Paracaseicola WFP1-1 and a compound microbial agent containing the same and application thereof

Abstract

The application discloses a strain of paracasei lactobacillus WFP1-1, a compound microbial inoculant and application thereof, and belongs to the technical field of microorganisms. The strain of paracasei lactobacillus WFP1-1 is classified as Lacticaseibacillus paracesei, is preserved in the Guangzhou Provincial Microorganism Culture Collection Center, and has a preservation number of GDMCC No.62587. The strain of paracasei lactobacillus WFP1-1 has good inhibiting effects on Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis. Meanwhile, the application also provides a compound microbial inoculant containing the strain of paracasei lactobacillus WFP1-1 and Bifidobacterium longum WFP05. The Bifidobacterium longum WFP05 has a preservation number of GDMCC No.62592. The compound microbial inoculant has obvious comprehensive inhibiting effects on harmful bacteria in the oral cavity, has a self-agglutination rate of 88.2%, and has a co-agglutination rate with the Streptococcus mutans of 86.53%. Through animal experiment verification, the compound microbial inoculant in a live form or a killed form has the effect of preventing dental caries, is beneficial to preventing dental caries, periodontitis and other oral diseases, and has a wide application prospect in medicines or daily chemical products with the effect of freshening breath.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus paracasei WFP1-1 strain and a composite bacterial agent consisting of the strain and Bifidobacterium longum WFP05, as well as applications of the Lactobacillus paracasei WFP1-1 and the composite bacterial agent. Background Art

[0002] Common oral diseases include caries and periodontal disease. Surveys show that 78.9% of children under 12 in my country suffer from caries, while the prevalence of periodontal disease in adults is as high as 85%. It is generally believed that caries is caused by Streptococcus mutans, the primary bacteria that ferments carbohydrates in the mouth, leading to demineralization of dental hard tissues. This is primarily due to their strong adhesion to tooth surfaces and high acid production. Periodontal disease is primarily caused by a combination of Gram-negative anaerobic bacteria, including Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia. Antibiotics are currently the primary method for preventing and treating caries and periodontal disease, but long-term use can easily lead to an imbalance in the oral microbiome and the development of drug-resistant bacteria.

[0003] Probiotics can inhibit the growth of oral pathogens by aggregation, adhesion, and immune regulation, maintaining a balanced oral microbiome and safeguarding oral health. Currently, probiotics with significant inhibitory effects on oral pathogens include Lactobacillus rhamnosus LGG, Lactobacillus paracasei LPC48, and Lactobacillus reuteri. Studies have shown that the intake of Lactobacillus rhamnosus LGG or certain specific strains of Lactobacillus reuteri can inhibit the growth of Streptococcus mutans, a dental caries pathogen, and reduce the number of Streptococcus mutans in the mouth. Specific strains of Lactobacillus reuteri can also effectively reduce the incidence of periodontal disease and bad breath.

[0004] my country is rich in traditional fermented food resources, and many lactic acid strains have not yet been discovered or systematically studied. Currently, most of the strains claimed to have beneficial effects on oral health are foreign strains. Therefore, it is of great significance to explore, screen, and develop strains with independent intellectual property rights for the prevention and treatment of oral diseases. In addition, in the production of strains, a high yield of live bacteria must be guaranteed at each stage to have a cost advantage. In the circulation of probiotics, products in the form of live bacteria must ensure good stability in order to exert their health effects. Once the bacteria are inactivated or inactivated during production, processing, transportation, sales, etc., they often cannot exert their corresponding effects. In addition, milk is the largest carrier for probiotic consumption. Whether probiotics can adapt to the production of yogurt and milk beverages is a key factor in evaluating their industrialization scale and potential economic benefits in the consumer market. However, most probiotics on the market currently do not meet all of the above conditions.

[0005] Therefore, it is very important to provide a bacterial strain and agent that has the effects of preventing and treating tooth decay and freshening breath, while also having excellent properties that meet the convenience of application in multiple links of production, circulation and consumption, in order to gain a competitive advantage in market competition. Summary of the Invention

[0006] (1) Technical problems solved

[0007] In view of the shortcomings of currently available probiotics in terms of efficacy characteristics and activity maintenance in production, circulation, consumption and other links, in particular, the shortcomings in maintaining high functional activity in multiple links of production, circulation and consumption while having excellent efficacy in preventing and treating tooth decay and freshening breath. The present invention provides a composite bacterial agent containing Lactobacillus paracasei WFP1-1 and Bifidobacterium longum WFP05, so that the composite bacterial agent can meet the application requirements in production, circulation and consumption while ensuring the significant efficacy in preventing and treating tooth decay and freshening breath.

