CN104561227A - Analysis method of rejection capability of lactobacillus plantarum metabolite and application of lactobacillus plantarum metabolite - Google Patents
Analysis method of rejection capability of lactobacillus plantarum metabolite and application of lactobacillus plantarum metabolite Download PDFInfo
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Abstract
The invention discloses ab analysis method of rejection capability of lactobacillus plantarum metabolite and application of the lactobacillus plantarum metabolite. The effective extract of lactobacillus plantarum FB-T9 contains micromolecular bacteriocin substances and hydrogen peroxide ingredients, so that bacteriostatic activity for streptococcus mutans in floating state is provided and relatively bacteriostatic activity can be provided under biological membrane state to destroy the integrity of the biological membrane structure. The lactobacillus plantarum FB-T9 is applied to fermented food or pharmaceutical compositions containing metabolite of the lactobacillus plantarum FB-T9, is used for preventing, decreasing or treating tooth decay and has wide application prospect.
Description
Technical field
The present invention relates to a kind of Lactobacillus plantarum metabolite chosen to the analytical method of Streptococcus mutans biomembrane inhibition, the metabolite of this lactobacillus and composition thereof can reduce the quantity of Streptococcus mutans in oral cavity by inhibit activities, for prevention or treatment dental caries, belong to microbial technology field.
Background technology
Dental caries is the common bacterial infection diseases of the mankind, can cause the progressive destruction of dental hard tissue, cause dental caries (also known as " dental caries " or " decayed tooth "), serious harm oral health.60th World Health Assembly's resolution is pointed out, by prevention oral disease and must promote that oral hygiene includes chronic disease comprehensive prevention and treatment planning in.Along with the development of oral microorganism thing, immunology and biochemical research, the cariogenic viewpoint of relatively generally acknowledging at present is tetrad factor theory, its basic concept comprises: dental caries to enter after human oral cavity in dental plaque due to the effect of the cariogenic bacterias such as Streptococcus mutans containing polysaccharide food (especially sucrose), utilize substrate, fermentation and acid, through the regular hour, caused by the inorganic matter dissolved destruction of tooth.Four required factors should be had: (1) cariogenic bacteria in dental caries pathogenic process; (2) food (saccharide or carbohydrate); (3) host's susceptible tooth; (4) dental plaque, and the pH of inherence maintains the regular hour at critical below pH5.
Current large quantity research confirms, Streptococcus sanguis and Streptococcus mutans are the most normal isolated strains in dental caries lesion, and Streptococcus mutans is because have some important virulence factors, is acknowledged as important cariogenic bacteria.Its virulence factor relates to and sticks, produces factor that is sour, acidproof and aspects such as synthesis extracellular polysaccharide etc., is the cariogenic material base of antibacterial.Streptococcus mutans can stick by the non-sucrose dependency of mediating bacterial to dental surface, in antibacterial initially sticking and promoting to play an important role in the gathering between thalline dental surface.In addition, it can also utilize sucrose and glucosyltransferase (GTFs) synthesis to have very strongly adherent water-insoluble extracellular polysaccharide (Loesche WJ, waits people, Microbiol Rev.50:353-380,1986).
From the bacillary cariogenic cause of disease of dental caries, the many angles played a role from minimizing Streptococcus mutans of current prevention and therapy dental caries.As reduced the quantity of Streptococcus mutans, medicament conventional is at present hibitane collutory etc.; Reduce the absorption of sugar, especially sucrose etc., the sweeting agent of the difficult metabolism of conventional Streptococcus mutans at present, as sorbitol, xylitol, aspartame etc.; Remove dental plaque, conventional is that machinery is removed at present.
Traditional view thinks that lactobacillus is the main cariogenic bacteria of dental caries, but study discovery now, the initiating effect of lactobacillus in dental caries pathogenic process is not obvious, it not the pathogen of first bringing out dental caries, but formed at dental caries, work in short dental caries development, especially play an important role in the development of deep dentin caries.Once dental caries infringement is formed, the lactobacillus counting of disease damage locally and in saliva just increases sharply, so predict the development trend of dental caries usually through measuring lactobacillus quantity in saliva, can be used as " the dental caries marker fungus " of Explosive Epidemic Disease investigation.These lactobacilluss are generally separated in dental plaque, have certain cariogenicity.
