A postbiotic having an improved effect on oral diseases and use thereof

Abstract

This invention provides the application of a probiotic composition in the preparation of products for improving oral diseases. The probiotic composition includes *Lactiplantibacillus plantarum* RH03147, *Lactiplantibacillus plantarum* HCS03-001, and *Limosilactobacillus reuteri* HCS02-001. The probiotic composition disclosed in this invention can effectively inhibit *Streptococcus mutans* and *Porphyromonas gingivalis*.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the preparation of a probiotic composition and its application in the preparation of medicines for the prevention or treatment of oral diseases. Background Technology

[0002] Metabiotics are non-living microorganisms and / or their components (including metabolites, cellular components, or cell-free mixtures) that are beneficial to host health. They include many different components such as short-chain fatty acids, microbial cellular components, extracellular polysaccharides, cell lysates, teichoic acid, peptidoglycans, bioactive lipids, and organic acids. Studies have demonstrated that metabiotics possess biological activities such as anti-inflammatory, antibacterial, immunomodulatory, antioxidant, anti-obesity, antihypertensive, lipid-lowering, hepatoprotective, and wound-healing promotion. Some metabiotics can also positively influence the homeostasis of the host's gut microbiota or the expression of metabolic and signaling pathways. Anti-inflammatory and antibacterial activities are the most common biological activities of metabiotics.

[0003] The oral microecology is an important ecosystem in the human body, mainly composed of the oral microbiota and the host's inherent oral environment. Under physiological conditions, the oral microbiota and the host are in dynamic equilibrium, maintaining oral health. When this balance is disrupted, it can induce the proliferation of harmful bacteria in the oral cavity, causing oral diseases such as tooth decay, periodontal disease, oral cancer, and oral mucositis.

[0004] Dental caries and periodontal disease are highly prevalent oral diseases, primarily caused by oral pathogens such as *Streptococcus mutans* and *Porphyromonas gingivalis*. Taking *Streptococcus* as an example, its growth process produces a biofilm rich in polysaccharides, giving it strong adhesive properties. Other cariogenic bacteria adhere to this biofilm, forming plaque and leading to tooth decay. If the number of bacteria at the gingival margin increases, causing gingivitis, it can develop into gingivitis. Adjusting the oral microecological balance and inhibiting the growth of harmful bacteria can reduce the occurrence of dental caries and other oral diseases. Traditional treatments aim to completely eliminate cariogenic bacteria in the mouth, with antibiotics and antibacterial agents becoming the first choice for sterilization. However, this approach leads to antibiotic overuse, further disrupts the oral microecology, causes bacterial resistance and other side effects, and generally has limited therapeutic effects. Therefore, using probiotics with antibacterial capabilities and the ability to enhance the body's resistance to treat oral diseases has gradually become a research hotspot.

[0005] Lactobacillus plantarum is a lactic acid bacterium widely found in nature, especially in various types of fermented foods. Colonizing the intestines of humans and animals, Lactobacillus plantarum possesses numerous beneficial functions, including inhibiting pathogenic bacteria, improving the intestinal environment, regulating intestinal flora, and enhancing immunity. It is widely used in food, medicine, and feed additives. (Patent CN)

[0006] Patent application 109957530A discloses that Lactobacillus plantarum HCS03-001 has strong antibacterial activity against oral pathogens and possesses laxative and antidiarrheal effects. The invention also discloses the application of this strain as an additive with good antibacterial properties, particularly strong antibacterial activity against oral pathogens, in the preparation of food, health food, or pharmaceutical compositions.

