CN118345002A - Bifidobacterium longum and application thereof in preparation of caries-resistant products - Google Patents
Bifidobacterium longum and application thereof in preparation of caries-resistant products Download PDFInfo
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- CN118345002A CN118345002A CN202410448970.1A CN202410448970A CN118345002A CN 118345002 A CN118345002 A CN 118345002A CN 202410448970 A CN202410448970 A CN 202410448970A CN 118345002 A CN118345002 A CN 118345002A
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- caries
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- streptococcus mutans
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Abstract
The invention relates to the technical field of microorganisms, in particular to bifidobacterium longum and application thereof in preparing products for resisting decayed teeth. The bifidobacterium longum DH182 provided by the invention has the function of copolymerizing with streptococcus mutans, and can effectively reduce the quantity of streptococcus mutans in the oral environment. The strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 13348. It can maintain microecological balance of oral cavity, and has dental caries preventing effect.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bifidobacterium longum and application thereof in preparing products for resisting decayed teeth.
Background
Bacterial-dominant oral diseases mainly include caries and periodontal disease, etc., wherein the main pathogenic bacteria of caries are: streptococcus mutans (Streptococcus mutans), streptococcus sanguis (Streptococcus sanguis), actinomyces viscosus (Actinomyces viscous) and Lactobacillus acidophilus (Lactobacillus acidophilus); the periodontal pathogen is mainly Porphyromonas gingivalis (Porphyromonas gingivalis), treponema pallidum (Treponema denticola), fusarium (TANNERELLA FORSYTHIA), actinobacillus symbiotic actinobacillus (Aggregatibacter actinomycetemcomitans). Wherein the streptococcus mutans has the greatest effect on dental caries.
The principle of caries is that bacteria decompose organic acids (especially lactic acid) produced by saccharides, reproducibly erode enamel surrounding the tooth, and some bacteria can convert the saccharides into complex polysaccharides such as Streptococcus mutans, which are the main causative bacteria of caries, and can produce viscous polysaccharide bodies attached to the tooth surface to form dental plaque. Plaque is a thin film that adheres to the surface of teeth and is particularly prone to occur on teeth that are not clean in oral hygiene. The presence of plaque tends to cause the attachment of sugars, harmful bacteria, and acidic substances to the tooth surface, which can cause damage to the tooth surface. Most of decayed teeth occur in the sulcus and concave parts of the large molar teeth and the small molar teeth, and if dental plaque exists, the decay of the smooth surface of the teeth is accelerated; if the food contains sugar, the acid produced by the bacteria can be eroded into the sulcus of the occlusal surfaces of the molar teeth in a lateral direction. After caries, hard tissues of teeth are damaged, and serious people may influence dental nerves, so that the teeth are painful, the ability of the oral cavity to chew food is reduced, the gastrointestinal tract is dysfunctional, and food digestion and nutrient absorption are affected. Further, the children can be caused to grow and develop abnormally, malnutrition, physical function reduction in young and strong years, tooth pain influence the working efficiency, and the conditions of osteoporosis, immunity reduction, constipation and the like of the middle-aged and the elderly are caused.
The experimental results of Ishikawa and the like are taught to find that probiotics (Lactobacillus salivariusTI2711, LS 1) have bactericidal effects on pathogenic bacteria causing periodontal disease, and have the effects of preventing periodontal disease, halitosis and the like. The administration of cow's milk containing probiotics (LGG) for 7 months at university of helsinki in finland for children aged 1-6 years showed that LGG was able to reduce the incidence of tooth decay and reduce the number of streptococcus mutans. Tan Chaoyin et al found a prospect of Lactobacillus paracasei WFP1-1 in the prevention and treatment of dental caries. In addition, streptococcus salivarius K12 has been commercially used as a buccal tablet by American company. However, although probiotic studies have become a research hotspot in China at present, oral probiotics with anti-caries effects are relatively few.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a bifidobacterium longum and its application in preparing anti-caries products.
The invention provides bifidobacterium longum (Bifidobacterium longum subsp. Longum) with the preservation number of CGMCC No. 13348.
The invention also provides a microbial agent or a microecological preparation, which comprises pharmaceutically acceptable auxiliary materials and at least one of the following components:
I) The live bifidobacterium longum is obtained,
II) the bacterial powder of the live bifidobacterium longum,
III) the fermentation product of said bifidobacterium longum,
IV) the inactivated product of bifidobacterium longum.
