CN110339217A - Lactobacillus paracasei L9 is used to preventing or treating mouth disease and adjust the purposes of oral cavity flora - Google Patents
Lactobacillus paracasei L9 is used to preventing or treating mouth disease and adjust the purposes of oral cavity flora Download PDFInfo
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- CN110339217A CN110339217A CN201910535701.8A CN201910535701A CN110339217A CN 110339217 A CN110339217 A CN 110339217A CN 201910535701 A CN201910535701 A CN 201910535701A CN 110339217 A CN110339217 A CN 110339217A
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- lactobacillus paracasei
- saliva
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- oral cavity
- significantly
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Abstract
The purposes of oral cavity flora is used to preventing or treating mouth disease and adjusted the invention discloses lactobacillus paracasei L9, L9 can be used for preparing prevention or treat the pharmaceutical composition and food of saprodontia, research shows that L9 can significantly alleviate the occurrence degree of E grades of saprodontias of occlusal surface, it takes containing after L9, oral cavity flora structure change is significant, the especially significant up-regulation of actinomyces door, the actinomyces door of L9 group is obvious to be lowered, and significantly lowered in Klebsiella, Aerococcus, Gemella, corynebacteria and the pseudomonas aeruginosa belonged in categorization levels, reduce the risk of mouth disease.Take L9 can saliva species richness significantly lower, the relative abundance of the mouth diseases correlation bacterium such as streptococcus salivarius section, 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 field that prevents, treats of mouth disease, in particular to lactobacillus paracasei L9 is for preventing or controlling
It treats mouth disease and adjusts the purposes of oral cavity flora.
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, is one plant of isolated probiotic strain from long lived elder excrement, has experiment
It proves 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 in the inhibition adhesion of oral cavity pathogen, and monitors secondary dry
Lactobacillus paracasei L9 to the adjustment effect of oral cavity flora, thus for prevent and control mouth disease provide effective measures and it is theoretical according to
According to.
The purpose of the present invention is what is be achieved through the following technical solutions:
Lactobacillus paracasei L9 is used to prepare prevention or treats the purposes of mouth disease product, which is characterized in that the pair
Lactobacillus casei L9 deposit number is CGMCC No.9800.
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 mouth disease includes saprodontia, halitosis and periodontosis.
Further, the lactobacillus paracasei L9 is bacterial strain and the thallus culture of active bacterial strain or deactivation
Object or their extract.
Further, the product includes pharmaceutical composition, food, oral cleaning product.The food includes but unlimited
In dairy drink, tea, coffee, buccal tablet, soft sweets；The dairy drink includes acidified milk, Yoghourt, cheese and milk powder.Oral cavity is clear
Clean product including but not limited to toothpaste, mouthwash, breath freshening are spraying, apply fluorine agent, tooth-cleaning powder.
Further, the thallus of lactobacillus paracasei L9 or thalline culture or their extraction in described pharmaceutical composition
The content of object is 15wt%-20wt%.
Further, in the food and oral cleaning product the thallus of lactobacillus paracasei L9 or thalline culture or it
Extract content be 5wt%-10wt%.
Another aspect of the present invention:
Lactobacillus paracasei L9 is used to adjust the purposes of oral cavity flora, and the lactobacillus paracasei L9 deposit number is
CGMCC No.9800。
Further, lactobacillus paracasei L9 is by lowering actinomyces door, streptococcus salivarius section, Actinomy cetaceae, digestion chain
Coccaceae, cilium Cordycepps, Fusobacterium section and Flavobacterium section adjust oral cavity flora to reach prevention or treat the mesh of mouth disease
's.
Further, the actinomyces door is false comprising Klebsiella, Aerococcus, Gemella, corynebacteria and verdigris
Monad.
