A marine-derived Lactobacillus plantarum HJ-S2 strain capable of improving oral health and its application

Abstract

The present invention belongs to the field of microbial technology, and in particular relates to a strain of Lactobacillus plantarum from marine beaked whales that can prevent and treat periodontal disease and dental caries, and its application. The present invention provides a biological product, comprising Lactobacillus plantarum HJ‑S2, which is screened from the intestine of a beaked whale. It can effectively inhibit the activity and biofilm formation of periodontitis pathogenic bacteria Porphyromonas gingivalis and dental caries pathogenic bacteria Streptococcus mutans, has the prospect of preventing and treating periodontitis and dental caries, and can tolerate the oral environment, bind to and remove pathogenic bacteria, thereby alleviating the symptoms of periodontitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a strain of Lactobacillus plantarum derived from marine beaked whales and capable of preventing and treating periodontal disease and dental caries, and an application thereof. Background Art

[0002] Periodontal disease is an inflammatory infectious disease caused by periodontal pathogens. It is the primary cause of tooth loss in adults, accompanied by symptoms such as swollen and bleeding gums, loose teeth, and chewing dysfunction. Porphyromonas gingivalis is the main pathogen causing chronic periodontal disease. It can destroy the oral ecological balance of the host through virulence factors such as lipopolysaccharide, gingival protease, pili, and capsule, and form dental plaque biofilm on the surface of the teeth, which can make bacteria resist the killing effect of the host immune system and antibacterial substances. At present, mechanical treatment and antibiotic treatment are used clinically for periodontal disease: mechanical treatment uses physical means such as ultrasonic cleaning, supragingival cleaning, subgingival scaling, and root planing, aiming to remove plaque and tartar on teeth and periodontal tissues, optimize the periodontal environment, and promote inflammation relief; while antibiotic treatment uses antibacterial drugs to inhibit or kill periodontal pathogens such as Porphyromonas gingivalis to reduce inflammation and promote tissue healing. However, all of the above treatments have limitations. Mechanical treatments (manual scaling and ultrasonic scaling) are used to remove plaque and tartar in the oral cavity, but it is difficult to completely eliminate pathogenic bacteria; antibiotic therapy not only destroys the ecological balance of oral microorganisms, but also brings about bacterial resistance.

[0003] Caries is a bacterial infection that occurs on the hard tissues of teeth. Streptococcus mutans is the main bacterium that causes caries. It produces acid during carbohydrate metabolism, which causes the pH value in the environment to decrease and leads to tooth demineralization. The main means of preventing and treating caries are mechanical plaque removal and drug treatment. Chlorhexidine and fluoride are commonly used drugs for caries treatment. Long-term use of chlorhexidine can lead to oral flora imbalance. Improper use of fluoride can easily lead to fluoride poisoning, posing a potential threat to human health.

[0004] Since periodontal disease and dental caries are closely related to oral flora imbalance, probiotic therapy has been used to adjust and improve the balance of oral microecology in recent years. Inhibiting the activity of oral pathogens is an important way to improve oral health. Lactic acid bacteria are the most widely used probiotics. They are a type of Gram-positive, facultative anaerobic or microaerobic rod-shaped and non-spore-forming bacteria. Lactic acid bacteria are not only important members of human and animal microbial communities, but also closely related to host health. The mechanisms of lactic acid bacteria in the oral cavity mainly include the production of antibacterial substances, competition for the attachment sites of pathogens, and immune regulation.

[0005] After searching, there are related applications disclosed in the prior art for methods of using lactic acid bacteria to prevent and treat oral diseases. However, they are all isolated from terrestrial sources and have relatively simple functions. The Chinese patent application with publication number CN202110171827.9 discloses a kimchi water source plant lactobacillus CCFM1137, which only has a preventive and / or therapeutic effect on periodontal disease. According to the application text, there is no clear explanation on whether there is a preventive or curative effect on caries; although the plant lactobacillus N13 involved in the publication number CN202111137085.4 has the function of preventing or treating caries and periodontal disease, the application text only describes the inhibitory power against unformed Streptococcus mutans and Porphyromonas gingivalis biofilms, and the removal effect on mature Porphyromonas gingivalis biofilms is not further described. The kimchi-derived plant lactobacillus VHProbo 010 strain in the publication number CN202210607143.3 has a good antibacterial effect on Streptococcus mutans and Actinomyces viscosus, but the inhibitory effect on Porphyromonas gingivalis is relatively weak. For example, the publication number CN202110397440.5 discloses a corn stalk-derived plant lactobacillus LP220 that can inhibit the growth of Staphylococcus aureus and Streptococcus mutans, but the function is relatively single and is only effective for dental caries, an oral disease. Therefore, it is still necessary to find a lactic acid bacterium that can both inhibit the formation of periodontal disease pathogens (Porphyromonas gingivalis) and reduce the amount of pathogen colonization in mature Porphyromonas gingivalis biofilms.

[0006] As can be seen from the above, according to the existing methods, multiple strains need to be added in combination to achieve different beneficial effects, and the combined effect needs to be evaluated, which consumes high manpower and production costs. In addition, most of the existing oral probiotic strains are terrestrial, and Lactobacillus plantarum that has an inhibitory effect on Porphyromonas gingivalis and Streptococcus mutans has not yet been found from the ocean. Therefore, based on the limitations of the existing technology in solving oral problems, new products with low cost and better effects need to be developed. Summary of the invention

[0007] In order to solve the above problems, the first aspect of the present invention provides an application of Lactobacillus plantarum in the preparation of products for preventing or treating periodontal disease and dental caries, wherein the Lactobacillus plantarum is Lactobacillus plantarum HJ-S2, which is screened from the intestines of beaked whales. The Lactobacillus plantarum HJ-S2 involved in this patent has multiple functions of preventing and treating periodontal disease and dental caries at the same time, and has lower cost in actual application scenarios.

[0008] The second aspect of the present invention provides a biological product for preventing or treating periodontal disease and dental caries. The components of the biological product prepared by Lactobacillus plantarum HJ-S2 as described in the first aspect include fermentation broth, fermentation supernatant and lyophilized powder of Lactobacillus plantarum; optionally, the fermentation broth of Lactobacillus plantarum HJ-S2 is obtained by fermenting and culturing Lactobacillus plantarum HJ-S2 activated overnight at 37°C for 24-72 hours; the fermentation supernatant is obtained by sterilizing the fermentation broth with a 0.22 μm sterile filter after centrifugation; and the lyophilized powder is the bacterial mud obtained after HJ-S2 centrifugation or the fermentation supernatant obtained after freeze drying in a freeze dryer.

[0009] Furthermore, the biological product is at least one of medicine, food, and daily chemical product.

[0010] Furthermore, the ingredients of the medicine also include a pharmaceutically acceptable carrier, and the carrier is a pharmaceutically acceptable filler.

[0011] Furthermore, the pharmaceutically acceptable filler is one or more of starch, lactose, sucrose, and inorganic salts.

[0012] Furthermore, the medicine comprises various pharmaceutically acceptable dosage forms, which are one or more of gel, gargle, lozenge, microsphere, ointment, and tablet.

[0013] Furthermore, the daily chemical product is one or more of probiotic toothpaste, tooth stickers, tooth cleaning powder, breath freshening spray, antibacterial liquid, probiotic mouthwash, tooth gel, tooth wax, and periodontal gel.

