhamnosus, and its fingerprint was shown in Figure 1 .

[0068] 3.3 Protein spectrum identification

[0069] Use a toothpick to pick up a single colony of the ZY7 strain on the plate and place it on the protein spectrum plate. Then use a toothpick to evenly spread the bacterial sludge on the disc on the mass spectrometry plate. The sludge does not need to be too thick. Then, according to the instructions of the protein spectrum kit, add 1 μL of the matrix solution in the mass spectrometry sample pretreatment box to cover the sample and dry it naturally at room temperature. After drying, place the mass spectrometry plate on the Motitof protein spectrometer for identification.

[0070] Protein spectrum identification results Figure 2 As shown, strain ZY7 was identified as Lactobacillus rhamnosus with a matching rate of 61%.

[0071] 3.4 Whole-genome sequencing

[0072] The bacterial liquid of ZY7 strain was inoculated into 500ml MRS broth medium at a volume ratio of 1%, cultured at 37℃ for 22h, and then centrifuged at 8000rpm for 10min to collect the bacteria. The bacteria were sent to the sequencing center to obtain the full gene sequence of the bacteria. The full gene sequence was uploaded to the NCBI gene database, and the GenBank number is CP095384.

[0073] The colony morphology and physiological and biochemical characteristics of strain ZY7 were compared, and the results of molecular biological identification were combined to determine that strain ZY7 was a new type of Lactobacillus rhamnosus, which was named Lacticaseibacillus rhamnosus VHProbi M14.

[0074] Example 3: Tolerance of Lactobacillus rhamnosus VHProbi M14 to artificial gastric juice and artificial intestinal juice

[0075] 1. Preparation of bacterial solution:

[0076] The cryopreserved Lactobacillus rhamnosus VHProbi M14 strain was streaked into MRS solid culture medium and cultured at 37°C for 24-48 hours. After subcultured once in MRS liquid culture medium, Lactobacillus rhamnosus VHProbi M14 was inoculated into fresh MRS liquid culture medium at an inoculum rate of 5% and cultured with shaking at 40°C for 24-48 hours to obtain a fresh bacterial solution.

[0077] 2. Preparation of artificial gastric juice

[0078] Weigh 5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl respectively, add 1000ml of distilled water, adjust pH to 3.0 with dilute hydrochloric acid, and then sterilize at 115℃ for 20min. Then add 3.2g of porcine mucosal pepsin before use, shake well to dissolve, and place in a 37℃ water bath shaker for 1h to simulate human body temperature.

[0079] 3. Preparation of artificial intestinal fluid

[0080] Weigh 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH2PO4 and 3.0g of ox bile salt respectively, add 77mL of 0.2mol / L NaOH solution, dilute to 1000ml, adjust pH to 6.8±0.1 with dilute hydrochloric acid or sodium hydroxide solution, sterilize at 115℃ for 20min. Then add 1g of pancreatic enzyme before use, shake well to dissolve, and place in a 37℃ water bath shaker for 1h to simulate human body temperature.

[0081] 4. Test methods

[0082] Take 2mL of fresh bacterial liquid, centrifuge at 5000rpm for 5min to collect the bacteria, wash the bacteria 3 times with saline, and resuspend with 2mL of saline as the inoculum. Take 1mL of the inoculum and add it to 9mL of artificial gastric juice that has been warmed for 1h, place it in a 37℃ water bath shaker at 200rpm for 2h, take 1mL of samples at 0h and 2h, and detect the amount of live bacteria. Then take 1mL of artificial gastric juice after 2h of digestion, add it to 24mL of artificial intestinal juice, place it in a 37℃ water bath shaker (200rpm) for 3h, take 1mL of samples, and detect the amount of live bacteria.

[0083] The viable bacteria count method was carried out in accordance with the national standard GB4789.35-2016-Food Microbiology Inspection Lactic Acid Bacteria Inspection to determine the bacterial count. The viable bacteria count (LogCFU / mL) of the strain after passing through artificial gastric juice and artificial intestinal juice is shown in Table 2.

[0084] Table 2: Table of live bacteria after digestion of artificial gastrointestinal tolerance fluid

[0085]

[0086] As can be seen from Table 2, the logarithmic value of the bacterial count of Lactobacillus rhamnosus VHProbi M14 did not decrease after digestion with artificial gastric juice; after passing through artificial intestinal juice, the logarithmic value of the bacterial count decreased by 0.84, indicating that the Lactobacillus rhamnosus VHProbi M14 strain has a strong tolerance to artificial gastric juice and artificial intestinal juice. When consumed, a sufficient amount of live bacteria will reach the intestines, and it can be used as a potential oral probiotic.

