Application of Lactobacillus casei in preventing or treating periodontal disease and dental caries

Abstract

The present invention belongs to the field of microbial technology, and in particular relates to a strain of Lactobacillus casei from large yellow croaker that can prevent and treat periodontal disease and dental caries, and an application thereof. The Lactobacillus casei is Lactobacillus casei DS31 (Lacticaseibacillus casei). The Lactobacillus casei DS31 provided by the present invention can effectively inhibit the activity of Porphyromonas gingivalis and Streptococcus mutans, thereby effectively solving oral problems such as periodontal disease and dental caries; and the bacteria can be obtained by microbial culture using a culture medium, with low cost, and has great application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to application of Lactobacillus casei derived from marine large yellow croaker in preventing or treating periodontal disease and dental caries. Background Art

[0002] The human oral cavity is rich in more than 700 kinds of microorganisms, including bacteria, fungi, viruses, mycoplasma, etc. Studies have shown that oral flora imbalance is an important cause of oral problems such as periodontal disease and dental caries.

[0003] Periodontal disease is manifested by gingival bleeding, periodontal pocket formation, alveolar bone resorption and loose teeth. The inflammatory factors in serum increase and interact with a variety of diseases, such as infective endocarditis, cardiovascular disease, stroke, diabetes and respiratory diseases. Virulence factors released from dental plaque, such as lipopolysaccharide (LPS) and antigens, enter the gingival tissue to cause inflammation and immune response in the body, thereby leading to the activation of host defense cells; the cell activation, including inflammatory mediators such as cytokines, chemokines, arachidonic acid metabolites and proteolytic enzymes, promotes periodontal tissue destruction and bone resorption. Porphyromonas gingivalis, as a key pathogen in periodontal disease, not only induces inflammatory response in periodontal tissue, but also causes gingival bleeding, recession and periodontal pocket formation. In severe cases, it can cause loose teeth or even tooth loss. At present, the treatments for periodontal disease include mechanical therapy and antibiotic therapy: Mechanical therapy uses physical methods such as ultrasonic scaling, supragingival scaling, subgingival scaling and root planing to remove plaque and tartar on teeth and periodontal tissues, optimize the periodontal environment and promote inflammation relief; while antibiotic therapy uses antibacterial drugs to inhibit or kill periodontal pathogens such as Porphyromonas gingivalis to reduce inflammation and promote tissue healing. However, mechanical therapy is difficult to completely eliminate pathogens, and antibiotic therapy may destroy the ecological balance of oral microorganisms and induce bacterial resistance.

[0004] Caries is a bacterial infectious disease that occurs on the hard tissues of teeth. Caries is caused by the interaction of oral microorganisms, hosts, food and time, and oral flora plays an important role in the occurrence and development of caries. Streptococcus mutans is a key bacterium in the occurrence of caries. It can use carbohydrates to produce a large amount of acidic substances, thereby destroying tooth enamel. In addition, Streptococcus mutans can hydrolyze sucrose to produce an extracellular polysaccharide matrix, which facilitates the adhesion and accumulation of cariogenic bacteria on the tooth surface, thereby promoting the formation of dental plaque biofilm and aggravating the development of caries. At present, antibacterial agents such as chlorhexidine and fluoride are mainly used to treat caries. However, long-term use of chlorhexidine may cause pigment deposition on the tooth surface, affecting the appearance. Improper use of fluoride can easily cause fluoride poisoning, posing a potential threat to human health.

[0005] Patent CN117337896A relates to a sugar-free candy for preventing dental caries and a preparation method thereof, wherein the composite bacterial species is obtained by mixing Torulopsis, Candida lipolytica, Lactobacillus buchneri and Lactobacillus paracasei in a sterile environment at a volume ratio of 5:4:1:1. Although it is disclosed that Lactobacillus casei has a certain positive effect in preventing and treating dental caries, the specific source of Lactobacillus casei is not described in the patent, nor is its effect of treating dental caries verified separately. Patent CN117441817A relates to a probiotic soft candy, wherein the antibacterial component contains Lactobacillus casei powder, which has an inhibitory effect on Escherichia coli, Staphylococcus aureus and Candida albicans, Streptococcus mutans and Porphyromonas gingivalis. Since Streptococcus mutans is the main pathogenic bacteria causing dental caries, and Porphyromonas gingivalis is the main pathogenic bacteria causing periodontal disease, the probiotic soft candy proposed in the patent provides a new idea for treating dental caries and periodontal disease by inhibiting these two bacteria. Likewise, the patent does not describe the specific origin of Lactobacillus casei, nor does it independently verify its effectiveness in treating caries or periodontal disease.

