CN110448578A - It is a kind of for preventing or treating the pharmaceutical composition and food of mouth disease - Google Patents
It is a kind of for preventing or treating the pharmaceutical composition and food of mouth disease Download PDFInfo
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- CN110448578A CN110448578A CN201910535630.1A CN201910535630A CN110448578A CN 110448578 A CN110448578 A CN 110448578A CN 201910535630 A CN201910535630 A CN 201910535630A CN 110448578 A CN110448578 A CN 110448578A
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- saliva
- group
- lactobacillus paracasei
- staphylococcus
- food
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/02—Local antiseptics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Oncology (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of for preventing or treating the pharmaceutical composition and food of mouth disease, includes lactobacillus paracasei L9 and saliva staphylococcus LD11 in described pharmaceutical composition and food.Research shows that L9 and LD11 can significantly alleviate the occurrence degree of E grades of saprodontias of occlusal surface, it takes containing after L9 and LD11, oral cavity flora structure change is significant, the especially significant up-regulation of actinomyces door, and significantly lowered in Klebsiella, Aerococcus, Gemella, corynebacteria and the pseudomonas aeruginosa belonged in categorization levels, reduce the risk of mouth disease.Take L9 and LD11 can saliva species richness significantly lower, the relative abundance of the mouth diseases correlation bacterium such as saliva staphylococcaceae, Actinomy cetaceae, peptostreptococcus section is significantly lowered, and may have relaxation effect to periodontal disease；Plaque flora species diversity is significantly lowered, and the relative abundance of cilium Cordycepps, Fusobacterium section and Flavobacterium section significantly reduces, and may have relaxation effect to mouth diseases such as periodontosis, halitosis, saprodontias.
Description
Technical field
The invention belongs to the fields that prevents, treats of mouth disease, in particular to a kind of for preventing or treating mouth disease
Pharmaceutical composition and food.
Background technique
With the raising of China's quality of residents'life, oral health has become the publilc health that one is concerned and asks
Topic.For a long time, some oral cavity common diseases, such as dental caries, periodontosis and mucosal disease disease incidence rise year by year.Mouth disease is not
The oral health of people is only affected, some systemic diseases can be also caused, and oral cavity pathogen is the pass for causing these diseases
Keyness factor, therefore, control and prevention to pathogenic bacteria in oral cavity, which are sticked, to be had extremely to prevention oral cavity and other systemic diseases
Close important meaning.
Having a large amount of clinical researches proves, probiotics plays very important work in terms of preventing or reducing mouth disease
With.Wherein, Lactobacillus rhamnosus LGG is not only able to suppress the growth of oral cavity pathogen in vitro, while can also colonize in mouth
In chamber, the content of Streptococcus mutans is reduced to reduce the disease incidence of Pediatric Oral Emergency dental caries；Some researches show that by using prebiotic
Bacterium chewable tablets uses the adjuvant of toothpaste with fluoride as preschool child daily, it is possible to reduce the development of child's saprodontia.This research
Used bacterial strain lactobacillus paracasei L9 and saliva staphylococcus LD11 is isolated prebiotic from long lived elder excrement
Bacterial strain has experiments have shown that bacterial strain safety with higher, is not applied to the research in oral cavity also at present.
Summary of the invention
The purpose of the present invention is research lactobacillus paracasei L9 and saliva staphylococcus LD11 in the inhibition of oral cavity pathogen
Adhesion, and lactobacillus paracasei L9 and saliva staphylococcus LD11 are monitored to the adjustment effect of oral cavity flora, to be pre-
Anti- and control mouth disease provides effective measures and theoretical foundation.
The purpose of the present invention is what is be achieved through the following technical solutions:
It is a kind of for preventing or treating the pharmaceutical composition of mouth disease, include lactobacillus paracasei L9 in the composition
Thallus or thalline culture or their extract and saliva staphylococcus LD11 thallus or thalline culture or they
Extract, the deposit number of lactobacillus paracasei L9 is CGMCC No.9800, and the preservation of the saliva staphylococcus LD11 is compiled
Number be CGMCC No.17880.
Further, the lactobacillus paracasei L9 is by inhibiting Streptococcus mutans and Ge Shi is streptococcic sticks and press down
Sucrose dependence in the development process of Dental plaque biofilm processed sticks to prevent or treat mouth disease.
Further, the thallus of lactobacillus paracasei L9 or thalline culture or their extract in the composition
Content is 15wt%-20wt%, in the composition thallus of saliva staphylococcus LD11 or thalline culture or they mention
The content for taking object is 15wt%-20wt%.
Further, the mouth disease includes saprodontia, halitosis and periodontosis.
Further, the lactobacillus paracasei L9 and saliva staphylococcus LD11 is active bacterial strain or deactivation
Bacterial strain.
A kind of thallus or thallus training for preventing the food of mouth disease, in the food comprising lactobacillus paracasei L9
Object or their extract, and the thallus or thalline culture or their extract of saliva staphylococcus LD11 are supported, pair is dry
Lactobacillus paracasei L9 deposit number is CGMCC No.9800, and the deposit number of the saliva staphylococcus LD11 is CGMCC
No.17880。
Further, the thallus of lactobacillus paracasei L9 or thalline culture or their extract contain in the food
Amount is 5wt%-10wt%, the thallus of saliva staphylococcus LD11 or thalline culture or their extract in the food
Content is 5wt%-10wt%.
Further, the lactobacillus paracasei L9 and saliva staphylococcus LD11 is active bacterial strain or deactivation
Bacterial strain.
Biological deposits:
Lactobacillus paracasei L9 (Lactobacillus paracasei) of the invention, was protected on October 22nd, 2014
Ensconce China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
No. 3, Institute of Microorganism, Academia Sinica, postcode: 100101) (depositary institution is abbreviated as CGMCC), deposit number is
CGMCC No.9800。
Saliva staphylococcus LD11 (Streptococcus salivarius) of the invention, in quilt on May 31st, 2019
Preservation, deposit number are CGMCC No.17880.