[0087] Example 4: Hemolytic activity test of Lactobacillus rhamnosus VHProbi M14

[0088] 1. Preparation of blood cell culture medium:

[0089] Weigh various components of TBS basal culture medium, dissolve them, and sterilize them by high pressure at 121°C for 15 min. When the culture medium cools to 50°C, add 5% sterile defibrinated sheep blood, mix well, and pour into a plate.

[0090] 2. Line cultivation:

[0091] Streak the M14 strain onto the prepared blood cell plate, culture it in a 37°C incubator, and observe whether there is hemolysis after 24 to 48 hours.

[0092] 3. Experimental results:

[0093] There was no change in the blood cell plate, indicating that Lactobacillus rhamnosus VHProbi M14 did not produce hemolysin and could not lyse blood cells.

[0094] Example 5: Determination of the antioxidant capacity of Lactobacillus rhamnosus VHProbi M14

[0095] 1. Preparation of bacterial suspension

[0096] A single colony of Lactobacillus rhamnosus VHProbi M14 with good growth status was inoculated into 3 mL of MRS liquid culture medium, cultured at 37°C for 24 hours, and the culture liquid was used as the inoculum, inoculated into 50 mL of MRS liquid culture medium at a 2% inoculum amount, and cultured statically for 24 hours to obtain the culture liquid of the strain. After collecting the bacterial cells with 1 mL of pH 7.0 phosphate buffer, the bacterial cells were washed twice, and then 1 mL of buffer was added to resuspend the bacterial cells for use.

[0097] 2. Determination of hydroxyl free radical scavenging ability

[0098] 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate, 500 μL of deionized water and 200 μL of lactic acid bacteria suspension were mixed and then 100 μL of 3% hydrogen peroxide solution was added. After 15 min in a 37°C water bath, the supernatant was collected and the sample absorbance was measured at a wavelength of 510 nm. The hydroxyl radical scavenging rate was calculated according to the following formula.

[0099] Clearance % = (A sample - A control) / (A blank - A control) x 100%.

[0100] Wherein: A control is the absorbance of the mixed solution of ferrous sulfate, hydrogen peroxide and sodium salicylate, and A blank is the absorbance of the mixed solution of ferrous sulfate and sodium salicylate.

[0101] The results showed that the scavenging rate of hydroxyl radicals by Lactobacillus rhamnosus VHProbi M14 was 18.1%.

[0102] 3. Determination of DPPH free radical scavenging ability

[0103] Take 1mL of the bacterial suspension of the strain to be tested, add 1mL of the freshly prepared DPPH free radical solution of 0.4mM, mix well and then place it at room temperature for 30min under light shielding, then measure the absorbance of the sample at a wavelength of 517nm (A sample), and measure it 3 times in parallel. The control group sample is adjusted to zero with an equal volume of PBS solution and DPPH·ethanol mixture, and an equal volume of PBS bacterial suspension and ethanol mixture blank. The clearance rate is calculated according to the following formula: Clearance rate % = [1-(A sample-A blank) / A control]×100%.

[0104] The results showed that the scavenging rate of Lactobacillus rhamnosus VHProbi M14 on DPPH free radicals was 16.8%.

[0105] Example 6: Antibacterial effect test of Lactobacillus rhamnosus VHProbi M14 on oral pathogens

[0106] 1. Lysis buffer preparation

[0107] Inoculate Lactobacillus rhamnosus VHProbi M14 into MRS broth medium at a 1% inoculation rate (volume ratio), culture at 37°C for 24 hours and then stop culturing to obtain fresh bacterial liquid. Use an ultrasonic disruptor to ultrasonically disrupt part of the bacterial liquid for 20 minutes, and the ultrasonic conditions are as follows: power 30%, ultrasonic for 2 seconds, stop for 2 seconds; inactivate at 80°C for 60 minutes to obtain a lysate without viable bacteria.

[0108] Referring to the antibacterial experimental steps of the Oxford cup method described in Example 1, the antibacterial effects of the Lactobacillus rhamnosus VHProbiM14 bacterial solution and lysate on five common oral pathogens, namely, Streptococcus mutans (three strains), Porphyromonas gingivalis BNCC353909, Actinomyces viscosus ATCC27044, Fusobacterium nucleatum BNCC 336949 and Aggregatibacter actinomycetemcomitans BNCC336945, were determined respectively. The diameters of the inhibition zones are shown in Table 3.