The microbial inoculum or the microecological preparation is prepared by fermenting bifidobacterium longum as described above, is a fermented living bacterium, can also be a fermented supernatant, or can be prepared by further extraction and purification; the bacterial mud is prepared by freeze-drying after being mixed with auxiliary materials, or can be prepared by mixing living bacteria with a protective agent and freezing.
The microbial agent or the microecological preparation is at least one of powder, tablet, capsule, aqua, gel, paste, dripping pill, pill or granule.
The invention also provides application of the bifidobacterium longum, the microbial inoculum or the microecological preparation in preparing products for preventing and treating dental caries.
In the present invention, the control includes inhibition of the number of harmful bacteria and/or inhibition of adhesion of harmful bacteria.
In some embodiments, the deleterious bacteria are streptococcus mutans, streptococcus sanguis, actinomyces viscosus, and/or lactobacillus acidophilus. The adhesion includes adhesion to the gastrointestinal tract or the oral cavity.
In the present invention, the control includes reducing the extent of caries lesions.
In some embodiments, the caries lesions are enamel caries, shallow dentin caries and/or caries in dentin. It is a caries lesion of the smooth surface of the tooth or a caries lesion of the fossa of the tooth.
The invention also provides a caries control product comprising bifidobacterium longum as described above or a microbial or probiotic agent as described.
In some embodiments, the product is a lozenge, a dental patch, a toothpaste, or a mouthwash.
Further, the present invention also provides a method for preventing dental caries comprising administering a product according to the present invention.
For example, the method of preventing dental caries comprises rinsing a product as described above, chewing or taking a lozenge as described above, applying a dental patch as described above, or brushing with a toothpaste as described above.
The bifidobacterium longum DH182 provided by the invention has the function of copolymerizing with streptococcus mutans, and can effectively reduce the quantity of streptococcus mutans in the oral environment. The strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 13348. It can maintain microecological balance of oral cavity, and has dental caries preventing effect.
Description of biological Material
Biological material: the bifidobacterium longum DH182 is classified and named as Bifidobacterium longum subsp.longum of bifidobacterium longum, which is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 11 and 28 days of 2016, and has the address of CGMCC No.13348 of China academy of sciences of China, north Star Xila No.1, which is the Korean area of Beijing.
Detailed Description
The invention provides bifidobacterium longum and application thereof in preparing anti-caries products, and the person skilled in the art can properly improve the technological parameters by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
Examples
1. Isolated culture and identification of bifidobacterium longum DH182
1.1 Experimental materials
Human feces, MRS broth, MRS agar.
1.2 Laboratory apparatus
Electric heating pressure steam sterilizer, electric heating blast drying oven, electronic balance (sensing 0.01 g), PHS-3CpH meter, magnetic stirrer, ultraviolet visible spectrophotometer, and vortex mixer
1.3 Experimental methods
1.3.1 Sample preparation
All preparation procedures for 1.3.1.1 samples should follow aseptic procedures.
1.3.1.2A 25g (mL) sample was weighed in a sterile manner and placed in a sterile Erlenmeyer flask containing 225mL of saline to make a 1:10 sample homogenate.
1.3.2 Dilution and spread culture step
1.3.2.1 1A 1:10 sample homogenate is drawn by a 1mL sterile pipette or micropipette, 1.0mL is slowly injected into a sterile test tube filled with 9mL physiological saline along the tube wall (note that the tip of the pipette does not touch the diluent), the test tube is shaken or repeatedly blown by a 1-branch sterile pipette to mix uniformly, and a 1:100 sample homogenate is prepared.
1.3.2.2A 1mL sterile pipette or micropipette tip is additionally taken, 10-fold incremental sample homogenization is performed in the order of 1.3.2.1 operations, and 1mL sterile pipette or tip is replaced once for every incremental dilution.
1.3.3 According to the number of live bacteria of the bacteria to be identified, three continuous suitable dilutions are selected, each dilution absorbs 0.1mL of the dilution, and the dilution is inoculated on a bifidobacterium agar plate or an MRS agar plate, or 0.1mL of a sample homogenate with the suitable dilution is evenly coated on the bifidobacterium agar plate or the MRS agar plate. Anaerobic culture is carried out for 48 hours plus or minus 2 hours at 36 ℃ plus or minus 1 ℃, and the culture can be prolonged to 72 hours plus or minus 2 hours.
