CN115960741A - A strain of Lactobacillus saliva associated with prevention or treatment of dental caries and periodontal disease - Google Patents

Abstract

The invention provides a lactobacillus salivarius for preventing or treating dental caries and periodontal disease and application thereof, wherein the preservation number of the lactobacillus salivarius is CCTCC NO: m2022172. The saliva combined with the lactobacillus provided by the invention has obvious inhibition effects on streptococcus mutans and actinomyces viscosus which cause dental caries, and porphyromonas gingivalis, fusobacterium nucleatum and aggregatibacter semiactinomyces which cause periodontal diseases, and can be used as oral probiotics to effectively prevent and improve oral diseases. The lysate of saliva combined with lactobacillus 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 semiactinomycetes, and can be added into medicines, health products, toothpaste, mouthwash and other tooth care products for use. Meanwhile, the saliva combined with the lactobacillus has the capability of resisting artificial gastric juice and artificial intestinal juice, and can be planted in the intestinal tract and play a probiotic role.

Description

A strain of Lactobacillus saliva associated with prevention or treatment of dental caries and periodontal disease

technical field

The invention belongs to the technical field of screening and application of probiotics, and in particular relates to a strain of Lactobacillus saliva associated with preventing or treating dental caries and periodontal disease and its application.

Background technique

Oral health is an important part of human health, which directly or indirectly affects the health of the whole body. There are a variety of anaerobic and aerobic microorganisms in the oral cavity, such as Streptococcus salivarius, Lactobacillus fermentum, Streptococcus mutans, Streptococcus sanguis, etc., so that the microorganisms check and balance each other to maintain health. Once the pathogenic microorganisms multiply, they will Lead to various oral diseases, such as caries, periodontal disease, oral ulcers, bad breath, etc. The extracellular products secreted by pathogenic bacteria can also damage the hard tissue of the tooth and the supporting tissue around the tooth, in addition to affecting the functions of chewing, speech, and aesthetics , can also cause social communication difficulties and psychological barriers. 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., endanger the health of the whole body and affect the quality of life.

Caries is the most common chronic infectious disease in the human oral cavity. It is the main pathological cause of toothache and tooth loss. Its essence is the imbalance of tooth demineralization and remineralization cycle caused by various 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 teeth and the formation of biofilm.

Periodontal disease refers to diseases that occur in the periodontal tissue, including gingival disease that only involves the gingival tissue and periodontitis that affects the deep periodontal tissue (periodontal ligament, alveolar bone, cementum). Periodontal disease is a common oral disease, one of the main causes of adult tooth loss, and a major oral disease that endangers human teeth and general health. It has been recognized that bacterial plaque is the initiating factor of periodontal disease and the main pathogenic factor causing periodontal disease. The most common pathogenic bacteria are Porphyromonas gingivalis, Fusobacterium nucleatum and Platts intermedia caused by bacteria, etc.

Probiotics are a class 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 "living 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 effect of probiotics on the gastrointestinal tract, but in the field of stomatology (especially periodontal disease), it has 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 effect. Probiotics can compete with pathogenic bacteria for the total number of adhesion sites, nutrients and growth factors, produce antibacterial substances, destroy the biofilm formed by pathogenic bacteria, and inhibit the growth of pathogenic bacteria, so as to maintain oral health. At present, the screening of oral probiotics with good antibacterial properties is a research hotspot in this field.

Contents of the invention

The purpose of the present invention is to provide a strain of Lactobacillus saliva associated with prevention or treatment of dental caries and periodontal disease and its application, so as to make up for the deficiencies in the prior art.

The saliva-associated lactobacillus provided by the present invention is a saliva-associated lactobacillus VHProbi A17 (Lagilactobacillus salivarius VHProbi A17) strain, which was preserved in the Chinese Type Culture Collection Center of Wuhan, China and Wuhan University on March 1, 2022, and the preservation number is CCTCC NO: M2022172.

The 16s rDNA sequence of the provided Lactobacillus salivarius VHProbi A17 strain is SEQ ID NO:1.

The protein spectrum of the provided Lactobacillus saliva associated Lactobacillus VHProbi A17 strain is shown in Figure 1, and the Riboprinter fingerprint is shown in Figure 2.

Another aspect of the present invention also provides a use of the Lactobacillus salivarius VHProbi A17 strain in the preparation of products with antioxidant function.

Another aspect of the present invention also provides the application of the Lactobacillus salivarius VHProbi A17 strain in the preparation of products capable of preventing or treating oral diseases.

The oral disease is dental caries or periodontal disease.

The product is health product, medicine or oral care product.

The oral care article can be any one of dental gel, tooth powder, toothpaste, mouthwash, mouth spray, chewing gum, dental floss, tooth cleaning solution, and tooth cleaning foam.

Another aspect of the present invention is to provide a product for inhibiting oral pathogenic bacteria, which contains the live bacteria and/or lysate of the above-mentioned Lactobacillus saliva-associated Lactobacillus VHProbi A17 strain;

The lysate is obtained by ultrasonically disrupting the fresh bacterium liquid of Lactobacillus saliva-associated Lactobacillus VHProbi A17 strain, and then performing heat inactivation.

Salivary-associated Lactobacillus VHProbi A17 provided by the present invention has significant effects on Streptococcus mutans and Actinomyces viscosus causing dental caries, and Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregate semiactinobacillus causing periodontal disease. The inhibitory effect can be used as oral probiotics to effectively prevent and improve oral diseases. The lysate of Lactobacillus salivarius VHProbi A17 can also remove the biofilm formed by Streptococcus mutans and inhibit the growth of Streptococcus mutans, Actinomyces viscosus, Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregate semiactinobacillus Propagation, can be added to medicines, health care products, and dental care products such as toothpaste and mouthwash. At the same time, Lactobacillus saliva-associated Lactobacillus VHProbi A17 has the ability to resist artificial gastric juice and artificial intestinal juice, and can colonize the intestinal tract and play a probiotic role.

