Probiotic composition for preventing and / or inhibiting oral pathogens as well as derivative product, preparation method and application thereof

Abstract

The invention provides a probiotic composition for preventing and / or inhibiting oral pathogens as well as a derivative product, a preparation method and application of the probiotic composition, and belongs to the technical field of oral care. The probiotic composition comprises the following bacterial strains in parts by mass: 90 to 0.45 part of a lactobacillus rhamnosus bacterial strain A2114, 10 to 0.3 part of a plant lactobacillus bacterial strain A2124, 80 to 0.4 part of a pediococcus pentosaceus bacterial strain A2135, 250 to 0.25 part of a lactobacillus reuteri bacterial strain A21350, and 40 to 0.2 part of a plant lactobacillus bacterial strain A2109. The probiotic composition or the metabolite thereof can effectively inhibit oral pathogens such as streptococcus mutans, porphyromonas gingivalis, helicobacter pylori, escherichia coli and staphylococcus aureus. Therefore, the probiotic composition or the metabolite thereof shows a good application prospect in the aspects of preventing and relieving oral problems such as decayed teeth, periodontitis and ozostomia.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of oral care, and particularly relates to a probiotic composition for preventing and / or inhibiting oral pathogenic bacteria, a derivative product thereof, a preparation method and application. BACKGROUND

[0002] The oral cavity is an important part directly connected with the external environment, and its microecological system is extremely complex, being the second largest microbial community next to the intestinal tract. The microorganisms colonized in the oral cavity are various, mainly including bacteria, fungi, spirochetes and the like, among which bacteria are dominant. These microorganisms form a unique microenvironment in the oral cavity, and when the homeostasis is broken, such as due to lifestyle changes or invasion of exogenous pathogenic bacteria, it can cause the occurrence of oral problems, such as dental caries, periodontitis, halitosis and the like.

[0003] The traditional methods for treating oral problems mainly include mechanical cleaning and use of antibacterial agents. Mechanical cleaning such as tooth brushing and tooth washing can inhibit the formation of plaque and dental calculus in the short term, but it is difficult to completely remove the bacteria in hidden parts such as periodontal pockets, and it cannot fundamentally regulate the oral microecology. The use of antibacterial agents such as antibiotics can inhibit the growth of pathogenic bacteria, but long-term use can increase drug resistance and further affect oral health by disrupting the balance of normal flora in the oral cavity. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a probiotic composition for preventing and relieving dental caries, periodontitis, halitosis and other oral problems by inhibiting the activity of Streptococcus mutans and Porphyromonas gingivalis in the oral cavity, regulating the balance of the flora in the oral cavity; and at the same time, inhibiting the infection of Helicobacter pylori, Escherichia coli and Staphylococcus aureus in the gastrointestinal tract, thereby achieving the purpose of oral care from the inside to the outside.

[0005] The present application provides a probiotic composition, comprising the following strains in various mass fractions: Lactobacillus rhamnosu strain A21149 0-0.45 parts, Lactiplantibacillus plantarum strain A21241 0-0.3 parts, Pediococcus pentosaceus strain A21358 0-0.4 parts, Limosilactobacillus reuteri strain A21325 0-0.25 parts and Lactiplantibacillus plantarum strain A21094 0-0.2 parts.

[0006] The preservation number of the Lactobacillus rhamnosu strain A21149 is GDMCC No:62226.

[0007] The preservation number of the Lactobacillus plantarum strain A21241 is GDMCC No: 63771;

[0008] The preservation number of the Pediococcus pentosaceus strain A21358 is GDMCC No: 63040;

[0009] The preservation number of the Lactobacillus rhamnosus strain A21325 is GDMCC No: 63041;

[0010] The preservation number of the Lactobacillus plantarum strain A21094 is GDMCC No: 62961.

[0011] Preferably, the composition comprises: 0.28-0.32 parts of the Lactobacillus rhamnosus strain A21149, 0.008-0.012 parts of the Lactobacillus plantarum strain A21241, 0.08-0.12 parts of the Pediococcus pentosaceus strain A21358, and 0.08-0.12 parts of the Lactobacillus rhamnosus strain A21325.

[0012] Preferably, the composition comprises: 0.13-0.17 parts of the Lactobacillus rhamnosus strain A21149, 0.28-0.32 parts of the Pediococcus pentosaceus strain A21358, 0.03-0.05 parts of the Lactobacillus rhamnosus strain A21325, and 0.008-0.012 parts of the Lactobacillus plantarum strain A21094.

[0013] Preferably, the composition comprises: 0.08-0.12 parts of the Lactobacillus rhamnosus strain A21149, 0.08-0.12 parts of the Lactobacillus plantarum strain A21241, 0.08-0.12 parts of the Pediococcus pentosaceus strain A21358, 0.08-0.12 parts of the Lactobacillus rhamnosus strain A21325, and 0.08-0.12 parts of the Lactobacillus plantarum strain A21094.

[0014] The present application provides a preparation, wherein the active ingredient comprises the probiotic composition or metabolites produced by the probiotic composition.