Dental plaque biofilm is the pathogenetic initiation factors of dental caries, plays an important role in the generation of dental caries.Also there is no the metabolite of lactobacillus to the patent document of the biomembranous inhibition of Streptococcus mutans at present.Therefore, screen a strain to the inhibited lactobacillus of the Streptococcus mutans swum, and prove that the effective ingredient in its metabolite has good inhibitory action in biological film model, the practical use developing the effective ingredient of its metabolite just seems very necessary simultaneously.
Summary of the invention
The object of this part is some aspects of general introduction embodiments of the invention and briefly introduces some better embodiment.May do in the specification digest and denomination of invention of this part and the application a little simplify or omit with avoid making this part, specification digest and denomination of invention object fuzzy, and this simplification or omit and can not be used for limiting the scope of the invention.
In view of the analytical method of above-mentioned and/or existing Lactobacillus plantarum metabolite inhibition and Lactobacillus plantarum FB-T9 metabolite, Problems existing in the application in treatment and caries prevention, proposes the present invention.
Therefore, an object of the present invention is to provide a kind of Lactobacillus plantarum FB-T9 metabolite to the modeling of Streptococcus mutans biomembrane inhibition to set up the analytical method on biomembrane basis.
For solving the problems of the technologies described above, according to an aspect of the present invention, the invention provides following technical scheme: a kind of analytical method of Lactobacillus plantarum metabolite inhibition, it comprises, Lactobacillus plantarum FB-T9 metabolite is to the impact analysis of Streptococcus mutans biofilm formation, and it comprises: (a) is on the impact of Streptococcus mutans biofilm formation amount: prepare Streptococcus mutans bacteria suspension; When 0h, add Streptococcus mutans bacteria suspension in culture medium, these three time points of the 0h in cultivation, 6h, 12h, add Lactobacillus plantarum FB-T9 fermented supernatant fluid respectively, cultivate 24h together; Or Streptococcus mutans single culture 24h or 48h, the antibacterial of removing culture medium and swimming, then add Lactobacillus plantarum FB-T9 fermented supernatant fluid; Negative control group and positive controls add normal saline and the chlorhexidine acetate solution of equivalent respectively; After cultivation terminates, abandon free bacteria, deionized water wash, natural drying; Add crystal violet solution, dye under room temperature, make the antibacterial of combination painted; Incline after dyeing liquor, deionized water wash, add the ethanol/acetone mixed liquor colour developing of mixing after drying, microplate reader 600nm measures absorbance; B () is on Streptococcus mutans biofilm structure and active impact: prepared by biomembrane specimen: in culture dish, add Streptococcus mutans bacteria suspension and the TSB culture medium containing 0.25% sucrose, Lactobacillus plantarum FB-T9 fermented supernatant fluid is added respectively when incubation time is 0h, 6h, 12h, each time point cultivates 3 samples, Anaerobic culturel 24h, PBS rinses, remove surperficial planktonic bacteria, dye in camera bellows under room temperature immediately; Or first cultivate Streptococcus mutans, after forming 24h or 48h biomembrane, remove culture medium, PBS washs, and removes surperficial planktonic bacteria, adds Lactobacillus plantarum FB-T9 fermented supernatant fluid, Anaerobic culturel 24h, removes flcating germ, fluorescence staining; The preparation of fluorescent dye and dyeing: respectively viable bacteria, dead bacterium are dyeed with fluorescein based dye CFSE, PI, make antibacterial alive, extremely antibacterial send green fluorescence respectively, red fluorescence, thus can biofilm structure be observed, dyeing terminates rear PBS and washs, removing residual dye; Confocal laser scanning microscope: observe under the above-mentioned biomembrane specimen having completed fluorescence staining is placed on CLSM, object lens × 20, eyepiece × 10, the exciting light observing biofilm structure is 510/480.With sample peak signal point for focal plane, with this plane for reference point, carrying out 2um along Z axis is that step pitch carries out tomoscan, finally carries out three-dimensional reconstruction, obtain biomembrane stereochemical structure, process record biofilm thickness, the antibacterial gross area, viable bacteria area, dead bacterium area and calculating viable bacteria percentage ratio; Lactobacillus plantarum FB-T9 metabolite is to the impact analysis of extracellular polysaccharide in Streptococcus mutans biomembrane: adopt as the method in step (b) prepares biomembrane specimen, make extracellular polysaccharide send blue-fluorescence with fluorescent dye, the exciting light used when observing with CLSM is 400 ~ 490nm; Biofilm states lower variation streptococcus is synthesized to the impact analysis of insoluble extracellular polysaccharide, it comprises: in polystyrene cell culture dish, adds different streptococcus bacteria suspension and the TSB culture medium containing 0.25% sucrose, Anaerobic culturel; Add Lactobacillus plantarum FB-T9 fermented supernatant fluid during 0h, 6h, 12h in Streptococcus mutans 24h biofilm formation, remove culture medium after 24h, PBS washs, and removes the antibacterial swum in surface; Or add Streptococcus mutans bacteria suspension and the TSB culture medium containing 0.25% sucrose, Anaerobic culturel, form 24h or 48h biomembrane, remove culture medium, PBS washs, remove the antibacterial swum in surface, add Lactobacillus plantarum FB-T9 fermented supernatant fluid, Anaerobic culturel 24h, remove the liquid in hole, PBS washs, and removes the antibacterial swum in surface; Add NaOH, collect thalline, collect supernatant, precipitation with NaOH washing, merges supernatant, as the sample of water-insoluble extracellular polysaccharide again; The content of extracellular polysaccharide is surveyed with Anthrone-sulfuricacid method.