The present invention having the beneficial effect that compared with prior art
1, the present invention carries out that oral cavity is relevant to grind to the lactobacillus paracasei L9 and saliva staphylococcus LD11 for the first time
Study carefully, by constructing oral cavity model in vitro, establishes the quantitative detecting method of the pathogenic bacteria based on RT-PCR technology, and prove
Lactobacillus paracasei L9 and saliva staphylococcus LD11 significantly suppress Ge Shi streptococcus and Streptococcus mutans in hydroxyapatite
On stick, culture 2,6, after 18h, the amount of sticking of Ge Shi streptococcus and Streptococcus mutans significantly reduces, while secondary cheese
Lactobacillus L9 significantly reduces the thickness and density for the biomembrane that Streptococcus mutans are formed on hydroxyapatite；
2, the present invention utilizes Streptococcus mutans inducing mouse caries model, further probes into lactobacillus paracasei L9 to small
The prevention of mouse dental decayed tooth and adjustment effect to oral cavity flora, result of study are shown: compared with model group, L9 group is significantly alleviated
The occurrence degree of E grades of saprodontias of occlusal surface；By the analysis of the RT-PCR of a variety of cariogenic factors, show the pre- preventing caries function of L9 with
It is related that it inhibits the sucrose dependence in the development process of Streptococcus mutans initially sticked with Dental plaque biofilm to stick；Oral cavity
The 16srDNA high-flux sequence of flora the result shows that, model group oral cavity flora diversity indices is without significant difference, but Bacterial community
It is changed significantly, the especially significant up-regulation of actinomyces door, and the actinomyces of L9 group door is obviously lowered, and is belonging to gram in categorization levels
The primary Pseudomonas of thunder, Aerococcus, Gemella, corynebacteria and pseudomonas aeruginosa are significantly lowered, and the wind of mouth disease is reduced
Danger；
3, influence of the L9 to normal population saliva and plaque flora is detected by the method for high-flux sequence, and examined simultaneously
Survey the situation of change of VSC content in saliva pH and oral cavity.Result of study is shown, after two weeks edible L9 probiotics lozenges, saliva object
Species richness is significantly lowered, the relative abundance of the mouth diseases correlation bacterium such as streptococcus salivarius section, Actinomy cetaceae, peptostreptococcus section
It is significant to lower, there may be relaxation effect to periodontal disease；Plaque flora species diversity is significantly lowered, cilium Cordycepps, shuttle
The relative abundance of Bacteriaceae and Flavobacterium section significantly reduces, and may have alleviation to make to mouth diseases such as periodontosis, halitosis, saprodontias
With.
Detailed description of the invention
Fig. 1 is the standard curve of Streptococcus mutans and format streptococcus quantitative detection；
Fig. 2 is the flow chart of hydroxy-apatite stone model adhesion experiment；
Fig. 3 Streptococcus mutans copy number is with the result of variations figure for sticking the time；Wherein, (a) is prevention group, is (b) treatment
Group；
Fig. 4 is Ge Shi streptococcus copy number with the result of variations figure for sticking the time；Wherein, (a) is prevention group, (b) is to control
Treatment group；
Fig. 5 is that hydroxyapatite disk forms biofilm thickness and its schematic diagram；Wherein, (a) is hydroxyapatite disc thickness
Significance analysis, * indicate that the group and control group have significant difference (P < 0.05)；(b), (c), (d) are respectively control group, L9
The front schematic view of group, LD11 group biomembrane, (e), (f), (g) be respectively control group, L9 group, LD11 group biomembrane side show
It is intended to；
Fig. 6 is cariogenic factor amplified production electrophoretogram；
Fig. 7 is spap, luxs and CiaH gene relative expression quantity column diagram；
Fig. 8 is gtf gene relative expression quantity column diagram；
Fig. 9 is mouse plaque dilution curve figure；
Figure 10 is the relation schematic diagram of core species number and sample size；
Figure 11 is that door horizontal mouse plaque microbiologic population forms thermal map；
Figure 12 is that section (a) and category (b) horizontal mouse plaque microbiologic population form thermal map；
Figure 13 is the horizontal mouse plaque microorganism PCoA figure of OUT；
Figure 14 is to belong to horizontal model group and the analysis of control group mice plaque species difference；
Figure 15 is to belong to horizontal mouse plaque species difference to analyze schematic diagram；
Figure 16 is kind of a horizontal mouse plaque Streptococcus spp variance analysis schematic diagram；
Figure 17 is the multifarious experiment flow figure of human body oral cavity flora；
Figure 18 is the relation schematic diagram of total species number and core species number and sample size；Wherein, a is saliva, and b is tooth bacterium
Spot, test2 (4) indicate that the 2nd (4) are all, abscissa expression sample size, total OUT number that the expression of a1, b1 ordinate detects, a2,
B2 ordinate indicates core OUT number；
Figure 19 is crowd's saliva and plaque dilution curve figure；Wherein, a is saliva, and b is plaque, and abscissa indicates to survey
Sequence amount, ordinate indicate species diversity；
Figure 20 is that the group of saliva and plaque forms column diagram in door level；Wherein, a is saliva, and b is plaque, horizontal
Each door microorganism of coordinate representation proportion in this group of sample, ordinate indicate group；
Figure 21 is the PCoA of saliva and plaque figure in the OUT level based on bray_curtis algorithm；Wherein, a is saliva
Liquid, b are plaque, and horizontal, ordinate indicates that two selected principal coordinate ingredients, percentage indicate that principal coordinate ingredient forms sample
The contribution margin of difference.Horizontal, axis of ordinates scale is relative distance, no practical significance.Two sample points are closer, show two samples
Species composition is more similar；
Figure 22, Figure 23 are that saliva and plaque dominant species form disparity map in section's level；Wherein, * 0.01 < P≤
0.001 P≤0.001 < P≤0.01, * * * 0.05, * *；
In above-mentioned attached drawing, same letter indicates that there was no significant difference (p > 0.05), and it is poor that different alphabets are shown with conspicuousness
Different (p < 0.05).
Specific embodiment
Will be described various embodiments of the present invention below, and cooperate attached drawing as an example, in addition to these be described in detail other than, this
Invention can also be widely performed in other embodiments, and substitution easily, modification, the equivalence changes of any embodiment all include
Within the scope of the invention, and it is subject to claim.
Lactobacillus paracasei L9 is isolated from Bama of Guangxi long lived elder enteron aisle, using lactic acid bacteria MRS broth bouillon into
Row culture.Saliva staphylococcus LD11 is cultivated using standard streptococcus lactis M17 broth bouillon.With 20% skimmed milk shape
Formula is stored in -20 DEG C, takes 200 μ l to cultivate 12h in 37 DEG C of constant incubators in corresponding culture medium, and pass on.