[0015] Preferably, the probiotic composition further comprises an adjuvant; the adjuvant comprises the following components in at least 3 parts by mass per unit mass of the probiotic composition: resistant dextrin 0.1-1.2 parts, xylo-oligosaccharide 0.2-0.45 parts, erythritol 0.05-0.3 parts, xylitol 0.2-0.45 parts, alginate 0.01-0.08 parts, Luo Han Guo powder 0.001-0.005 parts, mint powder 0.02-0.05 parts, orange powder 0.3-1.4 parts, lime powder 0.08-0.2 parts, lactitol 0.4-0.6 parts, sorbitol 0.25-0.55 parts, polydextrose 0.2-0.6 parts, fructo-oligosaccharide 0.1-0.3 parts, magnesium stearate 0.01-0.05 parts, peppermint essence 0.01-0.02 parts, vitamin C 0.002-0.01 parts, sodium bicarbonate 0.22-0.46 parts, anhydrous citric acid 0.1-0.3 parts, rose powder 0.05-0.2 parts, mogroside 0.01-0.05 parts, kiwi powder 0.2-0.22 parts, and grapefruit powder 0.15-0.18 parts.

[0016] Preferably, the probiotic composition has a viable bacterial count of not less than 3×10 9 CFU / g.

[0017] The present application provides use of the probiotic composition, metabolites produced by the probiotic composition, or the preparation in the preparation of an oral care product.

[0018] Preferably, the oral care comprises inhibiting at least one of the following oral pathogenic bacteria: Streptococcus mutans, Porphyromonas gingivalis, Helicobacter pylori, Escherichia coli, and Staphylococcus aureus.

[0019] Preferably, the oral care product comprises at least one of the following: mouthwash, toothpaste, solid powder, fruit-flavored lozenge, and effervescent tablet.

[0020] The present application provides a probiotic composition, comprising the following strain composition in various mass parts: Lactobacillus rhamnosus strain A21149 0-0.45 parts, Lactobacillus plantarum strain A21241 0-0.3 parts, Pediococcus pentosaceus strain A21358 0-0.4 parts, Lactobacillus reuteri strain A21325 0-0.25 parts, and Lactobacillus plantarum strain A21094 0-0.2 parts; the preservation number of the Lactobacillus rhamnosus strain A21149 is GDMCC No:62226; the preservation number of the Lactobacillus plantarum strain A21241 is GDMCC No:63771; the preservation number of the Pediococcus pentosaceus strain A21358 is GDMCC No:63040; the preservation number of the Lactobacillus reuteri strain A21325 is GDMCC No:63041; the preservation number of the Lactobacillus plantarum strain A21094 is GDMCC No:62961; the Lactobacillus rhamnosus strain A21149, the Pediococcus pentosaceus strain A21358 and the Lactobacillus reuteri strain A21325 all have significant anti-Helicobacter pylori function, can effectively inhibit the growth and infection of Helicobacter pylori by multiple mechanisms such as enhancing agglutination, reducing urease activity, and regulating inflammatory factors, and can reduce the inflammatory response caused by Helicobacter pylori. The Lactobacillus plantarum strain A21241 has a broad-spectrum inhibitory effect on oral pathogenic bacteria, and also has good anti-inflammatory ability and biological safety, and can be used for preparing oral care products. The Lactobacillus plantarum strain A21094 has good strong acid and strong alkali tolerance, and can inhibit main intestinal pathogenic bacteria such as Escherichia coli and E. coli. The results of the examples show that the probiotic composition has a significant inhibitory effect on Streptococcus mutans, Porphyromonas gingivalis, Helicobacter pylori, Escherichia coli and Staphylococcus aureus, and the inhibitory ability of the probiotic agent of the present application on oral pathogenic bacteria is much higher than that of similar products. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The inhibition results of the combined probiotic bacteria on pathogenic bacteria in Example 1;

[0022] Figure 2 The sensory quality evaluation results of the oral care solid beverage prepared in Example 2;

[0023] Figure 3 The inhibition results of the oral care solid beverage prepared in Example 4 on pathogenic bacteria. DETAILED DESCRIPTION

[0024] The present application provides a probiotic composition comprising the following strain composition in various mass parts: Lactobacillus rhamnosus strain A21149 0-0.45 parts, Lactobacillus plantarum strain A21241 0-0.3 parts, Pediococcus pentosaceus strain A21358 0-0.4 parts, Lactobacillus rhamnosus strain A21325 0-0.25 parts, and Lactobacillus plantarum strain A21094 0-0.2 parts;

[0025] The Lactobacillus rhamnosus strain A21149 has a preservation number of GDMCC No: 62226;

[0026] The Lactobacillus plantarum strain A21241 has a preservation number of GDMCC No: 63771;

[0027] The Pediococcus pentosaceus strain A21358 has a preservation number of GDMCC No: 63040;

[0028] The Lactobacillus rhamnosus strain A21325 has a preservation number of GDMCC No: 63041;

[0029] The Lactobacillus plantarum strain A21094 has a preservation number of GDMCC No: 62961.

