CN114621895B

CN114621895B - Saliva-associated lactobacillus capable of inhibiting clostridium nucleatum and improving breath

Info

Publication number: CN114621895B
Application number: CN202210252791.1A
Authority: CN
Inventors: 张秋香; 蒋震涛; 崔树茂; 毛丙永; 唐鑫; 陆文伟; 赵建新; 陈卫
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-03-14
Filing date: 2022-03-14
Publication date: 2023-09-12
Anticipated expiration: 2042-03-14
Also published as: CN114621895A

Abstract

The invention discloses a saliva combined lactobacillus capable of inhibiting clostridium with improved breath, belonging to the technical field of microorganisms. The saliva combined lactobacillus (Ligilactobacillus salivarius) CCFM1215 is preserved in the microorganism strain preservation center of Guangdong province at 12 months and 17 days of 2021, the preservation address is the building 5 No. 59 of the university of Mitsui No. 100 of Guangzhou City, and the preservation number is GDMCC No. 62141. The bacterium can reduce the amount of the fusobacterium nucleatum biomembrane and the amount of volatile sulfide, and has the function of improving halitosis in vivo.

Description

Saliva-associated lactobacillus capable of inhibiting clostridium nucleatum and improving breath

Technical Field

The invention relates to a saliva combined lactobacillus capable of inhibiting clostridium with improved breath, belonging to the technical field of microorganisms.

Background

Bad breath, a third biggest oral health problem, affects the physical and mental health and quality of life of people. Epidemiological studies have shown that worldwide bad breath has a prevalence of 31.8%,80% to 90% of bad breath being associated with oral factors, known as oral bad breath. Bad breath odors are mainly due to the breakdown of sulfur-containing amino acids (cystine, cysteine, methionine) or serum by microorganisms in the oral cavity, resulting in metabolites, mainly volatile sulfides (Volatile Sulfur Compounds, VSCs), 90% of which are hydrogen sulfide and methyl mercaptan, but also dimethyl sulfide, etc. The bacteria producing VSCs are mainly fusobacterium nucleatum, porphyromonas gingivalis, praecox intermedia, solobacterium moorei, etc.

At present, a mouth wash product such as chlorhexidine and essential oil is used for treating halitosis, but the method has poor mildness and safety, the essential oil can only cover smell, and the concentration of VSCs is not reduced essentially, so that the effect is poor; the traditional Chinese medicine has slow and general effects. For oral bad breath caused by microorganisms, "symptomatic drug administration" should be adopted, and the problem caused by microorganisms is solved by a microorganism method. At present, probiotics are gradually applied to the field of oral cavity due to the characteristics of safety and mildness.

For methods of improving bad breath using probiotics, CN111991429a discloses lactobacillus reuteri which inhibits oral pathogenic bacteria, and similarly US2006171901 discloses streptococcus salivarius which inhibits anaerobic bacteria. In short, these probiotics all have bacteriostatic effects. However, in the oral cavity, especially in the subgingival specific environment, the halitosis anaerobe exists in the form of a biological film, the biological film has strong stress resistance, and only the probiotics with antibacterial effect can not play the roles of inhibiting bacteria and removing halitosis in the oral cavity. CN201911218939.4 discloses a lactobacillus plantarum targeted to reduce fusobacterium nucleatum biofilm, which can specifically inhibit or reduce the formation amount and density of fusobacterium nucleatum biofilm, but no experiment proves that the fusobacterium nucleatum biofilm can reduce the VSCs yield of fusobacterium nucleatum. In addition, CN201811441014.1 discloses that dendrobium candidum oligomannose is added as a prebiotic component, and a plurality of probiotic components are added to obtain a solid beverage for freshening breath, but no evaluation of in vivo effect of improving breath is made.