[0007] *Lactobacillus reuteri* is widely found in the intestines of humans and animals, making it a relatively abundant source. It has numerous probiotic effects, effectively preventing diarrhea by regulating intestinal flora, and inhibiting the growth of pathogenic microorganisms, thus reducing the incidence of intestinal diseases. *Lactobacillus reuteri* has many probiotic effects, one of which is its involvement in cholesterol metabolism. After ingestion, it enters the intestines through the digestive system and possesses acid- and bile-salt-resistant properties, exerting a beneficial effect on the body. Patent CN114990011A discloses that *Lactobacillus reuteri* HCS02001 has cholesterol-lowering and Gardnerella vaginalis-inhibiting effects. However, its application in maintaining oral health is not disclosed. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a probiotic composition that effectively inhibits Streptococcus mutans and Porphyromonas gingivalis, reducing the occurrence of oral diseases such as dental caries and periodontal disease. It effectively prevents a series of side effects caused by traditional treatment methods, such as bacterial resistance, adjusts the oral microecological balance, and maintains oral health.

[0009] On the one hand, the present invention provides an application of a probiotic composition in the preparation of probiotic preparations, beverages, dairy products, confectionery products, oral care products, and medicines for the prevention or treatment of oral diseases. The probiotic composition is composed of Lactobacillus plantarum RH03147, Lactobacillus plantarum HCS03-001, and Lactobacillus reuteri HCS02-001.

[0010] Specifically, the activation method for *Lactobacillus plantarum* RH03147, *Lactobacillus plantarum* HCS03-001, and *Lactobacillus reuteri* HCS02-001 is as follows:

[0011] (1) Take the cryopreservation tubes of *Lactobacillus plantarum* RH03147, *Lactobacillus plantarum* HCS03-001, and *Lactobacillus reuteri* HCS02-001 stored in the low temperature freezer, and immediately place them in a 37℃ water bath for 15-30 seconds until the liquid in the cryopreservation tubes is completely thawed to revive the strains.

[0012] (2) According to the inoculation amount of 10%, the recovered bacterial strains were inoculated into different culture media A and incubated at 37°C for 17 hours to obtain seed liquid;

[0013] (3) The seed liquid was inoculated into culture medium A at a rate of 5%, and cultured at 37°C for 17 hours to obtain the culture solutions of the three strains.

[0014] Specifically, the preparation of culture medium A is as follows: yeast peptone 12.0 g / L, glucose 25.0 g / L, yeast extract 8.0 g / L, sodium acetate 8.0 g / L, citric acid monohydrate 3.0 g / L, potassium dihydrogen phosphate 3.0 g / L, Tween 80 0.2 g / L, magnesium sulfate 0.8 g / L, with the remainder being purified water; weigh according to the formula ratio, heat to dissolve, sterilize at 115℃ for 30 min, and adjust the pH of the culture medium to 6.60 with 1 mol / L food-grade NaOH solution.

[0015] Specifically, the *Lactobacillus plantarum* RH03147, *Lactobacillus plantarum* HCS03-001, and *Lactobacillus reuteri* HCS02-001 are mixed in a bacterial count ratio of 2-4:1-4:1-2.

[0016] In some embodiments, the bacterial count mixing ratio of *Lactobacillus plantarum* RH03147, *Lactobacillus plantarum* HCS03-001, and *Lactobacillus reuteri* HCS02-001 is 3.5:3.5:1.

[0017] In some embodiments, the mixing ratio of *Lactobacillus plantarum* RH03147, *Lactobacillus plantarum* HCS03-001, and *Lactobacillus reuteri* HCS02-001 is 2:1:1.

[0018] Specifically, *Lactobacillus plantarum* RH03147, *Lactobacillus plantarum* HCS03-001, and *Lactobacillus reuteri* HCS02-001 were inoculated into culture medium B at an inoculation rate of 2-6% and incubated at 35-40°C.

[0019] Stir and culture for 17 hours to obtain fermentation broth.

[0020] More preferably, the fermentation temperature is 35°C; and the inoculum size is 5%.

[0021] Specifically, the culture medium B comprises 5-15.0 g / L yeast peptone, 15-20.0 g / L glucose, 5-12.0 g / L yeast extract, 3-5.0 g / L sodium acetate, 2.0-3.0 g / L citric acid monohydrate, 2.0-3.0 g / L potassium dihydrogen phosphate, 0.1-0.2 g / L Tween 80, 0.6-0.8 g / L magnesium sulfate, 0.20-0.25 g / L manganese sulfate, and the balance being purified water.