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。
The present invention having the beneficial effect that compared with prior art
1, the present invention carries out the relevant research in oral cavity to the lactobacillus paracasei L9 for the first time, by constructing mouth in vitro
Cavity mold type, establishes the quantitative detecting method of the pathogenic bacteria based on RT-PCR technology, and proves that lactobacillus paracasei L9 is significant
Inhibit Ge Shi streptococcus and Streptococcus mutans sticking on hydroxyapatite, culture 2,6, after 18h, Ge Shi streptococcus and change
The different streptococcic amount of sticking significantly reduces, while lactobacillus paracasei L9 significantly reduces Streptococcus mutans on hydroxyapatite
The thickness and density of the biomembrane of formation；
2, the present invention utilizes Streptococcus mutans inducing mouse caries model, further probes into lactobacillus paracasei L9 to mouse
The prevention of dental decayed tooth and adjustment effect to oral cavity flora, result of study are shown: compared with model group, L9 group, which is significantly alleviated, is stung
The occurrence degree of conjunction face E grades of saprodontia；By the analysis of the RT-PCR of a variety of cariogenic factors, show L9 pre- preventing caries function and its
The sucrose dependence in the development process of Streptococcus mutans initially sticked with Dental plaque biofilm is inhibited to stick related；Oral Bacteria
The 16srDNA high-flux sequence of group the result shows that, model group oral cavity flora diversity indices is without significant difference, but Bacterial community becomes
Change significantly, the especially significant up-regulation of actinomyces door, and the actinomyces of L9 group door is obviously lowered, and is belonging to the Cray in categorization levels
Primary Pseudomonas, Aerococcus, Gemella, corynebacteria and pseudomonas aeruginosa are significantly lowered, and the risk of mouth disease is reduced；
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) is respectively control group, L9 group, life
The front schematic view of object film, (d), (e) be respectively control group, L9 group biomembrane side schematic view；
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 the horizontal mouse plaque microorganism PCoA figure of OUT；
Figure 13 is to belong to horizontal mouse plaque species difference to analyze schematic diagram；
Figure 14 is the multifarious experiment flow figure of human body oral cavity flora；
Figure 15 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 16 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 17 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 18 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 19, Figure 20 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.- 20 DEG C are stored in the form of 20% skimmed milk, 200 μ l is taken to cultivate in 37 DEG C of constant incubators in corresponding culture medium
12h, and pass on.
Embodiment 1 --- lactobacillus paracasei L9 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 enzyme, 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, are moved back
Anneal 60s under fiery temperature 60 C, 72 DEG C of extension 30s, 40 circulations；72 DEG C of extension 5min.
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) make a variation chain
It is y=-3.5581x+58.998, R that coccus, 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, following detection has been carried out:
Hydroxy-apatite stone model adhesion experiment:
(1) saliva pre-processes
Saliva collection selects 10 24-40 years old healthy volunteers, collects saliva.Health adult uses after feed 2 hours
Clear water is gargled, and collects nonirritant saliva (mouth containing swab stick 10 minutes), the saliva being collected into using Salivette saliva collection pipe
Supernatant is taken with the revolving speed centrifugation 15min of 5500r/min at 4 DEG C using low-temperature and high-speed centrifuge immediately, with 0.22 μm of micropore
The saliva part of membrane filtration 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 in -80 for a long time
℃.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, is made
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.It will
Experiment is divided into two groups of prevention groups and treatment group: prevention group is outstanding for addition pathogenic bacteria after 125 μ l turning effort 1h of L9 suspension are added
125 μ l of supernatant liquid；Treatment group is that 125 μ l of L9 suspension is added after pathogenic 125 μ l turning effort 1h of bacterium suspension is added；Blank control
Group then replaces L9 suspension with the potassium chloride buffer of the bovine serum albumin containing 5mg/ml.Room temperature 6r/min rotates 2,6,18h, chlorination
Potassium buffer washes twice, and collects hydroxyapatite pearl and carries out quantitative PCR, collects buffer and measures pH, every group repeats three times.
Lactobacillus paracasei L9 to the Streptococcus mutans result sticked of prevention as shown in figure 3, compared with the control group, secondary cheese
Lactobacillus L9 is after being added 2h and 6h, and Streptococcus mutans content significantly reduces (P < 0.05), and L9 group reduces when sticking 2h
26.88% (0.93 × 109Copies), 6h reduces 33.26% (1.49 × 109Copies), after acting on 18h, the reality of L9 is added
Test the content (2.46 × 10 of Streptococcus mutans in group9Copies) it is substantially less than control group (4.26 × 109Copies) (P <
0.05)；And in treatment group, L9 significantly suppresses the content (P > 0.05) of Streptococcus mutans after acting on 2h, reduces
51.55% (3.0 × 109copies)。
Lactobacillus paracasei L9 shows Ge Shi streptococcus suppression result such as Fig. 4, the dagger-axe formula hammer with the control group that L9 is not added
Bacterial content compared to be added L9 and dagger-axe formula streptococcus interaction 2,6, after 18h, the Ge Shi streptococcus amount of sticking significantly reduces (P <
0.05) 51.19% (2.15 × 10, is reduced respectively9Copies), 56.09% (3.36 × 109Copies), 58.40% (3.51
×109Copies), sufficiently demonstrate L9 and significant prevention adhesion is all had to dagger-axe formula streptococcus.