Disclosure of Invention

In view of the above, the present invention aims to provide a lactobacillus salivarius (Ligilactobacillus salivarius) which can significantly inhibit or reduce the formation of fusobacterium nucleatum biofilm and reduce the VSCs production thereof, so as to reduce the risk of halitosis occurrence and development.

[ technical problem ]

In the prior art, most probiotics capable of relieving halitosis take bacteriostasis as a target, the VSC amount cannot be reduced in the oral environment, and the bacteria inhibition effect is only exerted when the addition amount of the probiotics is large.

Technical scheme

The invention provides a saliva combined lactobacillus (Ligilactobacillus salivarius) CCFM1215 which can reduce the biomembrane quantity of Fusobacterium nucleatum and the Volatile Sulfide (VSCs) production quantity, and has the function and effect of improving halitosis in vivo.

The colony of the Lactobacillus salivarius (Ligilactobacillus salivarius) CCFM1215 on the MRS culture medium is smooth, white and round small bulges.

The saliva combined with the lactobacillus CCFM1215 can prevent the formation of the fusobacterium nucleatum biomembrane, which means that the capacity of forming the biomembrane of the fusobacterium nucleatum can be obviously reduced in vitro compared with a control group.

The saliva combined with the lactobacillus CCFM1215 can reduce the amount of the fusobacterium nucleatum biological film, which means that the formation of the biological film of the fusobacterium nucleatum can be still inhibited or destroyed in the forming stage of the biological film.

The saliva combined with the lactobacillus CCFM1215 can inhibit the growth of the clostridium nucleatum, which means that the growth quantity of the clostridium nucleatum can be reduced.

The lactobacillus salivarius CCFM1215 can reduce the volatile sulfide production (VSCs) amount of the clostridium nucleatum, which means the supernatant of the lactobacillus salivarius CCFM 1215.

The saliva combined lactobacillus CCFM1215 can reduce the expression level of the gene Fn1220 of the clostridium nucleatum VSCs, and refers to supernatant fluid and heat-inactivated bacteria of the saliva combined lactobacillus CCFM 1215.

The saliva combined lactobacillus CCFM1215 can relieve halitosis, namely the saliva combined lactobacillus CCFM1215 living bacteria can effectively relieve the halitosis of people in vivo, and effectively reduce the VSCs of patients with halitosis.

The saliva combined lactobacillus CCFM1215 can be used for preparing products for inhibiting the formation of a biological film of the clostridium nucleatum, inhibiting the growth of the clostridium nucleatum, inhibiting the expressed gene Fn1220 of the clostridium nucleatum, reducing the amount of volatile sulfide generated by the clostridium nucleatum and/or improving halitosis. The products include, but are not limited to, live or dead cells or extracellular metabolites of lactobacillus salivarius (Ligilactobacillus salivarius) CCFM1215 as described above. Such dead cells include, but are not limited to, cells that are naturally inactive or cells that have been subjected to an inactivation treatment. Forms of the product include, but are not limited to, microbial preparations, solid beverages, breath fresheners, toothpastes, mouthwashes.

[ advantageous effects ]

The invention screens and obtains a saliva combined lactobacillus (Ligilactobacillus salivarius) CCFM1215 which has the capacity of inhibiting Fusobacterium nucleatum in vitro and has the function of relieving halitosis in vivo, and the specific steps are as follows:

(1) Can prevent the formation of F.nucleatum biofilm, and inhibit 71.07% of saliva combined with lactobacillus CCFM1215 when 10% of supernatant is added.

(2) Has certain inhibition on the growth of Fusobacterium nucleatum.

(3) Has good copolymerization capability with Fusobacterium nucleatum.

(4) Can inhibit production of H by Fusobacterium nucleatum ₂ S gene Fn1220.

(5) Can improve the breath value of volunteers, and reduce the VSCs value by 43.69%.

Preservation of biological materials

The saliva combined lactobacillus (Ligilactobacillus salivarius) CCFM1215 is preserved in the microorganism strain collection center of Guangdong province at 12 months and 17 days of 2021, the preservation address is the building 5 No. 59 of the 100 th university of Mitsui, guangzhou, and the preservation number is GDMCC No. 62141.

