A strain of salivary lactobacillus having the efficacy of preventing or treating dental caries and periodontal disease

Abstract

The present invention provides a saliva-associated lactobacillus having the function of preventing or treating dental caries and periodontal disease and its application. The deposit number of the saliva-associated lactobacillus is CCTCC NO: M2022172. The saliva-associated lactobacillus provided by the present invention has a significant inhibitory effect on mutans streptococci and viscosus actinomycetes that cause dental caries, and on porphyromonas gingivalis, fusobacterium nucleatum and semiactinomycetemcomedans that cause periodontal disease, and can be used as an oral probiotic to effectively prevent and improve oral diseases. The lysate of the saliva-associated lactobacillus can also remove the biofilm formed by mutans streptococci and inhibit the growth and reproduction of mutans streptococci, viscosus actinomycetes, porphyromonas gingivalis, fusobacterium nucleatum and semiactinomycetemcomedans, and can be added to medicines, health products, and toothpaste, mouthwash and other dental care products for use. At the same time, the saliva-associated lactobacillus has the ability to resist artificial gastric juice and artificial intestinal juice, and can be colonized in the intestine and play a probiotic role.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic screening and application, and specifically relates to a saliva-associated lactobacillus capable of preventing or treating dental caries and periodontal disease and an application thereof. Background Art

[0002] Oral health is an important part of human health, directly or indirectly affecting overall health. There are many anaerobic and aerobic microorganisms in the oral cavity, such as Streptococcus salivarius, Lactobacillus fermentum, Streptococcus mutans, Streptococcus sanguinis, etc. In this way, the microorganisms check and balance each other to maintain health. Once pathogenic microorganisms multiply in large numbers, they will cause various oral diseases, such as caries, periodontal disease, oral ulcers, halitosis, etc. The extracellular products secreted by pathogenic bacteria will also destroy the hard tissues of teeth and the supporting tissues around teeth. In addition to affecting functions such as chewing, speech, and aesthetics, it will also cause social interaction difficulties and psychological disorders. Some microorganisms exist in the oral cavity for a long time, which can cause or aggravate certain systemic diseases such as coronary heart disease, diabetes, etc., endangering overall health and affecting the quality of life.

[0003] Caries is the most common chronic infectious disease in the human oral cavity and the main pathological cause of toothache and tooth loss. Its essence is the imbalance of tooth demineralization and remineralization cycle caused by multiple cariogenic factors. Streptococcus mutans is the most common cariogenic microorganism. Exopolysaccharide production and acid production are the two main cariogenic virulence factors of Streptococcus mutans. In the biological process of sucrose-dependent adhesion, Streptococcus mutans can use glucosyltransferase to metabolize sucrose to produce glucan, which promotes its adhesion to the tooth and the formation of biofilm.

[0004] Periodontal disease refers to diseases that occur in the periodontal tissues, including gingival disease that only affects the gingival tissue and periodontitis that affects the deep periodontal tissues (periodontal ligament, alveolar bone, and cementum). Periodontal disease is a common oral disease and one of the main causes of tooth loss in adults. It is also a major oral disease that endangers human teeth and overall health. It is now recognized that plaque is the initiator of periodontal disease and the main pathogenic factor causing periodontal disease. The most common pathogens are Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia.

[0005] Probiotics are a type of microorganisms that are beneficial to the host. The Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) jointly define them as "live microbial preparations that are beneficial to the health of the host (human or animal) when taken in appropriate doses." There have been many studies on the effects of probiotics in the gastrointestinal tract, but in the field of oral medicine (especially periodontal disease), it has only just started in recent years. Studies have shown that the adhesion of probiotics to mucosal cells is the primary condition for probiotics to exert their mucosal protective effects. Probiotics can compete with pathogens for the total number of adhesion sites, nutrients and growth factors, produce antibacterial substances, destroy the biofilm formed by pathogens, and inhibit the growth of pathogens, thereby achieving the purpose of maintaining oral health. At present, screening oral probiotics with good antibacterial properties is a research hotspot in this field. Summary of the invention

[0006] The purpose of the present invention is to provide a saliva-associated lactobacillus capable of preventing or treating dental caries and periodontal disease and application thereof, thereby making up for the deficiencies of the prior art.

[0007] The salivary lactobacillus provided by the present invention is the salivary lactobacillus VHProbi A17 (Lagilactobacillus salivarius VHProbi A17) strain, which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on March 1, 2022, with a deposit number of CCTCC NO: M2022172.

[0008] The provided salivary lactobacillus VHProbi A17 strain has a 16s rDNA sequence of SEQ ID NO:1.

[0009] The protein spectrum of the salivary Lactobacillus VHProbi A17 strain provided is as follows Figure 1 As shown, the Riboprinter fingerprint is as follows Figure 2 shown.