[0006] Therefore, it is urgent to find a method or product that can improve oral health with good effect, high safety and low cost to treat oral problems such as periodontal disease and caries. At present, no marine strains with anti-oral pathogenic bacteria activity have been reported in the species of Lactobacillus casei. Summary of the invention

[0007] To solve the above problems, the first aspect of the present invention provides an application of Lactobacillus casei in preparing a product for preventing or treating periodontal disease and dental caries. The Lactobacillus casei is Lactobacillus casei DS31, which is screened from the intestine of large yellow croaker.

[0008] Furthermore, the periodontal disease and dental caries are diseases caused by specific pathogenic bacteria that endanger oral safety.

[0009] Furthermore, the pathogenic bacteria include at least one of Porphyromonas gingivalis and Streptococcus mutans.

[0010] The second aspect of the present invention provides a biological product for preventing or treating periodontal disease and dental caries, which is prepared from the Lactobacillus casei DS31 as described in the first aspect.

[0011] Furthermore, the biological product is at least one of daily chemical products, food, and medicine.

[0012] Furthermore, the daily chemical product comprises one or more of toothpaste, tooth powder, oral spray, mouthwash, oral gel, and periodontal gel.

[0013] Furthermore, the daily chemical product also contains a daily chemical product additive, and the daily chemical product additive includes xanthan gum, sorbitol and / or calcium carbonate.

[0014] Furthermore, the components of the oral spray include Lactobacillus casei DS31 fermentation broth, sodium bicarbonate, sodium benzoate, vitamin C, peppermint oil, lemon oil, hyaluronic acid, xanthan gum, glycerin, and hydroxypropyl methylcellulose.

[0015] Furthermore, the food comprises one or more of fermented milk, fermented milk beverage, fermented cream, chewing gum, ice cream, and oral probiotic powder.

[0016] Furthermore, the fermented milk is yogurt, the fermented milk beverage is yogurt beverage, and the components of the oral probiotic powder include Lactobacillus casei DS31 bacterial mud and a freeze-drying protective agent.

[0017] Furthermore, the freeze-drying protective agent contains 10-15% skim milk powder, 5-7% trehalose, 3-5% sucrose, and 3-5% sodium glutamate.

[0018] Furthermore, the food also contains food additives, and the food additives include pectin, carrageenan and / or xylitol.

[0019] Furthermore, the medicine contains bacterial polysaccharide.

[0020] Furthermore, the medicine also contains pharmaceutical excipients, and the pharmaceutical excipients include sodium carboxymethyl cellulose, starch and / or maltodextrin.

[0021] Furthermore, the medicine comprises various pharmaceutically acceptable dosage forms, which are one or more of powders, granules, capsules, suspensions, emulsions, gels, gargles, ointments, and tablets, and the tablets are buccal tablets.

[0022] The technical solution of the present invention has the following advantages: The Lactobacillus casei DS31 provided by the present invention can effectively inhibit the activity of Porphyromonas gingivalis and Streptococcus mutans, thereby effectively solving oral problems such as periodontal disease and caries; and the bacteria can be obtained by microbial culture using a culture medium, with low cost, and has great application prospects.