[0109] Table 3: Antibacterial effect of Lactobacillus rhamnosus VHProbi M14 on oral pathogens

[0110] Oral Pathogens Diameter of inhibition zone of M14 fermentation liquid The diameter of the inhibition zone of M14 lysis solution Streptococcus mutans 23mm 23mm Porphyromonas gingivalis 14mm 11mm Actinomycetes viscosus 15mm 14mm Fusobacterium nucleatum 12mm 11mm Aggregatibacter actinomycetemcomitans 10mm 10mm

[0111] From the results in Table 3, it can be seen that the Lactobacillus rhamnosus VHProbi M14 provided by the present invention has obvious inhibitory effects on five oral pathogens, namely, Streptococcus mutans, Porphyromonas gingivalis, Actinomyces viscosus, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans, and has a wide antibacterial spectrum. Among them, the strain has the strongest antibacterial effect on Streptococcus mutans, with an antibacterial zone diameter of 23 mm.

[0112] Example 7: Agglutination effect test of Lactobacillus rhamnosus VHProbi M14 on oral pathogens

[0113] Coaggregation is the interaction between cells and cell proteins. Probiotics can prevent dental caries by agglutinating with pathogenic bacteria to prevent their colonization and adhesion in the mouth. There are two types of agglutination tests. One is to determine the agglutination effect based on whether lactobacilli combine with pathogenic bacteria under certain conditions to form visible flocculent precipitation and the size of the precipitation. The other is to determine the coaggregation effect by measuring the agglutination rate.

[0114] 1. Preparation of bacterial suspension

[0115] The rhamnosus Lactobacillus VHProbi M14 was inoculated into MRS broth medium at an inoculation rate of 1% (volume ratio), and the culture was stopped after culturing at 37° C. for 24 hours to obtain a fresh bacterial solution; the fresh bacterial solution was centrifuged at 8000 rpm for 10 minutes to collect the bacterial cells; the bacterial cells were washed twice with pH 7.0 phosphate buffer, and then resuspended with pH 7.0 phosphate buffer until the initial absorbance OD600 of the bacterial suspension was between 0.5 and 0.6, and then used for later use.

[0116] 2. Preparation of pathogenic bacteria suspension:

[0117] Inoculate the mutans Streptococcus into BHI broth medium at an inoculation volume of 1% (volume ratio), culture at 37°C aerobically for 24 hours, then stop culturing to obtain fresh bacterial solution; inoculate Porphyromonas gingivalis, Aggregatibacterium semiactinomycetemcomitans and Fusobacterium nucleatum into BHI broth medium (adding 5% bovine serum) at an inoculation volume of 1% (volume ratio), culture at 37°C anaerobically for 48 hours, then stop culturing to obtain fresh bacterial solution;

[0118] The fresh bacterial solution was centrifuged at 8000 rpm for 10 min to collect the bacteria; the bacteria were washed twice with pH 7.0 phosphate buffer, and then resuspended with pH 7.0 phosphate buffer, and the initial absorbance OD600 of the bacterial suspension was adjusted to between 0.5 and 0.6 for use.

[0119] 3. Agglutination point test

[0120] Take 300 μL of the rhamnosus Lactobacillus VHProbi M14 bacterial suspension and add it to a 24-well plate, then add 300 μL of the pathogenic bacteria suspension as a reaction sample, and take an equal amount of the rhamnosus Lactobacillus VHProbi M14 bacterial suspension and buffer solution as a control, and set up 2 parallels for each control and sample. Place the 24-well plate in a microplate constant temperature oscillator at 400 rpm, room temperature, and oscillate and incubate. Oscillate for 30 minutes, stop for 30 minutes, and take photos to record the initial well plate status and the well plate status at each stop.

[0121] from Figure 3 The results show that within 2 hours, Lactobacillus rhamnosus VHProbi M14 and four pathogenic bacteria, Streptococcus mutans, Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter semiactinomycetemcomitans, all showed agglutination points. Among them, the agglutination with Streptococcus mutans and Fusobacterium nucleatum was multiple agglutination points, and the agglutination with Porphyromonas gingivalis and Aggregatibacter semiactinomycetemcomitans was a large agglutination point. This shows that Lactobacillus rhamnosus VHProbi M14 can effectively bind to common oral pathogens and significantly inhibit their adhesion to teeth or gums.

[0122] 4. Determination of self-agglutination rate and co-agglutination rate

[0123] (1) Determination of autoagglutination rate

[0124] 1 ml of the prepared Lactobacillus rhamnosus VHProbi M14 suspension was added to a 24-well plate and allowed to stand at room temperature. 100 μL was taken to measure the initial absorbance A0 at a wavelength of 600 nm. The upper layer of the bacterial suspension was aspirated every 2 hours in the following 6 hours, the absorbance At was measured, and the self-aggregation force (Rself) was calculated. The results are shown in Table 4.