[0022] The aforementioned care products may be mouthwash, mouthwash powder, toothpaste, tooth powder, or mouth spray.

[0023] The nursing products may also include other excipients, such as one or more of vitamins, minerals, sweeteners, acidulants, cooling agents, and fillers.

[0024] The dosage form of the medicine may be a mouthwash, spray, film, aerosol, ointment or paste.

[0025] The medicine may also include other pharmaceutically acceptable carriers or excipients, such as one or more of starch, dextrin, sucrose, lactose, mannitol, microcrystalline cellulose, crospovidone, crospovidone sodium carboxymethyl cellulose, and crospovidone sodium carboxymethyl starch.

[0026] On the other hand, the present invention provides the application of postbiotics in the preparation of oral care products and medicines for the prevention or treatment of oral diseases, wherein the postbiotics are prepared by the above-mentioned probiotic composition.

[0027] Specifically, the aforementioned probiotic composition is fermented, sterilized, and dried to obtain the product.

[0028] More specifically, the probiotic composition is inoculated into culture medium B, fermented at a constant temperature of 35-40℃, concentrated, and sterilized to obtain postbiotic liquid, and after adding excipients, spray-dried to obtain postbiotic powder.

[0029] Preferably, the fermentation temperature is 35°C.

[0030] Specifically, the fermentation broth was obtained by stirring and culturing at 35°C for 48 hours. The fermentation broth was then filtered to obtain a filtrate that was colorless to light yellow, transparent, and free of impurities. The supernatant was then concentrated by heating to twice its original volume and sterilized by boiling for 30 minutes to obtain the post-biotic solution.

[0031] Specifically, the excipients include maltodextrin, with an addition amount of 5-15%.

[0032] Preferably, the excipients include maltodextrin, and the addition amount is 8%.

[0033] Specifically, after adding maltodextrin to the post-biotic solution, the mixture is stirred until completely dissolved. The ambient humidity is ≤50%, and the mixture is placed in a spray dryer for spray drying. The inlet air temperature is 180℃ and the outlet air temperature is 110℃ to obtain post-biotic powder.

[0034] Specifically, the culture medium B comprises 5-15.0 g / L yeast peptone, 15-20.0 g / L glucose, 5-12.0 g / L yeast extract, 3-5.0 g / L sodium acetate, 2.0-3.0 g / L citric acid monohydrate, 2.0-3.0 g / L potassium dihydrogen phosphate, 0.1-0.2 g / L Tween 80, 0.6-0.8 g / L magnesium sulfate, 0.20-0.25 g / L manganese sulfate, and the balance being purified water.

[0035] The aforementioned care products may be mouthwash, mouthwash powder, toothpaste, tooth powder, or mouth spray.

[0036] The nursing products may also include other excipients, such as one or more of vitamins, minerals, sweeteners, acidulants, cooling agents, and fillers.

[0037] The dosage form of the medicine may be a mouthwash, spray, film, aerosol, ointment or paste.

[0038] The medicine may also include other pharmaceutically acceptable carriers or excipients, such as one or more of starch, dextrin, sucrose, lactose, mannitol, microcrystalline cellulose, crospovidone, crospovidone sodium carboxymethyl cellulose, and crospovidone sodium carboxymethyl starch.

[0039] On the other hand, the present invention provides the application of a probiotic composition in inhibiting Streptococcus mutans and / or Porphyromonas gingivalis, wherein the probiotic composition comprises Lactobacillus plantarum RH03147, Lactobacillus plantarum HCS03-001 and Lactobacillus reuteri HCS02-001, and the application is for non-disease diagnosis or treatment.

[0040] The present invention also provides the application of a metabiotic in inhibiting Streptococcus mutans and / or Porphyromonas gingivalis, wherein the metabiotic is prepared by the probiotic composition described above, and the application is for non-disease diagnosis or treatment.