Embodiment 1 --- lactobacillus paracasei L9 and saliva staphylococcus LD11 sticks the external inhibition of oral cavity pathogen
By quantitative PCR filter out can specific amplification Streptococcus mutans and Ge Shi streptococcus, but cannot expand secondary dry
The specific primer of Lactobacillus paracasei --- forward primer (5 ' -3 ') sequence: GCCTACAGCTCAGAGATGCTATTCT reversely draws
Object (5 ' -3 ') sequence: GCCATACACCACTCATGAATTGA.The reaction system and reaction condition of primer are as follows: 2.5 μ l DNA,
10 μ l Taq enzymes, 0.5 μ l upstream primer, 0.5 μ l downstream primer, 6.5 μ l ultrapure waters；95 DEG C of initial denaturation 2min；95 DEG C of denaturation
5s, anneal at 60 DEG C of annealing temperature 60s, 72 DEG C of extension 30s, 40 circulations；72 DEG C of 5 min of extension.
Quantitative pcr amplification is carried out to pathogenic bacteria using above-mentioned specific primer, product is subjected to agarose gel electrophoresis, is cut
Glue simultaneously using plastic recovery kit recycle DNA, measure standard DNA concentration and carry out 10 times gradient dilution 8 times, in this, as template
Quantitative PCR is carried out, finally using the logarithm of the copy Particle density of DNA in each gradient dilution liquid as abscissa, using Ct value as ordinate,
Standard curve is made, as shown in Figure 1, recurring number Ct value is as y-axis using DNA copy number log concentration value as x-axis.(a) it makes a variation
It is y=-3.5581x+58.998, R that streptococcus, which expands standard curve,2=0.9982；(b) Ge Shi streptococcus amplification standard curve is
Y=-3.693x+60.528, R2=0.9977.Pass through R2> 0.99 judges, can be used for Streptococcus mutans and Ge Shi is streptococcic fixed
Amount detection.
For the inhibition oral cavity pathogen Adhesion property for verifying lactobacillus paracasei L9 and saliva staphylococcus LD11, carry out
It detects below:
Hydroxy-apatite stone model adhesion experiment:
(1) saliva pre-processes
Saliva collection selects 10 24-40 years old healthy volunteers, collects saliva.Health adult is after feed 2 hours
It is gargled with clear water, collects nonirritant saliva (mouth containing swab stick 10 minutes) using Salivette saliva collection pipe, be collected into
Saliva takes supernatant at 4 DEG C using low-temperature and high-speed centrifuge immediately with the revolving speed centrifugation 15min of 5500r/min, with 0.22 μm
The saliva part of filtering with microporous membrane degerming, collection is mixed with the potassium chloride buffer 1: 1 of 0.1mol/L, for constructing saliva
Coated hydroxyapatite model, is partially mixed for biofilm formation with PBS 1: 1.Be stored in -20 DEG C it is spare, deposit for a long time
It is put in -80 DEG C.Volunteer requires as follows:
Without smoking history, does not feed, drinks water, drinks in 1 hour before collection；Not just it is sick, without
It takes drugs, does not especially take antibiotics in January；Yoghourt, probiotic beverage are not eaten in three days；Without dental caries, periodontal
The oral diseases such as disease.
(2) as shown in Fig. 2, precise 10mg hydroxyapatite (HA) pearl is in 24 well culture plates, every hole adds for operation
Enter soaked overnight in 200 μ l potassium chloride buffers, next day sucks buffer and the 100 processed salivas of μ l are added, at room temperature 6r/
Saliva is sucked after min rotation 1h, 200 μ l buffers wash twice and blot buffer, and the 100 μ l egg of cow's serum containing 5mg/ml is added
The potassium chloride buffer of white (BSA), 6r/min is blotted after rotating 30min at room temperature, and potassium chloride buffer washes twice, that is, makes
At the hydroxyapatite model of saliva coated.
The second generation bacterium room temperature 2500r/min of passage is centrifuged 15min, collects bacterium, and potassium chloride buffer washes bacterium twice, hangs
Float in the potassium chloride buffer containing 5mg/ml bovine serum albumin, it is 10 that bacteria concentration, which is made,7The bacteria suspension of cfu/ml is spare.
Experiment is divided into two groups of prevention groups and treatment group: prevention group is caused a disease to be added after 125 μ l turning effort 1h of LD11 suspension to be added
125 μ l of bacterium suspension；Treatment group is that 125 μ l of LD11 suspension is added after pathogenic 125 μ l turning effort 1h of bacterium suspension is added；It is empty
White control group then replaces LD11 suspension with the potassium chloride buffer of the bovine serum albumin containing 5mg/ml.Room temperature 6r/min rotation 2,6,
18h, potassium chloride buffer wash twice, collect hydroxyapatite pearl carry out quantitative PCR, collect buffer measure pH, every group three
Secondary repetition.
Lactobacillus paracasei L9 and saliva staphylococcus LD11 to the Streptococcus mutans result sticked of prevention as shown in figure 3,
Compared with the control group, for two probiotics after 2h and 6h is added, Streptococcus mutans content significantly reduces (P < 0.05), sticks
LD11 group and L9 group reduce 10.40% (0.36 × 10 respectively when 2h9) and 26.88% (0.93 × 10 copies9Copies),
6h reduces 61.61% (2.76 × 10 respectively9) and 33.26 % (1.49 × 10 copies9Copies), two plants of bacterium are to variation chain
The inhibiting effect of coccus be not present significant difference (P > 0.05), act on 18h after, the Streptococcus mutans content of LD11 group with it is right
According to group there was no significant difference (P > 0.05), and be added Streptococcus mutans in the experimental group of L9 content (2.46 ×
109Copies) it is substantially less than control group (4.26 × 109) and LD11 group (4.09 × 10 copies9Copies) (P < 0.05)；And
In treatment group, LD11 inhibits not significant (P > 0.05) to it, and the amount of sticking is all larger than 5 × 109Copies, L9 is being acted on instead
The content (P > 0.05) that Streptococcus mutans are significantly suppressed after 2h, reduces 51.55% (3.0 × 109copies)。
Lactobacillus paracasei L9 and saliva staphylococcus LD11 shows Ge Shi streptococcus suppression result such as Fig. 4, and benefit is not added
The dagger-axe formula hammer bacterial content of the control group of raw bacterium compared to be added two probiotics and dagger-axe formula streptococcus interaction 2,6, after 18h,
The Ge Shi streptococcus amount of sticking significantly reduces (P < 0.05), and wherein saliva staphylococcus LD11 reduces 27.62% (1.16 respectively
×109Copies), 74.79% (4.48 × 109Copies), 45.25 % (2.72 × 109Copies), lactobacillus paracasei L9
51.19% (2.15 × 10 is reduced respectively9Copies), 56.09% (3.36 × 109Copies), 58.40% (3.51 ×
109Copies), sufficiently demonstrate LD11 and L9 and significant prevention adhesion is all had to dagger-axe formula streptococcus.In interaction 2h
When, with positive control LD11 group (3.04 × 109Copies it) compares, lactobacillus paracasei L9 inhibition streptococcic to dagger-axe formula is made
With saliva staphylococcus LD11 (P < 0.05) is significantly higher than, Ge Shi streptococcus is reduced to 2.05 × 109copies.And in 6h
When the L9 group Ge Shi streptococcus amount of sticking (2.63 × 109Copies) it is significantly higher than LD11 group (1.51 × 109Copies), 18h two
There was no significant difference (P > 0.05) for the inhibition adhesion of group probiotic group；In treatment group such as Fig. 4 (b), control group with it is prebiotic
The Ge Shi streptococcus of bacterium L9 and LD11 group sticks result there are no significant difference (P > 0.05).