[0014] Furthermore, the probiotic toothpaste comprises silicon dioxide, a sorbitol aqueous solution, xanthan gum, a Lactobacillus plantarum HJ-S2 bacterial solution, sodium saccharin, sodium monofluorophosphate, a sodium laurate sulfate aqueous solution and ethylparaben.

[0015] Furthermore, the probiotic toothpaste comprises the following components by weight: 600 parts of silicon dioxide (ML-520A), 100 parts of silicon dioxide (ML-520B), 400 parts of sorbitol aqueous solution, 3 parts of xanthan gum, 1 part of Lactobacillus plantarum HJ-S2, 1 part of sodium saccharin, 5 parts of sodium monofluorophosphate, 1 part of sodium laurate sulfate aqueous solution, and 1 part of ethylparaben.

[0016] Furthermore, the probiotic mouthwash comprises water, glycerin, sucralose, cetylpyridinium chloride, propylene glycol, a preservative, methyl diisopropyl propionamide, a fragrance, peppermint essential oil, Lactobacillus plantarum HJ-S2 fermentation supernatant, xylitol and glucose oxidase.

[0017] Furthermore, the probiotic mouthwash comprises the following raw materials in parts by weight: Phase A: water 87.96%, glycerol 8%, sucralose 0.03%, cetylpyridinium chloride 0.05%; Phase B: propylene glycol 2%, preservative 0.25%, methyl diisopropyl propionamide 0.05%, fragrance 0.05%; Phase C: 1% peppermint essential oil, 0.5% supernatant of fermentation of Lactobacillus plantarum HJ-S2, 0.1% xylitol, 0.01% glucose oxidase. The aromatic agent is essence, and the preservative is PEG-40 hydrogenated castor oil.

[0018] Furthermore, the food is one or more of probiotic solid beverage, ice cream, lozenge, soft candy, jelly, probiotic chewing gum, oral probiotic powder, probiotic fermentation agent, probiotic fermented milk, etc.

[0019] Furthermore, the solid beverage formula is edible corn starch (balance), white sugar (10%), oligofructose (5-6%), oligomaltose (9%), citric acid, edible flavoring, and probiotic powder (3-5%).

[0020] Furthermore, the probiotic solid beverage comprises edible corn starch, white sugar, oligofructose, oligomaltose, citric acid, edible flavor and Lactobacillus plantarum HJ-S2 bacterial powder.

[0021] Furthermore, the probiotic chewing gum comprises Lactobacillus plantarum HJ-S2 bacterial powder, gum matrix, powdered sugar, maltose, calcium carbonate, glucose and flavoring.

[0022] Furthermore, the formula of the probiotic chewing gum comprises the following components in percentage by weight: 0.1% of Lactobacillus plantarum HJ-S2 powder, 29.9% of gum base, and 70% of sugar and other ingredients.

[0023] Furthermore, the probiotic powder comprises Lactobacillus plantarum HJ-S2 bacterial mud and a freeze-drying protective agent.

[0024] Furthermore, the freeze-drying protective agent contains 10-15% skim milk powder, 5-7% trehalose, 3-5% sucrose, and 3-5% sodium glutamate.

[0025] Furthermore, the probiotic fermentation agent comprises the original strain of Lactobacillus plantarum HJ-S2 and 10% skim milk.

[0026] Furthermore, the probiotic fermented milk contains Lactobacillus plantarum HJ-S2 starter, cow's milk and sucrose.

[0027] Furthermore, the sucrose is 6% sucrose.

[0028] The technical solution of the present invention has the following advantages: The present invention provides a strain of Lactobacillus plantarum HJ-S2 derived from the intestine of beaked whales, which exhibits effective inhibitory effects on dental caries and periodontal disease pathogens, and can therefore be directly used in the development of biological products for the prevention and treatment of these two oral diseases.

[0029] The invention provides a biological product of Lactobacillus plantarum HJ-S2, which can effectively inhibit the activity and biofilm formation of periodontitis pathogenic bacteria Porphyromonas gingivalis and dental caries pathogenic bacteria Streptococcus mutans, has the prospect of preventing and treating periodontitis and dental caries, and can tolerate the oral environment, bind to and eliminate pathogenic bacteria, thereby alleviating the symptoms of periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0031] Figure 1 : Histogram of “inhibition zone diameter” of Lactobacillus plantarum HJ-S2; Figure 2-1 : "Lysozyme concentration-absorbance" bar graph of Lactobacillus plantarum HJ-S2; Figure 2-2 : "Hydrogen peroxide concentration-absorbance" bar graph of Lactobacillus plantarum HJ-S2; Figure 3-1 : "Time-self-aggregation rate" bar graph of Lactobacillus plantarum HJ-S2; Figure 3-2 : The “time-coaggregation rate” bar graph of Lactobacillus plantarum HJ-S2 and Porphyromonas gingivalis; Figure 3-3 : The "time-coaggregation rate" bar graph of Lactobacillus plantarum HJ-S2 and Streptococcus mutans; Figure 4-1 : Gram-stained colony image of Lactobacillus plantarum HJ-S2 inhibiting the biofilm formation of Porphyromonas gingivalis, and inverted microscope image; Figure 4-2 :Scanning electron micrographs of the effect of Lactobacillus plantarum HJ-S2 on the biofilm formation of Porphyromonas gingivalis; Figure 4-3 :Confocal laser scanning microscopy images of the effect of Lactobacillus plantarum HJ-S2 on the biofilm formation of Porphyromonas gingivalis; Figure 5-1 :Gram-stained colony images and inverted microscope images of Lactobacillus plantarum HJ-S2 clearing mature Porphyromonas gingivalis biofilm; Figure 5-2 : Histogram of mature Porphyromonas gingivalis biofilms in each group detected by CV and TTC methods; Figure 6: The results of the bacterial solution showing the effect of Lactobacillus plantarum HJ-S2 on the biofilm formation of Streptococcus mutans; Figure 7-1 :The colony diagram of the effect of Lactobacillus plantarum HJ-S2 on the biofilm formation of mature mutans Streptococcus; Figure 7-2 : Bar graph showing the effect of Lactobacillus plantarum HJ-S2 treatment on biofilm formation of mature Streptococcus mutans; Figure 8 :Histogram of the inhibition of inflammatory factor secretion by Lactobacillus plantarum HJ-S2 in periodontitis cell model; Figure 9-1 : 3D restoration images of alveolar bones of rats in each group; Figure 9-2 : Histogram of CEJ-ABC distance of rats in each group; Figure 9-3 : Bar graph of alveolar bone density of rats in each group; Fig.10 : Schematic diagram of the secretion levels of various inflammatory factors in the gingival tissue of rats in each group. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with specific implementations and drawings, but the implementation modes of the present invention are not limited thereto, and the protection scope of the present invention is not limited thereto.

[0033] The strains involved in the following examples are from the following sources: The plant lactobacillus HJ-S2 involved in the following embodiments was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on May 7, 2019, with the deposit number CGMCC No.17720.

[0034] Porphyromonas gingivalis (ATCC33277) and Streptococcus mutans (ATCC25175) involved in the following examples were purchased from Guangdong Microbiological Culture Collection Center; Lactobacillus plantarum HJ-S2 was isolated and screened from the intestine of marine beaked whales by the Third Institute of Oceanography of the Ministry of Natural Resources.