Drawings

Fig. 1: effects of 10% different lactobacillus supernatants on fusobacterium nucleatum biofilm formation, note: * P <0.01, p <0.001 compared to MRS group

Fig. 2: effect of 5% lactobacillus supernatant on fusobacterium nucleatum biofilm formation, note: * p <0.05, < p <0.01, < p <0.001 compared to MRS group

Fig. 3: growth curve of Fusobacterium nucleatum

Fig. 4: the effect of the saliva on the formation of the fusobacterium nucleatum biofilm in combination with the lactobacillus CCFM1215 intervention at different time points was noted: * P <0.001 compared to MRS group.

Fig. 5: copolymerization ability of Lactobacillus and Fusobacterium nucleatum

Fig. 6: relative expression level of Fusobacterium nucleatum Fn1220 gene, note: * P <0.01, p <0.001, compared to MRS group.

Detailed Description

The following examples relate to the following media:

MRS medium: 5.0g/L of yeast powder, 10.0g/L of beef extract, 10.0g/L of peptone, 20.0g/L of glucose, 2.0g/L of anhydrous sodium acetate, 2.0g/L of diamine hydrogen citrate, 2.6g/L of dipotassium hydrogen phosphate, 0.25g/L of manganese sulfate monohydrate, 0.5g/L of magnesium sulfate heptahydrate, 1mL of tween-80 and pH of 6.2-6.4.

Food grade MRS medium (/ L): 10g of edible yeast extract (purchased from Angel Yeast Co., ltd.), 20g of edible glucose (purchased from Shandong Kogyo Biotechnology Co., ltd.), 10g of soybean peptone (purchased from Henan Wanbang chemical technology Co., ltd.), 0.1g of edible magnesium sulfate (purchased from Laiyou chemical Co., ltd.), 0.05g of edible manganese sulfate (purchased from Lithospermum erythrorhizon Taiwei food ingredient Co., ltd.), 5g of edible sodium acetate (purchased from Jiangsu Koronto food ingredient Co., ltd.), 2g of edible ammonium citrate (purchased from Jiangsu Koronto food ingredient Co., ltd.), 2g of edible dipotassium hydrogen phosphate (purchased from Xuzhou Fenrui Biotechnology Co., ltd.)

BHI medium: purchased from Qingdao sea blogs, 0.05% of hemin and 0.1% of vitamin K1 are additionally added, pH 7.2-7.4.

The preparation method of the supernatant involved in the following examples is as follows:

lactobacillus supernatant: inoculating in MRS liquid culture medium at 1% inoculum size, culturing in 37 deg.C incubator for 24 hr, centrifuging at 10,000r/min and 4deg.C for 10min, filtering with 0.22 μm filter membrane, sterilizing, short-term preserving at 4deg.C, and long-term preserving at-20deg.C.

Lactobacillus inactivated bacterial suspension: inoculating lactobacillus into MRS culture medium with 1% inoculum size, and culturing at 37deg.C for 24 hr to obtain culture solution; centrifuging the culture solution at 10,000r/min and 4 ℃ for 10min, and collecting bacterial sludge; washing the bacterial mud with sterile physiological saline, and re-suspending in sterile PBS buffer solution until bacterial solution concentration is 10 ⁹ CFU/mL, obtaining bacterial suspension; inactivating the bacterial suspension at 60 ℃ for 30min to obtain the lactobacillus inactivated bacterial suspension.

Streptococcus salivarius K12 supernatant: inoculating in MRS liquid culture medium at 1% (v/v), culturing in 37 deg.C incubator for 24 hr, centrifuging at 10,000r/min and 4deg.C for 10min, filtering with 0.22 μm filter membrane, sterilizing, short-term preserving at 4deg.C, and long-term preserving at-20deg.C.