[0041] The technical effects achieved by this invention are as follows: This invention provides a probiotic composition. Antibacterial test data show that *Lactobacillus plantarum* RH03147, *Lactobacillus plantarum* HCS03-001, and *Lactobacillus reuteri* HCS02-001 have a synergistic effect. The combined use of these strains is more effective than using them alone in inhibiting *Streptococcus mutans* and *Porphyromonas gingivalis*, thus reducing the occurrence of oral diseases such as dental caries and periodontal disease. It effectively prevents a series of side effects caused by traditional treatment methods, such as bacterial resistance, adjusts the oral microecological balance, and maintains oral health. Attached Figure Description

[0042] Figure 1 The graph shows the results of the inhibitory effect of the probiotic composition postbiotic on Streptococcus mutans.

[0043] Figure 2 The graph shows the results of the antibacterial effect of the probiotic composition postbiotic on Porphyromonas gingivalis. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0045] Example 1: Preparation of postbiotics from probiotic compositions

[0046] In this embodiment, the probiotics are: Lactobacillus plantarum RH03147 (accession number CGMCC NO.25775), Lactobacillus plantarum HCS03-001 (accession number CGMCC NO.16258), and Lactobacillus reuteri HCS02-001 (accession number CGMCC NO.19746).

[0047] The specific steps for preparing postbiotics from probiotic compositions are as follows:

[0048] (1) Take cryovials of *Lactobacillus plantarum* RH03147, *Lactobacillus plantarum* HCS03-001, and *Lactobacillus reuteri* HCS02-001 stored in a low-temperature freezer and immediately place them in a 37°C water bath for 15-30 seconds until the liquid in the cryovials is completely thawed to revive the strains. Inoculate the revive strains into different culture media A at a 10% inoculation rate and incubate them at 37°C for 17 hours to obtain seed culture. Inoculate the seed culture into culture media A at a 5% inoculation rate and incubate them at 37°C for 17 hours to obtain culture solutions of the three strains, so that the bacterial concentration in the final culture solutions of each strain is close.

[0049] (2) The culture media of Lactobacillus plantarum RH03147, Lactobacillus plantarum HCS03-001 and Lactobacillus reuteri HCS02-001 were inoculated into the same culture medium B at a ratio of 3.5:3.5:1, with an inoculation amount of 5%, and were cultured at 35℃ with stirring for 17h to obtain the primary fermentation broth.

[0050] (3) Immediately after step 2, the primary fermentation broth was transferred to culture medium B again with an inoculation rate of 5%, and cultured at 35°C with stirring for 48 hours to obtain the secondary fermentation broth.

[0051] (4) Filter the secondary fermentation broth to obtain a filtrate that is colorless to light yellow and transparent without impurities; heat and concentrate the supernatant twice, and then boil and sterilize for 30 minutes to obtain the post-fermentation liquid.

[0052] (5) Add 8% maltodextrin to the post-biotic solution, mix and stir until completely dissolved, keep the ambient humidity ≤50%, place it in a spray dryer for spray drying, with an inlet air temperature of 180℃ and an outlet air temperature of 110℃, to obtain post-biotic powder.

[0053] The culture medium A is prepared as follows: yeast peptone 12.0 g / L, glucose 25.0 g / L, yeast extract 8.0 g / L, sodium acetate 8.0 g / L, citric acid monohydrate 3.0 g / L, potassium dihydrogen phosphate 3.0 g / L, Tween 80 0.2 g / L, magnesium sulfate 0.8 g / L, with the remainder being purified water; weigh according to the formula ratio, heat to dissolve, sterilize at 115℃ for 30 min, and adjust the pH of the culture medium to 6.60 with 1 mol / L food-grade NaOH solution.

[0054] The culture medium B is prepared as follows: yeast peptone 15.0 g / L, glucose 20.0 g / L, yeast extract 12.0 g / L, sodium acetate 5.0 g / L, citric acid monohydrate 2.0 g / L, potassium dihydrogen phosphate 2.0 g / L, Tween 80 0.2 g / L, magnesium sulfate 0.6 g / L, manganese sulfate 0.25 g / L, with the remainder being purified water; weigh according to the formula ratio, heat to dissolve, sterilize at 115℃ for 30 min, and adjust the pH of the culture medium to 6.80 with 1 mol / L food-grade NaOH solution.