Description of drawings

Figure 1 is the protein spectrum of Lactobacillus salivarius associated Lactobacillus VHProbi A17;

Figure 2 is the Riboprinter fingerprint of Lactobacillus saliva associated Lactobacillus VHProbi A17;

Figure 3 is the agglutination test diagram of Lactobacillus saliva VHProbi A17 and four strains of pathogenic bacteria; A is the control of Lactobacillus saliva, B is Lactobacillus saliva + Streptococcus mutans; C is Lactobacillus saliva + Streptococcus mutans; C is Lactobacillus saliva + gingivalis Porphyrobacter; D is Lactobacillus salivarius + Fusobacterium nucleatum; E is Lactobacillus saliva + Aggregate semiactinobacillus;

Fig. 4 is the inhibitory growth curve of Streptococcus mutans of Lactobacillus salivarius VHProbi A17;

Fig. 5 is the graph of the inhibition adhesion test of saliva combined Lactobacillus VHProbi A17 to Streptococcus mutans, wherein A is the control group, and B is the experimental group;

Figure 6 is a picture showing that saliva combined with Lactobacillus VHProbi A17 inhibits Porphyromonas gingivalis from adhering to normal human gingival epithelial cells; where A is the control group and B is the experimental group;

Figure 7 is a picture of saliva combined with Lactobacillus VHProbi A17 inhibiting the adhesion of Fusobacterium nucleatum to normal human gingival epithelial cells; where A is the control group and B is the experimental group;

Figure 8 is a picture showing that saliva combined with Lactobacillus VHProbi A17 inhibits the adhesion of Aggregate semiactinomycetes to gingival epithelial cells; where A is the control group and B is the experimental group.

Detailed ways

According to multiphase taxonomy, the Lactobacillus saliva-associated Lactobacillus VHProbi A17 provided by the present invention is a newly discovered strain; it can be used as a source of food raw materials, and there will be no side effects and risks of overdose if taken for a long time. The saliva-associated lactobacillus VHProbi A17 of the invention can effectively inhibit common oral pathogens and has important application value.

The screened Lagilactobacillus salivarius VHProbi A17 (Lagilactobacillus salivarius VHProbi A17) VHProbi A17 was deposited on March 1, 2022 at the Chinese Type Culture Collection Center of Wuhan University, Wuhan, China, and its preservation number is CCTCC NO: M2022172.

The present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings.

The isolation and screening of embodiment 1 bacterial strain

1. Primary screening:

According to the 2019 version of the "Human Genetic Resource Bank Ethics Code", after signing the project commitment letter and informed consent form with the sample provider, in accordance with the standard operating specifications of the biobank, collect the feces of healthy infants and young children who have not consumed probiotics within half a year; For serial dilution, take 100 μL each of the three dilution gradients of 10 ^-1 , 10 ^-2 , and 10 ^-3 , spread them on the MRS selective medium, and culture anaerobically at 37°C for 48 hours; after a single colony grows on the plate, , through microscopic examination, pick out the lactic acid bacteria that shape is bacillus, altogether 23 strains, named as ZY1, ZY2, ..., ZY23 respectively.

2. Re-screening against Streptococcus mutans strains

(1) Prepare the bottom agar plate

Prepare the bottom 1.5% agar plate ahead of time and allow to dry.

(2) Preparation of Streptococcus mutans liquid

Streptococcus mutans ATCC 25175, CCTCC AB 99010, and BNCC700610 were streaked on the BHI plate respectively, and then a single colony was picked into the BHI broth medium, aerobically cultured at 37°C for 24 hours, and then transferred to the new strain at a ratio of 1%. In the BHI broth medium, cultured aerobically at 37°C for 24 hours to obtain a fresh bacterial solution, and the fresh bacterial solution was mixed according to an equal volume of 1:1:1 to obtain a Streptococcus mutans bacterial solution.

The Streptococcus mutans bacterium liquid described in the following examples is all the same as this example.

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

(3) Oxford cup antibacterial test

Prepare BHI medium with 0.7% agar content and sterilize it; wait until the temperature drops below 47°C, add 0.3% (volume ratio) mixed Streptococcus mutans bacteria solution, shake well; take 7mL and pour it on the bottom agar plate, etc. After solidification, put the Oxford cup, add 150 μL of the fermentation broth of lactobacillus, which was first screened, to the holes, incubate at 37°C for 48 hours, and observe whether there is a bacteriostatic zone.

The results showed that among the 23 strains of Lactobacillus obtained by the primary screening of the present invention, only 2 strains had an inhibitory effect on Streptococcus mutans, and an obvious inhibition zone appeared. Among them, the ZY3 strain has the most significant inhibitory effect on Streptococcus mutans, and the diameter of the inhibition zone produced by its fermentation broth reaches 27mm.

The identification of embodiment 2 ZY3 bacterial strains

1. Identification of colony morphology

The ZY3 strain was inoculated on MRS agar medium, and after anaerobic culture at 37°C for 24 hours, a single colony was milky white and shiny, the surface of the colony was smooth and moist, the edges were neat, and the diameter of the colony was 1-2mm. Microscopically short rod-shaped.

2. Carbon source metabolism test identification

The metabolic effects of ZY3 strain on 49 carbon sources were determined by API 50CHL reagent strip.