[0035] The culture medium involved in the following examples is as follows: MRS medium and BHI medium are commercial culture media purchased from Guangdong Huankai Microbiology Technology Co., Ltd.; TSB medium is a commercial medium purchased from Qingdao Haibo Biotechnology Co., Ltd.; DMEM medium is a commercial medium purchased from Wuhan Punosai Life Science Technology Co., Ltd.; TSB modified medium: TSB medium 30 g / L, yeast extract 5 g / L, L-cysteine ​​hydrochloride 0.5 g / L, hemin solution 5 mg / L, vitamin K1 solution 1 mg / L, pH 7.1-7.5.

[0036] TSA modified medium: TSB medium 30 g / L, yeast extract 5 g / L, L-cysteine ​​hydrochloride 0.5 g / L, hemin solution 5 mg / L, vitamin K1 solution 1 mg / L, agar powder 18 g / L, pH 7.1-7.5.

[0037] Complete culture medium: Use high-glucose DMEM medium 90%, fetal bovine serum 10%, penicillin / streptomycin 1%.

[0038] The preparation method of the bacterial suspension involved in the following examples is as follows: Porphyromonas gingivalis suspension: Porphyromonas gingivalis activated overnight was inoculated into TSB modified medium at a 1-3% (v / v) inoculum, and the concentration of the bacterial suspension was adjusted to 10 after anaerobic culture at 37°C. 5 -10 7 CFU / mL.

[0039] Mutans Streptococcus suspension: Inoculate mutans Streptococcus activated overnight into BHI medium at 1-3% (v / v) and culture aerobically at 37°C until the concentration of the bacterial suspension reaches 10 5 -10 7 CFU / mL.

[0040] Lactobacillus plantarum HJ-S2 bacterial suspension: Inoculate the overnight activated Lactobacillus plantarum HJ-S2 into MRS medium at an inoculum rate of 1-3% (v / v) and culture aerobically at 37°C for 24-72 h.

[0041] The preparation method of the fermentation supernatant involved in the following examples is as follows: The overnight activated Lactobacillus plantarum HJ-S2 was inoculated into MRS medium at an inoculum amount of 1-3% (v / v), cultured at 37°C for 24-72 h, and filtered and sterilized with a 0.22 μm sterile filter after centrifugation to obtain the fermentation supernatant.

[0042] The microbiological terminology involved in the following embodiments is as follows: The term "complete medium" refers to a culture medium to which various growth factors, nutrients, antibiotics and other substances are added to the basic culture medium. It is designed to meet all the nutritional requirements for the growth, reproduction and various life activities of microorganisms or cells. It is commonly used in cell culture and microbiology research.

[0043] The term "lysozyme" refers to a hydrolase that is widely present in a variety of organisms and can destroy and dissolve bacterial cell walls. It has natural anti-infective, anti-inflammatory, anti-inflammatory and antiviral effects and has a wide range of applications in the medical field.

[0044] The term "inhibition zone" refers to a circular area where no bacteria grow, formed around the colony in a bacterial culture dish due to the action of antibiotics or antibacterial substances. The diameter of the area can reflect the antibacterial ability of the antibacterial substance.

[0045] The term "Porphyromonas gingivalis lipopolysaccharide" refers to a preparation of purified lipopolysaccharide from the Gram-negative bacterium Porphyromonas gingivalis. It is an important virulence factor in the pathogenesis of periodontal disease. Lipopolysaccharide is a major component of Gram-negative bacteria that activates the innate immune system.

[0046] The term "scanning electron microscope" refers to a high-resolution electron microscope that uses a focused electron beam to scan the surface of a sample and forms an image of the sample by collecting and processing various physical signals excited.

[0047] The term "inverted microscope" refers to a microscope in which the objective is located below the stage, allowing samples to be observed from below. It is particularly suitable for observing cultured living cells and has phase contrast objectives. It is an important tool in biological and medical research.

[0048] The term "laser confocal microscope" refers to a microscope based on the principle of fluorescence microscopy imaging, equipped with a laser scanning device, which uses ultraviolet light or visible light to excite fluorescent probes, and uses computers, lasers and image processing technology to obtain fluorescent images of the microstructure inside cells or tissues. It can observe physiological signals and changes in cell morphology at the subcellular level.

[0049] The term "Gram staining kit" refers to a staining kit used to identify bacterial species. Through the steps of primary staining with crystal violet, mordanting with iodine solution, decolorizing with a decolorizer and re-staining with a red dye, bacteria are divided into Gram-positive bacteria and Gram-negative bacteria. It is widely used in bacteriological research and clinical testing.

[0050] The term "self-aggregation rate" refers to an indicator used to describe the ability of microorganisms or particles to self-aggregate under specific conditions. It is often used to evaluate the self-aggregation characteristics of microbial strains, cells or colloidal particles. In the field of microbiology, the determination of the self-aggregation rate usually involves centrifuging the cultured microbial suspension to remove the supernatant, then washing the microorganisms with saline and adjusting their concentration. Next, the self-aggregation rate can be calculated by measuring the absorbance of the microbial suspension at different time points. The calculation formula for the self-aggregation rate is generally: Self-aggregation rate (%) = (1-A t / A0)×100%, where At represents the absorbance at a specific time point t, and A0 represents the absorbance at the initial time.

[0051] The term "coaggregation rate" refers to an indicator used to describe the proportion or degree of coaggregation between different microorganisms or between different strains of the same microorganism under specific conditions. The coaggregation rate is a relative concept, and its magnitude is affected by many factors, such as the type of microorganism, the physicochemical properties of the cell surface, environmental factors, and the interaction mode between microorganisms. Therefore, when determining the coaggregation rate, the experimental conditions need to be strictly controlled to ensure the accuracy and reliability of the results.

[0052] The term "plate dilution method" refers to a series of gradient dilutions of bacterial solution, and then spreading the bacterial solutions of different dilutions on the surface of agar solid culture medium for cultivation. In a sufficiently diluted bacterial solution, the microorganisms that were originally aggregated together will be dispersed into single cells, thus forming a single colony on the surface of the culture medium.

[0053] The term "pro-inflammatory cytokines" refers to a class of cytokines that can promote inflammatory responses. Usually, the body releases a large number of pro-inflammatory cytokines in disease states, such as infection, autoimmune disease or tissue damage.

[0054] Example 1: Screening of strains with inhibitory effects on Porphyromonas gingivalis and Streptococcus mutans (1) Screening of strains that inhibit Porphyromonas gingivalis by agar punch method The TSA modified medium was cooled to about 55°C and mixed with the Porphyromonas gingivalis suspension in a certain proportion to make the number of viable pathogens in the system reach 10 5 -10 7 CFU / mL, and then quickly poured into a sterile culture dish. After the culture medium is cooled and solidified, 3 holes with a diameter of 10 mm are punched on each plate, and 100 μL of HJ-S2 fermentation broth, HJ-S2 fermentation supernatant, and MRS culture medium are added to each well. The plate is lightly covered and placed upright under anaerobic conditions at 37°C. After the appropriate incubation time, the diameter of the inhibition zone is observed and measured with a vernier caliper.

[0055] The results are as follows Figure 1 As shown, Lactobacillus plantarum HJ-S2 bacterial liquid and fermentation supernatant had a significant inhibitory effect on Porphyromonas gingivalis, indicating that this strain of bacteria can inhibit the growth of periodontitis pathogens, improve the environment of oral flora, and prevent chronic periodontitis.