The lactic acid bacteria referred to in the following examples are: streptococcus salivarius K12, CCFM215, lactobacillus salivarius 1, lactobacillus salivarius 2, lactobacillus salivarius 3, lactobacillus salivarius 4, and lactobacillus salivarius 5.

The Lactobacillus salivarius CCFM1215 used in the examples below was selected from feces, and the screening was performed using the production of the inhibition of Clostridium nucleatum VSCs as an index, and identified as Lactobacillus salivarius (Ligilactobacillus salivarius).

Example 1: effect of Lactobacillus supernatant on Fusobacterium nucleatum biofilm formation

Supernatant intervention group: add 10 to each well of 96 well plate ⁷ CFU/mL of Clostridium nucleatum suspension 180. Mu.L was added followed by 20. Mu.L of Lactobacillus supernatant to a total volume of 200. Mu.L, anaerobic culture at 37℃for 48h, each supernatant was provided with 6 parallel wells.

Negative control group: the lactobacillus supernatant was replaced with the same volume of MRS.

Blank Control group: the same volume of BHI was used instead of the Lactobacillus supernatant.

After incubation, the cells were washed with PBS for 2 times, then fixed with 99% methanol for 15min, and the supernatant was discarded and dried at room temperature. After drying is completed, 100 mu L of 0.1% crystal violet solution is added into each hole, dyeing is carried out for 5min, after dyeing is completed, cleaning is carried out for 2 times by using sterile water, and then the mixture is placed at room temperature to be dried completely. Finally, 200 mu L of 33% acetic acid solution is added for dissolution, after being blown and evenly mixed by a gun, 175 mu L of each well is removed and added into a new 96-well plate, and the absorbance value is read at 570nm of an enzyme-labeled instrument. The decrease after biofilm mediation was calculated as inhibition (%) = (negative control biofilm-supernatant intervention biofilm amount)/negative control biofilm.

As shown in FIG. 1, the experiment was conducted on 6 strains of Lactobacillus salivarius and 1 strain of Streptococcus salivarius K12, wherein the inhibition rate of F.nucleatum biofilm was 71.07% when only 10% (20. Mu.L/(180. Mu.L+20. Mu.L)) was added to the supernatant of Lactobacillus salivarius CCFM1215, which was the best among several strains of Lactobacillus salivarius.

Example 2: effect of 5% of Lactobacillus supernatant on Fusobacterium nucleatum biofilm formation

The addition amount of lactobacillus supernatant was changed to 5% according to the method of example 1, i.e., 10 was added to each well of a 96-well plate ⁷ CFU/mL of Clostridium nucleatum suspension 190. Mu.L was then added with 10. Mu.L of the filtered Lactobacillus supernatant to bring the total volume to 200. Mu.L, the remaining steps being identical.

Results As shown in FIG. 2, when only 5% of the sialyl-Lactobacillus CCFM1215 was added, there was still a significant inhibition of F.nucleatum biofilm, up to 39.8%, which was 15.45% higher than the positive control S.salivarius K12.

Example 3: effect of Lactobacillus supernatant on Fusobacterium nucleatum VSCs production

Will 10 ⁷ CFU/mL of the Clostridium nucleatum suspension and 15% of lactobacillus supernatant or Streptococcus salivarius K12 supernatant were added to Hengalit test tubes in total for 9h, and 2 50mL syringe needles were inserted into the test tubes, one end was connected to a Halimeer halitosis meter, and the other end was used to balance air pressure,and measuring while oscillating by an oscillator. Since the upper limit of detection by the halimer is 2000, the control group was subjected to gas dilution and then calculated. The specific operation is as follows: after shaking and mixing, 1mL of gas was sucked up by a 1mL syringe and immediately added to an empty hengambir test tube, and the mixture was measured while shaking. As shown in Table 2, the inhibition rate of Streptococcus salivarius K12 is 67.81%, the inhibition rate of Lactobacillus salivarius CCFM1215 reaches 92.53%, and the effect is better than Streptococcus salivarius K12.