[0023] The Lactobacillus casei DS31 provided by the invention belongs to oral probiotics and intestinal probiotics, has a high safety factor, and can be used for product development as a directly edible strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 : Histogram of “inhibition zone diameter” of Lactobacillus casei DS31; Figure 2-1 : "Lysozyme concentration-absorbance" bar graph of Lactobacillus casei DS31; Figure 2-2 : "Hydrogen peroxide concentration-absorbance" bar graph of Lactobacillus casei DS31; Figure 3-1 : Histogram of “time-self-aggregation rate” of Lactobacillus casei DS31; Figure 3-2 : Histogram of “time-coaggregation rate” of Lactobacillus casei DS31 and Porphyromonas gingivalis; Figure 3-3 : The histogram of “time-coaggregation rate” of Lactobacillus casei DS31 and Streptococcus mutans; Figure 4-1 :The Gram-stained colony image of Lactobacillus casei DS31 inhibiting the biofilm formation of Porphyromonas gingivalis; Figure 4-2 : Inverted microscope image after Gram staining of Lactobacillus casei DS31 inhibiting the biofilm formation of Porphyromonas gingivalis; Figure 4-3 :Scanning electron micrograph of the effect of Lactobacillus casei DS31 on the biofilm formation of Porphyromonas gingivalis; Figure 4-4 :Confocal laser scanning microscopy images of the effect of Lactobacillus casei DS31 on the biofilm formation of Porphyromonas gingivalis; Figure 5-1 :The Gram-stained colony images of Lactobacillus casei DS31 in 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 bacterial solution results of the effect of Lactobacillus casei DS31 on the biofilm formation of Streptococcus mutans; Figure 7-1 :The colony diagram of the effect of Lactobacillus casei DS31 on the treatment of mature mutans Streptococcus biofilm; Figure 7-2 : Bar graph showing the effect of Lactobacillus casei DS31 on the treatment of mature mutans Streptococcus biofilm; Figure 8 : Histogram showing that Lactobacillus casei DS31 inhibits the secretion of inflammatory factors in periodontal disease cell model; Figure 9-1 : 3D reconstruction 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

[0026] 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.

[0027] The strains involved in the following examples are from the following sources: The Lactobacillus casei DS31 involved in the following examples was deposited in the General Microbiological Center of China National Committee for Microbiological Culture Collection on November 28, 2014, with the address of No. 2, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.10073.

[0028] The Porphyromonas gingivalis (ATCC33277) and Streptococcus mutans (ATCC25175) involved in the following examples were purchased from Guangdong Microbiological Culture Collection Center. 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.

[0029] TSB medium is a commercial medium purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0030] DMEM high-glucose medium is a commercial medium purchased from Wuhan Pronocell Life Science Technology Co., Ltd.

[0031] 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.

[0032] 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.

[0033] Complete culture medium: Use high glucose DMEM medium 90%, fetal bovine serum 10%, penicillin / streptomycin 1%.

[0034] The preparation methods of the supernatant, bacterial suspension and bacterial powder involved in the following examples are as follows: 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 rate of 1-3% (v / v), and then cultured anaerobically at 37°C and the concentration of the bacterial suspension was adjusted to 10 5 -10 7 CFU / mL.

[0035] 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.

[0036] Lactobacillus casei DS31 bacterial suspension: Inoculate the overnight activated Lactobacillus casei DS31 into MRS medium at an inoculum size of 1-3% (v / v) and culture aerobically at 37°C for 24-72 h.

[0037] The preparation method of the fermentation supernatant involved in the following examples is as follows: The overnight activated Lactobacillus casei DS31 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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 / A 0 )×100%, where A t represents the absorbance value at a specific time point t, A 0 Indicates the absorbance value at the initial time.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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 order of magnitude, and then quickly poured into a sterile culture dish. After the culture medium cooled and solidified, 3 holes with a diameter of 10 mm were made on each plate, and 100 μL of DS31 bacterial solution, DS31 fermentation supernatant, and MRS culture medium were added to each well respectively; the plate was lightly covered and placed upright under anaerobic conditions at 37°C; after culturing for an appropriate time, the diameter of the inhibition zone was observed and measured with a vernier caliper.

[0051] The results are as follows Figure 1 As shown, the bacterial liquid and fermentation supernatant of Lactobacillus casei DS31 had a significant inhibitory effect on Porphyromonas gingivalis, indicating that this strain of bacteria can inhibit the growth of periodontal disease pathogens, improve the environment of oral flora, and prevent chronic periodontal disease.