[0125] The calculation formula of self-cohesion (Rself): R 自 =1-At / A0;

[0126] Wherein: R is the self-aggregation force, %; At is the absorbance at time t, t = 2, 4 and 6h; A0 is the initial absorbance of the Lactobacillus rhamnosus VHProbi M14 bacterial suspension.

[0127] Table 4 Self-agglutination rate of Lactobacillus rhamnosus VHProbi M14

[0128] Coagulation time Self-agglutination rate 2h 16.29％ 4h 30.04％ 6h 43.05％

[0129] From the results in Table 4, it can be seen that the self-agglutination rate of the Lactobacillus rhamnosus VHProbi M14 provided by the present invention reaches 43.05% at 6 hours, which shows that the Lactobacillus rhamnosus VHProbi M14 can be effectively colonized in the oral cavity.

[0130] (2) Coagulation rate determination

[0131] Take 100 μL of the rhamnosus Lactobacillus casei VHProbi M14 suspension and the pathogenic bacteria suspension to determine the initial absorbance OD600, then mix equal amounts of the rhamnosus Lactobacillus casei VHProbi M14 suspension and the pathogenic bacteria suspension, shake well, and let stand at room temperature. Each sample is repeated three times, and 100 μL of the upper suspension is taken every 2 hours to determine the absorbance OD600. Calculate the coagulation (R 共 ). The results are shown in Table 5.

[0132] R 共 =1-2At / (A0+B0);

[0133] Wherein: R is the copolymerization %; At is the absorbance at time t, t = 2h, 4h and 6h; A0 is the initial absorbance of the Lactobacillus rhamnosus VHProbi M14 bacterial suspension; B0 is the initial absorbance of the pathogenic bacteria suspension.

[0134] Table 5: Coaggregation effect of Lactobacillus rhamnosus VHProbi M14 on oral pathogens

[0135]

[0136] From the results in Table 5, it can be seen that the Lactobacillus rhamnosus VHProbi M14 provided by the present invention can effectively bind to four common oral pathogens, namely, Streptococcus mutans, Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter semiactinomycetemcomitans, and the coaggregation rate reaches 10.93%-51.83% within 2-6 hours. Among them, Lactobacillus rhamnosus VHProbi M14 has the best coaggregation effect on Streptococcus mutans and Aggregatibacter semiactinomycetemcomitans, and the coaggregation rate at 6 hours is as high as 42.27%-59.11%.

[0137] Lactobacillus rhamnosus VHProbi M14 can effectively bind to pathogenic bacteria and significantly reduce the number of pathogenic bacteria in the oral cavity through saliva flow and oral cleaning, thereby achieving the effect of reducing the load of pathogenic bacteria in the oral cavity.

[0138] Example 8: Antagonistic adhesion test of Lactobacillus rhamnosus VHProbi M14 against Streptococcus mutans

[0139] Streptococcus mutans is currently recognized as the main pathogen of dental caries. Its colonization and adhesion in the oral cavity are the main causes of dental caries. Oral probiotics can effectively reduce the occurrence of dental caries by forming agglutination with pathogenic bacteria, inhibiting their growth and adhesion.

[0140] 1. Preparation of bacterial suspension

[0141] Lactobacillus rhamnosus VHProbi M14 was inoculated into MRS broth medium at an inoculation rate of 1% (volume ratio), and the culture was stopped after culturing at 37°C for 24 hours to obtain a fresh bacterial solution; the fresh bacterial solution was centrifuged at 8000 rpm for 10 minutes to collect the bacteria; the bacteria were washed twice with pH 7.0 phosphate buffer, and then resuspended with the same volume of pH 7.0 phosphate buffer.

[0142] 2. Preparation of Streptococcus mutans suspension:

[0143] Prepare a mutans Streptococcus bacterial solution by referring to the method described in Example 1; centrifuge the fresh bacterial solution at 8000 rpm for 10 min to collect the bacterial cells; wash the bacterial cells twice with pH 7.0 phosphate buffer, and then resuspend them with the same volume of pH 7.0 phosphate buffer.

[0144] Mix 500 μL of Lactobacillus rhamnosus VHProbi M14 suspension and 500 μL of Streptococcus mutans suspension evenly, let stand for 5 minutes, take 500 μL of the upper solution and add it to the 24-well plate with sterile cell slides, culture at 37°C for 2 hours, remove the upper solution, wash with pH 7.0 phosphate buffer, add 0.5 mL of methanol to fix for 10 minutes, and then discard. Finally, add 300 μL of Giemsa stain for 10 minutes, then discard the stain, rinse with pH 7.0 phosphate buffer, and place the cell slide on a slide to observe the amount of bacteria on the cell slide.