[0056] (2) Agar punch method to screen strains that inhibit mutans streptococci Cool the BHI solid medium to about 55°C and mix it with the Streptococcus mutans suspension in a certain proportion so that the number of viable pathogens in the system is 10 5-10 7 CFU / mL, and then quickly poured into a sterile culture dish. After the culture medium cooled and solidified, three holes with a diameter of 10 mm were punched on each plate, and 100 μL of HJ-S2 fermentation broth, HJ-S2 fermentation supernatant, and MRS culture medium were added to each well. The culture was placed upright under aerobic conditions at 37°C, and the diameter of the inhibition zone was observed and measured with a vernier caliper.

[0057] The results are as follows Figure 1 As shown, both the fermentation broth and supernatant of Lactobacillus plantarum HJ-S2 had obvious inhibition zones against Streptococcus mutans, which preliminarily indicated that Lactobacillus plantarum HJ-S2 could inhibit the growth of Streptococcus mutans, regulate the balance of oral flora, and prevent dental caries.

[0058] Example 2: Oral Tolerance Evaluation of Lactobacillus plantarum HJ-S2 (1) Determination of lysozyme concentration The suspension of Lactobacillus plantarum HJ-S2 was inoculated into a 96-well plate, and different concentrations of lysozyme solution were added to make the final concentration of each well 100, 200, and 300 mg / mL, respectively. The blank group was replaced with sterile water. After culturing at 37℃ for 24 h, the OD 600 Measure the absorbance.

[0059] Figure 2-1 The figure shows the results of the tolerance of Lactobacillus plantarum HJ-S2 to lysozyme. HJ-S2 can not only tolerate the lysozyme concentration in the normal human oral environment (1-57 μg / mL), but also tolerate the lysozyme concentration in the severe oral conditions of periodontitis patients (200-300 μg / mL), indicating that Lactobacillus plantarum HJ-S2 has good adaptability to the human oral environment and has the ability to survive in the oral environment.

[0060] (2) Determination of resistance to hydrogen peroxide The suspension of Lactobacillus plantarum HJ-S2 was inoculated into a 96-well plate, and different concentrations of hydrogen peroxide were added to make the final concentrations in each well 0.08 mM and 0.8 mM, respectively. The blank group was replaced with sterile water. After culturing at 37°C for 24 h, the OD 600 Measure the absorbance.

[0061] Figure 2-2 Shown is the result of Lactobacillus plantarum HJ-S2's tolerance to hydrogen peroxide in the oral cavity. HJ-S2 can survive at 0.08 mM and 0.8 mM hydrogen peroxide, indicating that it has the conditions to adapt to hydrogen peroxide in the oral environment.

[0062] Example 3: Determination of the self-aggregation and co-aggregation abilities of Lactobacillus plantarum HJ-S2 (1) Determination of the self-aggregation ability of Lactobacillus plantarum HJ-S2 Take the cultured Lactobacillus plantarum HJ-S2 fermentation broth and centrifuge it, discard the supernatant, collect the precipitated bacteria and wash them with PBS for 3-5 times, resuspend them in PBS again, and adjust the OD 600 =1.0. PBS was used as blank control. Incubate at 37℃ for 2, 4, 6, and 8 h. Prepare 3 parallel tubes for each time period. Determine the optical density of the sample supernatant at 600 nm. Calculate the self-aggregation rate: Self-aggregation rate (%) = (1-A t / A0)×100% Where: A0 is the absorbance value at 0 h, i.e. 1.0; A t is the absorbance value at the time of sampling; from Figure 3-1 The results show that the Lactobacillus plantarum HJ-S2 provided by the present invention has a strong self-aggregation ability. As time goes by, the self-aggregation rate reaches 27.80% after 8 h.

[0063] (2) Determination of the coaggregation ability of Lactobacillus plantarum HJ-S2 and Porphyromonas gingivalis Take the cultured Lactobacillus plantarum HJ-S2 and Porphyromonas gingivalis culture liquid and centrifuge it, discard the supernatant, collect the precipitated bacteria and wash them with PBS for 3-5 times, resuspend them in PBS again and adjust the absorbance at 600 nm to 0.5 ± 0.05 and 0.6 ± 0.05, respectively. Take 500 μL of each and mix them, shake and mix them, incubate them at 37℃ for 2, 4, 6, and 8 hours, and make 3 parallel tubes for each time period. Determine the optical density of the sample supernatant at 600 nm. The formula for calculating the co-aggregation rate of the target strain and Porphyromonas gingivalis is: Coagulation rate (%) = (1-2C mix1 / (C0+C1))×100% Where: C mix1 OD after the mixture has been cultured for xh (x=2, 4, 6, 8) 600 Value; C0 is the initial OD of lactic acid bacteria 600 value; C1 is the initial OD of Porphyromonas gingivalis 600 value.

[0064] like Figure 3-2 As shown in the results, the plant lactobacillus HJ-S2 provided by the present invention has a strong copolymerization ability with Porphyromonas gingivalis, and the coaggregation rate can reach 34.50% at 8 hours. It shows that plant lactobacillus HJ-S2 can reduce the number of pathogenic bacteria in the oral cavity by combining with Porphyromonas gingivalis, and can reduce the colonization of Porphyromonas gingivalis in the oral cavity with the flow of saliva and gargling, thereby maintaining the balance of oral flora.

[0065] (3) Determination of the coaggregation ability of Lactobacillus plantarum HJ-S2 and Streptococcus mutans Take the cultured Lactobacillus plantarum HJ-S2 and Streptococcus mutans and centrifuge the supernatant, collect the precipitated bacteria and wash them with PBS for 3-5 times, resuspend them in PBS and adjust the absorbance at 600 nm to 0.5 ± 0.05 and 0.6 ± 0.05 respectively, take 500 μL of each, mix them and oscillate them, incubate them at 37℃ for 2, 4, 6, and 8 hours, and make 3 parallel tubes for each time period. Determine the optical density of the sample supernatant at 600 nm. The formula for calculating the co-aggregation rate of the target strain and Streptococcus mutans is: Coagulation rate (%) = (1-2C mix2 / (C0+C2))×100% Where: C mix2 The OD after the mixture was cultured for xh (x = 2, 4, 6, 8) 600 Value; C0 is the initial OD of lactic acid bacteria 600 Value; C2 is the initial OD of Streptococcus mutans 600 value.

[0066] like Figure 3-3 The results showed that as time went on, Lactobacillus plantarum HJ-S2 had the ability to copolymerize with Streptococcus mutans, and the coaggregation rate could reach 24.27% at 8 h, which indicated that Lactobacillus plantarum HJ-S2 could remove Streptococcus mutans through copolymerization and reduce the number of Streptococcus mutans in the oral cavity.

[0067] Example 4: Inhibitory effect of Lactobacillus plantarum HJ-S2 on biofilm formation of Porphyromonas gingivalis (1) Gram staining combined with inverted microscope observation Table 1 Comparison of the effects of different experimental groups on Porphyromonas gingivalis As shown in Table 1, the plant lactobacillus HJ-S2 bacterial suspension / supernatant and the gingival porphyromonas bacterial solution were added to a 24-well plate at a ratio of 1:1-3, and MRS medium was added as a negative control. The blank control group was added with MRS medium. After the culture was completed, the culture solution was discarded, and the plates were gently washed 2-3 times with sterile PBS, fixed with methanol, and dried at room temperature. After staining with crystal violet dye, the plates were washed with water, after staining with iodine solution, washed with water, after decolorization with ethanol, washed with water, after staining with safranin dye, washed with water, and finally dried, observed with an inverted microscope, and photographed and recorded.