1G of human body excrement sample is weighed and dissolved in 9ml of 0.85% sterile physiological saline, the mixture is fully and uniformly mixed by shaking, 200 mu L of mixed liquid is respectively coated on a commercial MRS solid culture medium, the mixture is cultured for 72 hours in a constant temperature incubator at 37 ℃, single bacteria with different colony forms are selected and respectively streaked on the solid culture medium for purity division, and the step is repeated for 2-3 times until colony characteristics are consistent. Then the purified single bacteria are inoculated in a corresponding liquid culture medium to be cultured for 18 hours at 37 ℃, and the purified single bacteria are preserved by 60 percent of glycerol and frozen in a refrigerator at the temperature of minus 80 ℃ for standby.
1.3.4 Pure culture: 3 or more individual colonies were picked and inoculated on either a bifidobacterium agar plate or an MRS agar plate. Anaerobic culture is carried out for 48 hours plus or minus 2 hours at 36 ℃ plus or minus 1 ℃, and the culture can be prolonged to 72 hours plus or minus 2 hours.
2. Identification of strains
The screened bifidobacterium longum DH182 is stored in China general microbiological culture Collection center with the preservation number of CGMCC No.13348, and is entrusted to the identification of microbiological research institute of China academy of sciences, and the identification result is Bifidobacterium longum subsp.
3. Determination of the self-aggregation Rate of the Strain
3.1 Purpose of test
The experiment selects bifidobacterium longum (DH 182, preservation number: CGMCC No. 13348) and lactobacillus paracasei DH068 with good culture characteristics, and the preservation number is: CGMCC NO.11225, bifidobacterium infantis DH231, and the preservation number is: CGMCC No.18372 and lactobacillus rhamnosus (DH-Lr-121, preservation number: CGMCC No. 13076) are used for researching the self-coagulation rate of the four lactobacillus strains in a normal natural growth state, and the high self-coagulation rate is beneficial to the field planting of the lactobacillus strains on the cell surface and the formation of a biological film and inhibits the adhesion of harmful bacteria to the gastrointestinal tract or the oral cavity.
3.2 Test materials and test instruments
3.2.1 Test materials
Test strain: bifidobacterium longum (DH 182, preservation number: CGMCC No. 13348), lactobacillus paracasei DH068, preservation number: CGMCC NO.11225, bifidobacterium infantis DH231, and the preservation number is: CGMCC No.18372 and lactobacillus rhamnosus (DH-Lr-121, preservation number: CGMCC No. 13076) are preserved in the China general microbiological culture Collection center. TSA medium, TSB medium, MRS broth medium, MRS agar medium.
3.2.2 Test instruments
Electric heating pressure steam sterilizer, electric heating blast drying oven, electronic balance (sensing 0.01 g), PHS-3CpH meter, magnetic stirrer, ultraviolet visible spectrophotometer, and vortex mixer
3.3 Test methods and test procedure
3.3.1 Test methods
The probiotics with the bacterial count of 1X 10 8 CFU/g are resuspended by using sterile PBS with the pH of 6.8, the bacterial suspension is diluted by using the sterile PBS with the pH of 6.8 until the OD600 (the absorbance of bacterial solution at the wavelength of 600 nm) is regulated to 0.6, the bacteria are kept stand for 4 hours and 8 hours at 37 ℃, and the OD600 of the upper liquid is measured.
Self-agglomeration rate/% = (A0-A1)/a0×100
Wherein: a0 is the initial OD600 of the bacterial suspension; a1 is OD600 of upper liquid of bacterial suspension after standing.
The experimental results are shown in the following table 1, the self-condensation rate of bifidobacterium longum DH182 is highest, and the self-condensation rate reaches 51.25% in 8 hours; secondly, lactobacillus rhamnosus DH-Lr-121 with self-aggregation rate of 49.17% in 8 hours; lactobacillus paracasei DH068 has a self-clotting rate of 46.81% at 8h, and the lowest self-clotting rate is bifidobacterium infantis DH231, with a self-clotting rate of 17.83% at 8 h. The high self-coagulation rate is favorable for the bacterial strain to colonize and form a biological film on the cell surface, and the adhesion of harmful bacteria to the gastrointestinal tract or the oral cavity is inhibited.