[0052] (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 order of magnitude, 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 DS31 bacterial solution, DS31 fermentation supernatant, and MRS culture medium were added to each well respectively; cultured upright at 37°C under aerobic conditions; observed and measured the diameter of the inhibition zone with a vernier caliper.

[0053] The results are as follows Figure 1 As shown, both the bacterial liquid and fermentation supernatant of Lactobacillus casei DS31 had obvious inhibition zones against Streptococcus mutans, which preliminarily indicated that Lactobacillus casei DS31 could inhibit Streptococcus mutans and prevent dental caries.

[0054] Example 2: Oral Tolerance Evaluation of Lactobacillus casei DS31 (1) Determination of lysozyme concentration The suspension of Lactobacillus casei DS31 was inoculated into a 96-well plate, and different concentrations of lysozyme solution were added to make the final concentrations 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.

[0055] Figure 2-1 The figure shows the results of Lactobacillus casei DS31's tolerance to lysozyme. DS31 can not only tolerate the lysozyme concentration in the normal human oral environment (50-100 μg / mL), but also tolerate the lysozyme concentration in the poor oral conditions of periodontal disease patients (200-300 μg / mL), indicating that Lactobacillus casei DS31 has good adaptability to the human oral environment and has the ability to survive in the oral environment.

[0056] (2) Determination of resistance to hydrogen peroxide The suspension of Lactobacillus casei DS31 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.

[0057] Figure 2-2 The figure shows the tolerance of Lactobacillus casei DS31 to hydrogen peroxide in the oral cavity. DS31 can survive in 0.08 mM and 0.8 mM hydrogen peroxide, indicating that it has the conditions to adapt to hydrogen peroxide in the oral environment.

[0058] Example 3: Determination of self-aggregation and co-aggregation abilities of Lactobacillus casei DS31 (1) Determination of the self-aggregation ability of Lactobacillus casei DS31 Take the cultured Lactobacillus casei DS31 fermentation broth and centrifuge it, discard the supernatant, collect the precipitated bacteria and wash them 3-5 times with PBS, 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 / A 0 )×100% Where: A 0 is the absorbance value at 0 h, i.e. 1.0; A tis the absorbance value at the time of sampling.

[0059] from Figure 3-1 The results show that the Lactobacillus casei DS31 provided by the present invention has a strong self-aggregation ability. As time goes by, the self-aggregation rate reaches 27.34% after 8 h.

[0060] (2) Determination of coaggregation ability of Lactobacillus casei DS31 and Porphyromonas gingivalis Take the cultured Lactobacillus casei DS31 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 / (C 0 +C 1 ))×100% Where: C mix1 The OD after the mixture was cultured for xh (x = 2, 4, 6, 8) 600 Value; C 0 is the initial OD of lactic acid bacteria 600 Value; C 1 is the initial OD of Porphyromonas gingivalis 600 value.

[0061] like Figure 3-2 The results show that the Lactobacillus casei DS31 provided by the present invention has a strong copolymerization ability with Porphyromonas gingivalis, and the coaggregation rate can reach 34.93% at 8 h, indicating that Lactobacillus casei DS31 can remove Porphyromonas gingivalis by copolymerization, thereby maintaining the balance of oral flora.

[0062] (3) Determination of coaggregation ability of Lactobacillus casei DS31 and Streptococcus mutans Take the cultured Lactobacillus casei DS31 and Streptococcus mutans and centrifuge the culture to discard 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 and mix them by oscillation, incubate at 37℃ for 2, 4, 6, and 8 h, 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 (%) = (1-2Cmix2 / (C 0 +C 2 ))×100% Where: C mix2 The OD after the mixture was cultured for xh (x = 2, 4, 6, 8) 600 Value; C 0 is the initial OD of lactic acid bacteria 600 Value; C 2 is the initial OD of Streptococcus mutans 600 value.