[0030] In the present invention, the probiotic composition preferably comprises the following composition: Lactobacillus rhamnosus strain A21149 0.3 parts, Lactobacillus plantarum strain A21241 0.01 parts, Pediococcus pentosaceus strain A21358 0.1 parts, Lactobacillus reuteri strain A21325 0.1 parts. The probiotic composition preferably comprises the following composition: Lactobacillus rhamnosus strain A21149 0.15 parts, Pediococcus pentosaceus strain A21358 0.3 parts, Lactobacillus reuteri strain A21325 0.04 parts, and Lactobacillus plantarum strain A21094 0.01 parts. The probiotic composition preferably comprises the following composition: Lactobacillus rhamnosus strain A21149 0.08 to 0.12 parts, Lactobacillus plantarum strain A21241 0.08 to 0.12 parts, Pediococcus pentosaceus strain A21358 0.08 to 0.12 parts, Lactobacillus reuteri strain A21325 0.08 to 0.12 parts, and Lactobacillus plantarum strain A21094 0.08 to 0.12 parts; it can be Lactobacillus rhamnosus strain A21149 0.1 parts, Lactobacillus plantarum strain A21241 0.1 parts, Pediococcus pentosaceus strain A21358 0.1 parts, Lactobacillus reuteri strain A21325 0.1 parts, and Lactobacillus plantarum strain A21094 0.1 parts.

[0031] In the present application, the Lactobacillus rhamnosus strain A21149, the Lactobacillus plantarum strain A21241, the Pediococcus pentosaceus strain A21358 and the Lactobacillus reuteri strain A21325 as well as the Lactobacillus plantarum strain A21094 are all human-derived probiotics isolated from fresh feces of centenarian healthy old people. The Lactobacillus rhamnosus strain A21149 is disclosed in the patent with the patent name of Lactobacillus rhamnosus strain A21149 for inhibiting Helicobacter pylori and its application, and the publication number CN114958658A. The Lactobacillus plantarum strain A21241 is disclosed in the patent with the patent name of Application of Lactobacillus plantarum strain A21241 and / or its metabolites in the preparation of products for inhibiting oral pathogenic bacteria, and the publication number CN119033826A. The Pediococcus pentosaceus strain A21358 is disclosed in the patent with the patent name of Pediococcus pentosaceus strain A21358 with anti-Helicobacter pylori effect and its application, and the publication number CN115851551A. The Lactobacillus reuteri strain A21325 is disclosed in the patent with the patent name of Lactobacillus reuteri strain A21325 for inhibiting Helicobacter pylori infection and its application, and the publication number CN116004456A. The Lactobacillus plantarum strain A21094 is described in the patent with the patent name of Lactobacillus plantarum strain with the functions of reducing triglyceride, reducing cholesterol and reducing fat and its application, and the publication number CN116144541A.

[0032] In the embodiments of the present application, six probiotic compositions are configured by different strain compositions and different mass fraction ratios, specifically combinations 1-6, and in vitro bacteriostatic experiments are carried out. In the inhibition ability tests on Streptococcus mutans, Porphyromonas gingivalis, Helicobacter pylori, Escherichia coli and Staphylococcus aureus, combinations 2, 4 and 6 all show significant inhibition effects on the above-mentioned oral pathogenic bacteria. The specific inhibition effects are as follows: the inhibition ability on Streptococcus mutans is ranked as 2>4>6>1>3>5; the inhibition ability on Porphyromonas gingivalis is ranked as 2>4>6>3>1>5; the inhibition ability on Helicobacter pylori is ranked as 2>6>4>1>3>5; the inhibition ability on Escherichia coli is ranked as 2>6>4>1>3>5; and the inhibition ability on Staphylococcus aureus is ranked as 6>4>2>1>3>5.

[0033] The present application provides a preparation, and the active ingredient includes the probiotic composition or the metabolite produced by the probiotic composition.

[0034] In the present application, when the probiotic composition is prepared into a preparation, it is called a probiotic agent. When the metabolite produced by the probiotic composition described above is prepared into a preparation with an active ingredient, it is called a probiotic preparation.

[0035] In the present application, the preparation preferably further comprises an auxiliary material; the auxiliary material comprises the following components in at least 3 parts by mass per unit mass of the probiotic composition: resistant dextrin 0.1-1.2 parts, xylo-oligosaccharide 0.2-0.45 parts, erythritol 0.05-0.3 parts, xylitol 0.2-0.45 parts, alginate 0.01-0.08 parts, Luo Han Guo powder 0.001-0.005 parts, peppermint powder 0.02-0.05 parts, orange powder 0.3-1.4 parts, lime powder 0.08-0.2 parts, lactitol 0.4-0.6 parts, sorbitol 0.25-0.55 parts, polydextrose 0.2-0.6 parts, fructo-oligosaccharide 0.1-0.3 parts, magnesium stearate 0.01-0.05 parts, peppermint essence 0.01-0.02 parts, vitamin C 0.002-0.01 parts, sodium bicarbonate 0.22-0.46 parts, anhydrous citric acid 0.1-0.3 parts, rose powder 0.05-0.2 parts, mogroside 0.01-0.05 parts, kiwi powder 0.2-0.22 parts, and grapefruit powder 0.15-0.18 parts.

[0036] In the present application, the viable bacterial count of the probiotic composition is preferably not less than 3×10 9 CFU / g, which can be 4×10 9 CFU / g, or 4×10 10 CFU / g, or 5×10 10 CFU / g.

[0037] The present application provides the use of the probiotic composition, metabolites produced by the probiotic composition, or the preparation in the preparation of an oral care product.

[0038] In the present application, the oral care preferably comprises inhibiting at least one of the following oral pathogenic bacteria: Streptococcus mutans, Porphyromonas gingivalis, Helicobacter pylori, Escherichia coli, and Staphylococcus aureus. Experiments show that the oral care product prepared by the present application has a significant inhibitory effect on Streptococcus mutans, Porphyromonas gingivalis, Helicobacter pylori, Escherichia coli, and Staphylococcus aureus, and the antibacterial activity of the product on other pathogenic bacteria is better than that of commercially available probiotics. The product can effectively inhibit common oral pathogenic bacteria and has a certain protective effect on oral health, and the effect is better than that of similar products on the market.