Under sterile conditions, take an appropriate amount of fresh bacterial liquid, centrifuge at 5000 rpm for 5 min, wash twice with pH 7.0 phosphate buffer, and resuspend with the same volume of buffer to obtain bacterial suspension. Add the fresh bacterial suspension to the culture medium of the API kit according to the addition amount of 10%, and then follow the operation of the kit, add the culture medium to the hole of the test strip, seal it with paraffin, and then put the test strip in a Inside the box, add about 10ml of sterile deionized water to the box substrate, cover the lid, place it in a 37°C incubator for 24-48 hours, and observe the color change. If the bacteria grow, the color will change from blue to yellow, and they will not grow. The color remains the same. Record the test results and upload them to the identification software API web.

The results showed that the ZY3 strain could utilize glucose, fructose, mannose, mannitol, sorbitol, N-acetylglucosamine, escin, maltose, sucrose, trehalose and D-arabinitol; Britol, D-arabinose, L-arabinose, D-xylose, L-xylose, ribose, galactose, D-calendol, β-methyl-D-xyloside, sorbose, 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, turonose, D-tower Glatose, Gluconate, L-arabitol, 2-Ketogluconate and 5-Ketogluconate.

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

3. Molecular biological identification

3.1 16s rDNA gene sequence analysis

3.1.1 Genomic DNA extraction

Pick a single colony of ZY3 strain and inoculate it in MRS liquid medium, incubate at 37°C for 24 hours, then take 500 μL of fermentation broth, and refer to the Genomic DNA Extraction Kit of Tienensis Bacteria (catalogue number: DP302) to obtain the genome of the strain.

3.1.2 16s rDNA gene amplification

(1) Primer sequence:

27F: AGAGTTTGATCCTGGCTCA;

1492R: GGTTACCTTGTTACGACTT.

(2) Reaction system (50μL)

Table 1: 6s rDNA PCR amplification system table

(3) Electrophoresis verification The nucleic acid electrophoresis result of the PCR product meets the requirements when it is about 1500 bp.

(4) PCR product sequencing

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 associated.

3.2 Protein profile identification

Use a toothpick to pick up a single colony of the ZY3 strain on the plate on the protein spectrum plate, and then use a toothpick to spread the sludge evenly on the disk on the mass spectrum plate. The coating of the sludge does not need to be too thick, and then follow the protein spectrum kit The instructions require adding 1 μL of the matrix solution in the mass spectrometry sample pretreatment box to cover the sample, and let it dry naturally at room temperature. The dried mass spectrometer plate was placed on a Motitof protein spectrometer for identification.

The results of protein spectrum identification are shown in Figure 1. The ZY3 strain was identified as Lactobacillus salivarius with a matching rate of 80.75%.

3.3 Riboprinter fingerprint

Pick up a single colony of the purified ZY3 strain from the agar medium plate with a bacterial removal stick, put it into the sample tube of the buffer solution, suspend it in the buffer solution with a hand-held stirring rod, and then mix the sample Put it into the Riboprinter system after being inactivated in a heater. After the sample undergoes DNA preparation, mold transfer, imaging detection and data processing, the bacterial identification result is obtained.

The identification results showed that the ZY3 strain was Lactobacillus salivarius associated, and its fingerprint is shown in Figure 2.

3.4 Whole Genome Sequencing

Inoculate the bacterium liquid of ZY3 strain into 500mL MRS broth medium at a volume ratio of 1%, incubate at 37°C for 22h, and then centrifuge at 8000rpm for 10min to collect the bacteria. The bacterium was sent to the sequencing center to obtain the whole gene sequence of the bacterium, and the gene sequence was uploaded to the NCBI gene database. The GenBank numbers are CP097165, CP097166, CP097167, CP097168 and CP097169. Wherein CP097165 is the sequence number of the genome, and CP097166, CP097167, CP097168 and CP097169 are the sequence numbers of the plasmids.

The colony morphology and physiological and biochemical characteristics of the ZY3 strain were compared, and the identification results of molecular biology were combined to determine that the ZY3 strain was a new strain of Lactobacillus salivarius, and named it as Lactobacillus salivarius VHProbi A17 (Lagilactobacillus salivarius VHProbi A17 ).

Example 3 The hemolytic test of saliva combined with Lactobacillus VHProbi A17

1. Blood cell culture medium preparation:

Weigh the various components of the TBS basal medium, dissolve, and autoclave at 121°C for 15 minutes. When the medium cools down to 50°C, add 5% sterile defibrinated sheep blood, mix well, and pour onto a plate.

2. Line cultivation:

Streak inoculation of the Lactobacillus saliva-associated Lactobacillus VHProbi A17 strain on the prepared blood cell plate, culture in a 37°C incubator, and observe whether there is hemolysis within 24 to 48 hours.

3. Experimental results:

The blood cell plate did not change, indicating that Lactobacillus salivarius VHProbi A17 does not produce hemolysin and cannot lyse blood cells.

Example 4 Determination of antioxidant capacity of saliva combined with Lactobacillus VHProbi A17 strain

1. Preparation of bacterial suspension

Inoculate a single colony of Lactobacillus salivarius VHProbi A17 with excellent growth status into 3 mL of MRS liquid medium, and cultivate it at 37°C for 24 hours. Use this culture liquid as the inoculum, and inoculate 50 mL of MRS liquid according to the inoculation amount of 2%. In the culture medium, static culture was carried out for 24 hours to obtain the culture solution of the bacterial strain. Aspirate 1mL of the bacterial liquid to collect the bacterial cells, wash the bacterial cells with 1mL of pH 7.0 phosphate buffer twice, and then add an equal volume of buffer solution to resuspend the bacterial cells for later use.