[0068] The results are as follows Figure 4-1As shown, the negative control group was a Porphyromonas gingivalis biofilm (negative bacteria), which was red after Gram staining; the HJ-S2 bacterial solution (positive bacteria) treatment group was purple, indicating that after the HJ-S2 bacterial solution treatment group, HJ-S2 replaced the Porphyromonas gingivalis biofilm and became the new dominant bacteria; the HJ-S2 supernatant group had no biofilm, indicating that there were active ingredients in the HJ-S2 supernatant treatment group that inhibited the formation of Porphyromonas gingivalis biofilm. In summary, both HJ-S2 bacterial solution and HJ-S2 fermentation supernatant had significant effects on the formation of Porphyromonas gingivalis biofilm. However, the effect of the HJ-S2 supernatant treatment group was better than that of the HJ-S2 bacterial solution treatment group. Therefore, Lactobacillus plantarum HJ-S2 has a beneficial effect on the prevention of periodontitis.

[0069] (2) Scanning electron microscopy observation of periodontitis model after HJ-S2 treatment The suspension / supernatant of Lactobacillus plantarum HJ-S2 and the bacterial solution of Porphyromonas gingivalis were added to a 24-well plate at a ratio of 1:1-3, and MRS medium was added as a negative control. The blank control group was added with MRS medium. After the culture was completed, the culture medium was discarded, and the plates were gently washed 2-3 times with sterile PBS, fixed with glutaraldehyde overnight, and the fixative was discarded, dried at room temperature, and gently washed with sterile PBS. After gradient dehydration with ethanol, the plates were dried at room temperature, sprayed with gold, and the biofilm morphology was observed by scanning electron microscopy and photographed and recorded.

[0070] The results are as follows Figure 4-2 As shown, it can be seen under the scanning electron microscope at 500, 2000, and 5000 times: in the absence of Lactobacillus plantarum HJ-S2 treatment, the Porphyromonas gingivalis biofilm is arranged tightly, but after treatment with Lactobacillus plantarum HJ-S2, the bacterial gaps can be clearly observed, and the number of bacteria in the form of biofilm is significantly reduced, especially after treatment with HJ-S2 supernatant, there are almost no bacteria. Therefore, the HJ-S2 bacterial solution treatment group and the HJ-S2 supernatant have a significant effect on the inhibition of Porphyromonas gingivalis biofilm formation, but the effect is better after treatment with HJ-S2 supernatant. Therefore, it shows that Lactobacillus plantarum HJ-S2 has a positive therapeutic effect on the prevention of periodontitis.

[0071] (3) Effect of HJ-S2 treatment on the biofilm formation activity of Porphyromonas gingivalis The suspension / supernatant of Lactobacillus plantarum HJ-S2 and the bacterial solution of Porphyromonas gingivalis were added to a 24-well plate at a ratio of 1:1-3, and MRS medium was added as a negative control. The blank control group was supplemented with MRS medium to the same volume. The culture medium was discarded, and sterile saline was added to gently wash, and SYTO 9 and PI stains were prepared. An appropriate amount of SYTO 9 and PI stains were added to saline to prepare a mixed stain and stained in the dark. After adding sterile saline to gently wash 3 times, it was observed under a laser confocal microscope.

[0072] The results are as follows Figure 4-3 As shown in the figure, the negative control group showed a large amount of green fluorescence and very little red fluorescence, indicating that the biofilm of Porphyromonas gingivalis was successfully constructed. There were no live bacteria in the biofilm after HJ-S2 bacterial solution treatment, indicating that the HJ-S2 bacterial solution treatment group had a good inhibitory effect on Porphyromonas gingivalis; there were a small number of green in the biofilm after HJ-S2 supernatant treatment, indicating that the HJ-S2 supernatant treatment group may contain antibacterial active substances, which can reduce the formation of Porphyromonas gingivalis biofilm and have a good effect on the prevention of periodontitis.

[0073] Example 5: Effect of Lactobacillus plantarum HJ-S2 on the removal of mature biofilm of Porphyromonas gingivalis (1) Gram staining combined with inverted microscope observation Add the cultured Porphyromonas gingivalis suspension to a 24-well plate and culture at 37°C anaerobically to form a mature Porphyromonas gingivalis biofilm. After the culture is completed, discard the culture medium, gently wash 2-3 times with sterile PBS, add 0.5-2 mL of plant lactobacillus HJ-S2 culture and fermentation supernatant, repeat three times for each group, culture at 37°C, anaerobically, and statically, and add an equal volume of culture medium as a negative control. After the culture is completed, discard the culture medium, add methanol to fix, discard the fixative, and dry at room temperature. Add crystal violet staining solution to stain and then wash with water, add iodine solution to stain and then wash with water; add decolorizing solution to decolorize and then wash with water; add safranin staining solution to stain and then wash with water, dry in the air, observe using an inverted microscope and take pictures to record the results.

[0074] The results are as follows Figure 5-1 As shown in the figure, the negative control group was red, which was a mature biofilm of Porphyromonas gingivalis; the red color decreased in the HJ-S2 bacterial solution treatment group, indicating that the HJ-S2 bacterial solution treatment group had an antagonistic effect on the mature Porphyromonas gingivalis biofilm and effectively reduced the colonization of Porphyromonas gingivalis; the HJ-S2 supernatant treatment group was light pink, indicating that the active substances in the HJ-S2 supernatant had a killing effect on the mature Porphyromonas gingivalis biofilm. In summary, both HJ-S2 bacterial solution and HJ-S2 supernatant were effective in treating the formed Porphyromonas gingivalis biofilm, so HJ-S2 had a good effect on the treatment of periodontitis.

[0075] (2) Determination of the removal rate of mature biofilm of Porphyromonas gingivalis by Lactobacillus plantarum HJ-S2 The cultured Porphyromonas gingivalis suspension was added to a 24-well plate and cultured anaerobically at 37°C to form a mature Porphyromonas gingivalis biofilm. After the culture was completed, the culture medium was discarded, and the plate was gently washed 2-3 times with sterile PBS. 0.5-2 mL of Lactobacillus plantarum HJ-S2 bacterial solution and fermentation supernatant were added, and the plate was cultured anaerobically at 37°C. The same volume of MRS culture medium was added as a negative control. After the culture was completed, the culture medium was discarded, and sterile PBS was added to gently wash 2-3 times to remove non-adherent bacteria. Use a sterile cotton swab to scrape off the biofilm as much as possible, break the cotton swab head and mix it in 0.9% saline, and vortex it; take the same volume of dilutions of different concentrations and apply them on Columbia blood plates. After anaerobic culture at 37°C, the number of Porphyromonas gingivalis was detected, and the removal rate of Porphyromonas gingivalis biofilm was calculated by the following formula: Removal rate (%) = (1-number of colonies in the experimental group / number of colonies in the negative control group) × 100% See Table 2 for specific results.