[0039] In the present application, the oral care product preferably comprises at least one of the following: mouthwash, toothpaste, solid powder, fruit-flavored lozenge, and effervescent tablet.

[0040] The solid powder preferably comprises the following components by mass fraction: Lactobacillus rhamnosus strain A211490.3 parts, Pediococcus pentosaceus strain A213580.1 part, Lactobacillus reuteri strain A213250.09 parts, Lactobacillus plantarum strain A212410.01 parts, resistant dextrin 0.967 parts, xylo-oligosaccharide 0.35 parts, erythritol 0.113 parts, xylitol 0.4 parts, alginate 0.01 parts, monk fruit powder 0.001 parts, peppermint powder 0.05 parts, orange powder 0.5 parts, and lime powder 0.1 parts.

[0041] The fruit-flavored lozenge preferably comprises the following components by mass fraction: Lactobacillus rhamnosus strain A211490.32 parts, Pediococcus pentosaceus strain A213580.12 parts, Lactobacillus reuteri strain A213250.09 parts, Lactobacillus plantarum strain A212410.012 parts, orange powder 1 part, lactitol 0.53 parts, sorbitol 0.47 parts, polydextrose 0.27 parts, resistant dextrin 0.2 parts, fructo-oligosaccharide 0.133 parts, magnesium stearate 0.053 parts, peppermint essence 0.01 parts, vitamin C 0.008 parts, monk fruit powder 0.00167 parts.

[0042] The effervescent tablet preferably comprises the following components by mass fraction: Lactobacillus rhamnosus strain A211490.3 parts, Pediococcus pentosaceus strain A213580.1 part, Lactobacillus reuteri strain A213250.09 parts, Lactobacillus plantarum strain A212410.01 parts, orange powder 1.067 parts, xylitol 0.45 parts, sorbitol 0.37 parts, sodium bicarbonate 0.37 parts, anhydrous citric acid 0.3 parts, lime powder 0.2 parts, rose powder 0.13 parts, magnesium stearate 0.04 parts, mogroside 0.033 parts, vitamin C 0.0067 parts.

[0043] The present application provides a probiotic composition for preventing and / or inhibiting oral pathogenic bacteria, and a preparation method and application thereof, which are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present application.

[0044] Example 1

[0045] Screening experiment of antibacterial ability of a probiotic composition

[0046] 1. Preparation of combined probiotic supernatant

[0047] The corresponding weight of bacterial powder was weighed into a 15 mL centrifuge tube according to Table 1, 3 mL of PBS was added and dissolved thoroughly, and then incubated at 37°C for 24 h, followed by centrifugation at 8000 rpm for 10 min. The collected solution was filtered with a 0.22 μm microporous filter membrane to obtain six kinds of combined probiotic supernatant.

[0048] Table 1 Probiotic composition components

[0049] No. A21094 A21149 A21241 A21358 A21325 1 0.3g 0.09g 0.06g 0.05g 0g 2 0g 0.3g 0.01g 0.1g 0.09g 3 0.05g 0g 0.3g 0.01g 0.14g 4 0.01g 0.15g 0g 0.3g 0.04g 5 0.1g 0.05g 0.05g 0g 0.3g 6 0.1g 0.1g 0.1g 0.1g 0.1g

[0050] Note: Strain A21094 is Lactobacillus plantarum, with accession number GDMCC No: 62961, described in the patent with publication number CN116144541A;

[0051] Strain A21149 is Lactobacillus rhamnosus, with accession number GDMCC No: 62226, described in the patent with publication number CN114958658A;

[0052] Strain A21241 is Lactobacillus plantarum, with accession number GDMCC No: 63771, described in the patent with publication number CN119033826A;

[0053] Strain A21358 is Pediococcus pentosaceus, with accession number GDMCC No: 63040, described in the patent with publication number CN115851551B;

[0054] Strain A21325 is Lactobacillus reuteri, with accession number GDMCC No: 63040, described in the patent with publication number CN116004456B.

[0055] 2. Preparation of culture medium

[0056] (1) MRS liquid medium: Proteose peptone 2.0 g / L, sodium acetate 5.0 g / L, beef infusion powder 40 g / L, Tween-80 0.1%, glucose 20.0 g / L, K2HPO4 2.0 g / L, yeast extract powder 4.0 g / L, MnSO4·4H2O 0.04 g / L, triammonium citrate 2.0 g / L, MgSO4 0.2 g / L, distilled water to 1000 mL, 121℃ high pressure sterilization for 15 min.

[0057] (2) MRS solid medium: Proteose peptone 2.0 g / L, sodium acetate 5.0 g / L, beef infusion powder 40 g / L, Tween-80 0.1%, glucose 20.0 g / L, K2HPO4 2.0 g / L, yeast extract powder 4.0 g / L, MnSO4·4H2O 0.04 g / L, triammonium citrate 2.0 g / L, MgSO4 0.2 g / L, agar powder 15.0 g / L, distilled water to 1000 mL, 121℃ high pressure sterilization for 15 min, pour plate.