[0063] like Figure 3-3 The results showed that as time went on, Lactobacillus casei DS31 had the ability to copolymerize with Streptococcus mutans, and the coaggregation rate could reach 25.73% at 8 h, which indicated that Lactobacillus casei DS31 could remove Streptococcus mutans through copolymerization and reduce the number of Streptococcus mutans in the oral cavity.

[0064] Example 4: Inhibitory effect of Lactobacillus casei DS31 on biofilm formation of Porphyromonas gingivalis (1) Gram staining kit combined with inverted microscope observation, the specific steps are as follows: Table 1 Comparison of the effects of different experimental groups on Porphyromonas gingivalis As shown in Table 1, the suspension / supernatant of Lactobacillus casei DS31 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 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 dried, and finally observed and photographed with an inverted microscope.

[0065] The results are as follows Figure 4-1 and 4-2 As shown in the figure, the negative control group was a biofilm of Porphyromonas gingivalis (negative bacteria), which was red after Gram staining. The DS31 bacterial solution (positive bacteria) treatment group was purple, indicating that after the DS31 bacterial solution treatment group, DS31 replaced the Porphyromonas gingivalis biofilm and became the new dominant bacteria. There was no biofilm in the DS31 supernatant group, indicating that there were active ingredients in the DS31 supernatant treatment group that inhibited the formation of Porphyromonas gingivalis biofilm. In summary, both DS31 bacterial solution and DS31 fermentation supernatant had significant effects on the formation of Porphyromonas gingivalis biofilm. However, the effect of the DS31 supernatant treatment group was better than that of the DS31 bacterial solution treatment group. Therefore, Lactobacillus casei DS31 has a beneficial effect on the prevention of periodontal disease.

[0066] (2) Scanning electron microscopy observation of the periodontal disease model after DS31 treatment. The specific steps are as follows: The suspension / supernatant of Lactobacillus casei DS31 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, dried at room temperature after gradient dehydration with ethanol, sprayed with gold, and the biofilm morphology was observed by scanning electron microscopy and photographed and recorded.

[0067] The results are as follows Figure 4-3 As shown, it can be seen under the scanning electron microscope at 500, 2000, and 5000 times: in the absence of Lactobacillus casei DS31 treatment, the Porphyromonas gingivalis biofilm is arranged tightly, while after treatment with Lactobacillus casei DS31, the bacterial gaps can be clearly observed, and the number of bacteria in the form of biofilm is significantly reduced, especially after treatment with DS31 supernatant, almost no bacteria exist. Therefore, the DS31 bacterial liquid treatment group and DS31 supernatant have significant effects on the inhibition of Porphyromonas gingivalis biofilm formation, but the effect is better after DS31 supernatant treatment. Therefore, it shows that Lactobacillus casei DS31 has a positive therapeutic effect on the prevention of periodontal disease.

[0068] (3) Effect of DS31 on the biofilm formation activity of Porphyromonas gingivalis Add the suspension / supernatant of Lactobacillus casei DS31 and the bacterial suspension of Porphyromonas gingivalis to a 24-well plate at a ratio of 1:1-3, and add MRS medium as a negative control. The blank control group is supplemented with MRS medium to the same volume. Aspirate the culture medium and add sterile saline to gently wash. Pipette an appropriate amount of SYTO 9 and PI stains and mix them with saline, vortex and mix to make SYTO 9 and PI mixed stains; add the mixed stains and stain in the dark, add sterile saline to gently wash 3 times, and observe under a laser confocal microscope.

[0069] The results are as follows Figure 4-4 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. After the DS31 bacterial solution was treated, there were no live bacteria in the biofilm, indicating that the DS31 bacterial solution treatment group had a good inhibitory effect on Porphyromonas gingivalis; after the DS31 supernatant was treated, there were a small number of green in the biofilm, indicating that the DS31 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 periodontal disease.

[0070] Example 5: Effect of Lactobacillus casei DS31 on the removal of mature biofilm of Porphyromonas gingivalis (1) Inverted microscope combined with Gram staining kit 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 with sterile PBS 2-3 times, add 0.5-2 mL of Lactobacillus casei DS31 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 with an inverted microscope, take pictures and record the results.