Probiotics is added successively affects sticking for pathogenic bacteria, should be the result shows that the suppression that L9 and LD11 are played in the oral cavity
Production is simple non-specific steric hindrance with not being, and being likely to L9 and LD11 can be with pathogenic bacteria competitive binding saliva
Protein receptor on liquid Acquired Pellicle, to reduce the amount of sticking of pathogenic bacteria.
Confocal laser microscope detects biomembrane:
Stick the result of study of model based on the above hydroxyapatite, carries out L9, LD11 and inhibit streptococcus mutans biomembrane shape
At experiment.Bacterium 37 DEG C of two generations of inoculation to growth logarithmic phase 4500r centrifugation 10min abandon supernatant, PBS (4 DEG C, 4500g,
It 10min) washs 2 times, is suspended in artificial saliva's culture medium containing 0.2% sucrose, it is 10 that bacteria concentration, which is made,7The bacterium of cfu/ml
Suspension is spare.Autoclaved disk is placed in the hole of 24 hole tissue culturing plates that (every Kong Zhongyi disk, disk can not contact holes
Wall), 37 DEG C are coated 4h with above-mentioned saliva (hole 1.5ml/), and later, 1.5ml PBS is rinsed 2 times.Bacterial suspension each 750 is added
μ l forms biomembrane in hydroxyapatite disc surfaces culture for 24 hours.
Disk is successively flushed three times with the sterile PBS of 1.5mL (soaking time rinsed every time, 10s), it is nonadherent to remove
Bacterium；Use the TCS SP2 Laser Scanning Confocal Microscope for the 488nm Ar/Ar-Kr laser scanning head being mounted on platform without friction
Carry out non-intrusion type co-focusing imaging.The object lens used are 20 ×, the amplification of image three times；Sample, which carries out LIVE/DEAD dyeing, to be made
WithBacterial action kit.Dyeing time is 9 ± 1min, and colored proportion 1: 1 is obtained in respective wavelength
(Syto9:515-530nm；Propidium iodide (PI): > 600nm) under best fluorescence signal；Random selection is covered with biomembrane
At least three independent and representative positions carry out these measurements (based on the stacking identified in being copolymerized burnt visual field on disk
Or " tower ").In each region, the thickness of most thick observation point is measured by determining top layer and the lowest level of biomembrane.
CLSM software is arranged to the z-axis scanning (8,1024 × 1024 pixels) using 1 μ m thick.In order to quantify the life in biomembrane
Object amount carries out analysis using NIS Viewer software program and is copolymerized burnt microphoto, the fluorescence of every kind of fluorescence color of manual setting
Intensity threshold.
Mature Dental plaque biofilm is the principal element for causing mouth disease to occur, from Fig. 5 (a) as can be seen that with
Control group is compared, and probiotic group (L9 and LD11) significantly reduces (P < in the biofilm thickness that hydroxyapatite surface is formed
0.05), and LD11 and the biofilm thickness of L9 group do not have significant difference.The average thickness of control group be 16.33 μm, L9 and
LD11 group average thickness is respectively 12.63 μm and 13.13 μm.Fig. 5 (b, c, d) shows that control group bacterium living beings film density is obviously high
In the experimental group that two kinds of probiotics are added, bacterium is layer upon layer of intensively at large stretch of cloud form, compares aggregation, arrangement is close, in the visual field
Gap range very little.And the experimental group bacterial clump that L9 and LD11 is added relatively disperses, black dull gap range is very big, bacterium point
Cloth is messy, and especially L9 group bacterium is presented fragmentary dotted, smaller than the bacterial density that LD11 group is formed.Therefore, secondary cheese cream bar
Bacterium L9 and saliva staphylococcus LD11 can reduce the generation of Dental plaque biofilm by inhibiting Streptococcus mutans sticking, from
And prevent the generation of mouth disease.
Embodiment 2 --- the influence of lactobacillus paracasei L9 and saliva staphylococcus LD11 to murine oral bacterial diversity
The Streptococcus mutans 1.2499 of -20 DEG C of preservations, lactobacillus paracasei L9 and saliva staphylococcus LD11 are existed respectively
37 DEG C of activation culture three generations in BHI, MRS and M17 broth bouillon of 10ml, 4500r/min remove supernatant after being centrifuged 10min
Liquid, Streptococcus mutans sterile saline redissolve bacterium mud, and lactobacillus paracasei L9 and saliva staphylococcus LD11 sucrose are molten
Liquid redissolves, adjusting bacterial concentration to 108Cfu/ml, 4 DEG C save backup.
Murine oral saprodontia modeling method:
The animal model of the present embodiment has selected purchase total from male SPF grades of BALB/c mouse of 3 week old of dimension tonneau China
28, every group 7, it is divided into control group, model group, lactobacillus paracasei L9 group and saliva staphylococcus LD11 group totally 4 groups.Purchase
Four groups are randomly divided into after the laundering period that the SPF grade mouse bought first passes around one week, mouse feeding sterile water and general during adaptation
Logical aseptic feed.Later, the sucrose solution and common aseptic feed of 10 % of control group mice feeding；Model group mouse feeding
10% sucrose solution and cariogenic feed, while continuous 4 days inoculation Streptococcus mutans, daily inoculation 1 time, inoculum concentration 1ml/
It is secondary, cleaning murine oral first is wiped with sterile PBS before inoculation；The mouse feeding of probiotic group contains the 10% of corresponding probiotics
Sucrose solution and cariogenic feed, inoculation are inoculated with probiotics 4 days after pathogenic bacteria 1 week, daily inoculation 1 time, inoculum concentration 108cfu/
It is secondary, all mouse are put to death after feeding 4w, 2h fasting before putting to death, with each position in oral cavity of aseptic cotton stick swab friction mouse after execution
Swab is cut and is placed in equipped with 1ml nuclease protection after acquiring mouse plaque sample by (including upper lower teeth, tongue, gum etc.)