[0145] The results are as follows Figure 4 As shown in the figure, the control group cell slides were full of mutans streptococci, while the experimental group cell slides added with Lactobacillus rhamnosus VHProbi M14 had very few mutans streptococci, indicating that Lactobacillus rhamnosus VHProbi M14 inhibited the adhesion of mutans streptococci to the cell slides. This shows that Lactobacillus rhamnosus VHProbi M14 can effectively prevent oral pathogens from adhering to teeth, which helps prevent the occurrence of dental caries.

[0146] Example 9: Effect of Lactobacillus rhamnosus VHProbi M14 on biofilm elimination of Streptococcus mutans

[0147] Mutans Streptococcus adheres and aggregates on the tooth surface to form a biofilm, which in turn produces acid and causes tooth decay. Therefore, removing the biofilm formed by mutans Streptococcus is very important for preventing tooth decay.

[0148] 1. Prepare fresh Streptococcus mutans liquid by referring to the method described in Example 1.

[0149] 2. Inoculate Lactobacillus rhamnosus VHProbi M14 into MRS broth medium at 1% inoculation volume (volume ratio), culture at 37°C for 24 hours, then stop culturing to obtain fresh fermentation liquid. Divide the fermentation liquid into three parts and perform the following treatments:

[0150] (1) centrifuging part of the fermentation broth at 8000 rpm for 10 min to collect the bacterial cells; washing the bacterial cells twice with pH 7.0 phosphate buffer, and then resuspending them with the same volume of pH 7.0 phosphate buffer to prepare a live bacterial suspension;

[0151] (2) using an ultrasonic disruptor to ultrasonically disrupt part of the fermentation broth for 20 min, the ultrasonic conditions were as follows: power 30%, ultrasonication for 2 s, and stop for 2 s; inactivation at 80° C. for 60 min to obtain a lysate without viable bacteria;

[0152] (3) Some fermentation broth is not treated in any way.

[0153] 3. Prepare a sterile 24-well plate with cell slides

[0154] 600 μL of Streptococcus mutans bacterial solution was added to each well. After culturing for 24 hours, the Streptococcus mutans adhered to the cell slide to form a biofilm. Then the culture medium and non-adherent bacteria in the wells were discarded and washed twice with pH 7.0 phosphate buffer. 600 μL of Lactobacillus rhamnosus VHProbi M14 fermentation liquid, bacterial suspension and non-viable bacteria lysis liquid were added to the wells respectively. Three parallels were made for each group, and an equal amount of MRS broth culture medium was used as a control.

[0155] The test sample and the biofilm formed by Streptococcus mutans were co-cultured at 37°C for 24 hours, and then the liquid in the wells was discarded, and 600 μL pH 7.0 phosphate buffer was added for washing. Each well was washed 3 times. During washing, continuous and strong vibration was required to remove non-adherent bacteria. The biofilm removal rate of the test sample was then determined by comparing the number of Streptococcus mutans on the cell slide.

[0156] The cell slide was placed in a sterile homogenizing bag and ultrasonically cleaned for 10 minutes to allow the bacteria to diffuse into the buffer solution. The buffer solution was gradient diluted, and 100 μL of the diluted buffer solution was applied to a light Streptococcus salivarius culture medium supplemented with 200 U / L bacitracin and aerobically cultured at 37°C for 24 hours to detect the number of mutans Streptococcus. The removal rate of mutans Streptococcus biofilm was calculated by the following formula.

[0157] Removal rate (%) = (1-bacterial amount in the experimental group / bacterial amount in the control group) × 100%.

[0158] Table 6: Effect of Lactobacillus rhamnosus VHProbi M14 on the removal of Streptococcus mutans biofilm

[0159] Grouping Streptococcus mutans count (CFU) Biofilm removal rate Control group 27700 - Fermentation Broth 0 100％ bacterial suspension 1600 99.42％ Lysis buffer 0 100％

[0160] From the data in Table 6, it can be seen that Lactobacillus rhamnosus VHProbi M14 can effectively remove the biofilm formed by Streptococcus mutans. Among them, the removal rate of the biofilm by the bacterial suspension, that is, the pure bacteria, is as high as 99.42%, indicating that there are binding sites for Streptococcus mutans on the bacterial cell membrane, and the pure bacteria themselves can also efficiently bind to Streptococcus mutans, and almost completely remove part of the biofilm in a short time; and the fermentation broth and lysate contain not only the bacteria themselves, but also more metabolites, which can more effectively remove the biofilm formed by Streptococcus mutans, with a removal rate of 100%.

[0161] Therefore, Lactobacillus rhamnosus VHProbi M14 can effectively prevent and treat dental caries caused by Streptococcus mutans, and its lysate can be widely used in oral care products such as toothpaste and mouthwash.