[0010] Another aspect of the present invention provides a use of the saliva-associated Lactobacillus VHProbi A17 strain, which is the use in preparing a product with antioxidant function.

[0011] In another aspect, the present invention also provides the use of the saliva-combined Lactobacillus VHProbi A17 strain in the preparation of a product having the function of preventing or treating oral diseases.

[0012] The oral disease is caries or periodontal disease.

[0013] The product is a medicine or an oral care product.

[0014] The oral care product can be any one of tooth gel, tooth powder, toothpaste, mouthwash, mouth spray, dental floss, tooth cleaning liquid and tooth cleaning foam.

[0015] Another aspect of the present invention is to provide a product for inhibiting oral pathogens, wherein the product comprises live bacteria and / or lysate of the above-mentioned saliva-associated Lactobacillus VHProbi A17 strain;

[0016] The lysate is obtained by ultrasonically disrupting saliva and fresh bacterial liquid of Lactobacillus VHProbi A17 strain, followed by heat inactivation.

[0017] The saliva-combined lactobacillus VHProbi A17 provided by the present invention has a significant inhibitory effect on Streptococcus mutans and Actinomyces viscosus that cause dental caries, and Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans that cause periodontal diseases, and can be used as oral probiotics to effectively prevent and improve oral diseases. The lysate of saliva-combined lactobacillus VHProbi A17 can also remove the biofilm formed by Streptococcus mutans and inhibit the growth and reproduction of Streptococcus mutans, Actinomyces viscosus, Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans, and can be added to medicines, and used in tooth care products such as toothpaste and mouthwash. At the same time, the saliva-combined lactobacillus VHProbi A17 has the ability to resist artificial gastric juice and artificial intestinal juice, and can be colonized in the intestine and play a probiotic role. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The protein spectrum of Lactobacillus salivarius VHProbi A17;

[0019] Figure 2 The Riboprinter fingerprint of salivary combined Lactobacillus VHProbi A17;

[0020] Figure 3 The agglutination test diagram of salivary combined Lactobacillus VHProbi A17 and four pathogenic bacteria; A is a control of salivary combined Lactobacillus, B is salivary combined Lactobacillus + Streptococcus mutans; C is salivary combined Lactobacillus + Porphyromonas gingivalis; D is salivary combined Lactobacillus + Fusobacterium nucleatum; E is salivary combined Lactobacillus + Aggregatibacter actinomycetemcomitans;

[0021] Figure 4 This is the inhibitory growth curve of saliva combined with Lactobacillus VHProbi A17 on Streptococcus mutans;

[0022] Figure 5 This is a graph showing the inhibition of adhesion of saliva combined with Lactobacillus VHProbi A17 on Streptococcus mutans, where A is the control group and B is the experimental group;

[0023] Figure 6 The picture shows that saliva combined with Lactobacillus VHProbi A17 inhibits the adhesion of Porphyromonas gingivalis to normal human gingival epithelial cells; A is the control group and B is the experimental group;

[0024] Figure 7 The picture shows that saliva combined with Lactobacillus VHProbi A17 inhibits the adhesion of Fusobacterium nucleatum to normal human gingival epithelial cells; A is the control group and B is the experimental group;

[0025] Figure 8 The picture shows that saliva combined with Lactobacillus VHProbi A17 inhibits the adhesion of Aggregatibacter actinomycetemcomitans to gingival epithelial cells; A is the control group and B is the experimental group. DETAILED DESCRIPTION

[0026] The polyphasic taxonomic identification shows that the salivary lactobacillus VHProbi A17 provided by the present invention is a newly discovered strain, and long-term use will not cause side effects and overdose risks. The salivary lactobacillus VHProbi A17 provided by the present invention can effectively inhibit common oral pathogens and has important application value.

[0027] The screened Lagilactobacillus salivarius VHProbi A17 VHProbi A17 was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on March 1, 2022, and its deposit number is CCTCC NO: M2022172.

[0028] The present invention is described in detail below in conjunction with embodiments and drawings.

[0029] Example 1 Isolation and screening of strains

[0030] 1. Initial screening:

[0031] According to the 2019 version of the Human Genetic Resource Bank Ethics Code, after signing the project commitment and informed consent form with the sample provider, the feces of healthy infants and young children who have not consumed probiotic preparations within half a year were collected in accordance with the standard operating procedures of the biological sample bank; the feces were serially diluted and 10 -1 , 10 -2 , 10 -3 100 μL of each of the three dilution gradients were spread on MRS selective culture medium and cultured anaerobically at 37°C for 48 h. After single colonies grew on the plates, lactic acid bacteria in the shape of rods were selected through microscopic examination. There were 23 strains in total and were named ZY1, ZY2, ..., ZY23.