L9 is added successively affects sticking for pathogenic bacteria, should be the result shows that the inhibiting effect that L9 is played in the oral cavity is not
It is simple non-specific steric hindrance, and being likely to L9 can be with the egg on pathogenic bacteria competitive binding Salivary Acquired Pellicle
Polymeric immunoglobulin receptor, 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 the reality that L9 inhibits streptococcus mutans biomembrane to be formed
It tests.Bacterium abandons supernatant in 37 DEG C of two generations of inoculation to growth logarithmic phase 4500r centrifugation 10min, and PBS (4 DEG C, 4500g, 10min) is washed
It 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 bacteria suspension of cfu/ml is standby
With.Autoclaved disk is placed in the hole of 24 hole tissue culturing plates (every Kong Zhongyi disk, disk can not contact hole wall), and 37
DEG C with above-mentioned saliva (hole 1.5ml/) be coated 4h, later, 1.5ml PBS rinse 2 times.Each 750 μ l of bacterial suspension is added, in hydroxyl
Apatite disc surfaces culture forms biomembrane 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 L9 group significantly reduces (P < 0.05) in the biofilm thickness that hydroxyapatite surface is formed, and control group is averaged
With a thickness of 16.33 μm, L9 group average thickness is respectively 12.63 μm.Fig. 5 (b, c) shows that control group bacterium living beings film density is obvious
Higher than the experimental group that L9 is added, bacterium is layer upon layer of intensively at large stretch of cloud form, compares aggregation, arrangement is close, the gap in the visual field
Range very little.And the experimental group bacterial clump that L9 is added relatively disperses, black dull gap range is very big, and Multiple drug resistance is messy, L9 group
Bacterium presentation is fragmentary dotted, and bacterial density is small.Therefore, lactobacillus paracasei L9 can by inhibiting Streptococcus mutans sticking,
The generation for reducing Dental plaque biofilm, to prevent the generation of mouth disease.
Embodiment 2 --- influence of the lactobacillus paracasei L9 to murine oral bacterial diversity
By the Streptococcus mutans 1.2499 of -20 DEG C of preservations, lactobacillus paracasei L9 respectively in BHI, MRS and M17 of 10ml
37 DEG C of activation culture three generations in broth bouillon remove supernatant after 4500r/min centrifugation 10min, and Streptococcus mutans are with sterile
Physiological saline redissolves bacterium mud, and lactobacillus paracasei L9 is redissolved with sucrose solution, adjusting bacterial concentration to 108Cfu/ml, 4 DEG C of guarantors
It deposits spare.
Murine oral saprodontia modeling method:
The animal model of the present embodiment has selected purchase total from male SPF grades of BALB/c mouses of 3 week old of dimension tonneau China
28, every group 7, it is divided into control group, model group, lactobacillus paracasei L9 group totally 3 groups.The SPF grade mouse bought is first
Four groups were randomly divided into after one week laundering period, mouse feeding sterile water and common aseptic feed during adaptation.Later, it compares
The sucrose solution and common aseptic feed of group mouse feeding 10%；The sucrose solution of model group mouse feeding 10% and cariogenic feeding
Material, while continuous 4 days inoculation Streptococcus mutans, daily inoculation 1 time, inoculum concentration are 1ml/ times, are first wiped with sterile PBS before inoculation
Clean murine oral；The mouse feeding of L9 group contains 10% sucrose solution and cariogenic feed of L9, and inoculation is followed by for pathogenic bacteria 1 week
Kind L94 days, daily inoculation 1 time, inoculum concentration 108Cfu/ times, all mouse are put to death after feeding 4w, 2h fasting before putting to death is put to death
Afterwards with each position in oral cavity (including upper lower teeth, tongue, gum etc.) of aseptic cotton stick swab friction mouse, mouse plaque is acquired
After sample, swab is cut and is placed in the centrifuge tube equipped with 1ml nuclease protection liquid, -20 DEG C of preservations.
The quantitative detection of the cariogenic factor
7 kinds of common cariogenic factors and its specific primer are filtered out by searching for document, primer sequence is 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 profilings, 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), in lactobacillus paracasei L9 group
The expression of spap and ciaH has had been lowered to the expression of control group, though the expression quantity of luxs gene substantially less than model
Group (p < 0.05), but compared than control group, relative expression quantity still increases, the luxs relative expression quantity and control group of L9 group
Or what there were significant differences.