[0008] (2) Technical solution

[0009] In order to achieve the above-mentioned purpose of the invention, the technical solutions provided by the present invention are as follows:

[0010] A strain of Lactobacillus paracesei WFP1-1, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 30, 2022, with the preservation number GDMCC No. 62587.

[0011] Preferably, the Lactobacillus paracasei WFP1-1 has an inhibitory effect on Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis.

[0012] Another object of the present invention is to provide a composite bacterial agent, which is composed of the above-mentioned Lactobacillus paracasei WFP1-1 and Bifidobacterium longum WFP05.

[0013] Preferably, the Bifidobacterium longum WFP05 was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 30, 2022, with the deposit number GDMCC No.62592.

[0014] Preferably, the self-agglutination rate of the Bifidobacterium longum WFP05 is not less than 50%.

[0015] Preferably, the coaggregation rate of the composite bacterial agent and Streptococcus mutans is not less than 85%.

[0016] Preferably, the self-aggregation rate of the composite bacterial agent is not less than 85%.

[0017] Preferably, in the composite bacterial agent, the ratio of the cell numbers of Bifidobacterium longum WFP05 and Lactobacillus paracasei WFP1-1 is 1:(5-10); the cell number is the number of viable bacteria or the number of inactivated bacterial cells.

[0018] Preferably, the composite bacterial agent exists in the form of live bacteria or inactivated cells; when it exists in the form of live bacteria, the number of live bacteria in the composite bacterial agent is not less than 1×10 8 CFU / g; when in the inactivated cell state, the number of inactivated bacterial cells in the composite microbial agent is not less than 1×10 9 cell / g.

[0019] The composite bacterial agent can be used in the preparation of medicines or daily chemicals that have the effects of preventing dental caries and periodontitis and refreshing breath.

[0020] (3) Beneficial effects

[0021] Compared with the prior art, the Lactobacillus paracasei WFP1-1 and its composite bacterial agent provided by the present invention have the following beneficial effects:

[0022] 1. Lactobacillus paracasei WFP1-1 provided by the present invention is screened from Tibetan yak yogurt, and its source is natural and safe. Simultaneously, this bacterial strain has a significant inhibitory effect on oral harmful bacteria, can effectively inhibit Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis, and the inhibition zone of this bacterial strain to the above-mentioned oral pathogens is significantly greater than the inhibition zone of positive control strains Lactobacillus rhamnosus LGG and Lactobacillus paracasei LPC48. This shows that this bacterial strain has a more excellent effect of inhibiting oral pathogens relative to existing Lactobacillus rhamnosus LGG and Lactobacillus paracasei LPC48.

[0023] 2. The Bifidobacterium longum WFP05 provided by the present invention exhibits good adhesion, with a self-aggregation rate of 53.97%, which is higher than that of the positive control strain Lactobacillus rhamnosus LGG. It can be combined with Lactobacillus paracasei WFP1-1 to synergistically enhance the adhesion of the composite bacterial agent and the co-aggregation rate against Streptococcus mutans, which is significantly higher than that of the positive control strains Lactobacillus rhamnosus LGG and Lactobacillus paracasei LPC48.

[0024] 3. The composite bacterial agent provided by the present invention can be either live bacteria (i.e., live bacteria type) or inactivated bacteria (i.e., inactivated type). Both can improve the severity of dental caries in rats in animal experiments. Compared with live bacteria agents, inactivated bacteria agents offer greater safety, a longer shelf life, and do not require a cold chain during transportation, storage, and sales, resulting in lower costs and a wider range of product applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a plate colony morphology diagram of the Lactobacillus paracasei WFP1-1 provided by the present invention.

[0026] Figure 2 Microscopic examination images of Lactobacillus paracasei WFP1-1 and Bifidobacterium longum WFP05 provided by the present invention;

[0027] Among them, a is Lactobacillus paracasei WFP1-1; b is Bifidobacterium longum WFP05.