In the centrifuge tube of liquid, -20 DEG C of preservations.
The quantitative detection of the cariogenic factor
Filter out 7 kinds of common cariogenic factors and its specific primer by searching for document, primer sequence as shown in table 1,
Using RecA gene as reference gene, target gene is normalized to the comparison of identical copies number using the internal reference, it is cariogenic to 7 kinds
The factor carries out relative quantification detection, and amplification system is that 20 μ l include: 2.5 μ l DNA templates, 0.5 upstream and downstream μ l primer, 10 μ l
SYBR enzyme and the sterile no enzyme water of 6.5 μ l carry out 40 times by 95 DEG C of initial denaturations 3min, 95 DEG C of 10s, 53 DEG C of 30s and 72 DEG C of 20s
Circulation.
1 primer sequence table of table
The amplified production of 7 target gene is subjected to agarose gel electrophoresis, as a result as shown in fig. 6, each primer pair is corresponding
Gene all has good specificity, can be used for the quantitative detection of each gene.
Quantitative result to the cariogenic factor in mouse plaque is as shown in fig. 7, it can be seen from the figure that with control group phase
Than, the relative expression quantity of spap, luxs, ciaH in model group are significantly increased (p < 0.05), lactobacillus paracasei L9 group and
The expression of spap and ciaH has had been lowered to the expression of control group in saliva staphylococcus LD11 group, luxs gene
Though expression quantity substantially less than model group (p < 0.05), is compared, relative expression quantity still increases, especially L9 than control group
Still there were significant differences for the luxs relative expression quantity and control group of group.
As can be seen from Figure 8, compared with the control group, the relative expression quantity of gtfB, gtfD in model group significantly mention
High (p < 0.05), especially gtfB, though two kinds of genes are in the expression quantity of lactobacillus paracasei L9 and saliva staphylococcus LD11 group
It increases than control group, but its relative expression quantity is substantially less than model group (p < 0.05), only the gtfB of LD11 group
Still there were significant differences with control group for expression.In addition, not significant (the p > of the relative expression quantity difference of the gtfC between four groups
0.05).This result and the conclusion of Wu Dicong et al. are slightly different, theirs the result shows that Streptococcus mutans 3 kinds of gtf genes
Expression quantity chronic up-regulation in saprodontia forming process.The soluble sugar that the reduction of gtfD expression quantity may be such that GTF-D synthesizes is reduced,
To reduce the offer of GTF-B metabolism substrate.So the reduction of gtfB, D gene relative expression quantity shows lactobacillus paracasei
L9 significantly suppresses the formation and development of Dental plaque biofilm.
Then carry out PCR and high-flux sequence:
(1) DNA of bacteria extracts
Using Qiagen DNA of bacteria extracts kit according to the bacterial genomes for illustrating to extract mouse plaque of manufacturer
DNA.DNA concentration is detected using 6 fluorometric quantification of Qubit R, and estimates molecular size using agarose gel electrophoresis.
(2) PCR amplification
For the accuracy and reliability for guaranteeing subsequent data analysis, two conditions need to be met: 1) using low circulation as far as possible
Number amplification；2) guarantee that the recurring number of each sample amplification is consistent.It randomly selects representative sample and carries out preliminary experiment, it is ensured that
Most samples are enable to amplify the suitable product of concentration in minimum recurring number.
16s rRNA is with primer B341F (5 '-CCTACGGGNGGCWGCAG-3 ') and B785R (5 '-
GACTACHVGGGTATCTAATCC-3 ') PCR amplification is carried out to the region general target V3-V4, target fragment length is 450bp and carries out
Annealing.Temperature is 55 DEG C.In first round PCR amplification, the reaction that 10ng template DNA is added to final 25 μ L volume is mixed
In object, contain 0.25 every kind of primer of μ L (25 μM), 12.5 Mix and PCR grades of Ready of HiFi HotStart of μ L2 × KAPA
Sterile no enzyme water.Cycling condition are as follows: in 95 DEG C of preincubate 3min, 25 circulations, 30s is denaturalized at 95 DEG C, at 55 DEG C
30s anneals, and 30s is carried out at 72 DEG C and is extended, and then carries out finally extending 5min at 72 DEG C and is maintained at 4 DEG C.
2 μ LPCR products are detected to detect target segment by 2% agarose gel electrophoresis, and use 1X AMPure XP Beads
(Beckman Coulter, Inc.) purifying.In second of PCR, the 2.5 above-mentioned PCR products of μ L are added to final 25 μ L body
In long-pending reaction mixture, wherein containing 0.25 every kind of primer of μ L (25 μM), 12.5 μ L 2 × KAPA HiFi HotStart
The sterile no enzyme water of Ready Mix and PCR grades of 9.5 μ l.PCR reaction is carried out to each sample in T100TM thermal cycler as follows:
Then 95 DEG C of initial denaturations 3min, 95 DEG C of 30s, 55 DEG C of 30s and 72 DEG C of 30s progress, 8 circulations finally extend at 72 DEG C
5min is simultaneously maintained at 4 DEG C.Merge the PCR reaction from same sample, separated using 2% Ago-Gel and extract progress and is pure
Change.
(3) fluorescent quantitative PCR
Target fragments are recycled by using QIAquick gel extraction kit, and use the library KAPA quantification kit
Quantitative detection.
(4) Miseq library construction
Illumina official joint sequence is added to target area outer end by PCR；It is cut using gel reclaims kit
Glue recycles PCR product；The elution of Tris-HCl buffer, the detection of 2% agarose electrophoresis；Sodium hydroxide denaturation, generates single stranded DNA piece
Section.
(5) Miseq is sequenced
One end of DNA fragmentation is complementary with primer base, is fixed on chip；Using DNA fragmentation as template, fixed on chip
Base sequence is that primer carries out PCR synthesis, and target DNA fragmentation to be measured is synthesized on chip；After denaturation, annealing, DNA piece on chip
The other end of section is also fixed at random with another neighbouring Primers complementary, is formed " bridge "；PCR amplification generates DNA cluster；
The chemical conversion of DNA cloning sub-line is single-stranded.The archaeal dna polymerase being transformed and the dNTP with 4 kinds of fluorescent markers is added, recycles every time
Only synthesize a base；Plate surface is reacted with laser scanning, every template sequence first round is read and reacts the core polymerizeing up
Thuja acid type；By " fluorophor " and " terminating group " chemical cleavage, restores 3 ' end viscosity, continue to polymerize second nucleotide；
Every fluorescence signal being collected into of taking turns is counted as a result, knowing the sequence of template DNA segment.