[0058] (3) Columbia liquid medium: Columbia finished product medium 29.0 g / L.

[0059] (4) Columbia blood agar medium: Columbia finished medium 29.0 g / L, agar 15.0 g / L, distilled water to 1000 mL, 121°C high pressure sterilization for 15 min, add sterile defibering sheep blood at 5% addition amount when about 50°C, pour plate.

[0060] (5) LB liquid medium: tryptone 10.0 g / L, yeast extract powder 5.0 g / L, sodium chloride 10.0 g / L, distilled water to 1000 mL, 121°C high pressure sterilization for 15 min.

[0061] (6) LB solid medium: tryptone 10.0 g / L, yeast extract powder 5.0 g / L, sodium chloride 10.0 g / L, agar powder 15.0 g / L, distilled water to 1000 mL, 121°C high pressure sterilization for 15 min, pour plate.

[0062] 3. Culture of oral pathogenic bacteria

[0063] (1) Streptococcus mutans

[0064] Take out the Streptococcus mutans glycerol preservation tube from the -80°C refrigerator, take 100 μL and smear on the MRS plate, and culture the plate in the 37°C anaerobic workstation for 48 h, subculture twice, scrape single colony and inoculate into MRS liquid medium, continue to culture for 24 h to obtain bacterial liquid. Centrifuge the bacterial liquid at 8000 r / min for 5 min to obtain bacterial bodies, wash twice with PBS and resuspend to OD 600 = 0.8 ± 0.05, that is, Streptococcus mutans suspension is obtained.

[0065] (2) Porphyromonas gingivalis

[0066] Take out the Porphyromonas gingivalis glycerol preservation tube from the -80°C refrigerator, take 100 μL and smear on the Columbia blood agar plate, and culture the plate in the 37°C anaerobic workstation for 48 h, subculture twice, scrape single colony and inoculate into Columbia liquid medium, and add fetal bovine serum at 5% addition amount, continue to culture for 48 h to obtain bacterial liquid. Centrifuge the bacterial liquid at 8000 r / min for 5 min to obtain bacterial bodies, wash twice with PBS and resuspend to OD 600 = 0.6 ± 0.05, that is, Porphyromonas gingivalis suspension is obtained.

[0067] (3) Helicobacter pylori

[0068] Take out the H. pylori glycerol preservation tube from the -80°C refrigerator, take 100 μL and coat on the Columbia blood agar plate, invert the plate and incubate in a 37°C three-gas incubator (5% oxygen, 10% carbon dioxide, 85% nitrogen) for 72 h, subculture twice, scrape the H. pylori from the blood plate into PBS and mix to obtain a bacterial solution, centrifuge the bacterial solution at 8000 r / min for 5 min to obtain bacterial cells, wash twice with PBS and resuspend to OD 600 = 1.0 ± 0.05, i.e. to obtain an H. pylori suspension.

[0069] (4) E. coli

[0070] Take out the E. coli glycerol preservation tube from the -80°C refrigerator, use a sterile inoculation loop to take the bacterial solution and streak on an LB plate, invert the plate and incubate in a 37°C conventional incubator for 24 h; inoculate a single colony from the plate into LB liquid medium and continue to culture for 24 h to obtain a bacterial solution. Centrifuge the bacterial solution at 8000 r / min for 5 min to obtain bacterial cells, wash twice with PBS and resuspend to OD 600 = 0.2 ± 0.05, i.e. to obtain an E. coli suspension.

[0071] (5) S. aureus

[0072] Take out the S. aureus glycerol preservation tube from the -80°C refrigerator, use a sterile inoculation loop to take the bacterial solution and streak on an LB plate, invert the plate and incubate in a 37°C conventional incubator for 24 h; inoculate a single colony from the plate into LB liquid medium and continue to culture for 24 h to obtain a bacterial solution. Centrifuge the bacterial solution at 8000 r / min for 5 min to obtain bacterial cells, wash twice with PBS and resuspend to OD 600 = 0.2 ± 0.05, i.e. to obtain an S. aureus suspension.

[0073] 4. Dilution spot plate experiment

[0074] (1) Mixing: mix the bacterial suspensions of the above cultured and OD-adjusted S. mutans, P. gingivalis, H. pylori, E. coli and S. aureus with the supernatants of the six combined probiotics in a 1:1 volume ratio, for example, mix 0.5 mL of the pathogenic bacterial suspension with 0.5 mL of the supernatant to obtain a mixed stock solution;

[0075] (2) Incubation: incubate the mixed stock solution at 37°C for 0-8 h according to the characteristics of each pathogenic bacterial strain;

[0076] (3) Dilution: gradient dilute the incubated stock solution to obtain concentrations of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10-5 Diluent;

[0077] (4) Plate spotting: Take 7 μL of the diluted solution and spot it onto the corresponding square plate culture medium;

[0078] (5) Incubation: After the liquid has dried, invert the culture medium in the square dish and place it in the corresponding incubator for incubation;

[0079] (6) Observe and record: observe the growth of Escherichia coli and Staphylococcus aureus after 24 hours, Streptococcus mutans after 48 hours, Porphyromonas gingivalis and Helicobacter pylori after 72 hours, and take photos to record.