TABLE 1 self-agglutination rates of different strains
4 Screening of Lactobacillus strains with Streptococcus mutans copolymerization
4.1 Purpose of test
The experiment selects bifidobacterium longum (DH 182, preservation number: CGMCC No. 13348), lactobacillus paracasei (DH 068, preservation number: CGMCC No. 11225) and bifidobacterium infantis DH231 with good culture characteristics, and the preservation number is: CGMCC No.18372. Lactobacillus rhamnosus (DH-Lr-121, preservation number: CGMCC No. 13076), researching the copolymerization of the four lactobacillus strains to streptococcus mutans, selecting the lactobacillus strain with the strongest co-aggregation capability with the streptococcus mutans, and providing target strain and basis for developing probiotic oral health products in the next step.
4.2 Test materials and test instruments
4.2.1 Test materials
Test strain: streptococcus mutans NCTC 10449; bifidobacterium longum DH182, lactobacillus paracasei DH068, bifidobacterium infantis DH231 and lactobacillus rhamnosus DH-Lr-121 are preserved in the China general microbiological culture Collection center. TSA medium, TSB medium, MRS broth medium, MRS agar medium.
4.2.2 Test instruments
Electric heating pressure steam sterilizer, electric heating blast drying oven, electronic balance (sensing 0.01 g), PHS-3CpH meter, magnetic stirrer, ultraviolet visible spectrophotometer, and vortex mixer
4.3 Test methods and test procedure
4.3.1 Test methods
Culturing streptococcus mutans: inoculating Streptococcus mutans strains to a TSA slant culture medium for resuscitation at 37 ℃ under anaerobic condition for 48 hours, picking out a loop of lawn, inoculating to a TSB liquid culture medium, culturing for 18-24 hours under anaerobic condition at 37 ℃, checking a smear to be pure culture, and inoculating to the TSB liquid culture medium for culturing for 48 hours according to 10% (V/V).
Culturing lactobacillus: inoculating lactobacillus strain to MRS agar inclined plane for resuscitation under 37 ℃ anaerobic condition for 48 hours, picking out a loop of lawn, inoculating to MRS broth culture medium, culturing for 18-24 hours under 37 ℃ anaerobic condition, inoculating to MRS broth culture medium according to 10% (V/V) after smear inspection as pure culture, and culturing for 24 hours.
After centrifugation of the bacterial liquid, washing once with distilled water, re-suspending, and adjusting the concentration with sterile PBS (pH 7.0) at room temperature; the probiotic concentration was adjusted to od600=1 and the streptococcus mutans concentration was adjusted to od600=1. The ratio of the two bacterial solutions is 1:1, the bacterial solutions are added into a 2mL centrifuge tube at the same time, uniformly mixed for 30s on a vortex mixer, the supernatant is sucked by a suction tube, absorbance is measured under the condition of 600nm of an ultraviolet spectrophotometer, and the flocculation activity of the culture solution is determined by taking the solution without the culture solution as a control. If flocculation does not occur within 20 minutes, it is considered to be non-reactive. The flocculation rate was calculated as follows:
flocculation rate = (a-B)/ax100%
Wherein A is the absorbance of the control supernatant and B is the absorbance of the sample supernatant.
4.4 Test results and analysis
From the experimental results, the bifidobacterium longum DH182, the lactobacillus paracasei DH068, the bifidobacterium infantis DH231 and the lactobacillus rhamnosus DH-Lr-121 all have certain flocculation effects on the streptococcus mutans, wherein the flocculation effects of the bifidobacterium longum DH182 are most obvious, the co-coagulation rate of the bifidobacterium longum DH182 and the streptococcus mutans is highest, the co-coagulation rate of the bifidobacterium longum DH182 and the streptococcus mutans can reach 88.67% in 8 hours according to the following table, and the co-coagulation rate of the lactobacillus rhamnosus DH-Lr-121 and the streptococcus mutans is 79.46% in 8 hours, the co-coagulation rate of the lactobacillus paracasei DH068 and the streptococcus mutans is 73.25% in 8 hours, and the co-coagulation rate of the bifidobacterium infantis DH231 and the streptococcus mutans is 53.14% in 8 hours. The co-aggregation rate of the bifidobacterium longum and the streptococcus mutans is highest, which shows that the bifidobacterium longum can effectively inhibit the streptococcus mutans in the aspects of adhesiveness and bacteriostasis and reduce the occurrence rate of dental caries.