[0032] 2. Rescreening against Streptococcus mutans strains

[0033] (1) Prepare the bottom agar plate

[0034] Prepare the bottom 1.5% agar plate in advance and let it dry.

[0035] (2) Preparation of Streptococcus mutans liquid

[0036] 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.

[0037] The mutans streptococcus bacterial liquid described in the following examples is the same as that in this example.

[0038] Using three different mutans Streptococcus as indicator strains can better reflect the antibacterial ability of the screened lactic acid bacteria against mutans Streptococcus.

[0039] (3) Oxford cup antibacterial experiment

[0040] 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.

[0041] The results showed that among the 23 lactobacilli obtained by the initial screening of the present invention, only 2 strains had an inhibitory effect on Streptococcus mutans, with obvious inhibition zones. Among them, the ZY3 strain had the most significant inhibitory effect on Streptococcus mutans, with the diameter of the inhibition zone produced by its fermented bacterial liquid reaching 27 mm.

[0042] Example 2 Identification of ZY3 strain

[0043] 1. Colony morphology identification

[0044] The ZY3 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. They appeared as short rods under a microscope.

[0045] 2. Carbon source metabolism test identification

[0046] API 50CHL reagent strips were used to determine the metabolism of 49 carbon sources by strain ZY3.

[0047] 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.

[0048] The results showed that the ZY3 strain could utilize glucose, fructose, mannose, mannitol, sorbitol, N-acetylglucosamine, esculin, maltose, sucrose, trehalose and D-arabitol; but could not utilize glycerol, erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, ribose, galactose, D-aconitol, β-methyl-D-xyloside, sorbitol, α-methyl-D-glucoside, amygdalin, phellodendron, rhamnose, salicin, cellobiose, dulcitol, inositol, α-methyl-mannoside, lactose, melibiose, raffinose, starch, glycogen, xylitol, D-lyxose, D-fucose, L-fucose, inulin, melezitose, gentiobiose, toulonose, D-tagatose, gluconate, L-arabitol, 2-ketogluconate and 5-ketogluconate.

[0049] API identification results: ZY3 strain is Lactobacillus salivarius, ID = 99.8%, T value = 0.43.

[0050] 3. Molecular Biology Identification

[0051] 3.1 16S rDNA gene sequence analysis

[0052] 3.1.1 Genomic DNA extraction

[0053] A single colony of the ZY3 strain was picked and inoculated into MRS liquid culture medium and cultured at 37° C. for 24 h. 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.

[0054] 3.1.2 16S rDNA gene amplification

[0055] (1) Primer sequence:

[0056] 27F:AGAGTTTGATCCTGGCTCA;

[0057] 1492R: GGTTACCTTGTTACGACTT.

[0058] (2) Reaction system (50 μL)

[0059] Table 1: 6s rDNA PCR amplification system

[0060]

[0061] (3) Electrophoresis verification: The PCR product meets the requirements when the nucleic acid electrophoresis result is about 1500 bp.

[0062] (4) PCR product sequencing

[0063] The 16s rDNA sequence of the ZY3 strain was obtained by sequencing as SEQ ID NO: 1, and the sequence was compared in the NCBI database to determine that the ZY3 strain was Lactobacillus salivarius.

[0064] 3.2 Protein spectrum identification

[0065] Use a toothpick to pick up a single colony of the ZY3 strain on the plate and place it on the protein spectrometry plate. Then use the 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 spectrometry 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.

[0066] Protein spectrum identification results Figure 1 As shown, the ZY3 strain was identified as Lactobacillus salivarius, with a matching rate of 80.75%.

[0067] 3.3Riboprinter fingerprint

[0068] Use a bacterial stick to pick up a single colony of the purified ZY3 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.

[0069] The identification results showed that strain ZY3 was Lactobacillus salivais, and its fingerprint was Figure 2 .

[0070] 3.4 Whole-genome sequencing

[0071] The bacterial liquid of ZY3 strain was inoculated into 500mL MRS broth medium at a volume ratio of 1%, cultured at 37°C 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, which was uploaded to the NCBI gene database with GenBank numbers of CP097165, CP097166, CP097167, CP097168 and CP097169. CP097165 is the genome sequence number, and CP097166, CP097167, CP097168 and CP097169 are the sequence numbers of the plasmids.

[0072] The colony morphology and physiological and biochemical characteristics of the ZY3 strain were compared, and combined with the results of molecular biological identification, the ZY3 strain was determined to be a new type of salivarius Lactobacillus, and was named Lagilactobacillus salivarius VHProbi A17.

[0073] Example 3 Hemolytic test of saliva combined with Lactobacillus VHProbi A17

[0074] 1. Preparation of blood cell culture medium:

[0075] 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.