[0055] Example 2: Postbiotic inhibition test of Streptococcus mutans

[0056] The inhibitory effect of postbiotics on Streptococcus mutans was verified using the double-layer agar diffusion method.

[0057] (1) Take the post-natal vitamin powder sample and dilute it 5 times (by mass) with sterile physiological saline. After it is completely dissolved, it is ready for use.

[0058] Pour 2% water agar medium into a sterile plate, just enough to cover the bottom of the plate, and wait for the agar to solidify.

[0059] (2) After thawing the cryovial of Streptococcus mutans strain, add 1 mL of the cryovial culture to 100 mL of BHI liquid medium and anaerobic culture at 37°C for 18-24 h; place 4 Oxford cups evenly on a water agar plate, and take 100 μL of Streptococcus mutans culture (concentration approximately 1×10⁻⁶) 8 Mix (CFU / mL) with 10 mL of 1.2% agar medium and pour into a Petri dish. After the agar solidifies, remove the Oxford cup to create a sample well with a diameter of 8 mm.

[0060] (3) Add 100 μL of postbiotic sample to three wells of the plate, and add MRS sterilized medium to the remaining well as a blank control. Incubate anaerobically at 37℃ for 24 h. Measure the diameter of the inhibition zone with calipers. The inhibition zone of the Oxford cup with postbiotic added reached 23.17 mm. Figure 1 As shown.

[0061] BHI (synthetic medium): tryptone 1.0%, anhydrous glucose 0.2%, sodium chloride 0.5%, beef heart powder 0.5%, disodium hydrogen phosphate dodecahydrate 0.25%, solid agar powder 2%, pH 7.4±0.2.

[0062] MRS medium: yeast peptone 10 g / L, beef meal 3 g / L, yeast extract 4 g / L, potassium dihydrogen phosphate 2 g / L, citric acid monohydrate 2 g / L, sodium acetate 5 g / L, anhydrous glucose 20 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, Tween 80 0.6 g / L, tomato juice 1% (v / v), pH 6.5.

[0063] Example 3: Postbiotic inhibition test of Porphyromonas gingivalis

[0064] The inhibitory effect of postbiotics on *Porphyromonas gingivalis* was verified using the double-layer agar diffusion method.

[0065] (1) Take the post-natal vitamin powder sample and dilute it 5 times (by mass) with sterile physiological saline. After it is completely dissolved, it is ready for use.

[0066] (2) Pour 2% water agar medium into a sterile plate, just enough to cover the bottom of the plate, and wait for the agar to solidify.

[0067] (3) After thawing the cryovial of *Porphyromonas gingivalis* strain, add 1 mL of the cryovial culture to 100 mL of TSB liquid medium and anaerobic culture at 37°C for 18-24 h; place 4 Oxford cups evenly on a water agar plate, and take 100 μL of *Porphyromonas gingivalis* (concentration approximately 1 × 10⁻⁶) 8 Mix (CFU / mL) with 10 mL of 1.2% agar medium and pour into a Petri dish. After the agar solidifies, remove the Oxford cup to create a sample well with a diameter of 8 mm.

[0068] (4) Add 100 μL of post-biotic sample to three wells of the plate, and add MRS sterilized medium to the remaining well as a blank control. Incubate anaerobically at 37℃ for 24 h. Measure the diameter of the inhibition zone with calipers. The inhibition zone in the Oxford cup with the added post-biotic solution reached 23.00 mm. Figure 2 As shown.

[0069] TSB medium: 17 g / L tryptone, 3 g / L plant peptone, 5 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L anhydrous glucose, 20 g / L solid agar powder, pH 7.3±0.2, after sterilization and cooling, add 5% sterile defibrinated sheep blood.