[0071] The results are as follows Figure 5-1 As shown in the figure, the negative control group is red, which is a mature biofilm of Porphyromonas gingivalis; the red color is reduced in the DS31 bacterial solution treatment group, indicating that the DS31 bacterial solution treatment group has an antagonistic effect on the mature Porphyromonas gingivalis biofilm and effectively reduces the colonization of Porphyromonas gingivalis; the DS31 supernatant treatment group is light pink, indicating that the active substances in the DS31 supernatant have a killing effect on the mature Porphyromonas gingivalis biofilm. In summary, both DS31 bacterial solution and DS31 supernatant are effective in treating the formed Porphyromonas gingivalis biofilm, so DS31 has a good effect on the treatment of periodontal disease.

[0072] (2) Colony counting by plate dilution spreading method 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 casei culture 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 and gently washed 2-3 times to remove non-adherent bacteria. A sterile cotton swab was used to scrape off the biofilm as much as possible, and the cotton swab head was broken off and mixed in 0.9% saline. The same volume of dilutions of different concentrations was applied to the Columbia blood plate, and the number of Porphyromonas gingivalis was detected after anaerobic culture at 37°C. 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.

[0073] Table 2 Clearance rate of mature biofilm mediated by Lactobacillus casei DS31 As can be seen from the data in Table 2, compared with the negative control group, the lactobacillus casei DS31 bacterial solution and supernatant treatment groups all reduce the number of mature biofilms of Porphyromonas gingivalis, and the treatment effect of DS31 supernatant is slightly better than that of DS31 bacterial solution treatment group. The DS31 bacterial solution treatment group has a clearance rate of 89.20% for mature biofilms of Porphyromonas gingivalis, indicating that the thalline may be effectively combined with pathogenic bacteria to reduce the colonization of pathogenic bacteria; the DS31 supernatant treatment group has a clearance rate of 93.52% for mature biofilms of Porphyromonas gingivalis, indicating that there are antibacterial active substances in the metabolites of DS31 that have a very strong killing effect on Porphyromonas gingivalis biofilms. Therefore, the lactobacillus casei DS31 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.

[0074] (3) Combined CV and TTC method to detect the effect of DS31 on 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 casei DS31 culture liquid 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 OD 595 , 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 .

[0075] The results are as follows Figure 5-2 As shown, compared with the negative control group, the DS31 supernatant treatment group can significantly reduce the number of live bacteria in the mature Porphyromonas gingivalis biofilm, indicating that the DS31 supernatant treatment can significantly reduce the number of Porphyromonas gingivalis.

[0076] Example 6: Inhibition of biofilm formation of Streptococcus mutans after treatment with Lactobacillus casei DS31 Add the suspension / supernatant of Lactobacillus casei DS31 and the bacterial solution of Streptococcus mutans to a 24-well plate at a ratio of 1:1-3, add MRS medium as a negative control, and take photos and record after the culture is completed.

[0077] 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 casei DS31, there was almost no Streptococcus mutans; after treatment with DS31 bacterial solution, the number of Streptococcus mutans was significantly reduced, and DS31 became the dominant bacteria.

[0078] Example 7: Effect of Lactobacillus casei DS31 treatment on mature Streptococcus mutans biofilm Add the mutans Streptococcus cultured overnight to a 24-well plate and incubate at 37°C to form a mature mutans Streptococcus biofilm. After the incubation is completed, discard the culture medium, gently wash 2-3 times with sterile PBS, add 0.3-2 mL of Lactobacillus casei DS31 culture 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 incubation is completed, discard the culture medium; take photos and record. After fixing the biofilm, discard the methanol, let the biofilm stand at room temperature to dry, add crystal violet solution to each well, stain the biofilm, wash it twice with PBS after staining, add ethanol to dissolve it, and read the OD 595 .