[0080] 5. Experimental Results

[0081] Colony growth is used as a key indicator to assess the inhibitory effect of probiotic combinations on pathogens. Specifically, the degree of inhibition of colony growth is positively correlated with the antibacterial activity of the probiotic combination; that is, the weaker the colony growth, the stronger the inhibitory effect of the probiotic combination on pathogens; conversely, the stronger the colony growth, the weaker the inhibitory effect.

[0082] Depend on Figure 1 It was found that in the inhibitory ability tests against Streptococcus mutans, Porphyromonas gingivalis, Helicobacter pylori, Escherichia coli, and Staphylococcus aureus, probiotic compositions 2, 4, and 6 all showed significant inhibitory effects against the aforementioned oral pathogens. The specific inhibitory effects are as follows: the inhibitory ability against Streptococcus mutans was ranked as follows: 2>4>6>1>3>5; against Porphyromonas gingivalis, the ranking was: 2>4>6>3>1>5; against Helicobacter pylori, the ranking was: 2>6>4>1>3>5; against Escherichia coli, the ranking was: 2>6>4>1>3>5; and against Staphylococcus aureus, the ranking was: 6>4>2>1>3>5. Among these, except for Staphylococcus aureus, where composition 6 was the optimal inhibitory group, the optimal inhibitory group for the other pathogens was composition 2. Therefore, considering the comprehensive inhibitory effects against multiple pathogens, composition 2 was selected as the preferred strain combination for subsequent product development.

[0083] Example 2

[0084] Preparation of probiotics

[0085] A solid beverage for preventing and / or inhibiting oral pathogens, comprising the following probiotics and excipients, accurately weighed by weight:

[0086] Sample 1: Lactobacillus rhamnosus strain A2114 18g, Pediococcus pentosaceus strain A2135 86g, Lactobacillus reuteri strain A2132 55.4g, Lactobacillus plantarum strain A2124 10.6g, resistant dextrin 49.5g, xylo-oligosaccharide 27g, erythritol 10g, xylitol 24g, alginate 0.6g, monk fruit powder 0.06g, peppermint powder 3g, orange powder 30g, lime powder 6g.

[0087] Sample 2: Lactobacillus rhamnosus strain A2114 18g, Pediococcus pentosaceus strain A2135 86g, Lactobacillus reuteri strain A2132 55.4g, Lactobacillus plantarum strain A2124 10.6g, resistant dextrin 59.5g, xylo-oligosaccharide 25g, erythritol 12g, xylitol 24g, alginate 0.6g, monk fruit powder 0.06g, peppermint powder 3g, lime powder 6g, kiwi powder 12g.

[0088] Sample 3: Lactobacillus rhamnosus strain A2114 18g, Pediococcus pentosaceus strain A2135 86g, Lactobacillus reuteri strain A2132 55.4g, Lactobacillus plantarum strain A2124 10.6g, resistant dextrin 62.5g, xylo-oligosaccharide 33g, erythritol 11g, xylitol 18g, alginate 0.6g, monk fruit powder 0.06g, peppermint powder 3g, orange powder 18g, grapefruit powder 9g.

[0089] Preparation method, the specific steps are as follows:

[0090] (1) The purchased raw materials are unpacked in the unpacking room, and the outer packaging is removed;

[0091] (2) The raw materials with the outer packaging removed in step (1) are placed in the material buffer room, and the inner packaging is sterilized by irradiation with ultraviolet lamp for 0.5-1h, and the raw materials are sent to the clean workshop;

[0092] (3) Strictly according to the product production formula and process, the probiotics and auxiliary materials corresponding to the weight are added in the mixer, and the materials are mixed for 30min to make the materials fully mixed and uniform, and the corresponding production record is made;

[0093] (4) The inner packaging machine is sterilized in advance for 30min, the mixed materials in step (3) are put into the packaging machine for sub-packaging, the net content is 1-3g per strip, the net content and the packaging sealing property are randomly sampled and tested to ensure that the product quality is qualified;

[0094] (5) Box, code, plastic seal.

[0095] Steps (2)-(4) are completed in a ten-thousand-level clean environment.

[0096] Example 3

[0097] Sensory evaluation of probiotic agent

[0098] A panel of 60 sensory trained professionals were selected to evaluate the sensory quality of the probiotic agent prepared in Example 2, which was consumed as a ready-to-eat product. The sensory evaluation criteria are shown in Table 2.

[0099] Table 2 Sensory evaluation criteria

[0100]

[0101]

[0102] Table 3 Sensory evaluation results

[0103] No. Item Sample 1 (parts) Sample 2 (parts) Sample 3 (parts) 1 A. Good taste 27 15 26 2 B. Average taste 14 21 12 3 C. Poor taste 0 5 3 4 A. Appropriate sour / sweet 21 12 13 5 B. Sweetish 13 19 24 6 C. Sourish 7 7 1 7 D. Not sweet enough 0 3 3 8 A. Good palatability 34 30 31 9 B. Poor palatability 5 9 9 10 C. Other 2 2 1 11 Overall score (good and above) 42 26 32

[0104] Note: Table 3 results are based on 60 servings, 41 of which were returned, and 19 people dropped out of the evaluation.