[0028] Figure 3 This is a diagram showing the effect of Lactobacillus paracasei WFP1-1 and compound bacteria in inhibiting harmful oral bacteria;

[0029] Among them, a is the effect diagram of inhibiting Streptococcus mutans, b is the effect diagram of inhibiting Fusobacterium nucleatum, and c is the effect diagram of inhibiting Porphyromonas gingivalis.

[0030] Figure 4 This is a diagram showing the adhesion effects of different bacterial strains on HT-29 cells;

[0031] Among them, a is the live bacteria of the composite bacterial agent; b is the inactivated bacteria of the composite bacterial agent; c is the single bacteria of Lactobacillus paracasei WFP1-1; d is Bifidobacterium longum WFP05; e is Lactobacillus paracasei LPC48; f is Lactobacillus rhamnosus LGG.

[0032] Figure 5 This is a diagram showing the effects of live bacteria composite bacterial agents and inactivated composite bacterial agents on reducing dental caries in rats (LPC48 control group omitted);

[0033] Among them, the 1st to 3rd columns are E-level, D-level, s Grade and D m The first to fifth rows are photos of rat caries in the blank group, model group, inactivated experimental group, live bacteria experimental group, and LGG control group, respectively. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0035] Strain Source: Lactobacillus paracasei WFP1-1 and Bifidobacterium longum WFP05 used in the following examples were obtained from naturally fermented yak milk products grown in Tibetan homes in Ngari, Tibet. After screening, isolation, and purification, the 16S rDNA nucleotide sequence of Lactobacillus paracasei is shown in SEQ ID NO. 1, and the 16S rDNA nucleotide sequence of Bifidobacterium longum is shown in SEQ ID NO. 2.

[0036] Example 1 Isolation, culture and identification of Lactobacillus paracasei WFP1-1

[0037] 1. Isolation and Culture of Strain

[0038] The naturally fermented yak milk from the Tibetan homes in Ali area of ​​Tibet was used as the sample. 1g of the sample was weighed and dissolved in 9mL of 0.85% sterile saline. The sample was shaken to mix thoroughly. 200μL of the mixture was applied to the commercial MRS solid culture medium and cultured in a constant temperature incubator at 37℃ for 72h. Single bacteria with different colony morphology were picked and then streaked on the solid culture medium for purification. This step was repeated 2-3 times until the colony characteristics were consistent. The purified single bacteria were then inoculated into the corresponding liquid culture medium and cultured at 37℃ for 18h. They were preserved with 60% glycerol and frozen in a -80℃ refrigerator for later use. The isolated colony images and microscopic examination images are shown as follows. Figure 1 and Figure 2 shown.

[0039] Cultivation characteristics: The optimum production temperature is 37°C, it is facultatively anaerobic, and the curd is fine and not sticky.

[0040] 2. Identification of strain 16S rDNA

[0041] The 16s rDNA nucleotide sequence of the strain (see sequence listing SEQ ID NO.1) was compared with the NCBI GenBank database for homology analysis, indicating that the strain had over 99.9% homology with Lacticaseibacillus paracesei. Therefore, it was identified as Lactobacillus paracasei and named Lactobacillus paracasei WFP1-1.

[0042] Example 2 Lactobacillus paracasei WFP1-1 and composite bacterial agent inhibit oral pathogens

[0043] In the sterilized MRS medium containing 1% agar (cooled to below 50°C), 6×10 7 Three indicator bacteria, Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis, were mixed by shaking and poured into a sterile culture dish. After the culture medium solidified, a sterile filter paper was evenly placed on the previously poured agar medium containing indicator bacteria. 200 μL of the corresponding bacterial solution (Lactobacillus paracasei WFP1-1, a composite bacterial agent with a bacterial cell count ratio of 9:1 of Lactobacillus paracasei: Bifidobacterium longum, and positive control strains Lactobacillus rhamnosus LGG and Lactobacillus paracasei LPC48) were added. The culture was placed in a 4°C refrigerator for diffusion for 8 hours, and then placed in a 37°C incubator for 16 hours. The size of the inhibition zone was observed and recorded.