As a kind of preferred version of the analytical method of Lactobacillus plantarum metabolite inhibition of the present invention, wherein: to described Lactobacillus plantarum FB-T9 metabolite to the impact analysis of Streptococcus mutans biofilm formation before, also comprise: with the fermented supernatant fluid for the treatment of of different temperature Lactobacillus plantarum FB-T9, the situation of change of its biocidal property is observed again by bacteriostatic experiment, obtain effective antibacterial substance and there is heat stability, get rid of the possibility of macro-molecular protein as antibacterial substance; The pH of the fermented supernatant fluid of Lactobacillus plantarum FB-T9 is transferred to different values, then observes the situation of change of its biocidal property by bacteriostatic experiment, obtain effective antibacterial substance and have pH dependency, namely pH is lower, and fungistatic effect is better; With the fermented supernatant fluid of trypsin, papain and E.C. 3.4.21.64 process Lactobacillus plantarum FB-T9, the situation of change of its biocidal property is observed again by bacteriostatic experiment, obtain fungistatic effect significantly to decline, prove in its effective antibacterial substance main containing micromolecule polypeptide bacterioid element; With the fermented supernatant fluid of hydrogen peroxide ferment treatment Lactobacillus plantarum FB-T9, then observed the situation of change of its biocidal property by bacteriostatic experiment, obtain fungistatic effect and decline, illustrate in its effective antibacterial substance containing hydrogen peroxide.
Another object of the present invention is to provide the application of metabolite in treatment and caries prevention of a kind of Lactobacillus plantarum FB-T9.It is for safeguarding oral health, and caries prevention provides new approach and solution.
Beneficial effect of the present invention: the analytical method of Lactobacillus plantarum metabolite inhibition of the present invention is the inhibitory action of Lactobacillus plantarum FB-T9 metabolite to cariogenic cause of disease Streptococcus mutans from biomembranous angle analysis; And the application of the metabolite of Lactobacillus plantarum FB-T9 in treatment and caries prevention, excavated the function of probiotic bacteria further, for safeguarding oral health, caries prevention provides new approach and solution.
Accompanying drawing explanation
Fig. 1 is that Lactobacillus plantarum FB-T9 metabolite is to the inhibitory action of Streptococcus mutans biofilm formation amount;
Fig. 2 is the impact of Lactobacillus plantarum FB-T9 metabolite on the antibacterial gross area in Streptococcus mutans biomembrane;
Fig. 3 is the impact of Lactobacillus plantarum FB-T9 metabolite on the extracellular polysaccharide gross area in Streptococcus mutans biomembrane;
Fig. 4 is the impact of Lactobacillus plantarum FB-T9 metabolite on extracellular polysaccharide content insoluble in Streptococcus mutans biomembrane.
Detailed description of the invention
For enabling above-mentioned purpose of the present invention, feature and advantage become apparent more, are described in detail below by the specific embodiment of the present invention.
Set forth a lot of detail in the following description so that fully understand the present invention, but the present invention can also adopt other to be different from alternate manner described here to implement, those skilled in the art can when without prejudice to doing similar popularization when intension of the present invention, therefore the present invention is by the restriction of following public specific embodiment.
Embodiment 1: Lactobacillus plantarum FB-T9 metabolite is tested the inhibitory action of the Streptococcus mutans of floating state
The mutants streptococcus strain that this example uses is ATCC 25175 (containing center available from Chinese Academy of Sciences's microorganism).By described strain culturing in the pancreas peptone soybean broth culture medium (TSBY) being added with 0.6% yeast extract.TSBY culture medium is the culture medium that those skilled in the art person knows, and it contains tryptone, soy peptone, yeast extract, sodium chloride, glucose and dipotassium hydrogen phosphate, pH 7.1 ~ 7.5.The Streptococcus mutans of incubated overnight regulates OD by fresh TSBY culture medium
600be 0.5, clump count is about 1 × 10
8cfu/ml.