2. Determination of the ability of bacterial strains to scavenge DPPH free radicals

Take 1mL of the bacterial suspension of the strain to be tested, add 1mL of 0.4mM freshly prepared DPPH free radical solution, mix evenly, then place it at room temperature for 30min in the dark, then measure the absorbance of the sample at a wavelength of 517nm A sample, measure 3 times parallel. The samples of the control group were adjusted to zero with an equal volume of phosphate buffer solution and DPPH·ethanol mixture, and an equal volume of bacterial suspension and ethanol mixture. The clearance rate was calculated according to the following formula.

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

The results showed that the scavenging rate of DPPH free radicals by Lactobacillus salivarius VHProbi A17 was 20.9%.

3. Determination of the ability of bacterial strains to scavenge hydroxyl radicals

Mix 100μL 5mM sodium salicylate-ethanol solution, 100μL 5mM ferrous sulfate, 500μL deionized water and 200μL lactic acid bacteria suspension, add 100μL 3% hydrogen peroxide solution, bathe in 37℃ water for 15min, then centrifuge at 4000rpm for 10min The supernatant was collected and the absorbance of the sample was measured at a wavelength of 510 nm. The hydroxyl radical scavenging rate was calculated according to the following formula.

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

Among them: 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.

The results showed that the scavenging rate of Lactobacillus salivarius VHProbi A17 on hydroxyl radicals was 10.6%.

Example 5 Tolerance of saliva combined with Lactobacillus VHProbi A17 to artificial gastric juice and artificial intestinal juice

1. Bacterial liquid preparation:

Streak-inoculate the cryopreserved Lactobacillus saliva-associated Lactobacillus VHProbi A17 strain in MRS solid medium, culture at 37°C for 24-48 hours, and subculture once in MRS liquid medium, then inoculate saliva-associated lactobacillus with 5% inoculum Bacillus VHProbi A17 was inoculated into fresh MRS liquid medium and cultured with shaking at 40°C for 24-48 hours to obtain fresh bacterial liquid.

2. Preparation of artificial gastric juice

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

3. Preparation of artificial intestinal juice

Weigh 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH ₂ PO ₄ and 3.0g of ox bile salt, add 77ml of 0.2mol/l NaOH solution, make the volume to 1000ml, and use dilute hydrochloric acid or sodium hydroxide solution Adjust the pH to 6.8±0.1, and sterilize at 115°C for 20 minutes. Then add 1g of trypsin before use, shake well to dissolve, and place it in a water bath shaker at 37°C for 1 hour to simulate the temperature of the human body.

4. Test method

Take 2 mL of fresh bacterial solution, centrifuge at 5000 rpm for 5 minutes to collect the bacterial cells, wash the bacterial cells with normal saline for 3 times, and then resuspend with 2 mL of normal saline as the inoculum. Take 1 mL of the inoculum solution, add it to 9 mL of artificial gastric juice that has been warmed for 1 h, place it in a water bath shaker at 37 °C at 200 rpm for 2 h, and take 1 mL samples at 0 h and 2 h, respectively, to detect the amount of viable bacteria. Then take 1 mL of artificial gastric juice digested for 2 hours, add it to 24 mL of artificial intestinal juice, place it in a water-bath shaker (200 rpm) at 37°C for 3 hours, sample 1 mL, and test the amount of viable bacteria.

The counting method of viable bacteria was determined according to the national standard "GB4789.35-2016-Food Microbial Examination of Lactic Acid Bacteria".

Table 2: Scale of viable bacteria after artificial gastrointestinal digestion

It can be seen from Table 2 that after digestion with artificial gastrointestinal juice, the number of live bacteria of Lactobacillus saliva-associated Lactobacillus VHProbi A17 decreased only slightly, which indicated that the strain had strong tolerance to artificial gastric juice and artificial intestinal juice.

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

1. Preparation of lysate

According to the inoculum amount (volume ratio) of 1%, the Lactobacillus salivarius associated Lactobacillus VHProbi A17 was inoculated into the MRS broth medium, cultured at 37°C for 24 hours, and then the culture was stopped to obtain fresh bacterial liquid. Part of the bacterial liquid was ultrasonically crushed with an ultrasonic breaker for 20 minutes, ultrasonic conditions: power 30%, ultrasonic 2 s, stop 2 s; inactivation at 80°C for 60 min, to obtain a lysate without live bacteria.

With reference to the Oxford cup method bacteriostasis test procedure described in Example 1, measure respectively saliva associated lactobacillus VHProbiA17 bacterium liquid and lysate to Streptococcus mutans (three strains), Actinomyces viscosus ATCC27044, Porphyromonas gingivalis BNCC353909, Bacteriostatic effects of five common oral pathogens, Aggregate actinomycetes BNCC336945 and Fusobacterium nucleatum BNCC 336949. Among them, the upper medium of Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregate semiactinobacterium was replaced with Columbia blood agar medium; the diameter of the inhibition zone is shown in Table 3.

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

oral pathogens The diameter of the inhibition zone of A17 fermentation broth The diameter of the inhibition zone of A17 lysate Streptococcus mutans 27mm 26mm Porphyromonas gingivalis 14.5mm 10mm Actinomyces viscosus 23.5mm 22mm Aggregate actinomycetes 11mm 10mm Fusobacterium nucleatum 12.5mm 11mm

From the results in Table 3, it can be seen that the saliva-associated lactobacillus VHProbi A17 provided by the present invention is virulent to five kinds of oral cavity, including Streptococcus mutans, Porphyromonas gingivalis, Actinomyces viscosus, Aggregate actinomycetes and Fusobacterium nucleatum. Bacteria have obvious inhibitory effect, and have a broad antibacterial spectrum. Among them, the strain has the strongest antibacterial effect on Streptococcus mutans and Actinomyces viscosus, the diameter of the antibacterial zone reaches 22-27mm, and the antibacterial effect of the fermentation liquid is slightly better than that of the lysate, which has achieved unexpected technology Effect.