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 increase in the expression quantity of lactobacillus paracasei L9 group than control group,
But its relative expression quantity is substantially less than model group (p < 0.05).In addition, the relative expression quantity difference of the gtfC between four groups is not shown
It writes (p > 0.05).The soluble sugar that the reduction of gtfD expression quantity may be such that GTF-D synthesizes is reduced, to reduce GTF-B metabolism
The offer of substrate.So the reduction of gtfB, D gene relative expression quantity shows that lactobacillus paracasei L9 significantly suppresses plaque
The formation and development of biomembrane.
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, 10ng template DNA is added to the reaction mixture of final 25 μ L volume
In, contain 0.25 every kind of primer of μ L (25 μM), 12.5 μ L2 × KAPA HiFi HotStart Mix and PCR grades of nothings of Ready
Bacterium is without enzyme water.Cycling condition are as follows: in 95 DEG C of preincubate 3min, 25 circulations, 30s is denaturalized at 95 DEG C, the 30s at 55 DEG C
It anneals, 30s is carried out at 72 DEG C and is extended, then carry out finally extending 5min at 72 DEG C and be maintained at 4 DEG C.Pass through
2% agarose gel electrophoresis detects 2 μ LPCR products to detect target segment, and uses 1XAMPure XP Beads (Beckman
Coulter, Inc.) purifying.In second of PCR, the reaction that the 2.5 above-mentioned PCR products of μ L are added to final 25 μ L volume is mixed
Close object in, wherein contain 0.25 every kind of primer of μ L (25 μM), 12.5 μ L 2 × KAPA HiFi HotStart Ready Mix and
The sterile no enzyme water of 9.5 PCR grades of μ l.PCR reaction: 95 DEG C of initial denaturations is carried out to each sample in T100TM thermal cycler as follows
3min, 95 DEG C of 30s, 55 DEG C of 30s and 72 DEG C of 30s carry out 8 circulations, then finally extend 5min at 72 DEG C and be maintained at 4
℃.Merge the PCR reaction from same sample, is purified using the separation of 2% Ago-Gel and extraction.
(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 11, domain by smallest sample sequence number
32, 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 detects 346 OUT, and lactobacillus paracasei treatment group detects 327 OUT.Compared with the control group, model group
The total OUT number detected 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 species composition of L9 group and control group are closer, with model group difference
It is larger.
Figure 12 is the PCoA analysis based on bray-curtis algorithm, be shown in figure the horizontal model group of OTU with compare group
Falling composition, there were significant differences；L9 group and model group sample group form significant difference, especially on PC1 (38.34%) axis.
As shown in figure 13, compared with the control group；Staphylococcus (Staphylococcus) in model group murine oral, gram
The primary Pseudomonas of thunder (Klebsiella) is significantly raised；Compared with model group, pasteurella, Gemella, mycoplasma in L9
(Mycoplasma), corynebacteria (Corynebacterium_1) is significantly lowered.
In summary:
(1) compared with the control group, model group E grades of dental caries significantly increase；Compared with model group, E grades of saprodontias of L9 group are significantly reduced；
Simultaneously between three groups DS, DM grades of dental caries without significant difference.
(2) compared with the control group, the cariogenic factor spap, luxs, ciaH, gtfB, gtfD expression quantity is significant in model group
Up-regulation；Lactobacillus paracasei L9 addition significantly reduces the expression of this five kinds of cariogenic factors.
(3) compared with the control group, model group oral cavity flora diversity indices is without significant difference, but Bacterial community variation is aobvious
It writes, especially actinomyces door (Actinobacteria) significantly raises；And significant changes have occurred in group's composition of L9 group.
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 14 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 10 as required in 4 weeks afterwards9Cfu/ piece), it collects again after two weeks
Sample be sequenced and 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 in 1h
It is inside immersed 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 probiotics tablets (L9 containing lactobacillus paracasei 109Cfu/ piece) saliva sample after 2 weeks
This OUT number reduces, and several litres of OUT of plaque sample high, all in all, compared to saliva sample, the richness of plaque sample
Want low.