[0076] 2. Line cultivation:

[0077] The saliva and Lactobacillus VHProbi A17 strain were streaked onto the prepared blood cell plate, cultured in a 37°C incubator, and observed for hemolysis for 24 to 48 hours.

[0078] 3. Experimental results:

[0079] There was no change in the blood cell plate, indicating that the saliva-combined Lactobacillus VHProbi A17 did not produce hemolysin and could not lyse blood cells.

[0080] Example 4 Determination of the antioxidant capacity of saliva combined with Lactobacillus VHProbi A17 strain

[0081] 1. Preparation of bacterial suspension

[0082] A single colony of Lactobacillus salivais VHProbi A17 with good growth status was inoculated into 3 mL of MRS liquid culture medium, cultured at 37°C for 24 hours, and the culture solution was used as the inoculum, inoculated into 50 mL of MRS liquid culture medium at a 2% inoculum amount, and cultured for 24 hours to obtain the culture solution 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 an equal volume of buffer was added to resuspend the bacterial cells for use.

[0083] 2. Determination of the ability of strains to scavenge DPPH free radicals

[0084] Take 1mL of the bacterial suspension of the strain to be tested, add 1mL of the freshly prepared 0.4mM DPPH free radical solution, 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, measure 3 times in parallel. The control group sample is adjusted to zero with an equal volume of phosphate buffer and DPPH·ethanol mixture, and an equal volume of bacterial suspension and ethanol mixture. The clearance rate is calculated according to the following formula.

[0085] Clearance % = [1-(A sample-A blank) / A control] x 100%.

[0086] The results showed that the scavenging rate of DPPH free radicals by saliva combined with Lactobacillus VHProbi A17 was 20.9%.

[0087] 3. Determination of the ability of strains to scavenge hydroxyl radicals

[0088] 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.

[0089] Clearance % = (A sample - A control) / (A blank - A control) x 100%.

[0090] 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.

[0091] The results showed that the scavenging rate of hydroxyl radicals by saliva combined with Lactobacillus VHProbi A17 was 10.6%.

[0092] Example 5 Tolerance of Saliva-Lactobacillus VHProbi A17 to Artificial Gastric and Intestinal Fluids

[0093] 1. Preparation of bacterial solution:

[0094] The frozen saliva-associated Lactobacillus VHProbi A17 strain was streaked and inoculated into MRS solid culture medium, cultured at 37°C for 24-48 hours, and then subcultured once in MRS liquid culture medium. Then, the saliva-associated Lactobacillus VHProbi A17 was inoculated into fresh MRS liquid culture medium at an inoculum size of 5% and cultured with shaking at 40°C for 24-48 hours to obtain a fresh bacterial solution.

[0095] 2. Preparation of artificial gastric juice

[0096] 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.

[0097] 3. Preparation of artificial intestinal fluid

[0098] Weigh 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH2PO4 and 3.0g of ox bile salt, 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.

[0099] 4. Test methods

[0100] 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.

[0101] The live 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 LOG (CFU / mL) of the live bacteria count of the strain after passing through artificial gastric juice and artificial intestinal juice is shown in Table 2.

[0102] Table 2: Table of live bacteria after artificial gastrointestinal digestion

[0103]

[0104] As can be seen from Table 2, after the saliva combined with Lactobacillus VHProbi A17 was digested by artificial gastrointestinal fluid, the amount of live bacteria decreased only slightly, which shows that the strain has a strong tolerance to artificial gastric juice and artificial intestinal juice.

[0105] Example 6 Antibacterial effect test of saliva combined with Lactobacillus VHProbi A17 on oral pathogens

[0106] 1. Lysis buffer preparation

[0107] Saliva combined with Lactobacillus VHProbi A17 was inoculated into MRS broth medium at 1% inoculation volume (volume ratio), and cultured at 37°C for 24 hours before stopping the culture to obtain fresh bacterial liquid. Part of the bacterial liquid was ultrasonically disrupted for 20 minutes using an ultrasonic disruptor, and the ultrasonic conditions were as follows: power 30%, ultrasonication for 2 seconds, and stop for 2 seconds; inactivation 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 saliva combined with Lactobacillus VHProbiA17 bacterial solution and lysate on five common oral pathogens, namely, Streptococcus mutans (three strains), Actinomyces viscosus ATCC27044, Porphyromonas gingivalis BNCC353909, Aggregatibacter actinomycetemcomitans BNCC336945 and Fusobacterium nucleatum BNCC 336949, were respectively determined. The upper culture medium of Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans was replaced with Columbia blood agar medium; the diameter of the inhibition zone is shown in Table 3.