TABLE 2 Co-aggregation Rate of different strains of Streptococcus mutans
5 Animal evaluation experiment of the anti-caries Effect of Probiotics
5.1 Purpose of test
The experiment selects a Wistar rat, and the caries-causing word stock is fed to the oral cavity of the rat to manufacture a caries model, simulate the oral cavity hygiene habit of human beings, treat the oral cavity of the rat with the medicine, finally take a jawbone specimen, detect the related indexes for measuring the caries disease, judge the caries prevention and treatment effects of bifidobacterium longum DH182, lactobacillus paracasei DH068, bifidobacterium infantis DH231 and lactobacillus rhamnosus DH-Lr-121, and provide target strains and basis for the next development of the probiotic oral cavity health products.
5.2 Test materials and test instruments
5.2.1 Test materials
Test strain: streptococcus mutans NCTC 10449; bifidobacterium longum DH182; lactobacillus paracasei DH068; bifidobacterium infantis DH231, lactobacillus rhamnosus DH-Lr-121; TSA medium, TSB medium, MRS broth medium;
Keyes 2000# (56% sucrose, 6% whole wheat flour, 28% refined milk powder, 3% alfalfa powder, 1% dehydrated whole liver powder, 4% yeast, 2% salt)
Test animals: wistar rat
Test animal production license: SCXK (robust) 20190003
5.2.2 Test instruments
CX41-FR biomicroscope; a carbon dioxide incubator; a biosafety cabinet.
5.3 Test methods and test procedure
5.3.1 Test methods
The experiment selects a Wistar rat, and the oral cavity of the rat is inoculated with bacteria, caries-causing word materials are fed to prepare a caries model, so that the oral cavity of the rat is simulated to simulate the oral hygiene habit of human beings, bifidobacterium longum DH182, lactobacillus paracasei DH068, bifidobacterium infantis DH231 and lactobacillus rhamnosus DH-Lr-121 are treated, finally, a jawbone specimen is taken, and relevant indexes for measuring caries are detected, so that the caries prevention and treatment effect of probiotics is evaluated.
5.3.2 Test procedure
SPF-grade rats of 3 weeks of age were randomly divided into 6 groups according to body weight, 8 groups each, and were set as a blank control group (blank group), a caries model group (model group), and an experimental group (Bifidobacterium longum DH182, lactobacillus paracasei DH068, bifidobacterium infantis DH231, lactobacillus rhamnosus DH-Lr-121). And wiping the streptococcus mutans bacterial liquid of the model group and the experimental group every day 10d before the start of the experiment, and wiping the bifidobacterium longum DH182, the lactobacillus paracasei DH068, the bifidobacterium infantis DH231 and the lactobacillus rhamnosus DH-Lr-121 bacterial liquid 1 time every day until the end of the experiment at week 8 after the success of the model group and the experimental group.
(1) Grouping
A: blank group, 8 rats;
b: model group (negative control group), 8 rats;
c: rats were brushed with 10 8 CFU/mL bifidobacterium longum DH182 solution per day, 8 rats;
D: the rats are brushed with 10 8 CFU/mL lactobacillus paracasei DH068 liquid every day, 8 rats;
e: the rats were brushed with 10 8 CFU/mL bifidobacterium infantis DH231 solution per day, 8 rats;
f: rats were brushed daily with 10 8 CFU/mL Lactobacillus rhamnosus DH-Lr-121, 8 rats.
(2) Culturing streptococcus mutans: inoculating Streptococcus mutans strains to a TSA slant culture medium for resuscitation under anaerobic condition at 37 ℃ for 48 hours, picking a loop of lawn, inoculating to a TSB liquid culture medium, culturing for 18-24 hours under anaerobic condition at 37 ℃, inoculating to the TSB liquid culture medium according to 10% (V/V) after smear examination as pure culture, centrifuging at 8000rpm for 5 minutes after culture is finished, collecting thalli, repeatedly washing 3 times with normal saline, and re-suspending to adjust the concentration of the thalli to 1X 10 8 CFU/mL for later use.