[0162] Example 10: Growth inhibition test of Lactobacillus rhamnosus VHProbi M14 on Streptococcus mutans

[0163] 1. Lysis buffer preparation

[0164] The cell wall of the fresh bacterial solution of Lactobacillus rhamnosus VHProbi M14 was broken by an ultrasonic cell disruptor, and then treated at 80°C for 60 minutes, and insoluble matter was removed by filtration with a 0.22 μL microporous filter membrane to prepare a lysate supernatant.

[0165] 2. Preparation of inoculum

[0166] Fresh mutans Streptococcus bacteria were washed twice with pH 7.0 phosphate buffer, resuspended in the same volume, and then diluted 5 times to serve as inoculum.

[0167] 3. 96-well plate culture

[0168] Add 50 μL BHI broth medium and 10 μL mutans Streptococcus liquid to each well, and add 20 μL lysate supernatant to the experimental group and 20 μL sterile water to the control group; then add sterile water to each well to make the volume 200 μL, and add 50 μL liquid paraffin. Set up 4 parallels for each group. Place the 96-well plate in a 37°C microplate reader, measure OD600 every 10 minutes, and measure for 40 hours to obtain the growth curve of mutans Streptococcus, as shown in the figure. Figure 5 shown.

[0169] from Figure 5 It can be seen that the mutans Streptococcus in the control group began to grow rapidly 10 hours after inoculation and reached a stable period around 20 hours; while in the experimental group added with the supernatant of the lysate of Lactobacillus rhamnosus VHProbi M14, the mutans Streptococcus had almost no growth within 40 hours, indicating that Lactobacillus rhamnosus VHProbi M14 can effectively inhibit the growth and reproduction of Streptococcus mutans.

[0170] Example 11: Toxicity test of Lactobacillus rhamnosus VHProbi M14 on normal human gingival epithelial cells

[0171] 1. Cell pre-culture

[0172] Normal human gingival epithelial cells were revived in liquid nitrogen and cultured in a carbon dioxide incubator to the required amount. When the cell density grew to about 80%, they were digested with trypsin to form a single cell suspension and counted on a hemocytometer. The cell number was 5×10 5 cells / mL, and then 500 μL of cell suspension was inoculated into a 24-well plate at a seeding density of 2.5×10 5 cells / well, and subsequent experiments were performed after cell culture for 24 h.

[0173] 2. Preparation of inactivated bacteria solution

[0174] The fresh bacterial liquid of Lactobacillus rhamnosus VHProbi M14 was inactivated in a water bath at 80°C for 20 min, then washed three times with pH 7.0 phosphate buffer, resuspended with cell culture medium, and the bacterial concentration was adjusted to between OD600=0.4-0.5.

[0175] 3. Cell culture

[0176] Experimental group: the inactivated bacterial solution was added to the cells in a 24-well plate at a MOI (Multiplicity of Infection) value of 10;

[0177] Blank control group: add cell culture medium of the same volume as the inactivated bacteria solution.

[0178] The culture was continued in a carbon dioxide incubator for 24 h.

[0179] Add MTT solution to each cell culture well to be tested to a final concentration of 0.3 g / L; place the 24-well plate in a carbon dioxide incubator and incubate for 3 hours, and carefully discard the supernatant; add 500 μL DMSO to each 24-well cell culture well and incubate at 37°C for 30 minutes to fully dissolve the purple crystals; detect the absorbance value at a wavelength of 490 nm.

[0180] The results showed that the absorbance value of the blank control group was 0.668, while the absorbance value of the experimental group was 0.668. The results showed that Lactobacillus rhamnosus VHProbi M14 had no significant effect on the proliferation activity of normal human gingival epithelial cells, had good safety, and had no cytotoxicity.

[0181] Example 12: Adhesion of Lactobacillus rhamnosus VHProbi M14 to normal human gingival epithelial cells

[0182] Adhesion is an important prerequisite for probiotics to colonize in the oral cavity, and high adhesion confers a competitive advantage in this ecosystem.

[0183] 1. Cell pre-culture

[0184] Normal human gingival epithelial cells were revived in liquid nitrogen and cultured to the required amount. When the cell density grew to about 80%, they were digested with trypsin to form a single cell suspension and counted on a hemocytometer. The cell number was 5×10 5 Then 500 μL of cell suspension was inoculated into a 24-well plate with a cell slide, with an inoculation density of 2.5×10 5 cells / well. After the cells were cultured overnight until they were completely attached to the wall, the culture medium was discarded and the cells were rinsed twice with fresh culture medium for later use.