Example 7: The agglutination effect test of saliva combined with Lactobacillus VHProbi A17 on oral pathogenic bacteria

Coaggregation is an interaction between cells and cellular proteins. Probiotics can prevent dental caries by agglutinating with pathogenic bacteria and preventing their colonization and adhesion in the oral cavity. The agglutination test is divided into two types, one is to judge the agglutination according to whether the lactobacilli combine with the pathogenic bacteria under certain conditions to form a visible flocculent precipitate and the size of the precipitate, and the other is to determine the coagulation by measuring the agglutination rate Effect.

1. Preparation of bacterial suspension

According to the inoculum amount (volume ratio) of 1%, inoculate into MRS broth medium, stop cultivating after 37 ℃ of cultures 24h, obtain fresh bacterial liquid; Centrifuge 10min with the rotating speed of fresh bacterial liquid 8000rpm, collect bacterial body; .0 phosphate buffer for two washes, then resuspend the bacteria with pH 7.0 phosphate buffer until the initial absorbance OD600 of the bacteria suspension is between 0.5-0.6, and set aside.

2. Preparation of pathogenic bacteria suspension:

According to 1% inoculum size (volume ratio), inoculate the Streptococcus mutans bacterium liquid into BHI broth medium, stop cultivating after 24 hours of 37 ℃ of aerobic cultures, get fresh bacterium liquid; According to 1% inoculum size (volume ratio) Porphyromonas gingivalis, Aggregate semiactinobacillus and Fusobacterium nucleatum were inoculated into BHI broth medium (with 5% bovine serum added), cultured anaerobically at 37°C for 48 hours, and the culture was stopped to obtain fresh bacterial solution ;

Centrifuge the fresh bacterial solution at 8000rpm for 10min to collect the bacterial cells; wash the bacterial cells twice with pH 7.0 phosphate buffer, then resuspend the bacterial cells with pH 7.0 phosphate buffer, and adjust the initial absorbance OD600 of the bacterial suspension to 0.5 Between -0.6, spare.

3. Coagulation point test

Take 300 μL of Lactobacillus saliva VHProbi A17 suspension and add it to a 24-well plate, then add 300 μL of pathogenic bacteria suspension as a reaction sample, and take another equal amount of Lactobacillus saliva VHProbi A17 suspension and buffer as a control, each The control and samples were set in 2 parallels. Place the 24-well plate on a microplate constant temperature shaker, 400rpm, room temperature, and shake for incubation. Oscillate for 30 minutes, stop for 30 minutes, take pictures and record the initial state of the orifice plate and the state of the orifice plate at each stop.

From the results in Figure 3, it can be seen that within 2 hours, the four pathogenic bacteria of Lactobacillus VHProbi A17 and Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregate semiactinobacillus had agglutination points. Among them, there are small but many agglutination points for Streptococcus mutans, Porphyromonas gingivalis and Fusobacterium nucleatum, and a large agglutination point for Aggregates semiactinobacteria, thus indicating that Lactobacillus saliva associated Lactobacillus VHProbi A17 It can effectively bind common oral pathogens and significantly inhibit their adhesion to teeth or gums.

4. Determination of self-agglutination rate and co-agglutination rate

(1) Determination of self-agglutination rate

Take 1ml of the prepared Lactobacillus saliva-associated Lactobacillus VHProbi A17 suspension and add it to a 24-well plate, and let it stand at room temperature. Take 100 μL to measure the initial absorbance value A0 at a wavelength of 600nm, absorb the upper layer of the bacterial suspension every 2h within the next 6h, and measure the absorbance value At at a wavelength of 600nm. Calculate self-cohesion (Rself). The results are shown in Table 4.

R _from = 1-At/A0;

In the formula: R is _self -cohesion, %; At is the absorbance at time t, t=2, 4 and 6h; A0 is the initial absorbance of Lactobacillus salivarius VHProbi A17 bacterial suspension.

Table 4: Self-aggregation rate table of Lactobacillus saliva-associated Lactobacillus VHProbi A17

coagulation time autoagglutination rate 2 hours 12.81% 4h 27.53% 6 hours 41.08%

As can be seen from the results in Table 4, the self-agglutination rate of the saliva-associated Lactobacillus VHProbi A17 provided by the present invention reached 41.085% at 6 hours. Thus, the salivary-associated Lactobacillus VHProbi A17 can effectively colonize the oral cavity.

(2) Determination of coagulation rate

Take 100 μL of saliva combined with Lactobacillus VHProbi A17 suspension and pathogenic bacteria suspension to measure the initial absorbance value OD600, then mix the same amount of saliva combined with Lactobacillus VHProbi A17 suspension and pathogenic bacteria suspension, shake well, and let it stand at room temperature. Each sample was placed in three parallels, and 100 μL of the upper layer suspension was taken every 2 hours to measure the absorbance value OD600. Calculate the cohesion force ( _Rco ) according to the following formula. The results are shown in the table.

R _total =1-2At/(A0+B0);

In the formula: R _is the copolymerization force, %; At is the absorbance at time t t=2, 4 and 6h; A0 is the initial absorbance of Lactobacillus salivarius VHProbi A17; B0 is the initial absorbance of the pathogenic bacteria suspension.