TABLE 2 Effect of Lactobacillus supernatant on Fusobacterium nucleatum VSCs production

Example 4: effect of Lactobacillus on Fusobacterium nucleatum growth

Add 10 to each well of 96 well plate ⁷ CFU/mL of Clostridium nucleatum suspension 190. Mu.L was added with 10. Mu.L of the filtered Lactobacillus supernatant and 10. Mu. L, MRS 10. Mu.L of the filtered Streptococcus salivarius K12 supernatant, and the mixture was subjected to anaerobic culture at 37℃with a Tecan infinite F50 instrument, and absorbance was measured at 600nm every 30min to prepare F.nucleatum growth curves, respectively. As a result, as shown in FIG. 3, when the stationary phase was reached, the growth inhibition of Lactobacillus salivarius CCFM1215 against Fusobacterium nucleatum was 23.06%, and the inhibition rate was only 23.65% that of Lactobacillus salivarius 6.

Example 5: influence of saliva on Fusobacterium nucleatum biofilm formation by Lactobacillus salivarius CCFM1215 intervention at different times

Combining the results of lactobacillus inhibition biofilm, growth and VSCs, the selection of salivary combined lactobacillus CCFM1215 further explored the effect of lactobacillus supernatant intervention at different times on fusobacterium nucleatum biofilm formation. Based on the procedure of example 1, intervention was performed by adding 5% of lactobacillus supernatant after 6h, 12h, 24h of fusobacterium nucleatum biofilm formation. As a result, as shown in FIG. 4, the effect was decreased as the supernatant was added earlier, that is, the effect of intervention was good before the formation of a biofilm, the effect was decreased at the initial stage (6 h) of the formation of a biofilm, 63.37%, 71.07% lower than 0h, and the effect was gradually decreased at two time points from the initiation of the formation of a biofilm to the formation (12, 24 h), with the inhibition rates of 40.87% and 27.89%, respectively. The positive control streptococcus salivarius K12 has obvious effect up to 24.24% after 6 hours, and has no obvious inhibition effect at 12 hours and 24 hours.

Example 6: copolymerization ability of Lactobacillus salivarius CCFM1215 and Fusobacterium nucleatum

The copolymerization ability of lactobacillus and pathogenic bacteria is also the key for exerting the oral cavity probiotics characteristics, the cultured bacterial liquid of the clostridium nucleatum (or lactobacillus) is centrifugated for 10min at 6000r/min and 4 ℃, the supernatant is discarded, the collected bacterial body is resuspended and washed by PBS buffer solution, 6000r/min and 4 ℃ and centrifugated for 10min, the two times are repeated, the PBS buffer solution with the original bacterial liquid volume is added into the finally collected bacterial body and evenly mixed, the bacterial suspension absorbance is measured at 600nm wavelength, the absorbance value at the moment is recorded, and the lactobacillus is A ₁ Fusobacterium nucleatum is A ₂ 。

Mixing lactobacillus suspension and clostridium nucleatum suspension in equal volume, culturing at 37deg.C for 2 hr, 4 hr, 8 hr, measuring absorbance of mixed bacterial suspension at 600nm wavelength, and recording absorbance value as A _xh ，A _xh Is the absorbance of the bacterial suspension supernatant after incubation at rest for xh. Copolymerization ratio C (%) = [1-2A _xh /(A ₁ +A ₂ )]X 100. The results in FIG. 5 show that the saliva combined with Lactobacillus CCFM1215 has a strong copolymerization ability with Fusobacterium nucleatum, and the copolymerization rate is 72.17%.