[0185] 2. Preparation of bacterial suspension

[0186] The fresh bacterial culture of Lactobacillus rhamnosus VHProbi M14 was washed twice with pH 7.0 phosphate buffer, and then resuspended with the same volume of 1640 culture medium containing 10% calf serum, and the absorbance was adjusted to OD600 = 0.4-0.5.

[0187] 3. Cell culture

[0188] 500 μL of bacterial suspension was added to the prepared 24-well plate containing primary human gingival epithelial cells, and the cells were symbiotically cultured in a carbon dioxide incubator for 2 h; the cells were washed three times with pH 7.0 phosphate buffer to remove non-adherent bacteria.

[0189] 4. Microscopic examination

[0190] The cell slides were fixed with methanol for 15 minutes, then stained with Giemsa stain for 5 minutes, washed with pH 7.0 phosphate buffer, and then the cell slides were taken out and placed on a glass slide. Observed and counted under a microscope, 50 cells were randomly selected, and the number of Lactobacillus rhamnosus on their visible cell surfaces was calculated. The mean and standard deviation of the adhesion index were calculated using statistical methods.

[0191] Adhesion index = number of adhered bacteria / number of cells.

[0192] The results showed that only about one bacterium could be seen adhering to five cells, and the adhesion index was 0.17, indicating that Lactobacillus rhamnosus VHProbi M14 had good adhesion to gingival epithelial cells and could effectively colonize in the oral cavity.

[0193] Example 13: Antagonistic adhesion test of Lactobacillus rhamnosus VHProbi M14 against periodontal pathogens at the cellular level

[0194] 1. Cell pre-culture

[0195] Normal human gingival epithelial cells were revived in liquid nitrogen and cultured to the required amount. When the cell density grew to about 80%, they were digested with trypsin to form a single cell suspension and counted on a hemocytometer. The cell number was 5×10 5 Then, 500 μL of cell suspension was inoculated into a 24-well plate with a cell slide, and cultured overnight until the cells were completely attached to the wall. Then, the culture medium was discarded, and the cells were rinsed twice with fresh culture medium for later use.

[0196] 2. Preparation of bacterial suspension

[0197] The fresh bacterial culture of Lactobacillus rhamnosus VHProbi M14 was washed twice with pH 7.0 phosphate buffer, and then resuspended with the same volume of 1640 culture medium containing 10% calf serum, and the absorbance was adjusted to OD600 = 0.4-0.5.

[0198] The bacterial suspensions of Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacterium semiactinomycetemcomitans were prepared by the same sampling method.

[0199] 3. Cell culture

[0200] Experimental group: Lactobacillus rhamnosus VHProbi M14 bacterial suspension and pathogenic bacteria suspension were mixed at a volume ratio of 1:1, and then 500 μL was added to the prepared 24-well plate;

[0201] Control group: Mix the pathogenic bacteria suspension and phosphate buffer at a volume ratio of 1:1, and then add 500 μL into the prepared 24-well plate.

[0202] The 24-well plate was placed in a carbon dioxide incubator for symbiotic culture for 2 h, the culture was terminated, and the plate was washed three times with pH 7.0 phosphate buffer to remove non-adherent bacteria.

[0203] 4. Microscopic examination

[0204] Each well was fixed with 500 μL methanol for 15 min, then stained with Giemsa stain for 5 min, washed with pH 7.0 phosphate buffer, and the cells in the well were taken out and climbed onto a glass slide. Observed and counted under a microscope, 50 cells were randomly selected, and the number of pathogens on their visible cell surface was calculated. The mean and standard deviation of the adhesion index were calculated using statistical methods. The specific results are shown in Table 7. The microscopic examination results are shown in Table 7. Figure 6-8 As shown, where adhesion index = number of adhered bacteria / number of cells.

[0205] Table 7: Antagonistic adhesion effect of Lactobacillus rhamnosus VHProbi M14 on periodontal pathogens

[0206]

[0207] from Figure 6-8 As can be seen from the data in Table 7, compared with the control group, the adhesion index of Aggregatibacterium semiactinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum to gingival epithelial cells in the experimental group added with the suspension of Lactobacillus rhamnosus VHProbiM14 generally decreased by 43%-96%, among which the adhesion inhibition effect on Aggregatibacter semiactinomycetemcomitans was the strongest, and the adhesion index was the lowest, only 1.38. This shows that Lactobacillus rhamnosus VHProbi M14 can significantly inhibit the adhesion of common periodontal pathogens to gingival epithelial cells, achieving unexpected technical effects.