High-flux sequence result:
Library is mixed, after denaturation, is added to Illumina MiSeq microarray dataset and carries out high-flux parallel sequencing.Through
16s rDNA sequencing, obtains optimization totally 1364420 in 28 mouse plaque samples, and average sequence length is
449.13；
The above-mentioned sequence of acquisition is subjected to OUT cluster, take out flat, Copolymer to 1 domain 1 by smallest sample sequence number
32, a boundary, 70 guiding principles of door, 135 Ge Mu228Ge section 404 belongs to 549 kinds of 781 OUT, and wherein control group detects 424
OUT, caries model group detect 346 OUT, and lactobacillus paracasei treatment group detects 327 OUT.Compared with the control group, mould
Total OUT number that type group detects reduces, and lactobacillus paracasei group seems that the OUT number detected is closer to model group.
As can be seen from Figure 9 as the aromatic index of increase (Shannon index) of sample sequencing amount is substantially equal to
Balance, shows that the sample sequencing amount of the present embodiment is enough, coverage is all larger than 0.99, representative to sample full sequence.
The core species number detected in Figure 10 gradually tends towards stability with the increase of sample size, shows the sample size foot of this research detection
It is enough.
Clustering mouse plaque flora classification by OTU is that 5 bacterium doors are as shown in figure 11, is Firmicutes respectively
(Firmicutes), Proteobacteria (Proteobacteria), actinomyces door (Actinobacteria), without wall bacterium door
(Tenericutes) and Bacteroidetes (Bacteroidetes), wherein Firmicutes and Proteobacteria are dominant bacteria door；With it is right
It is compared according to group, the obvious up-regulation of actinomyces door in model group, and the closer control group of L9 group, LD11 group group form with model group more
Close, actinomyces relative abundance is in higher level；The species composition of L9 and LD11 group and control group are closer, with model group
It differs greatly.
In section and belong to horizontal upper each group plaque group composition thermal map such as Figure 12, model group can be clearly seen that from figure
Middle Corynebacteriaceae (Corynebacteriaceae) and corynebacterium (Corynebacterium_1) are apparently higher than control
Group is horizontal, and LD11 processing group level changes unobvious compared with model group, but L9 processing group has significantly turned down the relatively rich of them
Degree, is on close level with control group.It can be seen that the Bifidobacterium (Bifidobacterium) of model group is relatively rich in Figure 12 (b)
Degree increased than control group, and the Bifidobacterium up-regulation effect of LD11 group is even more than model group, and different is L9 group
The relative abundance level and control group of Bifidobacterium maintain an equal level.The result of study of Lisi T et al. shows, periodontal disease and chronic resistance
There is significant correlations for plug property tuberculosis, meanwhile, pseudomonas aeruginosa recall rate is increased compared with the control group in disease group
Add.Figure 12 shows P. aeruginosa Cordycepps (Pseudomonas) and P. aeruginosa Pseudomonas (Pseudomonas) in model group
There is apparent up-regulation, and the bacterium of LD11 and L9 group is obviously restored to control group level.The addition of lactobacillus paracasei L9 makes
Bacillus acidi lactici obviously increases, and pseudomonas aeruginosa significantly reduces, and certain prevention effect is played to periodontosis.
Figure 13 is the PCoA analysis based on bray-curtis algorithm, and section (a) is shown in figure and belongs to (b) horizontal upper model
There were significant differences for group and control group group composition；LD11, L9 group and model group sample group form significant difference, especially in PC1
(38.34%) on axis, while LD11 group sample can be also clearly separated in figure with L9 group.Lactobacillus paracasei L9 significantly has adjusted
Plaque Bacterial community.
As shown in figure 14, on belonging to level, compared with the control group；Staphylococcus in model group murine oral
(Staphylococcus), Klebsiella (Klebsiella) and Aerococcus (Aerococcus) relative abundance are significantly high
In control group；Lactobacillus paracasei L9 significantly reduces the relative abundance of Klebsiella and Aerococcus, to reduce trouble
The risk of the diseases such as dental caries and septicemia.
Streptococcus oralis (Streptococcus) is the flora in normal oral, LD11 and L9 group hammer in Figure 15
Pseudomonas dramatically increases, especially lactobacillus paracasei L9, while detecting that increased hammer strain is not as seen from Figure 16
Oral cavity pathogen belongs to the normal streptococcus in oral cavity, and we have found that saliva staphylococcus (Streptococcus_
Salivarius_subsp_salivarius relative abundance) is apparently higher than control group and model group in LD11 and L9 group,
L9 group has been even more than saliva staphylococcus LD11 group, and bacteriocin caused by saliva staphylococcus, which is able to suppress, some leads to mouth
The growth of smelly bacterium, therefore L9 and LD11 have the function of alleviating halitosis.Figure 15 shows pasteurella (Pasteurella)
It is significantly reduced in the relative abundance of probiotic group, especially L9 group, it appears that antithesis with streptococcus variation.The addition of L9 and LD11
Considerably reduce the relative abundance of Gemella.The reduction of L9 group saliva staphylococcus and Gemella illustrates lactobacillus paracasei L9
Have the function of alleviating halitosis and mouth neoplasm.
Embodiment 3 --- influence of the lactobacillus paracasei L9 to human mouth bacterial diversity
Carry out personnel recruitment:
(1) it is included in standard
1. the subject of no active dental caries
2. without any systemic disease
3. without the subject of any prevention dental treatment history
(2) exclusion criteria
1. receiving the people of antibiotic treatment in the course of the research
2. in the course of the research, using the people of any other Probiotic supplement (Yoghourt or other tonics containing probiotics)
3. experiment start before 1 week and in the course of the research using xylitol products subject
4. with the subject of intraoral operation history in 6 months before experiment starts
5. smoking at present or experiment start the previous year smoker
(3) requirement during testing to subject
It is required that subject respectively in morning (before 9:00) (11:00-13:00) evening (after 17:00) one after each meal one
Probiotics tablets, subject, which need to contain to chew again after at least 2-3min, to swallow, and to avoid Unilateral chew；It brushes the teeth twice daily, respectively
Once in the morning and once at night, it can not brush teeth at least 2h after taking.
As shown in figure 17 filters out 15 volunteers according to above-mentioned standard, and (period is not edible after 1 week emptying phase
With any xylitol products or prebiotic replenishers), collect the saliva of subject, plaque is mentioned using DNA of bacteria extracts kit
It takes DNA, carries out 16srDNA sequencing, and measure the VSC in implication (volatile sulfur compounds) and saliva pH, as baseline index, it
Subject eats probiotics lozenge (L9 containing lactobacillus paracasei and saliva staphylococcus LD11 10 as required in 4 weeks afterwards9cfu/
Piece), it collects sample again after two weeks and be sequenced simultaneously testing index.