[0076] Table 2 Clearance rate of mature biofilm mediated by Lactobacillus plantarum HJ-S2 As can be seen from the data of Table 2, compared with the negative control group, the plant lactobacillus HJ-S2 bacterial solution and supernatant treatment groups all reduce the number of mature biofilms of Porphyromonas gingivalis, and the treatment effect of HJ-S2 supernatant is slightly better than that of HJ-S2 bacterial solution treatment group. The clearance rate of mature biofilm of Porphyromonas gingivalis in the HJ-S2 bacterial solution treatment group reaches 89.20%, indicating that the thalline may be effectively combined with pathogenic bacteria to reduce the colonization of pathogenic bacteria; The clearance rate of mature biofilm of Porphyromonas gingivalis in the HJ-S2 supernatant treatment group reaches 91.36%, indicating that there are antibacterial active substances in the metabolites of HJ-S2, which have a very strong clearing effect on Porphyromonas gingivalis biofilm. Therefore, the plant lactobacillus HJ-S2 provided by the present invention can effectively improve the periodontal condition of patients with chronic periodontitis, and can be widely used in food, medicine, daily chemical products, etc.

[0077] (3) Effect of HJ-S2 treatment on the activity of mature Porphyromonas gingivalis biofilm Add the Porphyromonas gingivalis suspension to a 24-well plate and culture at 37°C in an anaerobic, static manner until a mature Porphyromonas gingivalis biofilm is formed. After the culture is completed, measure the OD 600 , discard the culture medium, gently wash 2-3 times with sterile PBS, add 0.3-2mL of Lactobacillus plantarum HJ-S2 culture medium and fermentation supernatant, MRS medium, repeat three times for each group, continue to culture at 37℃, anaerobic, static. Take out two 24-well plates and measure OD 600 , take one of the 24-well plates and discard the culture medium, add crystal violet (CV) to measure the OD595 , remove another 24-well plate and discard the culture medium, add 2,3,5-triphenyltetrazolium chloride (TTC) solution and incubate at 37°C, add DMSO to resuspend the biofilm, and measure the OD 500 .

[0078] The results are as follows Figure 5-2 As shown, compared with the negative control group, the HJ-S2 supernatant treatment group can significantly reduce the number of live bacteria in the mature Porphyromonas gingivalis biofilm, indicating that the HJ-S2 supernatant treatment can significantly reduce the number of Porphyromonas gingivalis.

[0079] Example 6: Inhibition of biofilm formation of Streptococcus mutans by treatment with Lactobacillus plantarum HJ-S2 Add the bacterial suspension / supernatant of Lactobacillus plantarum HJ-S2 and the bacterial solution of Streptococcus mutans to a 24-well plate at a ratio of 1:1-3, add MRS culture medium as a negative control, and take photos and record after the culture is completed.

[0080] The results are as follows Figure 6 As shown in the figure, the growth of Streptococcus mutans in the negative control group was excellent. After treatment with the supernatant of Lactobacillus plantarum HJ-S2, there was almost no Streptococcus mutans; after treatment with HJ-S2 bacterial solution, the number of Streptococcus mutans was significantly reduced, and HJ-S2 became the dominant bacteria.

[0081] Example 7: Effect of Lactobacillus plantarum HJ-S2 treatment on mature mutans Streptococcus biofilm Add the mutans Streptococcus culture solution that has been cultured overnight to a 24-well plate and incubate at 37°C to form a mature mutans Streptococcus biofilm. After the culture is completed, discard the culture solution, gently wash 2-3 times with sterile PBS, add 0.3-2 mL of Lactobacillus plantarum HJ-S2 culture solution and fermentation supernatant, repeat three times for each group, incubate at 37°C, anaerobic, and statically, and add an equal volume of MRS culture medium as a negative control. After the culture is completed, discard the culture solution, wash the biofilm 2-3 times with PBS, and take photos and record. Add methanol to fix the biofilm and discard the methanol. After the biofilm is dried at room temperature, add crystal violet solution to each well to stain the biofilm. After staining, wash it twice with PBS, add ethanol to dissolve it, place it on a shaker, and then measure the OD 595 .

[0082] The results are as follows Figure 7-1 and 7-2As shown in the figure, the negative control group had mature mutans streptococcus biofilm, and after treatment with Lactobacillus plantarum HJ-S2 bacterial solution and supernatant, there was almost no biofilm, indicating that HJ-S2 had a good effect in removing mutans streptococcus biofilm. The results of the microplate crystal violet method showed that both HJ-S2 bacterial solution and supernatant had the effect of removing mature mutans streptococcus biofilm, and the supernatant of HJ-S2 had a better effect.

[0083] Example 8: Effects of Lactobacillus plantarum HJ-S2 on TNF-α, IL-1β, and IL-6 levels in periodontitis cell models (1) Construction of periodontitis cell model: Mouse macrophages RAW264.7 were inoculated in DMEM medium containing 10% (v / v) fetal bovine serum, cultured and activated in a cell culture incubator at 37°C with 5% (v / v) CO2 in the gas phase, and then inoculated into 24-well plates. After pre-incubation of the 24-well plates at 37°C for 0.5-2 h, Porphyromonas gingivalis lipopolysaccharide was added to each well.

[0084] (2) Experimental groups: as shown in Table 3, they were blank control group, model group, Lactobacillus plantarum HJ-S2 bacterial solution group, and Lactobacillus plantarum HJ-S2 supernatant group, and each group was repeated 3 times; Table 3 Construction of periodontitis models in different experimental groups (3) Experimental treatment: Equal volumes of HJ-S2 bacterial solution and HJ-S2 supernatant were added to the periodontitis cell model. The model group was the group that added equal volumes of complete culture medium to the periodontitis cell model, and the group that only added RAW264.7 cells was the blank control group.

[0085] Test results such as Figure 8 As shown in the figure, compared with the blank control group, all inflammatory factors in the periodontitis cell model group increased; however, after treatment with the bacterial solution and supernatant of Lactobacillus plantarum HJ-S2, all inflammatory factors could be reduced, among which IL-1β had a significant down-regulation effect.

[0086] Example 9: Effect of Lactobacillus plantarum HJ-S2 on alveolar bone in rats with periodontitis (1) Construction of rat periodontal disease model and drug administration Fifteen male and female SPF Wistar rats, 2 months old and weighing 200 g, were selected and randomly divided into a healthy group and a modeling group. The modeling group was uniformly modeled by combining four methods: silk thread ligation, infection with Porphyromonas gingivalis, injection of Porphyromonas gingivalis lipopolysaccharide, high-sugar feed and high-sugar drinking water. The 15 rats were randomly divided into 5 groups, with 3 rats in each group. The 5 groups were the healthy group, the model group, the Lactobacillus plantarum HJ-S2 bacterial solution group, the Lactobacillus plantarum HJ-S2 supernatant group, and the positive control group. Except for the healthy group and the model group, the other groups were fed with 0.1-2 mL of HJ-S2 bacterial solution, HJ-S2 supernatant, and compound chlorhexidine gargle by gavage, and fasted for half an hour after the end of the administration.