[0044] The experimental results are as follows Figure 3As shown, the composite bacterial agent prepared from Lactobacillus paracasei WFP1-1 and Bifidobacterium longum WFP05 had the best synergistic antibacterial effect, which was significantly higher than that of Lactobacillus paracasei WFP1-1 and the two positive control bacteria. Secondly, Lactobacillus paracasei WFP1-1 had a significantly higher inhibitory effect on Fusobacterium nucleatum and Porphyromonas gingivalis than the two positive control bacteria, while there was no significant difference in the inhibitory effect of Lactobacillus paracasei WFP1-1 and Lactobacillus paracasei LPC48 on Streptococcus mutans.

[0045] Example 3 Determination of strain autoaggregation rate

[0046] The number of bacteria was 1×10 8 CFU / g of Lactobacillus paracasei WFP1-1, Bifidobacterium longum WFP05, composite bacterial agent (Lactobacillus paracasei / Bifidobacterium longum viable count ratio of 9:1) and positive control strains Lactobacillus rhamnosus LGG and Lactobacillus paracasei LPC48 were resuspended, and then pH 6.8 sterile PBS was used to adjust the OD value of the bacterial suspension to 0. 600 Adjust to 0.6, let it stand at 37℃ for 4 or 8 hours, and measure the OD value of the upper liquid. 600 .

[0047] Self-aggregation rate / %=(A0-A1) / A0×100

[0048] Where: A0 is the initial OD of bacterial suspension 600 ; A1 is the OD of the upper liquid of the bacterial suspension after standing 600 .

[0049] The experimental results are shown in Table 1 below. The composite bacterial preparation prepared with Lactobacillus paracasei WFP1-1 and Bifidobacterium longum WFP05 had the highest self-aggregation rate, reaching 88.2% after 8 hours. Bifidobacterium longum WFP05 was second, with a self-aggregation rate of 53.97% after 8 hours, both significantly higher than the positive control strains Lactobacillus rhamnosus LGG and Lactobacillus paracasei LPC48. This high self-aggregation rate helps the strains colonize and form biofilms on the cell surface, inhibiting the adhesion of harmful bacteria to the gastrointestinal tract or oral cavity. The self-aggregation rate of the composite bacterial preparation was significantly higher than that of the other four bacterial strains alone. Even at 8 hours, the self-aggregation rate of the composite bacterial preparation exceeded the sum of the self-aggregation rates of the two individual strains of Lactobacillus paracasei WFP1-1 and Bifidobacterium longum WFP05, indicating that the two strains have a synergistic effect on the self-aggregation rate of the strains, which may be related to the adhesive properties of Bifidobacterium longum WFP05.

[0050] Table 1. Autoaggregation rate of different strains

[0051]

[0052] Example 4 Determination of the Adhesion of Different Strains and Composite Bacterial Agents to HT-29 Cells

[0053] HT-29 cells were passaged to the third generation, digested with 0.25% trypsin, centrifuged at 1000 rpm for 5 min, and resuspended in 5% DMEM medium containing 10% fetal bovine serum (with double antibody) until the cells were single-cell suspension. An appropriate amount of cells was counted using a hemocytometer and diluted to 1×10 cells using DMEM medium containing 10% fetal bovine serum (with double antibody). 6 cells / mL, 1 mL was inoculated into a 12-well cell culture plate with a cell slide, and cultured at 37°C and 5% CO2 for 2 days.

[0054] After activation of Lactobacillus paracasei WFP1-1, Bifidobacterium longum WFP05, composite bacterial agent (Lactobacillus paracasei / Bifidobacterium longum ratio of 9:1) and positive control strains Lactobacillus rhamnosus LGG and Lactobacillus paracasei LPC48 second generation, 10 mL of bacterial solution was centrifuged at 4000 × g for 10 min to collect the cells, and resuspended in DMEM complete medium (without double antibodies) containing 10% fetal bovine serum to adjust the cell concentration to 2 × 10 8 CFU / mL. Take 5mL of the compound bacterial agent adjustment solution and inactivate it at 95℃ for 5min, as the inactivated compound bacterial agent experimental group. Take 1mL of each of the above corresponding bacterial adjustment solutions and inoculate it into a 12-well cell culture plate with a cell slide placed on it, and culture it in a carbon dioxide incubator at 37℃ for 2h. After completion, slowly aspirate the culture medium, wash it 3 times with sterile PBS, fix it with 100% methanol for 8min, take out the cell slide and let it stand for 20min, seal it with neutral resin after Gram staining, and observe it under an optical microscope. The results are as follows Figure 4 shown.