Table 5: Coagulation effect of saliva combined with Lactobacillus VHProbi A17 on oral pathogenic bacteria

As can be seen from the results in Table 5, the saliva-associated lactobacillus VHProbi A17 provided by the present invention can be combined with four common oral pathogenic bacteria, Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregate semiactinobacillus. Bacteria are effectively combined, and the coagulation rate reaches 14.71%-58.63% within 2-6 hours. Among them, Lactobacillus salivarius VHProbi A17 has the best coagulation effect on Streptococcus mutans, Fusobacterium nucleatum and Aggregate semiactinobacillus, and the coagulation rate is as high as 48.05%-58.63% at 6 hours, which is unexpected. technical effect.

The salivary-associated Lactobacillus VHProbi A17 provided by the present invention can significantly reduce the number of pathogenic bacteria in the oral cavity through effective combination with pathogenic bacteria, along with the flow of saliva and oral cleaning, thereby reducing the load of pathogenic bacteria in the oral cavity Effect.

Embodiment 8: the inhibition growth test of saliva combined lactobacillus VHProbi A17 to Streptococcus mutans

1. Preparation of lysate supernatant

The fresh cell wall of Lactobacillus saliva associated Lactobacillus VHProbi A17 was crushed with an ultrasonic cell disruptor, then treated at 80°C for 60 min, and filtered with a 0.22 μL microporous membrane to remove insoluble matter, and the supernatant of the lysate was prepared.

2. Preparation of inoculum solution

Wash the fresh Streptococcus mutans solution twice with pH 7.0 phosphate buffer, and resuspend with the same volume. Then it was diluted 5 times and used as the inoculum.

3. 96-well plate culture

Add 50 μL BHI broth medium and 10 μL Streptococcus mutans solution to each well, add 20 μL lysate supernatant to the experimental group, and add 20 μL sterile water to the control group; then supplement the volume to 200 μL with sterile water, and add 50 μL liquid paraffin. Set up 4 parallels for each group. The 96-well plate was placed in a microplate reader at 37°C, and the OD600 was set to be measured every 10 minutes for 24 hours.

It can be seen from Figure 4 that the Streptococcus mutans in the control group began to grow rapidly after inoculation for 10 hours, and reached a stable phase at 20 hours; while in the experimental group added with the supernatant of the lysate of Lactobacillus saliva associated Lactobacillus VHProbi A17, the Streptococcus mutans inoculated within 20 hours There is almost no growth, which shows that the lysate of saliva combined with Lactobacillus VHProbi A17 can efficiently inhibit the growth and reproduction of Streptococcus mutans.

Embodiment 9: Antagonistic adhesion test of saliva combined Lactobacillus VHProbi A17 to Streptococcus mutans

Streptococcus mutans is currently recognized as the main caries-causing bacteria, and its colonization and adhesion in the oral cavity is the main cause of caries formation. Therefore, oral probiotics can inhibit the growth and adhesion of pathogenic bacteria by forming agglutination with pathogenic bacteria, thereby effectively reducing oral diseases.

1. Preparation of bacterial suspension

According to the inoculation amount (volume ratio) of 1%, inoculate the Lactobacillus saliva associated Lactobacillus VHProbi A17 into the MRS broth medium, culture at 37°C for 24 hours and then stop the cultivation to obtain fresh bacterial liquid; centrifuge the fresh bacterial liquid at 8000rpm for 10 min to collect the bacterial cells ; The cells were washed twice with pH 7.0 phosphate buffer, and then resuspended with the same volume of pH 7.0 phosphate buffer.

2. Preparation of Streptococcus mutans suspension:

With reference to the method described in Example 1, prepare the Streptococcus mutans bacterial liquid; centrifuge the fresh bacterial liquid at 8000 rpm for 10 min, and collect the thalline; wash the thalline twice with pH7.0 phosphate buffer, and then use the same volume of pH7.0 phosphate buffer solution resuspended.

Mix 500 μL of saliva-associated Lactobacillus VHProbi A17 suspension and 500 μL of Streptococcus mutans suspension evenly. After standing for 5 minutes, take 500 μL of the upper layer solution and add it to a 24-well plate that has been placed in a sterile cell slide. Cultivate for 2 hours at a temperature of °C, remove the upper layer solution, wash with pH 7.0 phosphate buffer, add 0.5 mL of methanol to fix for 10 minutes, and then discard it. Finally, 300 μL of Giemsa staining solution was added for staining for 10 minutes, then the staining solution was discarded, washed with pH 7.0 phosphate buffer, and the cell slides were placed on glass slides to observe the amount of bacteria on the cell slides.

The results are shown in Figure 5. The cell slides of the control group were covered with Streptococcus mutans, while the experimental group added Lactobacillus salivarius VHProbi A17 had very few Streptococcus mutans on the slides, almost invisible in the visible range. It was found that Lactobacillus salivarius VHProbi A17 can effectively inhibit the adhesion of Streptococcus mutans to the cell slide. Thus, the saliva-associated Lactobacillus VHProbi A17 provided by the present invention can effectively prevent pathogenic bacteria from adhering to teeth, thereby preventing the occurrence of dental caries.

Embodiment 10: the biofilm elimination action of saliva associated lactobacillus VHProbi A17 to Streptococcus mutans

The adhesion and accumulation of Streptococcus mutans on the tooth surface will form a layer of biofilm, and then produce acid substances to cause dental caries. Therefore, the removal of biofilm formed by Streptococcus mutans is very important for the prevention of dental caries.