By Lactobacillus plantarum FB-T9 bacterial strain with 2% inoculum concentration be inoculated in MRS culture medium, 37 DEG C of quiescent culture, in stable phase latter stage, collect culture fluid, 4 DEG C, centrifugal 20min under 12000rpm, get supernatant, after 0.22 μm of microporous filter membrane, obtain lactobacillus cell free fermentation supernatant.MRS culture medium is the culture medium that those skilled in the art person knows, and it contains tryptone, yeast extract, Carnis Bovis seu Bubali cream, glucose, sodium acetate, DisodiumHydrogen Citrate, dipotassium hydrogen phosphate, manganese sulfate, magnesium sulfate, tween 80, pH6.2 ~ 6.4.
Described bacteriostatic experiment operation is as follows, 100 μ l Streptococcus mutans bacteria suspensions are mixed homogeneously with the aseptic TSBY culture medium that 20ml contains 1.5% agar, pour in culture dish, solidify rear tweezers and aseptic Oxford cup (internal diameter is 6mm) is placed on culture dish.Drawing lactic acid bacteria fermentation supernatant 200 μ l joins in the cup of Oxford, first at 4 DEG C, balances 2h, then 37 DEG C of quiescent culture 48h, measures inhibition zone size.
From measurement result, Lactobacillus plantarum FB-T9 metabolite has stronger inhibitory action to the Streptococcus mutans under this state, and its antibacterial circle diameter is 21.64 ± 0.36mm, can be considered to have stronger bacteriostatic activity.
Embodiment 2: to the preliminary inquiry experiment of Lactobacillus plantarum FB-T9 antibacterial substance
The fermented supernatant fluid 1ml of extracting lactic acid bacterium, processes 20min respectively, arranges blank in 37 DEG C, 60 DEG C, 80 DEG C, 100 DEG C water-baths, and inspection bacteriostatic activity, probes into the impact of heat treatment on antibacterial substance activity with this; Regulate the pH value of supernatant to pH3.0,4.0,5.0,6.0,6.5,7.0 respectively with the sodium hydroxide solution of 1.0mol/L and the hydrochloric acid solution of 1.0mol/L, get each 1ml of the supernatant after adjustment, filter, inspection bacteriostatic activity, probes into the impact of pH on antibacterial substance activity with this; Get four parts of lactic acid bacteria fermentation supernatant, regulate pH to be respectively 8.0,6.0,7.0, add trypsin, papain, E.C. 3.4.21.64 successively, make its final concentration be 1mg/mL, 37 DEG C of water-bath 2h, then pH is adjusted to 4.0, filter.With not enzyme-added sample for contrast, inspection bacteriostatic activity, probes into the impact of protease on antibacterial substance activity with this; Extracting lactic acid bacterium fermented supernatant fluid 1mL, regulates pH6.5, adds catalase, make its final concentration be 1mg/mL, 37 DEG C of water-bath 2h, then pH is adjusted to 4.0, filters.With not enzyme-added sample for contrast, whether inspection bacteriostatic activity, probe in antibacterial substance containing hydrogen peroxide with this.
Its result of the test is as shown in table 1,2,3,4.
Table 1 heat treatment is on the impact of antibacterial substance effective in Lactobacillus plantarum FB-T9 metabolite
Table 2pH value is on the impact of antibacterial substance effective in Lactobacillus plantarum FB-T9 metabolite
Table 3 Protease Treatment is on the impact of antibacterial substance effective in Lactobacillus plantarum FB-T9 metabolite
Table 4 catalase is on the impact of antibacterial substance effective in Lactobacillus plantarum FB-T9 metabolite
As can be seen from Table 1, the antibacterial substance that Lactobacillus plantarum produces has heat stability, eliminates the possibility of macro-molecular protein; As can be seen from Table 2, the bacteriostatic activity of its antibacterial substance has pH dependency, and namely pH is lower, and bacteriostatic activity is better; As can be seen from Table 3, may containing micromolecule polypeptide bacterioid element in its antibacterial substance; As can be seen from Table 4, hydrogen peroxide is contained in its antibacterial substance.