[0055] from Figure 4 It can be seen that the inactivated and live bacteria of the composite bacterial agent prepared from Lactobacillus paracasei WFP1-1 and Bifidobacterium longum WFP05 showed excellent adhesion ability in the HT-29 cell model experiment. The number of single-cell adhesion to the inactivated composite bacterial agent reached 18 bacteria, and the number of single-cell adhesion to the live bacterial composite bacterial agent reached 13 bacteria, which was higher than the adhesion of the two single bacteria (single-cell adhesion to Lactobacillus paracasei WFP1-1 was 8.3, and adhesion to Bifidobacterium longum WFP05 was 9.8). At the same time, they were significantly higher than the positive control strains Lactobacillus rhamnosus LGG (single-cell adhesion 2.75) and Lactobacillus paracasei LPC48 (single-cell adhesion 4.5).

[0056] Example 5 Coagglutination rate of different strains with Streptococcus mutans

[0057] The number of bacteria was 1×10 8CFU / g Lactobacillus paracasei WFP1-1, Bifidobacterium longum WFP05, composite bacterial agent (the ratio of Lactobacillus paracasei / Bifidobacterium longum cell number is 8:1) and positive control strains Lactobacillus rhamnosus LGG and Lactobacillus paracasei LPC48 were resuspended, and then the OD of Streptococcus mutans and the test bacterial suspension was mixed with sterile PBS at pH 6.8. 600 Adjust to 0.6, mix evenly in equal proportions, and then measure OD 600 The upper liquid OD was measured at 37℃ for 4 or 8 hours. 600 .

[0058] Coagulation rate / % = (B0-2B1) / B0×100

[0059] Where: B0 is the initial OD of bacterial suspension 600 ; B1 is the OD of the upper liquid of the bacterial suspension after standing 600 .

[0060] The experimental results are shown in Table 2 below. The composite bacteria prepared by Lactobacillus paracasei WFP1-1 and Bifidobacterium longum WFP05 have the highest coaggregation rate with Streptococcus mutans, which can reach 86.53% in 8 hours. The second is Lactobacillus paracasei WFP1-1, which has a coaggregation rate of 50.25% with Streptococcus mutans in 8 hours, which is significantly higher than the positive controls Lactobacillus rhamnosus LGG and Lactobacillus paracasei LPC48.

[0061] The co-aggregation rate of the composite bacterial agent and Streptococcus mutans was high, indicating that the two strains of bacteria could synergistically and effectively inhibit Streptococcus mutans in terms of adhesion and antibacterial properties, thereby reducing the incidence of dental caries.

[0062] Table 2. Coaggregation rates of different strains with Streptococcus mutans

[0063]

[0064] Example 6. Production yield of composite bacterial agent

[0065] Cryopreserved tubes of Lactobacillus paracasei WFP1-1 and Bifidobacterium longum WFP05 were inoculated with a 2% inoculum size into commercial MRS liquid medium for activation and passage three times. Then, they were inoculated with a 4% inoculum size into modified MRS liquid medium (2% glucose, 2% sucrose, 1% peptone, 2% yeast extract, 0.3% dipotassium hydrogen phosphate, 0.1% Tween 80, and 0.1% L-cysteine ​​hydrochloride) for expansion culture. Ammonia water was used as the neutralizing solution to maintain a constant pH of 5.5 at 37°C for 10 h. The bacterial sludge was harvested at 15°C and 120 L / h. The bacterial sludge was thoroughly mixed with a lyoprotectant (18% skim milk powder, 4% glycerol, 10% trehalose, and 5% inulin) at a mass ratio of 1:1.7 to prepare an emulsion. After vacuum freeze-drying, the bacterial powder of the two strains was obtained.

[0066] The results of bacterial powder production are shown in Table 3. 1000 kg of fermentation liquid can produce 1310 kg of 1×10 10 CFU / g Lactobacillus paracasei WFP1-1 or 1×10 10 CFU / g Bifidobacterium longum WFP05 powder, the composite bacterial agent prepared with Lactobacillus paracasei WFP1-1 as the main ingredient has high yield and excellent economic benefits.