[0105] The proportion of people who found the probiotic agent of Sample 1 to be good in taste was 65.9%, the proportion of people who found the probiotic agent of Sample 1 to be sweet and sour was 51.2%, the proportion of people who found the probiotic agent of Sample 1 to be good in palatability was 82.9%, and the proportion of people who found the probiotic agent of Sample 1 to be excellent was 63.4%. The proportion of people who found the probiotic agent of Sample 2 to be good in taste was 36.6%, the proportion of people who found the probiotic agent of Sample 2 to be sweet and sour was 29.3%, the proportion of people who found the probiotic agent of Sample 2 to be good in palatability was 73.2%, and the proportion of people who found the probiotic agent of Sample 2 to be excellent was 26.8%. The proportion of people who found the probiotic agent of Sample 3 to be good in taste was 63.4%, the proportion of people who found the probiotic agent of Sample 3 to be sweet and sour was 31.7%, the proportion of people who found the probiotic agent of Sample 3 to be good in palatability was 75.6%, and the proportion of people who found the probiotic agent of Sample 3 to be excellent was 36.6%. In summary, the probiotic agent of Sample 1 was more popular than the probiotic agents of Samples 2 and 3 (Example 3), and therefore, the flavor of Sample 1 was preferred for subsequent product formulations. Figure 2

[0106] Example 4

[0107] Antibacterial ability of probiotic agent

[0108] 1. Solid beverage formulation

[0109] A solid beverage for preventing and / or inhibiting oral pathogenic bacteria, comprising the following probiotics and auxiliary materials by weight:

[0110] Lactobacillus rhamnosus strain A2114 918g, Pediococcus pentosaceus strain A2135 86g, Lactobacillus reuteri strain A2132 55.4g, Lactobacillus plantarum strain A2124 10.6g, resistant dextrin 58.02g, xylo-oligosaccharide 21g, erythritol 6.78g, xylitol 24g, alginate 0.6g, monk fruit powder 0.06g, mint powder 3g, orange powder 30g, lime powder 6g. Mix evenly using a mixer, and use a powder packaging machine to package and seal the mixture, with a packaging specification of 3g per bag, and the theoretical number of live bacteria should not be less than 1.67 x 10​10 CFU / g.

[0111] 2. Preparation of supernatant of product of the present application / commercial competitor

[0112] (1) Preparation of supernatant of probiotic agent of the present application:

[0113] Take 3g of the above solid beverage into a 15mL centrifuge tube, add 6mL of PBS and dissolve thoroughly, incubate at 37°C for 24h, then centrifuge at 8000rpm / min for 10min, filter the collected solution with a 0.22μm microporous filter, and the supernatant of probiotic agent of the present application is obtained.

[0114] (2) Preparation of supernatant of commercial probiotic agent:

[0115] The theoretical viable count of the commercial probiotic agent is 1x10 11 CFU / g, take 1g of the commercial probiotic agent into a 15mL centrifuge tube, add 6mL of PBS and dissolve thoroughly, incubate at 37°C for 24h, then centrifuge at 8000rpm / min for 10min, filter the collected solution with a 0.22μm microporous filter, and the supernatant of commercial probiotic agent is obtained.

[0116] 3. Preparation of culture medium as in Example 1.

[0117] 4. Cultivation of oral pathogenic bacteria as in Example 1.

[0118] 5. Dilution spot plate experiment

[0119] (1) Mixing: mix the Streptococcus mutans, Porphyromonas gingivalis, Helicobacter pylori, Escherichia coli, Staphylococcus aureus bacterial liquid respectively with the supernatant of probiotic agent of the present application or the supernatant of commercial probiotic agent (probiotic powder purchased from Beijing Tongren Tang) at a volume ratio of 1:1, mix uniformly to obtain a mixed stock solution, and PBS buffer is used as a blank control;

[0120] (2) Incubation: incubate the stock solution at 37°C for 0-8h according to the characteristics of the pathogenic bacterial strain;

[0121] (3) Dilution: gradient dilute the incubated stock solution to obtain dilution liquids with concentrations of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 ;

[0122] (4) Spot plate: take 7μL of the dilution liquid to perform spot plate on the corresponding square dish culture medium;

[0123] (5) Cultivation: After the liquid is dried, the square dish culture medium is placed in the corresponding incubator for cultivation;

[0124] (6) Observation and recording: the growth of E. coli, S. aureus after 24 hours, S. mutans after 48 hours, P. gingivalis, and H. pylori after 72 hours can be observed, and photographs are recorded.

[0125] 6. Experimental results

[0126] The experimental results are shown in Table 1. Figure 3 The experimental results show that, compared with the PBS blank control group, the oral care probiotic agent of the present application has a significant inhibitory effect on S. mutans, P. gingivalis, H. pylori, E. coli, and S. aureus. In addition, under the same experimental conditions, the inhibitory effect of the probiotic agent of the present application on the above-mentioned pathogenic bacteria is significantly better than that of the commercially available probiotic agent, and only the inhibitory ability of S. mutans is comparable. The experiment shows that the probiotic combination product of the present application can effectively inhibit common oral pathogenic bacteria and has a certain protective effect on oral health, and the effect is better than that of similar products on the market.

[0127] Example 5

[0128] A tablet candy for preventing and / or inhibiting oral pathogenic bacteria, the following probiotics and auxiliary materials are accurately weighed by weight:

[0129] Lactobacillus rhamnosus strain A2114 919.2g, Pediococcus pentosaceus strain A2135 86.4g, Lactobacillus reuteri strain A2132 55.76g, Lactobacillus plantarum strain A2124 10.64g, orange powder 60g, lactitol 31.58g, sorbitol 28g, polydextrose 16g, resistant dextrin 12g, fructooligosaccharide 8g, magnesium stearate 3.2g, peppermint essence 0.64g, vitamin C 0.48g and momordica grosvenori powder 0.1g. Mix evenly with a mixer, use a tablet press to press the mixture into tablet candy particles, then package and seal.