[0087] (2) Effect of Lactobacillus plantarum HJ-S2 on the gingival index (GI) of rats with periodontitis After the administration, the rats were anesthetized by intramuscular injection of 200 mg / kg of ketamine hydrochloride, and the gingival index was evaluated by periodontal pocket probing. The gingiva was scored using the examination method revised by Loe and Silness in 1967. The scoring criteria are as follows: 0 = healthy gingiva; 1 = mild gingival inflammation: the gingiva has a slight change in color and mild edema, and no bleeding on probing; 2 = moderate gingival inflammation: the gingiva is red, edematous and shiny, and bleeding on probing; 3 = severe gingival inflammation: the gingiva is obviously red and swollen or ulcerated, and there is a tendency to bleed automatically. The specific results are shown in Table 4, and the results are expressed as mean ± standard deviation.

[0088] Table 4 Gingival index of rats in each group Note: The absence of the same lowercase letters between the two numbers in the table indicates that there is a significant difference between the two numbers.

[0089] (3) Effects of Lactobacillus plantarum HJ-S2 on CEJ-ABC distance and bone density of alveolar bone in rats with periodontitis The rat maxilla was fixed in 4% paraformaldehyde for 36-48 h, then washed and dried with PBS buffer solution. X-ray scanning and imaging of rat maxilla samples were performed using an in vivo Micro-CT imaging system with the following parameters: voltage, 90 kV; current, 88 μA; field of view, 18 μm; acquisition time, 14 min; camera mode, high resolution. Each sample was rotated 360 degrees and all images were imported into Mimics 21.0 (Materialise NV, Leuven, Belgium). The three-dimensional images of the maxilla were reconstructed for analysis, and the alveolar bone was separated with a fixed threshold. The following parameters were measured in the three-dimensional reconstruction views of each group: ① CEJ-ABC distance: the distance from the cementoenamel junction of the second molar to the alveolar crest; ② bone density: the density of minerals contained in the bone per unit volume.

[0090] according to Figure 9-1 , 9-2 , 9-3 It can be seen that the rat model group with periodontitis showed symptoms such as alveolar bone resorption of the second molar and severe root exposure compared with the healthy group, and the alveolar bone density was lower than that of the healthy group; the root exposure area after treatment with HJ-S2 bacterial solution and supernatant and compound chlorhexidine mouthwash was relatively small, and the bone loss was relatively weakened. In summary, the HJ-S2 bacterial solution and supernatant can alleviate the symptoms of rats with periodontitis.

[0091] Example 10: Effects of Lactobacillus plantarum HJ-S2 on inflammatory factors TNF-α, IL-1β, IL-6, and IL-8 in gingival tissue of rats with periodontitis 0.1-0.3 g of rat gingival tissue was added to 0.9 mL of RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors for lysis. After being fully disrupted by a tissue disruptor, the tissue was centrifuged at 9500 rpm for 10 min, the precipitate was discarded, the supernatant was aspirated, and the protein concentration was determined according to the instructions of the BCA protein concentration kit. At the same time, the TNF-α, IL-1β, IL-6, and IL-8 in rat gingival tissue were determined according to the instructions of the ELISA kits for rat TNF-α, IL-1β, IL-6, and IL-8. The amount of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) / total protein amount = relative expression of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8).

[0092] according to Fig.10It can be seen that compared with the blank group, the four inflammatory indicators of TNF-α, IL-1β, IL-6, and IL-8 in the gingival tissue of rats with periodontitis were significantly upregulated (P < 0.05). For indicators such as TNF-α, IL-1β, IL-6, and IL-8, the bacterial liquid and supernatant of Lactobacillus plantarum HJ-S2 can reduce the secretion of TNF-α, IL-1β, IL-6, and IL-8 to a certain extent.

[0093] Example 11: Preparation of biological products using Lactobacillus plantarum HJ-S2 (1) Preparation of oral probiotic powder using Lactobacillus plantarum HJ-S2 Preparation of plant lactobacillus HJ-S2 bacterial sludge: Pick a fresh plant lactobacillus HJ-S2 single colony into 10 mL MRS liquid culture medium and culture it in a shaking incubator at 37°C overnight for activation. Inoculate the activated bacterial solution into 100 mL MRS liquid culture medium at a 1-3% inoculation amount and culture it in a shaking incubator at 37°C for 24-72 h to obtain plant lactobacillus HJ-S2 bacterial solution. After centrifugation of the obtained bacterial solution, discard the supernatant, collect the bacterial precipitate, and rinse the bacterial body twice with sterile physiological saline to obtain plant lactobacillus HJ-S2 bacterial sludge.

[0094] Preparation of freeze-dried protective agent: The freeze-dried protective agent contains 10-15% skim milk powder, 5-7% trehalose, 3-5% sucrose, and 3-5% sodium glutamate. The above protective agent is dissolved in water and sterilized at 110°C for 15 min for later use.

[0095] Preparation of Lactobacillus plantarum HJ-S2 powder: The prepared Lactobacillus plantarum HJ-S2 bacterial slurry was pre-frozen overnight at -80°C to freeze evenly on the inner wall of the container, and then vacuum freeze-dried. After drying for 24 hours, Lactobacillus plantarum HJ-S2 powder was obtained.

[0096] (2) Preparation of probiotic fermentation agent using Lactobacillus plantarum HJ-S2 The original strain of Lactobacillus plantarum HJ-S2 was inoculated into 10% skim milk (sterilized at 115℃ for 20 min), cultured aerobically at 37℃ for 24 h until curdling, and activated for two generations continuously as the mother starter. The mother starter was inoculated into 10% skim milk (sterilized at 115℃ for 20 min) at an inoculum rate of 1%-5%, and cultured at 37℃ for 24 h until curdling, thus obtaining the Lactobacillus plantarum HJ-S2 starter.

[0097] (3) Preparation of probiotic fermented milk using Lactobacillus plantarum HJ-S2 The conventionally pretreated milk was heated to about 60°C, 6% sucrose was added, and after being fully dissolved, homogenized under a pressure of 25 MPa. Then, after heat treatment at 95°C for 10 min, it was cooled to 38°C. The plant lactobacillus HJ-S2 starter was inoculated at a 5% inoculum, and fermented at 37°C for 20-24 h until the milk curdled. After cooling, it was refrigerated at 4°C to obtain the functional fermented milk of Lactobacillus HJ-S2.

[0098] (4) Preparation of probiotic solid beverage using Lactobacillus plantarum HJ-S2 The steps of preparing a probiotic solid beverage with Lactobacillus plantarum HJ-S2 are as follows: First, prepare the probiotic powder of Lactobacillus plantarum HJ-S2 with reference to (1). Second, according to the solid beverage formula: edible corn starch (balance), white sugar (10%), oligofructose (5-6%), oligomaltosaccharide (9%), citric acid, edible flavoring, and probiotic powder (3-5%) are added and mixed evenly to ensure that the number of live bacteria of Lactobacillus plantarum is 10-15 g per small package, and 10 small packages are packaged into a large package.

[0099] (5) Preparation of probiotic chewing gum using Lactobacillus plantarum HJ-S2 Provided is a chewing gum sheet containing highly active Lactobacillus plantarum, which is composed of the Lactobacillus plantarum HJ-S2 bacterial powder described in (1) and gum matrix, powdered sugar, maltose, calcium carbonate, glucose, flavors and other ingredients. Except for the gum matrix and bacterial powder, the components necessary in the preparation process of the chewing gum sheet are all auxiliary materials, which is the same as in the prior art. The formula includes the following components in weight percentage: 0.1% Lactobacillus plantarum HJ-S2 bacterial powder, 29.9% gum matrix, and 70% sugar and other ingredients.