Sampling method:
(1) saliva (sample time: before early 9:00)
The swab stick 2min that saliva acquisition requires to chew after gargling in saliva collection pipe is completely wet to swab stick, to avoid inclined side
Chewing, two sides chew time will be averaged, be placed in centrifuge tube, deposit in -20 DEG C for extracting DNA；
(2) plaque (sample time: before early 9:00)
With buccal swab, other are not easy clearly on corona, the gap between teeth, gum, gingival sulcus, oral pocket position and tooth when sampling
The positions such as clean nest ditch, crack, adjacent surface, cavity surface rub back and forth puts 4 for swab after 1min and returns in swab pipe, and
It is immersed in 1h in RNA later and deposits in 4 DEG C for extracting DNA；
(3) implication value measurement (sample time: before early 10:30)
Subject's detection does not use armaticity beverage, the lipstick of obvious smell or lip gloss in first 24 hours and other are gargled
Water, the higher food of the sulfur-bearings such as edible garlic, leek, radish, does not brush teeth from morning, does not gargle, does not feed.
The implication value that record every patient is detected using Hialmeter instrument, is first returned to zero with air, subject is silent using preceding
After 3min, the intracavitary about 4cm of the air collecting pipe insert port of Hialmeter is placed in back at 1/3 top 0.5cm, detection process
Middle mouth keeps pico- and opens, and breathes with the nose, and when numerical value rises to peak value and begins to decline, removes suction pipe, record volatility vulcanization
Object peak concentration.Detection 3 times is repeated, results are averaged.
PCR and high-flux sequence are carried out according to identical method described in embodiment 2.
High-flux sequence result:
Library is mixed, after denaturation, is added to Illumina MiSeq microarray dataset and carries out high-flux parallel sequencing.Through
16s sequencing, obtains the sequence of 2946563 high quality, average sequence length 449.12,48 altogether in 48 saliva samples
The sequence of 2088899 high quality, average sequence length 459.44 are obtained in a plaque sample altogether.
The above-mentioned sequence of acquisition is subjected to OUT cluster, takes out and puts down by smallest sample sequence number, 1 is detected in saliva sample
1, domain, 23, boundary door, 43 guiding principles, 102 Ge Mu180Ge section, 442 categories, 786 kinds of 1125 OUT, wherein 0 week detects 941
OUT detects 766 OUT on the 2nd week, detects 808 OUT within the 2nd week；1,1 domain, 14, boundary is detected in plaque sample
Door 27 45 Ge Mu77Ge sections of guiding principle, 166 categories, 334 kinds of 643 OUT detect 589 on the 2nd week wherein measuring within 0 week 415 OUT
A OUT detects 549 OUT on the 4th week, eats the probiotics tablets (staphylococcus of saliva containing LD11 LD11 109Cfu/ piece) after 2 weeks
Saliva sample OUT number reduces, and several litres of OUT of plaque sample high, all in all, compared to saliva sample, plaque sample
Richness wants low.
The increase with detection sample size is shown in Figure 18, and the horizontal and shared OUT level of total OUT is not further added by, says
Bright experiment saliva, plaque sample size are all sufficient, and the total OUT of saliva is reduced after edible probiotics tablets, and plaque is total
OUT number obviously rises.It can be seen that increase saliva and the plaque sample OUT water with sample sequencing amount from Figure 19 dilution curve
Flat aromatic index (shannon) tends to balance, and no longer rises, i.e., species diversity is not further added by, it was demonstrated that this crowd experiment
Sample sequencing amount is enough.
From table 2 it can be seen that compared with the control group, group's diversity index of the 2nd week saliva sample (sobs, ace,
Chao) extremely significant downward, group's richness reduces, and other diversity index are without significant changes；Table 3 shows edible probiotics
After piece 2,4w, plaque community diversity index (Shannon) is significantly lowered, and Simpson index significantly raises, group's multiplicity
Property reduce；The significant up-regulation of group's diversity index (ace, chao), group's richness increase；Saliva, the covering of plaque sample
Degree (coverage) is all larger than 0.99 close to 1, it was demonstrated that this sequencing result is able to reflect the truth of microorganism in sample, tool
It is representative.
2 saliva sample alpha diversity indices table of table
Note: 0.001 P≤0.001 < P≤0.01, * * * * 0.01 < P≤0.05, * *
3 plaque sample alpha diversity indices table of table
Note: 0.001 P≤0.001 < P≤0.01, * * * * 0.01 < P≤0.05, * *
If the microorganism detected in Figure 20 a saliva sample mainly includes 8 doors, Firmicutes (Firmicutes) become
Shape bacterium door (Proteobacteria), burgdorferi strain door (Saccharibacteria), Bacteroidetes (Bacteroidetes),
Actinomyces door (Actinobacteria), SR1, Cyanophyta (Cyanobacteria), Fusobacterium door (Fusobacteria), separately
It is not annotated into there are one outer, wherein the 0th and 4 week Firmicutes are dominant bacteria door, and the 2nd week Proteobacteria significantly raises
As dominant bacteria, burgdorferi strain door is remarkably decreased, and furthermore Bacteroidetes relative abundance gradually increases；Plaque sample is (as schemed
6 doors, Firmicutes (Firmicutes), Proteobacteria (Proteobacteria), Bacteroidetes are detected in 20b)
(Bacteroidetes), actinomyces door (Actinobacteria), Fusobacterium door (Fusobacteria), burgdorferi strain door
(Saccharibacteria), wherein Firmicutes are dominant bacteria door, and actinomyces door accounting obviously increases, and Bacteroidetes is obvious
It is few.
Saliva and plaque sample PCoA Figure 21 show, whether saliva or plaque, and the 2nd, 4 week sample and 0 week exist
It is obviously separated in figure, shows to have significantly affected the distribution of oral microbial community after taking in probiotics tablets.Saliva PCoA figure
In the 2nd week sample and 0 week it is slightly close, the 4th week is then far apart with 0 week sample, and wherein saliva PC1 and PC2 explanation degree divide
Not Wei 40.87% and 9.76%, while it can be seen that the distribution of the 0th week saliva sample is more loose in figure, and process LD11 saliva
After staphylococcus LD11 bacterium piece is adjusted, sample distribution aggregation in the 2nd, 4 week；The the 2nd, 4 week plaque sample is several in plaque PCoA figure
It can not separate, be only significantly separated with 0 week sample, PC1 and PC2 explanation degree is respectively 18% and 14.56%.