[0130] Example 6

[0131] A micro-effervescent tablet for preventing and / or inhibiting oral pathogenic bacteria, the following probiotics and auxiliary materials are accurately weighed by weight:

[0132] Lactobacillus rhamnosus strain A2114 921.6g, Pediococcus pentosaceus strain A2135 87.2g, Lactobacillus reuteri strain A2132 56.48g, Lactobacillus plantarum strain A2124 10.72g, orange powder 64g, xylitol 27g, sorbitol 22g, sodium bicarbonate 22g, anhydrous citric acid 18g, lime powder 12g, rose powder 8g, magnesium stearate 2.4g, mogroside 2g and vitamin C 0.4g. The mixture is mixed evenly by a mixer, and then the mixture is tabletted by a tablet machine to form micro-effervescent tablet granules, and then the micro-effervescent tablet granules are packaged and sealed.

[0133] The above merely describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A probiotic composition, characterized in that, The sample includes multiple samples of the following strains in varying weights: Lactobacillus rhamnosu strain A211490–0.45 parts, Lactiplantibacillus plantarum strain A212410–0.3 parts, Pediococcus pentosaceus strain A213580–0.4 parts, Limosilactobacillus reuteri strain A213250–0.25 parts, and Lactiplantibacillus plantarum strain A21094 0–0.2 parts; the number of each strain may not be 0 at the same time. The preservation number of the Lactobacillus rhamnosus strain A21149 is GDMCC No:62226; The preservation number of the *Lactobacillus plantarum* strain A21241 is GDMCC No:63771; The preservation number of the Pediococcus pentosaceus strain A21358 is GDMCC No:63040; The preservation number of the Lactobacillus reuteri strain A21325 is GDMCC No:63041; The preservation number of the *Lactobacillus plantarum* strain A21094 is GDMCC No:62961.

2. The probiotic composition according to claim 1, characterized in that, The composition includes the following components: Lactobacillus rhamnosus strain A21149 0.28–0.32 parts, Lactobacillus plantarum strain A21241 0.008–0.012 parts, Pediococcus pentosus strain A21358 0.08–0.12 parts, and Lactobacillus reuteri strain A21325 0.08–0.12 parts.

3. The probiotic composition according to claim 1, characterized in that, The composition includes the following components: Lactobacillus rhamnosus strain A21149 0.13–0.17 parts, Pediococcus pentosus strain A21358 0.28–0.32 parts, Lactobacillus reuteri strain A21325 0.03–0.05 parts, and Lactobacillus plantarum strain A21094 0.008–0.012 parts.

4. The probiotic composition according to claim 1, characterized in that, The composition includes the following components: Lactobacillus rhamnosus strain A21149 (0.08–0.12 parts), Lactobacillus plantarum strain A21241 (0.08–0.12 parts), Pediococcus pentosus strain A21358 (0.08–0.12 parts), Lactobacillus reuteri strain A21325 (0.08–0.12 parts), and Lactobacillus plantarum strain A21094 (0.08–0.12 parts).

5. A formulation, characterized in that, The active ingredient includes the probiotic composition according to any one of claims 1 to 4 or metabolites produced by the probiotic composition.

6. The formulation according to claim 5, characterized in that, It also includes auxiliary materials; The excipients, based on the unit mass parts of the probiotic composition, include at least three of the following components: 0.1–1.2 parts resistant dextrin, 0.2–0.45 parts xylooligosaccharide, 0.05–0.3 parts erythritol, 0.2–0.45 parts xylitol, 0.01–0.08 parts algal polysaccharide, 0.001–0.005 parts monk fruit powder, 0.02–0.05 parts peppermint powder, 0.3–1.4 parts orange powder, 0.08–0.2 parts lime powder, 0.4–0.6 parts lactitol, 0.25–0.55 parts sorbitol, 0.2–0.6 parts polydextrose, 0.1–0.3 parts fructooligosaccharide, 0.01–0.05 parts magnesium stearate, 0.01–0.02 parts peppermint flavoring, and vitamin C. 0.002–0.01 parts, sodium bicarbonate 0.22–0.46 parts, anhydrous citric acid 0.1–0.3 parts, rose powder 0.05–0.2 parts, mogroside 0.01–0.05 parts, kiwi powder 0.2–0.22 parts, and grapefruit powder 0.15–0.18 parts.

7. The formulation according to claim 5 or 6, characterized in that, The probiotic composition has a live bacteria count of not less than 3 × 10⁻⁶. 9 CFU / g.

8. The use of the probiotic composition of any one of claims 1 to 4, the metabolites produced by the probiotic composition, or the formulation of any one of claims 5 to 7 in the preparation of oral care products.

9. The application according to claim 8, characterized in that, The oral care includes inhibiting at least one of the following oral pathogens: Streptococcus mutans, Porphyromonas gingivalis, Helicobacter pylori, Escherichia coli, and Staphylococcus aureus.

10. The application according to claim 8, characterized in that, The oral care products include at least one of the following: mouthwash, toothpaste, solid powder, fruit-flavored lozenges, and effervescent tablets.

Images