[0067] Table 3. Production results of Lactobacillus paracasei WFP1-1 and Bifidobacterium longum WFP05

[0068]

[0069] Example 7. Effect of the composite bacterial agent on reducing dental caries in rats infected with Streptococcus mutans

[0070] Three-week-old SPF rats were randomly divided into four groups according to body weight, with 8 rats in each group. They were set up as a blank control group (blank group), a caries model group (model group), experimental groups (live bacteria composite microbial agent experimental group and inactivated bacteria composite microbial agent experimental group), and a positive control group (Lactobacillus rhamnosus LGG and Lactobacillus paracasei LPC48). Ten days before the start of the experiment, the model group, experimental group, and positive control group were swabbed with Streptococcus mutans every day. After the model group, experimental group, and positive control group were confirmed to have successfully established the model, experimental group, and positive control group, the two experimental groups were swabbed with the live bacteria composite microbial agent or the inactivated bacteria composite microbial agent once a day until the end of the eighth week of the experiment. The two positive control groups were swabbed with Lactobacillus rhamnosus LGG or Lactobacillus paracasei LPC48 every day.

[0071] During the experiment, cariogenic mutans Streptococcus was cultured at 37°C for 12 h, centrifuged at 8000×g for 5 min, collected, washed three times with saline, and resuspended to adjust the bacterial concentration to 1×10 8 CFU / mL. Live bacteria compound agent, Lactobacillus rhamnosus LGG and Lactobacillus paracasei LPC48 powder were adjusted to a bacterial concentration of 1×10 8 CFU / mL, the inactivated composite bacterial powder was adjusted to a bacterial concentration of 1×10 9 Cell / mL, 0.2mL was dipped into a sterile cotton swab and rubbed on the rat molars for 15 seconds. During the experiment, the blank control group was fed with normal feed and drinking water, while the other groups were maintained with cariogenic feed and 5% sucrose drinking water for 8 weeks.

[0072] The rats were killed at the end of the 2nd, 4th, 6th, and 8th weeks of the experiment, and their molars were removed and sterilized at 115°C for 20 minutes. The soft tissue was removed and the molars were naturally dried. The molars were then immersed in 0.4% murexide for 12 hours, rinsed with clean water, and naturally dried. The degree of caries in the rat molars was observed under a stereo microscope. The caries scores of the rat teeth were calculated according to the Keyes method (E grade: caries only involves the enamel; Ds grade: caries involves the enamel and the outer 1 / 4 of the dentin; Dm grade: caries involves between 1 / 4 and 3 / 4 of the dentin thickness; Dx grade: caries involves more than 3 / 4 of the dentin thickness). The results are shown in Table 4 and Figure 4 (LPC48 control group omitted).

[0073] From Table 4 and Figure 4 Compared with the dental caries model group, both the experimental and positive control groups significantly reduced enamel caries, superficial dentin caries, and intermediate dentin caries, with the experimental group showing significantly greater efficacy in reducing caries than the positive control group. In the experimental group, the inactivated bacterial compound was slightly more effective than the live bacterial compound in reducing enamel caries, superficial dentin caries, and intermediate dentin caries. Overall, both live and inactivated bacterial compounds effectively reduced the severity of dental caries in rats.

[0074] Table 4. Rats dental caries scores

[0075]

[0076] Note: The caries degree in the above test did not reach D x Grade, so no D x level data.

[0077] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A composite bacterial agent, characterized in that: It is composed of Lactobacillus paracesei WFP1-1 and Bifidobacterium longum WFP05; the Lactobacillus paracesei WFP1-1 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 30, 2022, with a preservation number of GDMCC No. 62587; the Bifidobacterium longum WFP05 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 30, 2022, with a preservation number of GDMCC No. 62592.

2. The composite bacterial agent according to claim 1, characterized in that In the composite bacterial agent, the ratio of the cell numbers of Bifidobacterium longum WFP05 and Lactobacillus paracasei WFP1-1 is 1:(5-10); the cell number is the number of viable bacteria or the number of inactivated bacterial cells.

3. Use of the composite bacterial agent according to any one of claims 1 to 2 in the preparation of medicines with the efficacy of preventing dental caries and daily chemicals with the efficacy of freshening breath.

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