[0079] The results are as follows Figure 7-1 and 7-2 As shown in the figure, the mutans streptococci in the negative control group have formed a dense mature biofilm. After being treated with the bacterial solution and supernatant of Lactobacillus casei DS31, there is almost no mutans streptococcus biofilm, indicating that the antibacterial substances in DS31 can remove the mature biofilm of mutans streptococci.

[0080] Example 8: Effect of Lactobacillus casei DS31 on TNF-α, IL-1β, and IL-6 levels in periodontal disease cell models (1) Construction of periodontal disease cell model: Mouse macrophages RAW264.7 were inoculated in DMEM medium containing 10% (v / v) fetal bovine serum and incubated at 37°C with 5% (v / v) CO 2 The cells were cultured and activated in a cell culture incubator and then inoculated into a 24-well plate. The 24-well plate was pre-incubated at 37°C for 0.5-2 h, and then Porphyromonas gingivalis lipopolysaccharide was added to each well.

[0081] (2) Experimental groups: blank control group, model group, Lactobacillus casei DS31 bacterial solution group, and Lactobacillus casei DS31 supernatant group, each group was repeated 3 times.

[0082] Table 3 Construction of periodontitis models in different experimental groups (3) Experimental treatment: Equal volumes of DS31 bacterial solution and DS31 supernatant were added to the periodontal disease cell model. The model group was the group to which equal volumes of complete culture medium were added, and the group to which only RAW264.7 cells were added was the blank control group.

[0083] Test results such as Figure 8 As shown, compared with the blank control group, the pro-inflammatory factors such as TNF-α, IL-1β, and IL-6 in the periodontal disease cell model group increased; and after treatment with the bacterial solution and supernatant of Lactobacillus casei DS31, various inflammatory factors could be reduced, among which TNF-α had a significant down-regulating effect.

[0084] Example 9: Effect of Lactobacillus casei DS31 on alveolar bone in rats with periodontal disease (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 silk thread ligation, infection with Porphyromonas gingivalis, injection of Porphyromonas gingivalis lipopolysaccharide, high-sugar feed and drinking water. The 15 rats were randomly divided into 5 groups, with 3 rats in each group. The 5 groups were healthy group, model group, Lactobacillus casei DS31, Lactobacillus casei DS31 supernatant group, and positive control group. Except for the healthy group and the model group, the other groups were fed with 0.1-2 mL DS31 bacterial solution, DS31 supernatant, and compound chlorhexidine gargle by gavage, and were fasted and deprived of water for half an hour after the end of administration.

[0085] (2) Effect of Lactobacillus casei DS31 on the gingival index (GI) of rats with periodontal disease 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.

[0086] 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.

[0087] (3) Effects of Lactobacillus casei DS31 on CEJ-ABC distance and bone density of alveolar bone in rats with periodontal disease The rat maxilla was fixed in 4% paraformaldehyde for 36-48 h, then washed and dried with PBS buffer solution. X-ray scanning imaging of rat maxilla samples was 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. The following parameters were measured: ① CEJ-ABC distance: the distance from the enamel-cementum junction of the second molar to the alveolar ridge top; ② bone density: the density of minerals contained in the bone per unit volume.

[0088] according to Figure 9-1 , 9-2 , 9-3, it can be seen that the teeth of the healthy group rats were tightly arranged, the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) was relatively short, and the root exposure area was relatively small. The model group showed obvious symptoms of alveolar bone resorption and severe root exposure. The root exposure area after treatment with DS31 bacterial solution and supernatant and compound chlorhexidine mouthwash was relatively small, indicating that DS31 bacterial solution and supernatant can alleviate the symptoms of periodontal disease rats.

[0089] Example 10: Effects of Lactobacillus casei DS31 on inflammatory factors TNF-α, IL-1β, IL-6 and IL-8 in gingival tissue of rats with periodontal disease 0.1-0.3g rat gingival tissue was added to 0.9 mL RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors for lysis. After being fully disrupted by a tissue disruptor, it 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. 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).

[0090] according to Fig.10 It 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 periodontal disease were significantly upregulated (P < 0.05). Both the bacterial liquid and supernatant of Lactobacillus casei can reduce the secretion of TNF-α, IL-1β, IL-6, and IL-8 to a certain extent.