The increase with detection sample size is shown in Figure 15, 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 the total OUT of plaque
Number is obvious to be risen.It can be seen that from Figure 16 dilution curve as the increase saliva of sample sequencing amount and plaque sample OUT are horizontal
Aromatic index (shannon) tend to balance, no longer rise, i.e., species diversity is not further added by, it was demonstrated that this crowd test sample
This 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 tablets
2, after 4w, plaque community diversity index (Shannon) is significantly lowered, and Simpson index significantly raises, and community diversity subtracts
It is few；The significant up-regulation of group's diversity index (ace, chao), group's richness increase；Saliva, plaque sample coverage
(coverage) it 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, has
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 17 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 17b)
(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 18 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 it is newborn to pass through secondary cheese
After bacillus L9 bacterium piece is adjusted, sample distribution aggregation in the 2nd, 4 week；The the 2nd, 4 week plaque sample almost can not in plaque PCoA figure
It separates, is 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 19,25 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
Section's relative abundance that kind does not annotate is remarkably decreased, the variation of the 4th week saliva and the 2nd week not consistent, melaninogenicus, purple list
Born of the same parents bacterium, campylobacter, pasteurella, Fusobacterium are significantly raised, occur such result may be due to salivary flow compared with
Greatly, the microorganism in saliva updates very fast, and L9 is difficult to stop for a long time in saliva, is only capable of playing tune to saliva in a short time
Section effect, therefore maintain the effect time shorter；Totally 16 such as Figure 20, streptococcus are the advantage Cordycepps detected in plaque sample
Most advantage Cordycepps, and the 2nd, 4 week sample microorganism group be at close, in addition to Actinomy cetaceae significantly raises, cilium Cordycepps, purple unit cell
Bacterium, Lachnospira section, Fusobacterium section, Flavobacterium section relative abundance significantly reduce.In addition, Wei Rong bacterium, melaninogenicus, titanium dioxide
Carbon 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, furthermore
Bacteroidetes relative abundance gradually increases at any time；
(2) after eating L9 lozenge, plaque flora species diversity is significantly lowered, and richness significantly raises, species composition
It is changed significantly, actinomyces door accounting obviously increases, and Bacteroidetes significantly reduces, and the 2nd week and species composition in the 4th week are without significance difference
It is different.In section's categorization levels, after edible mushroom piece in addition to actinomyces, cilium bacterium, purple monad, Lachnospira, Fusobacterium, Flavobacterium section,
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, lactobacillus paracasei L9 deposit number are CGMCC No.9800.The thallus is active
Thallus, the content of the thallus is 16wt% in composition, and the quantity of lactobacillus paracasei L9 is 103CFU or more, preferably
Person, the quantity of lactobacillus paracasei are 106CFU or more.
Embodiment 5
It present embodiments provides a kind of for preventing or treating the dairy drink of mouth disease, pair is included in the composition
The thallus of Lactobacillus casei L9, lactobacillus paracasei L9 deposit number are CGMCC No.9800.The thallus is the bacterium of inactivation
Body, the content of the thallus is 5wt% in composition, and the quantity of lactobacillus paracasei L9 is 103CFU or more, preferably, pair is dry
The quantity of Lactobacillus paracasei is 106CFU or more.
Claims (10)
1. lactobacillus paracasei L9 is used to prepare prevention or treats the purposes of mouth disease product, which is characterized in that described secondary dry
Lactobacillus paracasei L9 deposit number is CGMCC No.9800.
2. purposes according to claim 1, which is characterized in that the lactobacillus paracasei L9 is by inhibiting Streptococcus mutans
Stick with the sucrose dependence in the streptococcic development process sticked and inhibit Dental plaque biofilm of dagger-axe formula to prevent or control
Treat mouth disease.
3. purposes according to claim 1, which is characterized in that the mouth disease includes saprodontia, halitosis and periodontosis.
4. purposes according to claim 1, which is characterized in that the lactobacillus paracasei L9 be active bacterial strain or
The bacterial strain and thalline culture or their extract of deactivation.
5. purposes according to claim 1, which is characterized in that the product includes pharmaceutical composition, food, oral cleaning
Product.
6. purposes according to claim 5, which is characterized in that the thallus of lactobacillus paracasei L9 in described pharmaceutical composition
Or thalline culture or the content of their extract are 15wt%-20wt%.
7. purposes according to claim 5, which is characterized in that lactobacillus paracasei in the food and oral cleaning product
The thallus or thalline culture of L9 or the content of their extract are 5wt%-10wt%.
8. the purposes that lactobacillus paracasei L9 is used to adjust oral cavity flora, which is characterized in that the lactobacillus paracasei L9 preservation
Number is CGMCC No.9800.
9. purposes according to claim 8, which is characterized in that lactobacillus paracasei L9 is by lowering actinomyces door, saliva
Streptococcaceae, Actinomy cetaceae, peptostreptococcus section, cilium Cordycepps, Fusobacterium section and Flavobacterium section adjust oral cavity flora to reach
To the purpose for preventing or treating mouth disease.
10. purposes according to claim 9, which is characterized in that the actinomyces door includes Klebsiella, aerococcus
Category, Gemella, corynebacteria and pseudomonas aeruginosa.
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