The advantage Cordycepps detected in saliva sample totally 28 kinds such as Figure 22,25 in addition to Wei Rong bacterium, Neisseria, actinomyces
A section has the variation of significant difference, especially the 2nd week the most significant, cud Cordycepps, Halomonas section, leaf nodule Cordycepps, quasi-
Bacteriaceae and the fine extremely significant rising of Cordycepps relative abundance, streptococcus, Family_XIII, Fusobacterium section, peptostreptococcus and
Three kinds of section's relative abundances not annotated are remarkably decreased, the variation of the 4th week saliva and the 2nd week it is not consistent, melaninogenicus,
Purple monad, campylobacter, pasteurella, Fusobacterium are significantly raised, and such result occur may be due to salivary flow
Property it is larger, microorganism in saliva updates very fast, and L9 is difficult in saliva to stop for a long time, is only capable of in a short time playing saliva
To adjustment effect, therefore maintain the effect time shorter；The advantage Cordycepps detected in plaque sample totally 16 such as Figure 23, chain
Coccus is most advantage Cordycepps, and the 2nd, 4 week sample microorganism group be at close, in addition to Actinomy cetaceae significantly raises, cilium Cordycepps, purple
Monad, Lachnospira section, Fusobacterium section, Flavobacterium section relative abundance significantly reduce.In addition, Wei Rong bacterium, melaninogenicus, two
Carbonoxide Cytophaga was significantly reduced in significant decrease in the 2nd week and Neisseria and corynebacteria at the 4th week.
Saliva, plaque sample coverage are high, and sequencing result is able to reflect the truth of microorganism in sample, have generation
Table.The reduction of saliva sample group richness and plaque community diversity illustrates edible L9 probiotics tablets to oral cavity flora
There is positive adjustment.Our result of study shows that streptococcus seems the synchronous increase in patch with the relative abundance of actinomyces
And it is reduced in saliva.This variation may be related with bacterial adhesion.
In summary:
(1) after short-term (2w) eats L9 probiotics lozenge, saliva species richness is significantly lowered, and species composition occurs significant
Variation, Proteobacteria, which significantly raises, becomes dominant bacteria, and burgdorferi strain door is remarkably decreased, but the 4th circumferential original level is replied, this
Outer Bacteroidetes relative abundance gradually increases at any time；
(2) after eating L9 probiotics lozenge, plaque flora species diversity is significantly lowered, and richness significantly raises, object
Kind composition is changed significantly, and actinomyces door accounting obviously increases, and Bacteroidetes significantly reduces, and the 2nd week and species composition in the 4th week
Without significant difference.In section's categorization levels, after edible mushroom piece in addition to actinomyces, cilium bacterium, purple monad, Lachnospira, Fusobacterium, Huang
Bacteriaceae, Wei Rong bacterium, melaninogenicus, carbon dioxide Cytophaga, Neisseria and corynebacteria relative abundance significantly reduce；
(3) use of lactobacillus paracasei L9 probiotics lozenge does not show the VSC content in crowd oral cavity and saliva pH influence
It writes, but (2w) can reduce pH of saliva and VSC content in a short time.
Embodiment 4
It present embodiments provides a kind of for preventing or treating the pharmaceutical composition of mouth disease, includes in the composition
The thallus of lactobacillus paracasei L9 and saliva staphylococcus LD11.The thallus is active thallus, the bacterium in composition
The content of body is 16wt%, and the quantity of lactobacillus paracasei L9 is 106CFU or more, preferably, the quantity of lactobacillus paracasei
It is 1010CFU or more；The quantity 10 of saliva staphylococcus LD115CFU or more, preferably, the quantity of saliva staphylococcus LD11
It is 106CFU or more.
Embodiment 5
It present embodiments provides a kind of for preventing the dairy drink of mouth disease, secondary cheese is included in the dairy drink
The thallus of lactobacillus L9 and saliva staphylococcus LD11.The thallus is the thallus of inactivation, and the content of the thallus is in composition
5wt%, and the quantity of lactobacillus paracasei L9 and saliva staphylococcus LD11 are 103CFU or more, preferably, secondary cheese cream bar
The quantity of bacterium and saliva staphylococcus LD11 are 106CFU or more.
Claims (8)
1. a kind of for preventing or treating the pharmaceutical composition of mouth disease, which is characterized in that comprising secondary dry in the composition
Thallus or the thallus training of the thallus or thalline culture or their extract and saliva staphylococcus LD11 of Lactobacillus paracasei L9
Object or their extract are supported, the deposit number of lactobacillus paracasei L9 is CGMCC No.9800, the saliva staphylococcus
The deposit number of LD11 is CGMCC No.17880.
2. pharmaceutical composition according to claim 1, which is characterized in that the lactobacillus paracasei L9 is by inhibiting variation
Sucrose dependence in streptococcus and the streptococcic development process sticked and inhibit Dental plaque biofilm of Ge Shi, which is sticked, to be come in advance
Anti- or treatment mouth disease.
3. pharmaceutical composition according to claim 1, which is characterized in that the bacterium of lactobacillus paracasei L9 in the composition
Body or thalline culture or the content of their extract are 15wt%-20wt%, saliva staphylococcus in the composition
The thallus or thalline culture of LD11 or the content of their extract are 15wt%-20wt%.
4. pharmaceutical composition according to claim 1, which is characterized in that the mouth disease includes saprodontia, halitosis and tooth
All diseases.
5. pharmaceutical composition according to claim 1, which is characterized in that the lactobacillus paracasei L9 and saliva grape ball
Bacterium LD11 is the bacterial strain of active bacterial strain or deactivation.
6. a kind of for preventing the food of mouth disease, which is characterized in that include the bacterium of lactobacillus paracasei L9 in the food
Body or thalline culture or their extract, and saliva staphylococcus LD11 thallus or thalline culture or they
Extract, lactobacillus paracasei L9 deposit number are CGMCC No.9800, and the deposit number of the saliva staphylococcus LD11 is
CGMCC No.17880。
7. food according to claim 6, which is characterized in that the thallus or thallus of lactobacillus paracasei L9 in the food
The content of culture or their extract is 5wt%-10wt%, the thallus of saliva staphylococcus LD11 or bacterium in the food
The content of body culture or their extract is 5wt%-10wt%.
8. food according to claim 6, which is characterized in that the lactobacillus paracasei L9 and saliva staphylococcus LD11
For the bacterial strain of active bacterial strain or deactivation.
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