[0109] Table 3: Antibacterial effect of saliva combined with Lactobacillus VHProbi A17 on oral pathogens

[0110] Oral Pathogens Diameter of inhibition zone of A17 fermentation liquid The diameter of the inhibition zone of A17 lysate Streptococcus mutans 27 mm 26mm Porphyromonas gingivalis 14.5mm 10mm Actinomycetes viscosus 23.5mm 22mm Aggregatibacter actinomycetemcomitans 11mm 10mm Fusobacterium nucleatum 12.5mm 11mm

[0111] From the results in Table 3, it can be seen that the saliva-combined lactobacillus VHProbi A17 provided by the present invention has a significant inhibitory effect on five oral pathogens, namely, Streptococcus mutans, Porphyromonas gingivalis, Actinomyces viscosus, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum, and has a wide antibacterial spectrum. Among them, the strain has the strongest antibacterial effect on Streptococcus mutans and Actinomyces viscosus, with an inhibition zone diameter of 22-27 mm, and the antibacterial effect of the fermented bacterial solution is slightly better than that of the lysate, achieving an unexpected technical effect.

[0112] Example 7: Experiment on the Agglutination Effect of Saliva Combined with Lactobacillus VHProbi A17 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] Inoculate into MRS broth medium at an inoculum volume of 1% (volume ratio), culture at 37°C for 24 hours and then stop culturing to obtain fresh bacterial liquid; centrifuge the fresh bacterial liquid at 8000 rpm for 10 minutes to collect the bacteria; wash the bacteria twice with pH 7.0 phosphate buffer, and then resuspend the bacteria with pH 7.0 phosphate buffer until the initial absorbance OD600 of the bacterial suspension is between 0.5 and 0.6, and set aside.

[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, Aggregatibacter actinomycetemcomitans 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 saliva combined with Lactobacillus VHProbi A17 suspension and add it to a 24-well plate, then add 300 μL of pathogen suspension as a reaction sample, and take an equal amount of saliva combined with Lactobacillus VHProbi A17 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 state and the well plate state at each stop.

[0121] from Figure 3The results show that within 2 hours, saliva-lactobacillus VHProbi A17 and four pathogenic bacteria, namely, Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans, all showed agglutination points. Among them, there were small and multiple agglutination points for Streptococcus mutans, Porphyromonas gingivalis and Fusobacterium nucleatum, and one large agglutination point for Aggregatibacter actinomycetemcomitans, indicating that saliva-lactobacillus VHProbi A17 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 saliva-combined Lactobacillus VHProbi A17 bacterial 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 h in the following 6 h, and the absorbance At was measured at a wavelength of 600 nm. The self-aggregation force (Rself) was calculated. The results are shown in Table 4.

[0125] R 自 =1-At / A0;

[0126] Where: R 自 is the self-aggregation force, %; At is the absorbance at time t, t = 2, 4 and 6h; A0 is the initial absorbance of the saliva-combined Lactobacillus VHProbi A17 bacterial suspension.

[0127] Table 4: Self-agglutination rate of saliva combined with Lactobacillus VHProbi A17

[0128] Coagulation time Self-agglutination rate 2h 12.81％ 4h 27.53％ 6h 41.08％

[0129] From the results in Table 4, it can be seen that the self-agglutination rate of the saliva combined with Lactobacillus VHProbi A17 provided by the present invention reaches 41.085% at 6 hours, which indicates that the saliva combined with Lactobacillus VHProbi A17 can effectively colonize in the oral cavity.

[0130] (2) Coagulation rate determination

[0131] Take 100 μL of saliva combined with Lactobacillus VHProbi A17 suspension and pathogenic bacteria suspension to determine the initial absorbance OD600, then mix equal amounts of saliva combined with Lactobacillus VHProbi A17 suspension and 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. The coagulation force (R) is calculated according to the following formula 共 ). The results are shown in the table.

[0132] R共 =1-2At / (A0+B0);

[0133] Where: R 共 is the coagulation force, %; At is the absorbance at time t = 2, 4 and 6h; A0 is the initial absorbance of saliva combined with Lactobacillus VHProbi A17; B0 is the initial absorbance of pathogenic bacteria suspension.

[0134] Table 5: Coaggregation effect of saliva combined with Lactobacillus VHProbi A17 on oral pathogens

[0135]

[0136] As can be seen from the results in Table 5, the saliva-combined lactobacillus VHProbi A17 provided by the present invention can effectively combine with four common oral pathogens, namely, Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans, and the coaggregation rate reaches 14.71%-58.63% within 2-6 hours. Among them, the saliva-combined lactobacillus VHProbi A17 has the best coaggregation effect on Streptococcus mutans, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans, and the coaggregation rate at 6 hours is as high as 48.05%-58.63%, achieving unexpected technical effects.