1. Prepare fresh Streptococcus mutans bacteria liquid with reference to the method described in Example 1.

2. Inoculate the Lactobacillus saliva associated Lactobacillus VHProbi A17 into the MRS broth medium according to the inoculum amount (volume ratio) of 1%, culture at 37°C for 24 hours, and then stop the culture to obtain fresh fermentation broth. The fermented liquid is divided into three parts and processed as follows:

(1) Part of the fermentation broth was centrifuged at 8000rpm for 10min to collect the thallus; the thallus was washed twice with pH 7.0 phosphate buffer, and then resuspended with the same volume of pH 7.0 phosphate buffer to prepare a live bacteria suspension;

(2) Part of the fermented liquid was sonicated with an ultrasonic breaker for 20 minutes, ultrasonic conditions: power 30%, ultrasonic 2 s, stop 2 s; 80 ° C inactivation for 60 min, to obtain a lysate without live bacteria;

(3) Part of the fermentation broth was not subjected to any treatment.

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

Add 600 μL of Streptococcus mutans bacteria solution to each well, and after culturing for 24 hours, Streptococcus mutans adheres to the cell slide to form a layer of biofilm, then discard the culture solution and non-adhered bacteria in the well, and use pH7.0 Wash twice with phosphate buffer; add 600 μL of Lactobacillus saliva-associated Lactobacillus VHProbi A17 fermentation broth, bacterial suspension and lysate without living cells into the wells, and each group is used to do 3 parallels, and the same amount of MRS broth medium is used as control.

Let the sample to be tested and the biofilm formed by Streptococcus mutans co-culture at 37°C for 24 hours, then discard the liquid in the well, add 600 μL pH7.0 phosphate buffer to wash, wash each well 3 times, and shake continuously when washing To remove unadhered bacteria, and then judge the biofilm removal rate of the sample to be tested by comparing the number of Streptococcus mutans on the cell slide.

Place the cell slide in a sterile homogeneous bag, ultrasonically clean it for 10 minutes to diffuse the bacteria into the buffer, and then through a series of dilutions, take 100 μL and apply it to Streptococcus salivarius added with 200U/L bacitracin 37 ℃ aerobic culture on the culture medium for 24h, detect the amount of Streptococcus mutans, calculate the removal rate of Streptococcus mutans biofilm by the following formula: removal rate (%)=(1-the amount of bacteria in the experimental group/the amount of bacteria in the control group )×100%. The specific results are shown in Table 6.

Table 6: Removal effect of saliva-associated Lactobacillus VHProbi A17 on Streptococcus mutans biofilm

From the data in Table 6, it can be seen that Lactobacillus salivarius VHProbi A17 can efficiently remove the biofilm formed by Streptococcus mutans. Among them, the bacterial suspension, that is, the removal rate of the simple bacteria to the biofilm is 40.79%, indicating that there are binding sites for Streptococcus mutans on the cell membrane of the bacteria, and the simple bacteria themselves can also combine with Streptococcus mutans. Remove part of the biofilm; while the fermentation broth and lysate contain not only the bacteria itself, but also more metabolites, 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-associated 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.

Embodiment 11: saliva associated lactobacillus VHProbi A17 to human normal gingival epithelial cells: toxicity test

1. Cell pre-culture

Resuscitate normal human gingival epithelial cells in liquid nitrogen, culture them in a carbon dioxide incubator to the required amount, and when the cell density grows to about 80%, trypsinize them into a single-cell suspension, count them on a hemocytometer, and the number of cells is 5×10 ⁵ cells/mL, and then 500 μL of the cell suspension was inoculated into a 24-well plate at a seeding density of 2.5×10 ⁵ cells/well, and the cells were cultured for 24 hours for subsequent experiments.

2. Preparation of inactivated bacteria solution

The fresh bacterial solution of Lactobacillus saliva associated Lactobacillus VHProbi A17 was inactivated in an 80°C water bath for 20 minutes, then washed three times with pH 7.0 phosphate buffer, resuspended with cell culture medium, and adjusted the bacterial concentration OD600 = 0.4 Between -0.5.

3. Cell culture

Experimental group: Add the inactivated bacteria solution to the cells in the 24-well plate according to the ratio of MOI (Multiplicity of Infection, multiplicity of infection) value to 10;

Blank control group: add cell culture medium equal to the volume of inactivated bacteria solution.

The culture was continued for 24 h in the carbon dioxide incubator.

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

The results showed that the absorbance value of the blank control group was 0.668, while the absorbance value of the experimental group was 0.680, basically the same. This shows that the salivary-associated Lactobacillus VHProbi A17 has no significant effect on the proliferation activity of normal human gingival epithelial cells, and has good safety and no cytotoxicity.

Example 12: Adhesion of saliva-associated lactobacillus VHProbi A17 to gingival epithelial cells

1. Cell pre-culture

Resuscitate human normal gingival epithelial cells in liquid nitrogen, culture them to the required amount, and when the cell density grows to about 80%, trypsinize them into a single-cell suspension, count them with a hemocytometer, and the cell number is 5×10 ⁵ cells/ mL. Then 500 μL of cell suspension was inoculated into a 24-well plate with cell slides at a seeding density of 2.5×10 ⁵ cells/well. After the cells were cultured overnight until they were completely attached, the culture medium was discarded, rinsed twice with fresh culture medium, and set aside.

2. Bacterial suspension preparation

Wash the fresh bacterial solution of Lactobacillus saliva associated Lactobacillus VHProbi A17 twice with pH7.0 phosphate buffer, then resuspend with the same volume of 1640 culture medium containing 10% calf serum, and adjust the absorbance to make OD600=0.4-0.5 .