[0208] The above cell experiment results show that Lactobacillus rhamnosus VHProbi M14 has no toxicity to normal human gingival epithelial cells, and can adhere to the cells, effectively colonize in the oral cavity, and help balance the oral flora. Lactobacillus rhamnosus VHProbi M14 can significantly inhibit the adhesion and growth of periodontal pathogens (Semiactinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum) on normal human gingival epithelial cells, effectively preventing and improving symptoms such as periodontitis and gingival bleeding caused by the pathogens. Sequence Listing <110> Qingdao Blue Biotechnology Co., Ltd. Qingdao Blue Biotechnology Group Co., Ltd. <120> A strain of Lactobacillus rhamnosus and its application in preventing or treating dental caries and periodontal disease <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1351 <212> DNA <213> Lacticaseibacillus rhamnosus <400> 1 ccggcttcgg gtgttacaaa ctctcatggt gtgacgggcg gtgtgtacaa ggcccgggaa 60 cgtattcacc gcggcgtgct gatccgcgat tactagcgat tccgacttcg tgtaggcgag 120 ttgcagccta cagtccgaac tgagaatggc tttaagagat tagcttgacc tcgcggtctc 180 gcaactcgtt gtaccatcca ttgtagcacg tgtgtagccc aggtcataag gggcatgatg 240 atttgacgtc atccccacct tcctccggtt tgtcaccggc agtcttacta gagtgcccaa 300 ctaaatgctg gcaactagtc ataagggttg cgctcgttgc gggacttaac ccaacatctc 360 acgacacgag ctgacgacaa ccatgcacca cctgtcattt tgcccccgaa ggggaacctg 420 atctctcagg tgatcaaaag atgtcaagac ctggtaaggt tcttcgcgtt gcttcgaatt 480 aaaccacatg ctccaccgct tgtgcgggcc cccgtcaatt cctttgagtt tcaaccttgc 540 ggtcgtactc cccaggcgga atgcttaatg cgttagctgc ggcactgaag ggcggaaacc 600 ctccaacacc tagcattcat cgtttacggc atggactacc agggtatcta atcctgttcg 660 ctacccatgc tttcgagcct cagcgtcagt tacagaccag acagccgcct tcgccactgg 720 tgttcttcca tatatctacg catttcaccg ctacacatgg agttccactg tcctcttctg 780 cactcaagtt tcccagtttc cgatgcactt cctcggttaa gccgagggct ttcacatcag 840 acttaaaaaa ccgcctgcgc tcgctttacg cccaataaat ccggataacg cttgccacct 900 acgtattacc gcggctgctg gcacgtagtt agccgtggct ttctggttgg ataccgtcac 960 gccgacaaca gttactctgc cgaccattct tctccaacaa cagagtttta cgacccgaaa 1020 gccttcttca ctcacgcggc gttgctccat cagacttgcg tccattgtgg aagattccct 1080 actgctgcct cccgtaggag tttgggccgt gtctcagtcc caatgtggcc gatcaacctc 1140 tcagttcggc tacgtatcat tgccttggtg agccgttacc tcaccaacta gctaatacgc 1200 cgcgggtcca tccaaaagcg atagcttacg ccatctttca gccaagaacc atgcggttct 1260 tggatttatg cggtattagc atctgtttcc aaatgttatc ccccacttaa gggcaggtta 1320 cccacgtgtt actcacccgt ccgccactcg t 1351

Claims

1. A Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), characterized in that, The preservation number of the Lactobacillus rhamnosus is CCTCC NO: M2022171.

2. Use of the Lactobacillus rhamnosus described in claim 1 in preparing a product having the function of scavenging DPPH free radicals or scavenging hydroxyl free radicals.

3. Use of the Lactobacillus rhamnosus described in claim 1 in preparing a product having the function of inhibiting oral pathogens, wherein the oral pathogens are Streptococcus mutans, Porphyromonas gingivalis, Actinomyces viscosus, Fusobacterium nucleatum or Aggregatibacter actinomycetemcomitans.

4. A product for inhibiting oral pathogens, characterized in that: The product contains live bacteria and / or lysate of Lactobacillus rhamnosus according to claim 1; the oral pathogens are Streptococcus mutans, Porphyromonas gingivalis, Actinomyces viscosus, Fusobacterium nucleatum or Aggregatibacter actinomycetemcomitans.

5. The article according to claim 4, characterized in that The lysate is obtained by ultrasonically disrupting the fresh bacterial solution of Lactobacillus rhamnosus according to claim 1 and then performing heat inactivation.

6. The product according to claim 4 or 5, which is a medicine or an oral care product.

7. The article according to claim 6, characterized in that The oral care product is any one of tooth gel, tooth powder, toothpaste, dental floss, tooth cleaning liquid, mouthwash, mouth spray or tooth cleaning foam.

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