[0070] Example 4

[0071] The postbiotic was prepared according to the method in Example 1, wherein the bacterial counts of *Lactobacillus plantarum* RH03147, *Lactobacillus plantarum* HCS03-001, and *Lactobacillus reuteri* HCS02-001 were mixed in a ratio of 2:1:1. The culture medium B was prepared as follows: yeast peptone 5.0 g / L, glucose 15.0 g / L, yeast extract 5.0 g / L, sodium acetate 3.0 g / L, citric acid monohydrate 3.0 g / L, potassium dihydrogen phosphate 2.0 g / L, Tween 80 0.2 g / L, magnesium sulfate 0.8 g / L, manganese sulfate 0.2 g / L, with the remainder being purified water; the contents were weighed according to the formula, heated to dissolve, sterilized at 115°C for 30 min, and the pH of the culture medium was adjusted to 6.80 with 1 mol / L food-grade NaOH solution.

[0072] Using the above-mentioned metabiotic, antibacterial tests were conducted on Streptococcus mutans and Porphyromonas gingivalis, respectively, according to Examples 2 and 3. The test results are as follows: the diameter of the inhibition zone of the metabiotic against Streptococcus mutans was 23.22 mm; the diameter of the inhibition zone against Porphyromonas gingivalis was 23.10 mm.

[0073] Comparative Examples 1-10

[0074] The postgenes in the comparative examples were all prepared according to the method in Example 1.

[0075] Comparative Examples 1-5 were set up and experiments were conducted according to the method in Example 2. The setup of the comparative examples and the final inhibition zone results are as follows:

[0076]

[0077] Comparative Examples 6-10 were set up and experiments were conducted according to the method in Example 3. The setup of the comparative examples and the final inhibition zone results are as follows:

[0078]

Claims

1. The application of an epigenetic agent in the preparation of oral care products and pharmaceuticals for the prevention or treatment of oral diseases, characterized in that, The probiotic composition is inoculated into the culture medium, fermented at a constant temperature of 35-40℃, concentrated and sterilized to obtain postbiotic liquid, and after adding excipients, it is spray-dried to obtain postbiotic powder. The probiotic composition consists of Lactobacillus plantarum RH03147, Lactobacillus plantarum HCS03-001, and Lactobacillus reuteri HCS02-001. The *Lactobacillus plantarum* RH03147, *Lactobacillus plantarum* HCS03-001, and *Lactobacillus reuteri* HCS02-001 were mixed in a bacterial count ratio of 2-4:1-4:1-2.

2. The application according to claim 1, characterized in that, The bacterial count mixing ratio of *Lactobacillus plantarum* RH03147, *Lactobacillus plantarum* HCS03-001, and *Lactobacillus reuteri* HCS02-001 was 3.5:3.5:

1.

3. The application according to claim 1, characterized in that, The mixing ratio of *Lactobacillus plantarum* RH03147, *Lactobacillus plantarum* HCS03-001, and *Lactobacillus reuteri* HCS02-001 was 2:1:

1.

4. The application according to claim 1, characterized in that, The culture medium comprises yeast peptone 5-15.0 g / L, glucose 15-20.0 g / L, yeast extract 5-12.0 g / L, sodium acetate 3-5.0 g / L, citric acid monohydrate 2.0-3.0 g / L, potassium dihydrogen phosphate 2.0-3.0 g / L, Tween 80 0.1-0.2 g / L, magnesium sulfate 0.6-0.8 g / L, manganese sulfate 0.20-0.25 g / L, and the balance being purified water.

5. The use of a metabiotic in the preparation of products that inhibit Streptococcus mutans and / or Porphyromonas gingivalis, characterized in that, The probiotic composition is inoculated into the culture medium, fermented at a constant temperature of 35-40℃, concentrated and sterilized to obtain postbiotic liquid, and after adding excipients, it is spray-dried to obtain postbiotic powder. The probiotic composition consists of Lactobacillus plantarum RH03147, Lactobacillus plantarum HCS03-001, and Lactobacillus reuteri HCS02-001. The *Lactobacillus plantarum* RH03147, *Lactobacillus plantarum* HCS03-001, and *Lactobacillus reuteri* HCS02-001 were mixed in a bacterial count ratio of 2-4:1-4:1-2. The applications described are not for disease diagnosis or treatment.

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