Example 7: influence of Lactobacillus salivarius CCFM1215 on Fusobacterium nucleatum VSCs-producing genes

Fn1220 contributes more than 80% of the genes involved in the production of VSCs by F.nucleatum. And respectively examining the expression difference of genes Fn1220 related to VSCs in the fusobacterium nucleatum by adopting a fluorescence quantitative PCR method under the condition that whether lactobacillus supernatant or lactobacillus inactivated bacteria exist. Total RNA was extracted and reverse transcribed into cDNA, and the synthesized cDNA template was stored at-20℃for further use. The primers designed in Table 1 were used for fluorescent quantitative PCR, and the 16S rRNA gene was used as a reference gene. As shown in FIG. 6, both supernatant and dead cells of Lactobacillus salivarius CCFM1215 inhibited the production of VSCs by F.nucleatum by significantly reducing the expression level of Fn1220.

TABLE 1 primer sequences

Example 8: preparation of probiotic bacteria powder

Bacterial mud: the saliva is combined with lactobacillus CCFM1215 to be cultured for 24 hours by using a food grade MRS culture medium at 37 ℃, the supernatant is removed by centrifugation to collect bacterial sludge, and the bacterial sludge is removed by centrifugation after washing with sterile water.

Lyoprotectant: 200g/L of skim milk powder, 300g/L of mannitol, and the mass ratio of bacterial sludge to the freeze-drying protective agent is 1:3.

And (3) freeze-drying: and (3) uniformly mixing the bacterial mud with the freeze-drying protective agent, pre-cooling overnight at-80 ℃, and freeze-drying in a freeze dryer.

Bacterial powder: mixing the lyophilized powder with fructooligosaccharides so that 10 per gram is contained ⁹ CFU/g viable bacteria. Packaging, wherein each bag of fungus powder is 2g.

Placebo: 2g of fructo-oligosaccharide per bag is consistent with the bacterial powder in shape, smell, package and the like.

Example 9: crowd experiment for improving halitosis by probiotics powder

1. Volunteer recruitment and experimental procedure

Inclusion criteria for volunteer recruitment: halimeter detection >200ppb, no antibiotic used for 1 month; exclusion criteria: smoking, during pregnancy.

Volunteers were enrolled in a total of 30, randomized into 2 groups, 15 placebo group, and 15 probiotic group. The experiments were carried out for a total of 5 weeks, 4 weeks with the powder (placebo) and 30min after a day of meal, and 1 week with no powder (placebo). Day 1, days 7, 14, 28, 35 from the first day of administration of the powder required oral cavity examination with a halimeer halitosis meter.

2. Detection method

Halimer halitosis meter detection: the volunteer was asked to keep off water 2h from fasting before checking, the subject was closed for 3min before sampling, the end of the straw was inserted into the mouth of the patient to a depth of about 2.5cm, the lips were almost closed, a little clearance was allowed between the lips and the straw, and the lips and teeth could not be pressed against the straw. During sampling, breathing should be performed through the nose. When the value of the instrument display panel starts to decrease after the value of the instrument display panel is increased to the highest value and is stabilized, the highest value is recorded, and the average value is obtained by detecting three times, and the result is shown in Table 3.

TABLE 3 variation of oral gas values (ppb) before and after administration of the powder to volunteers

Note that: ^** p<0.01, the dry prognosis of the probiotic group compared to the baseline period, ^# placebo group compared to baseline period, p<0.05

3. Experimental results ^#

The baseline period was not significantly different for the placebo and probiotic groups, there was a significant decrease (p < 0.01) in the probiotic group's oral gas values after 1 week of administration of the powder, a 43.69% decrease, and no significant change in the placebo group. After two weeks of administration, the decrease in the mouth-gas value of the probiotic group was very pronounced (p < 0.01), and the placebo group was also decreased (p > 0.05), possibly due to psychological effects, since the mouth-gas was also physiologically and psychologically affected, but the decrease in the placebo group was transient, insignificant and unstable. The probiotics group is in a descending trend, after four weeks of taking, the mouth odor value is reduced by 46.50 percent, and the probiotics group has obvious effect (p < 0.01) after one week of eluting.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Claims

1. The saliva combined lactobacillus (Ligilactobacillus salivarius) CCFM1215 is preserved in the microorganism strain collection center of Guangdong province at 12 months and 17 days of 2021, the preservation address is the building 5 No. 59 of the 100 th university of Mitsui, guangzhou, and the preservation number is GDMCC No. 62141.