[0091] Example 11: Preparation of biological products using Lactobacillus casei DS31 (1) Preparation of oral probiotic powder using Lactobacillus casei DS31 Preparation of Lactobacillus casei DS31 bacterial sludge: Pick a single colony of fresh Lactobacillus casei DS31 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 rate and culture it in a shaking incubator at 37°C for 24-72 h to obtain Lactobacillus casei DS31 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 Lactobacillus casei DS31 bacterial sludge.

[0092] 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.

[0093] Preparation of Lactobacillus casei DS31 powder: The prepared Lactobacillus casei DS31 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 casei DS31 powder was obtained.

[0094] (2) Preparation of oral spray using Lactobacillus casei DS31 The weight components of the oral spray are: 9 parts of Lactobacillus casei DS31 fermentation liquid, 2 parts of sodium bicarbonate, 2 parts of sodium benzoate, 3 parts of vitamin C, 3 parts of peppermint oil, 1 part of lemon oil, 3 parts of hyaluronic acid, 2 parts of xanthan gum, 1 part of glycerin, and 1 part of hydroxypropyl methylcellulose.

[0095] Preparation method of oral spray: Lactobacillus casei DS31, sodium bicarbonate, sodium benzoate, vitamin C, hyaluronic acid, and glycerin are weighed in a beaker according to the composition ratio, and an appropriate amount of pure water is added and stirred evenly until all the ingredients are dissolved, and then peppermint oil, lemon oil, xanthan gum, and hydroxypropyl methylcellulose are added, and the above mixture is heated and stirred in a 50° C. water bath, and water is added to the required volume during the heating process until all the ingredients are completely dissolved. The resulting solution is the oral spray in this embodiment.

[0096] 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 casei in the preparation of products for preventing or treating periodontal disease and dental caries, characterized in that: The Lactobacillus casei is Lactobacillus casei DS31, which is screened from the intestine of large yellow croaker.

2. The use according to claim 1, characterized in that The periodontal disease and dental caries are diseases caused by specific pathogenic bacteria that endanger oral safety.

3. The use according to claim 2, characterized in that The pathogenic bacteria include at least one of Porphyromonas gingivalis and Streptococcus mutans.

4. A biological product for preventing or treating periodontal disease or dental caries, characterized in that: Prepared by Lactobacillus casei DS31 as claimed in claim 1.

5. The biological product according to claim 4, characterized in that The biological product is at least one of daily chemical products, food, and medicine.

6. The biological product according to claim 5, characterized in that The daily chemical product comprises one or more of toothpaste, tooth powder, oral spray, mouthwash, oral gel, and periodontal gel; and / or the daily chemical product further comprises a daily chemical product additive, and the daily chemical product additive comprises xanthan gum, sorbitol and / or calcium carbonate.

7. The biological product according to claim 6, characterized in that The components of the oral spray include Lactobacillus casei DS31 fermentation liquid, sodium bicarbonate, sodium benzoate, vitamin C, peppermint oil, lemon oil, hyaluronic acid, xanthan gum, glycerin and hydroxypropyl methylcellulose.

8. The biological product according to claim 5, characterized in that The food comprises one or more of fermented milk, fermented milk beverage, fermented cream, chewing gum, ice cream, and oral probiotic powder; the fermented milk is yogurt, the fermented milk beverage is yogurt beverage, and the components of the oral probiotic powder include Lactobacillus casei DS31 bacterial mud and a freeze-drying protective agent; and / or the food also contains food additives, and the food additives include pectin, carrageenan and / or xylitol.

9. The biological product according to claim 5, characterized in that The medicine contains bacterial polysaccharide; and / or the medicine further contains pharmaceutical excipients, and the pharmaceutical excipients contain sodium carboxymethyl cellulose, starch and / or maltodextrin.

10. The biological product according to claim 9, characterized in that The medicine comprises various pharmaceutically acceptable dosage forms, which are one or more of powder, granules, capsules, suspensions, emulsions, gels, gargles, ointments, and tablets, and the tablet is a lozenge.

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