[0137] The saliva-combined Lactobacillus VHProbi A17 provided by the present invention can effectively combine with pathogenic bacteria, and with the flow of saliva and oral cleaning, significantly reduce the number of pathogenic bacteria in the oral cavity, thereby achieving the effect of reducing the load of pathogenic bacteria in the oral cavity.

[0138] Example 8: Experiment on the inhibition of growth of Streptococcus mutans by saliva combined with Lactobacillus VHProbi A17

[0139] 1. Preparation of lysate supernatant

[0140] The saliva and fresh bacterial liquid of Lactobacillus VHProbi A17 were broken into cell walls by an ultrasonic cell disruptor, and then treated at 80° C. for 60 min. Insoluble matter was removed by filtration with a 0.22 μL microporous filter membrane to prepare a lysate supernatant.

[0141] 2. Preparation of inoculum

[0142] 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.

[0143] 3. 96-well plate culture

[0144] 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 4 parallels for each group. Place the 96-well plate in a 37°C microplate reader, set the OD600 to be measured every 10 minutes, and measure for 24 hours.

[0145] from Figure 4 As can be seen from the above, the mutans Streptococcus in the control group began to grow rapidly 10 hours after inoculation and reached a stable period at 20 hours; while in the experimental group added with the supernatant of saliva combined with Lactobacillus VHProbi A17 lysate, the mutans Streptococcus had almost no growth within 20 hours, indicating that the saliva combined with Lactobacillus VHProbi A17 lysate can effectively inhibit the growth and reproduction of mutans Streptococcus.

[0146] Example 9: Antagonistic adhesion test of saliva combined with Lactobacillus VHProbi A17 against Streptococcus mutans

[0147] 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. Therefore, oral probiotics can inhibit the growth and adhesion of pathogenic bacteria by forming agglutination with them, thereby effectively reducing oral diseases.

[0148] 1. Preparation of bacterial suspension

[0149] The saliva-combined Lactobacillus VHProbi A17 was inoculated into MRS broth medium at an inoculation volume 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.

[0150] 2. Preparation of Streptococcus mutans suspension:

[0151] 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.

[0152] 500 μL of saliva combined with Lactobacillus VHProbi A17 suspension and 500 μL of Streptococcus mutans suspension were mixed evenly, and after standing for 5 minutes, 500 μL of the upper solution was added to the 24-well plate with sterile cell slides, and cultured at 37°C for 2 hours, and then the upper solution was removed, washed with pH 7.0 phosphate buffer, and fixed with 0.5 mL of methanol for 10 minutes, and then discarded. Finally, 300 μL of Giemsa stain was added for 10 minutes, and then the stain was discarded, rinsed with pH 7.0 phosphate buffer, and the cell slide was placed on a slide to observe the amount of bacteria on the cell slide.

[0153] The results are as follows Figure 5 As shown, the control group cell slides were full of Streptococcus mutans, while the experimental group cell slides added with Lactobacillus saliva VHProbi A17 had very few Streptococcus mutans, which were almost invisible within the visual range, indicating that Lactobacillus saliva VHProbi A17 can effectively inhibit Streptococcus mutans from adhering to the cell slides. Thus, it is shown that the Lactobacillus saliva VHProbi A17 provided by the present invention can effectively prevent pathogens from adhering to teeth, thereby preventing the occurrence of dental caries.

[0154] Example 10: Effect of saliva combined with Lactobacillus VHProbi A17 on biofilm elimination of Streptococcus mutans

[0155] Streptococcus mutans adheres to and aggregates on the tooth surface to form a biofilm, which in turn produces acid substances that lead to tooth decay. Therefore, removing the biofilm formed by Streptococcus mutans is very important for preventing tooth decay.

[0156] 1. Prepare fresh Streptococcus mutans liquid by referring to the method described in Example 1.

[0157] 2. Inoculate saliva and Lactobacillus VHProbi A17 into MRS broth medium at 1% inoculation volume (volume ratio), culture at 37°C for 24 hours, and then stop culturing to obtain fresh fermentation liquid. Divide the fermentation liquid into three parts and perform the following treatments:

[0158] (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;

[0159] (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;

[0160] (3) Some fermentation broth is not treated in any way.

[0161] 3. Prepare a sterile 24-well plate with cell slides

[0162] 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 saliva combined with Lactobacillus VHProbi A17 fermentation liquid, bacterial suspension and lysis liquid without living cells were added to the wells. Three parallels were made for each group, and an equal amount of MRS broth culture medium was used as a control.

[0163] The sample to be tested 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. Then, the removal rate of the biofilm of the sample to be tested was determined by comparing the number of Streptococcus mutans on the cell slide.