3. Cell culture

Add 500 μL of bacterial suspension to the prepared 24-well plate containing primary human gingival epithelial cells, and co-culture for 2 hours in a carbon dioxide incubator; wash 3 times with pH 7.0 phosphate buffer to remove non-adhered bacteria .

4. Microscopic examination

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 removed and placed on glass slides. Observe and count under a microscope, randomly select 50 cells, and calculate the number of Lactobacillus saliva associated Lactobacillus VHProbi A17 on the visible cell surface, and use statistical methods to calculate the mean and standard deviation of the adhesion index.

Adhesion index = number of adhered bacteria/number of cells.

The results showed that about one Lactobacillus salivarius associated Lactobacillus VHProbi A17 adhered to five gingival cells, and the adhesion index was 0.2. This shows that Lactobacillus saliva-associated Lactobacillus VHProbi A17 has good adhesion performance and can be colonized in the oral cavity.

Example 13: Antagonistic adhesion test of salivary combined lactobacillus VHProbi A17 to periodontal pathogenic bacteria at the cellular level

1. Cell pre-culture

Resuscitate human normal gingival epithelial cells in liquid nitrogen, culture them to the required amount, and when the cell density grows to about 80%, trypsinize them into a single-cell suspension, count them with a hemocytometer, and the cell number is 5×10 ⁵ cells/ mL. Then take 500 μL of the cell suspension and inoculate it into a 24-well plate with cell slides, culture overnight until completely attached, then discard the culture medium, rinse twice with fresh culture medium, and set aside.

2. Bacterial suspension preparation

Wash the fresh bacterial solution of Lactobacillus saliva associated Lactobacillus VHProbi A17 twice with pH7.0 phosphate buffer, then resuspend with the same volume of 1640 culture medium containing 10% calf serum, and adjust the absorbance to make OD600=0.4-0.5 .

The suspensions of Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregate semiactinomycetes were prepared by the same sampling method.

3. Cell culture

Experimental group: mix the saliva-associated Lactobacillus VHProbi A17 suspension and the pathogenic bacteria suspension at a volume ratio of 1:1, and then take 500 μL and add it to the 24-well plate prepared above;

Control group: mix the pathogenic bacteria suspension and phosphate buffer at a ratio of 1:1 by volume, and then take 500 μL and add it to the 24-well plate prepared above.

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

4. Microscopic examination

Add 500 μL of methanol to each well to fix for 15 minutes, then stain with Giemsa stain for 5 minutes, wash and rinse with pH7.0 phosphate buffer solution, and take out the cells in the wells and slide them onto the glass slide. Observe and count under a microscope, randomly select 50 cells, and calculate the number of pathogenic bacteria on the visible cell surface, and use statistical methods to calculate the mean and standard deviation of the adhesion index. The specific results are shown in Table 7. Microscopic examination results are shown in Figure 6-8.

Adhesion index = number of adhered bacteria/number of cells.

Table 7: Antagonistic adhesion effect of saliva combined Lactobacillus VHProbi A17 on periodontal pathogenic bacteria

As can be seen from the data in Figures 6-8 and Table 7, compared with the control group, the experimental group that added the saliva-combined Lactobacillus VHProbiA17 bacterial suspension The adhesion index to gingival epithelial cells generally decreased by 87%-95%, and the adhesion inhibition to A. semiactinobacteria was the strongest, and the adhesion index was only 1.64. This shows that the combination of saliva and Lactobacillus VHProbi A17 can significantly inhibit the adhesion of common periodontal pathogens to gingival epithelial cells, and has achieved unexpected technical effects.

The results of the above cell experiments showed that Lactobacillus saliva-associated Lactobacillus VHProbi A17 had no toxicity to normal human gingival epithelial blood cells, and could significantly inhibit periodontal pathogenic bacteria (Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregate semiactinobacillus) Adhesive growth on gingival epithelial cells, effectively preventing and improving periodontitis, gingival bleeding and other symptoms.

Sequence Listing

<110> Qingdao Weilan Biological Co., Ltd.

  Qingdao Weilan Biological Group Co., Ltd.

<120> A strain of Lactobacillus salivarius associated with prevention or treatment of 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 (10)

1. A Lactobacillus salivarius, characterized in that the preservation number of the Lactobacillus saliva is CCTCC NO: M2022172. 2. Lactobacillus salivarius as claimed in claim 1, is characterized in that, the 16srDNA sequence of described Lactobacillus salivarius is SEQ ID NO:1. 3. The Lactobacillus salivarius as claimed in claim 1, wherein the protein spectrum of the Lactobacillus salivarius is as shown in Figure 1, and the Riboprinter fingerprint is as shown in Figure 2. 4. the application of the Lactobacillus salivarius associated with claim 1 in the preparation of products with antioxidant function. 5. The application of the Lactobacillus salivarius associated with claim 1 in the preparation of products with the function of preventing or treating oral diseases. 6. The application according to claim 6, characterized in that the oral disease is dental caries or periodontal disease. 7. The application according to any one of claims 4-6, wherein the product is medicine, health product or oral care product. 8. application as claimed in claim 7, is characterized in that, described oral care article is dental gel, tooth powder, toothpaste, mouthwash, mouth spray, chewing gum, dental floss, tooth cleaning liquid, tooth cleaning any kind of foam. 9. A product for inhibiting oral pathogenic bacteria, characterized in that the product contains the live bacteria and/or lysate of Lactobacillus salivarius associated with claim 1. 10. The product for inhibiting oral pathogenic bacteria as claimed in claim 9, wherein the lysate is subjected to heat extinguishing after the fresh bacterium liquid of saliva combined with Lactobacillus according to claim 1 is ultrasonically broken. Can live.

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