[0100] The preparation method is as follows: first, vinyl acetate, ester gum, butyryl phthalate butyl glycol ester, and chicle gum are prepared into chewing gum base. During preparation, each raw material is put into a kneader, and an emulsifier and a plasticizer are added at the same time. The kneading temperature is strictly controlled within the range of 80-120° C. to form a whole material. Then, a mixer is used to mix the chewing gum base that has been heated and dissolved, and then sugar, glucose, maltose, spices and other ingredients are added in a certain order, and the mixture is uniform in texture and has a moisture content of 2.1%. The raw materials are cut into rectangular small pieces of 8×25×5 cm by a block cutter, and then loaded into an extruder for extrusion. After extrusion, the pieces are pressed into sheets of a certain thickness by a multi-stage roller machine, and then cut into appropriate shapes by a cutting machine, and then kept in a conditioning room at a temperature of 26° C. and a relative humidity of 45-50% for 6 hours before packaging.

[0101] The number of viable bacteria in the chewing gum containing highly active Lactobacillus plantarum is 2.0×10 7CFU / g, it has the functions of cleaning the oral cavity, refreshing breath, and antagonizing Streptococcus mutans and Porphyromonas gingivalis.

[0102] (6) Preparation of probiotic mouthwash using Lactobacillus plantarum HJ-S2 A probiotic mouthwash composition having the efficacy of preventing and / or treating periodontitis and dental caries, comprising the following raw materials in parts by weight: Phase A: water 87.96%, glycerol 8%, sucralose 0.03%, cetylpyridinium chloride 0.05%; Phase B: propylene glycol 2%, preservative 0.25%, methyl diisopropyl propionamide 0.05%, fragrance 0.05%; Phase C: 1% peppermint essential oil, 0.5% supernatant of fermentation of Lactobacillus plantarum HJ-S2, 0.1% xylitol, 0.01% glucose oxidase. The aromatic agent is essence, and the preservative is PEG-40 hydrogenated castor oil.

[0103] A preparation method of a probiotic mouthwash composition with the efficacy of preventing and / or treating periodontitis and dental caries comprises the following steps: putting phase A into an emulsifying pot, heating to 80-82°C, stirring evenly, and cooling; pre-mixing phase B, stirring and dissolving evenly, and setting aside; cooling phase A in the emulsifying pot to 45°C, putting phase B into the emulsifying pot, and stirring until completely dissolved; adding phase C raw materials into the emulsifying pot, and stirring until completely dissolved; inspecting the discharged materials, aging and standing them, filling and packaging after passing the inspection, and storing after passing the inspection.

[0104] (7) Preparation of probiotic toothpaste using Lactobacillus plantarum HJ-S2 A probiotic toothpaste composition having the efficacy of preventing and / or treating periodontitis and dental caries comprises the following raw materials in parts by weight: The components are taken by weight: 600 parts of silicon dioxide (ML-520A), 100 parts of silicon dioxide (ML-520B), 400 parts of sorbitol aqueous solution, 3 parts of xanthan gum, 1 part of plant lactobacillus HJ-S2, 1 part of saccharin sodium, 5 parts of sodium monofluorophosphate, 1 part of sodium laurate sulfate aqueous solution, and 1 part of ethylparaben. The preparation method of the toothpaste for improving periodontitis and dental caries comprises the following steps: firstly, taking saccharin sodium, sodium monofluorophosphate, and sorbitol aqueous solution, adding saccharin sodium and sodium monofluorophosphate to the sorbitol aqueous solution, and stirring evenly; then adding xanthan gum, silicon dioxide (ML-520A), and silicon dioxide (ML-520B), and stirring evenly; then adding sodium laurate sulfate aqueous solution and ethylparaben, and stirring evenly; finally, adding plant lactobacillus HJ-S2 and water, stirring evenly, and conducting quality inspection on the obtained product. After passing the quality inspection, packaging is carried out to obtain the finished product.

[0105] The embodiments described above are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application, and should all be included in the protection scope of the present application.

Claims

1. An application of Lactobacillus plantarum in the preparation of products for preventing or treating periodontal disease and dental caries, characterized in that: The Lactobacillus plantarum is Lactobacillus plantarum HJ-S2, which is screened from the intestine of Beaked Whale.

2. A biological product for preventing or treating periodontal disease or dental caries, characterized in that: The components of the biological product prepared by Lactobacillus plantarum HJ-S2 as claimed in claim 1 are made of fermentation broth, fermentation supernatant, or lyophilized powder of Lactobacillus plantarum HJ-S2; optionally, the fermentation broth of Lactobacillus plantarum HJ-S2 is obtained by fermenting and culturing Lactobacillus plantarum HJ-S2 activated overnight at 37°C for 24-72 hours; the fermentation supernatant is obtained by sterilizing the fermentation broth with a 0.22 μm sterile filter after centrifugation; and the lyophilized powder is the bacterial mud obtained after HJ-S2 centrifugation or the fermentation supernatant obtained after freeze drying in a freeze dryer.

3. The biological product according to claim 2, characterized in that The biological product is at least one of medicine, food and daily chemical product.

4. The biological product according to claim 3, characterized in that The components of the medicine also include a pharmaceutically acceptable carrier, which is a pharmaceutically acceptable filler.

5. The biological product according to claim 4, characterized in that The pharmaceutically acceptable filler is one or more of starch, sucrose, lactose and inorganic salts.

6. The biological product according to claim 4, characterized in that The medicine comprises various pharmaceutically acceptable dosage forms, which are one or more of gel, gargle, lozenge, microsphere, ointment and tablet.

7. The biological product according to claim 3, characterized in that The daily chemical product is one or more of probiotic toothpaste, tooth stickers, tooth cleaning powder, breath freshening spray, antibacterial liquid, probiotic mouthwash, tooth gel, tooth wax, and periodontal gel.

8. The biological product according to claim 7, characterized in that The probiotic toothpaste comprises silicon dioxide, sorbitol aqueous solution, xanthan gum, Lactobacillus plantarum HJ-S2 bacterial solution, sodium saccharin, sodium monofluorophosphate, sodium laurate sulfate aqueous solution and ethylparaben; the probiotic mouthwash comprises water, glycerin, sucralose, cetylpyridinium chloride, propylene glycol, a preservative, methyl diisopropyl propionamide, a fragrance, peppermint essential oil, Lactobacillus plantarum HJ-S2 fermentation supernatant, xylitol and glucose oxidase.

9. The biological product according to claim 3, characterized in that The food is one or more of probiotic solid beverage, ice cream, lozenge, soft candy, jelly, probiotic chewing gum, oral probiotic powder, probiotic fermentation agent, probiotic fermented milk, etc.

10. The biological product according to claim 9, characterized in that The probiotic solid beverage comprises edible corn starch, white sugar, oligofructose, oligomaltose, citric acid, edible flavor and Lactobacillus plantarum HJ-S2 powder; the probiotic chewing gum comprises Lactobacillus plantarum HJ-S2 powder, gum matrix, powdered sugar, maltose, calcium carbonate, glucose and flavor; the probiotic powder comprises Lactobacillus plantarum HJ-S2 mud and freeze-drying protective agent; the probiotic fermentation agent comprises the original strain of Lactobacillus plantarum HJ-S2 and 10% skim milk; the probiotic fermented milk comprises Lactobacillus plantarum HJ-S2 fermentation agent, cow's milk and sucrose.

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