[0164] The cell slide was placed in a sterile homogenizing bag and ultrasonically cleaned for 10 minutes to diffuse the bacteria into the buffer. Then, after a series of dilutions, 100 μL was applied to a light salivarius Streptococcus culture medium supplemented with 200 U / L bacitracin and aerobically cultured at 37°C for 24 hours. The bacterial count of mutans streptococci was detected and the removal rate of mutans streptococcus biofilm was calculated by the following formula: removal rate (%) = (1- bacterial count of experimental group / bacterial count of control group) × 100%. The specific results are shown in Table 6.

[0165] Table 6: Effect of saliva combined with Lactobacillus VHProbi A17 on the removal of Streptococcus mutans biofilm

[0166] Grouping Streptococcus mutans count (CFU) Biofilm removal rate Control group 27700 - Fermentation Broth 0 100％ bacterial suspension 16400 40.79％ Lysis buffer 0 100％

[0167] From the data in Table 6, it can be seen that saliva combined with lactobacillus VHProbi A17 can efficiently remove the biofilm formed by Streptococcus mutans. Among them, the removal rate of the biofilm by the bacterial suspension, that is, the simple bacterial body, is 40.79%, indicating that there are binding sites for Streptococcus mutans on the bacterial cell membrane, and the simple bacterial body itself can also bind to Streptococcus mutans and remove part of the biofilm in a short time; and the fermentation liquid and the lysate contain more metabolites in addition to the bacterial body itself, which can more efficiently remove the biofilm formed by Streptococcus mutans, with a removal rate of up to 100%. Therefore, it is shown that the saliva combined with lactobacillus VHProbi A17 provided by the present invention can effectively prevent or treat dental caries caused by Streptococcus mutans, and can be widely used in oral care products such as toothpaste and mouthwash.

[0168] Example 11: Toxicity test of saliva combined with Lactobacillus VHProbi A17 on normal human gingival epithelial cells

[0169] 1. Cell pre-culture

[0170] 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.

[0171] 2. Preparation of inactivated bacteria solution

[0172] The fresh bacterial liquid of saliva-combined Lactobacillus VHProbi A17 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 OD600 = 0.4-0.5.

[0173] 3. Cell culture

[0174] 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;

[0175] Blank control group: add cell culture medium of the same volume as the inactivated bacteria solution.

[0176] The culture was continued in a carbon dioxide incubator for 24 h.

[0177] 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.

[0178] The results showed that the absorbance value of the blank control group was 0.668, while that of the experimental group was 0.680, which was basically the same. This shows that saliva combined with Lactobacillus VHProbi A17 has no significant effect on the proliferation activity of normal human gingival epithelial cells, has good safety, and is non-cytotoxic.

[0179] Example 12: Adhesion of saliva combined with Lactobacillus VHProbi A17 to gingival epithelial cells

[0180] 1. Cell pre-culture

[0181] 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.

[0182] 2. Preparation of bacterial suspension

[0183] The fresh bacterial liquid of saliva-associated Lactobacillus VHProbi A17 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.

[0184] 3. Cell culture

[0185] 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.

[0186] 4. Microscopic examination

[0187] 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 saliva-associated Lactobacillus VHProbi A17 on their visible cell surfaces was calculated. The mean and standard deviation of the adhesion index were calculated using statistical methods.

[0188] Adhesion index = number of adhered bacteria / number of cells.

[0189] The results showed that approximately one Lactobacillus salivae VHProbi A17 cell adhered to five gingival cells, and the adhesion index was 0.2, indicating that Lactobacillus salivae VHProbi A17 had good adhesion properties and could be colonized in the oral cavity.

[0190] Example 13: Antagonistic adhesion test of saliva combined with Lactobacillus VHProbi A17 against periodontal pathogens at the cellular level

[0191] 1. Cell pre-culture

[0192] 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 5×105 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.

[0193] 2. Preparation of bacterial suspension

[0194] The fresh bacterial liquid of saliva-associated Lactobacillus VHProbi A17 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.

[0195] The same sampling method was used to prepare bacterial suspensions of Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans.

[0196] 3. Cell culture

[0197] Experimental group: Mix the saliva-lactobacillus VHProbi A17 suspension and the pathogenic bacteria suspension in a volume ratio of 1:1, and then take 500 μL and add it to the prepared 24-well plate;

[0198] 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.

[0199] 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.

[0200] 4. Microscopic examination

[0201] 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. Figure 6-8 shown.

[0202] Adhesion index = number of adhered bacteria / number of cells.

[0203] Table 7: Antagonistic effect of saliva combined with Lactobacillus VHProbi A17 on periodontal pathogens

[0204]

[0205] from Figure 6-8As can be seen from the data in Table 7, compared with the control group, the adhesion index of Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans to gingival epithelial cells in the experimental group added with saliva combined with Lactobacillus VHProbiA17 bacterial suspension generally decreased by 87%-95%, among which the adhesion inhibition to Aggregatibacter actinomycetemcomitans was the strongest, with an adhesion index of only 1.64. This shows that saliva combined with Lactobacillus VHProbi A17 can significantly inhibit the adhesion of common periodontal pathogens to gingival epithelial cells, achieving unexpected technical effects.