2. Use of lactobacillus salivarius (Ligilactobacillus salivarius) CCFM1215 in the manufacture of a product for inhibiting fusobacterium nucleatum biofilm formation, for inhibiting fusobacterium nucleatum growth, for alleviating bad breath, for inhibiting fusobacterium nucleatum expression gene Fn1220, or for reducing the amount of volatile sulfide production by fusobacterium nucleatum, wherein the product comprises: microbial preparation, breath freshener, toothpaste and mouthwash.

3. A product for inhibiting fusobacterium nucleatum biofilm formation, comprising living, dead or extracellular metabolites of the saliva of claim 1 in combination with lactobacillus (Ligilactobacillus salivarius) CCFM1215, the product comprising: microbial preparation, breath freshener and mouthwash.

4. A product for inhibiting the growth of fusobacterium nucleatum comprising a living, dead or extracellular metabolite of the saliva of claim 1 in combination with lactobacillus (Ligilactobacillus salivarius) CCFM1215, the product comprising: microbial preparation, breath freshener and mouthwash.

5. A product for alleviating halitosis comprising a live, dead or extracellular metabolite of the saliva of the lactobacillus salivarius (Ligilactobacillus salivarius) CCFM1215, the product comprising: microbial preparation, breath freshener and mouthwash.

6. A product for inhibiting expression of gene Fn1220 by fusobacterium nucleatum, comprising living, dead or extracellular metabolites of the saliva of CCFM1215 of lactobacillus salivarius (Ligilactobacillus salivarius) as defined in claim 1, said product comprising: microbial preparation, breath freshener and mouthwash.

7. A product for reducing the amount of volatile sulfide production by fusobacterium nucleatum comprising living, dead or extracellular metabolites of the saliva of lactobacillus salivarius (Ligilactobacillus salivarius) CCFM1215 of claim 1, the product comprising: microbial preparation, breath freshener and mouthwash.

8. Toothpaste for inhibiting the formation of a biofilm of clostridium nucleatum, comprising dead cells or extracellular metabolites of the saliva of lactobacillus salivarius (Ligilactobacillus salivarius) CCFM1215 of claim 1.

9. Toothpaste for inhibiting the growth of fusobacterium nucleatum, comprising dead cells or extracellular metabolites of the saliva of claim 1 in combination with lactobacillus (Ligilactobacillus salivarius) CCFM 1215.

10. Toothpaste for use in the relief of bad breath, characterized in that the saliva according to claim 1 is combined with dead cells or extracellular metabolites of lactobacillus (Ligilactobacillus salivarius) CCFM 1215.

11. Toothpaste for inhibiting the expression of gene Fn1220 by fusobacterium nucleatum, comprising dead cells or extracellular metabolites of CCFM1215 of lactobacillus salivarius (Ligilactobacillus salivarius) as claimed in claim 1.

12. Toothpaste for reducing the amount of volatile sulphide produced by fusobacterium nucleatum, characterized in that it comprises dead cells or extracellular metabolites of lactobacillus salivarius (Ligilactobacillus salivarius) CCFM1215 according to claim 1.

13. The product according to any one of claims 2 to 12, wherein the extracellular metabolite is obtained by culturing lactobacillus salivarius (Ligilactobacillus salivarius) with MRS medium, CCFM1215, centrifuging, and filtering the supernatant obtained by centrifugation to remove the bacterial cells.

14. The product according to any one of claims 3 to 12, wherein the dead cells are cells after inactivation treatment.