(3) In the experimental process, bifidobacterium longum DH182, lactobacillus paracasei DH068, bifidobacterium infantis DH231 and lactobacillus rhamnosus DH-Lr-121 are inoculated in an MRS broth slant culture medium for resuscitation for 48 hours under the anaerobic condition at 37 ℃, one ring of fungus moss is selected to be inoculated in the MRS broth liquid culture medium, enrichment culture is carried out for 18-24 hours under the anaerobic condition at 37 ℃, smear examination is carried out to be pure culture, then the obtained product is inoculated in the MRS broth liquid culture medium for 48 hours according to 10% (V/V), after the culture is finished, the product is centrifuged at 8000X rpm for 5 minutes to collect thalli, and the obtained product is repeatedly washed for 3 times by using physiological saline to resuspend the concentration of the regulatory fungus to 1X 10 8 CFU/mL for standby. 0.2mL of the solution was dipped with a sterile cotton swab and rubbed over the molar teeth of the rat for 15s. During the experiment, the blank group was fed normal feed and drinking water, and the other groups were maintained for 8 weeks by applying caries feed and Streptococcus mutans bacteria liquid to teeth.
(4) The treatment method comprises the following steps: after successful molding, the bacteria solutions of different experimental groups are split-packed into EP pipes (800 mu L/branch) according to the concentration of 1X 10 8 CFU/mL, each rat is respectively used for one pipe, the left hand is used for holding the rat, the right hand is used for dipping the bacteria solution to be saturated, the molar teeth (tooth-engaging surface, cheek tongue surface) and the oral mucosa (cheek mucosa, hard palate and tongue back) of the rat are sequentially cleaned, the rest bacteria solution is injected into the oral cavity of the rat by using a blunt-end injector to rinse the mouth, the bacteria solution is ensured to be in place, the whole process is 1min, the diet is forbidden after 2 hours of treatment, the operation is repeated for 2 times per day (8:00 am, 8:00pm each time), and the treatment is continuously carried out for 5 weeks. At the beginning of the experiment, the body weight of the rats was recorded 2 times a week for the first 2 weeks, and 1 time a week later, while the health status of the rats was recorded by daily observation.
(5) Rat saliva and jaw specimen collection:
① At 64 days, rats were injected with Pi Luoka pieces (0.75 mg/100 g) and 100. Mu.L/mouse stimulating saliva was collected in 1.5mLEP tubes after the saliva spontaneously flowed out.
② At 66 days, the rats are sacrificed under ether anesthesia, the jawbone specimens are taken, washed and dried, soaked in 0.45% ammonium purple urea solution for 12 hours, dyed, taken out, washed clean and naturally dried. The teeth are half cut in the near-far middle direction along the teeth-grinding surface of the upper and lower jawbones by using an ultrathin silicon carbide piece with the thickness of 0.1mm and the diameter of 25mm, and the teeth-grinding surface is cleaned and dried.
(6) And (3) index observation:
① And (3) detecting streptococcus mutans: the kit method is adopted for detecting the streptococcus mutans in the oral cavity.
② Keyes score: the caries inhibition of the drug was assessed by scoring the smooth surface of the molar and caries lesions of the fossa in rats under the microscope according to the Keyes scoring method, which was done blindly by two experimenters (one record Keyes scoring, the other check).
Rat molar Keyes score:
The Keyes score is a measure of caries lesions in rats as individual dental caries lesions and recorded in four ways as probe depth, namely:
the affected enamel is noted as E (enamel caries);
let Ds (dentin shallow caries) refer to enamel dentin boundary to 1/4 of dentin surface;
the involvement of dentin surface 1/4 to dentin surface 3/4 is denoted as Dm (caries in dentin);
Exceeding dentin by 3/4 is noted as Dx (dentin deep caries).
Wherein the class E score reflects caries lesion involvement breadth, and the class Ds, dm/Dx scores reflect caries lesion involvement dentin depth, both together reflecting caries lesion severity. Caries extent unit record combines depth units, shallow unit record contains deep units (for example, the total number of shallow Ds level units contains middle Dm and deep Dx units, because the tooth surface units contained by middle Dm and deep Dx must pass through shallow Ds process), all weight distribution does not use half unit count, experimental result statistics needs to calculate caries reduction rate:
Caries reduction rate= (caries score of negative control group-caries score of experimental group)/caries score of negative control group x 100%.
(7) And (3) judging an experimental result:
the significant reduction in the number of Streptococcus mutans in saliva was effective in the treatment group.
The treatment of the group caries rate is obviously reduced.