[0206] The results of the above cell experiments showed that saliva combined with Lactobacillus VHProbi A17 had no toxicity to normal human gingival epithelial blood cells, and could significantly inhibit the adhesion and growth of periodontal pathogens (Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans) on gingival epithelial cells, effectively preventing and improving symptoms such as periodontitis and gingival bleeding. Sequence Listing <110> Qingdao Blue Biotechnology Co., Ltd. Qingdao Blue Biotechnology Group Co., Ltd. <120> A strain of salivary lactobacillus having the efficacy of preventing or treating dental caries and periodontal disease <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1389 <212> DNA <213> Lagilactobacillus salivariu <400> 1 ctttgggtgt tacaaactct catggtgtga cgggcggtgt gtacaaggcc cgggaacgta 60 ttcaccgcgg catgctgatc cgcgattact agcgattccg acttcgtgta ggcgagttgc 120 agcctacagt ccgaactgag aatggtttta agagattagc ttaacctcgc ggtctcgcga 180 ctcgttgtac catccattgt agcacgtgtg tagcccaggt cataaggggc atgatgattt 240 gacgtcgtcc ccaccttcct ccggtttgtc accggcagtc tcactagagt gcccaactta 300 atgctggcaa ctagtaataa gggttgcgct cgttgcggga cttaacccaa catctcacga 360 cacgagctga cgacaaccat gcaccacctg tcattctgtc cccgaaggga acctctaatc 420 tcttagactg tcagaagatg tcaagacctg gtaaggttct tcgcgtagct tcgaattaaa 480 ccacatgctc caccgcttgt gcgggccccc gtcaattctt ttgagtttca accttgcggt 540 cgtactcccc aggcggatta cttaatgcgt tagctgcagc actgaagggc ggaaaccctc 600 caacacttag taatcatcgt ttacggcatg gactaccagg gtatctaatc ctgttcgcta 660 cccatgcttt cgagcctcag cgtcagttgc agaccagaca gccgccttcg ccactggtgt 720 tcttccatat atctacgcat ttcaccgcta cacatggagt tccactgtcc tcttctgcac 780 tcaagtctcc cagtttccaa tgcacttctt cggttgagcc gaaggctttc acattagact 840 taaaagaccg cctgcgctcg ctttacgccc aataaatccg gataacgctt gccacctacg 900 tattaccgcg gctgctggca cgtagttagc cgtggctttc tggttaaata ccgtcactgg 960 gtaaacagtt actcttaccc acgttcttct ttaacaacag agctttacga gccgaaaccc 1020 ttcttcactc acgcggcgtt gctccatcag acttgcgtcc attgtggaag attccctact 1080 gctgcctccc gtaggagtct gggccgtgtc tcagtcccaa tgtggccgat taccctctca 1140 ggtcggctac gtatcactgc cttggtgagc ctttacctca ccaactagct aatacgccgc 1200 gggtccatcc agaagtgata gcagagccat cttttaaaag aaaaccatgc ggttttctct 1260 gttatacggt attagcatct gtttccaggt gttatcccct acttctgggc aggttaccca 1320 cgtgttactc acccgttcgc cactcacttc gtgttaaaat ctcaatcagt acaagtacgt 1380 cataatcaa 1389

Claims

1. A salivary lactobacillus, It is characterized in that The deposit number of the saliva-associated lactobacillus (Lagilactobacillus salivarius) is CCTCC NO: M2022172.

2. Use of the saliva-combined lactobacillus according to claim 1 in preparing a product with antioxidant function.

3. Use of the salivary lactobacillus according to claim 1 in the preparation of a product having the function of preventing or treating oral diseases; the oral diseases are dental caries or periodontal disease caused by Streptococcus mutans, Actinomyces viscosus, Porphyromonas gingivalis, Fusobacterium nucleatum or Aggregatibacter actinomycetemcomitans.

4. The use according to claim 2 or 3, It is characterized in that The product is a medicine or an oral care product.

5. The use according to claim 4, It is characterized in that The oral care product is any one of tooth gel, tooth powder, toothpaste, mouthwash, mouth spray, dental floss, tooth cleaning liquid and tooth cleaning foam.

6. A product for inhibiting oral pathogens, It is characterized in that The product contains the live bacteria of salivary-associated lactobacillus according to claim 1, and the oral pathogens are Streptococcus mutans, Actinomyces viscosus, Porphyromonas gingivalis, Fusobacterium nucleatum or Aggregatibacter actinomycetemcomitans.

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