5.4 Test results and analysis
5.4.1 Detection of inhibition of Streptococcus mutans in saliva
TABLE 3 Streptococcus mutans quantity in saliva
| Group of | Streptococcus viable count (10 8 CFU/ml) | Antibacterial rate% |
| Group A | 0.6±0.03 | / |
| Group B | 9.3±0.05 | / |
| Group C | 2.4±0.02 | 74.19 |
| Group D | 5.5±0.03 | 40.86 |
| Group E | 6.7±0.02 | 27.96 |
| Group F | 3.1±0.02 | 66.73 |
As shown in Table 3, the number of viable bacteria of Streptococcus mutans was reduced to various degrees after brushing teeth with the same concentration of the bacterial liquid, indicating that all four probiotics have inhibitory effects on Streptococcus mutans. Wherein, the bacteriostasis effect of the bifidobacterium longum DH182 is most obvious, and the bacteriostasis rate reaches 74.19 percent; secondly, lactobacillus rhamnosus DH-Lr-121 has the antibacterial rate of 66.73 percent; lactobacillus paracasei DH068 has a bacteriostasis rate of 40.86%; the antibacterial rate of the bifidobacterium infantis DH231 is 27.96 percent.
5.4.1 Keyes score results
TABLE 4 scoring results on smooth surface Keyes of molar in rats
| Group of | E | Ds | Dm | Dx |
| Group A | 0 | 0 | 0 | 0 |
| Group B | 9.26±0.44* | 8.35±1.44 | 1.25±1.04* | 0.72±0.48 |
| Group C | 4.65±1.24* | 1.25±0.44* | 0.65±0.84* | 0 |
| Group D | 6.25±0.94* | 3.65±1.78 | 0.95±1.12 | 0 |
| Group E | 7.23±0.54* | 5.85±1.65* | 2.05±0.64* | 0.52±0.78* |
| Group F | 5.34±0.75* | 2.55±0.44* | 0.80±0.88* | 0 |
Compared with the model group, has statistical significance, * P is less than 0.05
TABLE 5 scoring results for rat molar fossa caries lesions Keyes
Compared with the model group, has statistical significance, * P is less than 0.05
As can be seen from the experimental data in tables 4 and 5, caries was reduced to various degrees after brushing teeth with the same concentration of bacterial liquid in the oral cavity of the rat. Compared with the caries model group, the enamel caries (E), the dentin shallow caries (Ds) and the dentin medium caries (Dm) of the experimental group have obvious reduction effect, and after the bifidobacterium longum DH182 is treated, the streptococcus mutans number in saliva is obviously reduced, and the caries rate is most obviously reduced, and then the lactobacillus rhamnosus DH-Lr-121, the lactobacillus paracasei DH068 and the bifidobacterium infantis DH231 are further arranged.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. Bifidobacterium longum (Bifidobacterium longum subsp. Longum) with the preservation number of CGMCC No. 13348.
2. A microbial agent or microecological preparation comprising pharmaceutically acceptable excipients and at least one of the following:
i) The viable bifidobacterium longum of claim 1,
II) the viable bacteria of Bifidobacterium longum according to claim 1,
III) the fermentation product of Bifidobacterium longum according to claim 1,
IV), the inactivated bifidobacterium longum product of claim 1.
3. The microbial agent or the microecological preparation according to claim 2, wherein the preparation is at least one of powder, tablet, capsule, aqua, gel, paste, drop pill, pill or granule.
4. Use of bifidobacterium longum as claimed in claim 1 or a microbial agent or a probiotic as claimed in claim 2 or claim 3 in the manufacture of a product for the prevention and treatment of dental caries.
5. The use according to claim 4, wherein the control comprises inhibiting the number of harmful bacteria and/or inhibiting the adhesion of harmful bacteria.
6. The use according to claim 5, wherein the harmful bacteria is streptococcus mutans.
7. The use of claim 4, wherein said controlling comprises reducing the extent of caries.
8. The use according to claim 7, wherein the caries lesions are enamel caries, shallow dentin caries and/or caries in dentin.
9. A caries-controlling product comprising bifidobacterium longum as claimed in claim 1 or a bacterial agent or a probiotic as claimed in claim 2 or claim 3.
10. The product according to claim 9, characterized in that it is a lozenge, a dental paste, a toothpaste or a mouthwash.
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