Embodiment 3: Lactobacillus plantarum FB-T9 metabolite is tested the impact of Streptococcus mutans biofilm formation
Bacteria planting (0 ~ 6h), bacterial adhesion (6 ~ 12h), biomembrane basic structure substantially can be divided into form (12 ~ 24h) for the formation of early stage Dental plaque biofilm and biofilm forms (24 ~ 48h) these four Main Stage.Therefore in the present embodiment, at initial stage in bacteria planting stage (during 0h), initial stage in bacterial adhesion stage (during 6h), biomembrane basic structure starts the initial stage that formed (during 12h), biomembrane basic structure forms rear (during 24h) and biofilm structure is formed rear (during 48h), carries out mediation with the metabolite of Lactobacillus plantarum FB-T9.
A () is on the impact of Streptococcus mutans biofilm formation amount
First preparing bacteria concentration is 1 × 10
5the Streptococcus mutans bacteria suspension of cfu/ml.Then at 0h, every hole adds 100 μ l Streptococcus mutans bacteria suspensions, 37 DEG C of Anaerobic culturel 24h, these three time points of the 0h in cultivation, 6h, 12h, adds 100 μ l lactic acid bacteria fermentation supernatant in corresponding hole, cultivates 24h together; Or Streptococcus mutans single culture 24h or 48h, the antibacterial of removing the culture medium in hole and swimming, then add 200 μ l lactic acid bacteria fermentation supernatant.Negative control group and positive controls add normal saline and the chlorhexidine acetate solution of equivalent respectively.Finally cultivate after terminating, abandon free bacteria, every hole deionized water of 200 μ l softly washs 3 times, natural drying; Every hole adds the crystal violet solution of 50 μ l 10g/L, and dye under room temperature 15min, makes the antibacterial of combination painted; Incline after dyeing liquor, with deionized water wash more than 3 times; After dry, every hole adds the ethanol/acetone mixed liquor colour developing that 200 μ l mix, and microplate reader 600nm measures absorbance.Its result as shown in Figure 1.
As can be seen from accompanying drawing 1, the metabolite along with Lactobacillus plantarum FB-T9 starts the time of mediation more early in Streptococcus mutans biofilm formation process, and biofilm formation amount is fewer, larger relative to the slip of untreated fish group.
B () is on Streptococcus mutans biofilm structure and active impact
Prepared by biomembrane specimen: the sterile cover slips putting into 18mm × 18mm specification in the glass culture dish of diameter 6cm, adds Streptococcus mutans bacteria suspension 1ml and contains 0.25% sucrose TSB culture medium 3ml.Add 4ml lactic acid bacteria fermentation supernatant respectively when incubation time is 0h, 6h, 12h, each time point cultivates 3 samples.N
295%, CO
25%, 37 DEG C of Anaerobic culturel 24h, take out sheet glass, PBS rinses 2 times, removes surperficial planktonic bacteria, dyes immediately under room temperature in camera bellows.Or first cultivate Streptococcus mutans, after forming 24h or 48h biomembrane, remove culture medium in culture dish, PBS washs slide 2 times, remove surperficial planktonic bacteria, add lactic acid bacteria fermentation supernatant 4ml, Anaerobic culturel 24h, take out sheet glass, remove flcating germ, fluorescence staining.
The preparation of fluorescent dye and dyeing: dye to viable bacteria, dead bacterium respectively with fluorescein based dye CFSE, PI, they make antibacterial alive, dead antibacterial sends green fluorescence respectively, red fluorescence, thus can observe biofilm structure.The ultimate density of CFSE and PI is respectively 2uM and 4uM.Dyeing terminates rear PBS and washs, removing residual dye.
Laser confocal microscope (CLSM) is observed: observe under the above-mentioned biomembrane specimen having completed fluorescence staining is placed on CLSM.Object lens × 20, eyepiece × 10, the exciting light observing biofilm structure is 510/480.With sample peak signal point for focal plane, with this plane for reference point, 2um is that step pitch carries out tomoscan (carrying out along Z axis), finally carries out three-dimensional reconstruction, obtains biomembrane stereochemical structure.By professional software process record biofilm thickness, the antibacterial gross area, viable bacteria area, dead bacterium area and calculating viable bacteria percentage ratio.Its result of the test is as shown in accompanying drawing 2 and table 5.
Table 5 Lactobacillus plantarum FB-T9 metabolite is on the impact of Streptococcus mutans biomembrane activity
As can be seen from these results, in 24h and 48h normality biomembrane, viable bacteria is occupied an leading position, and only has a small amount of dead bacterium to exist, thalline be lumps compact siro spinning technology together, biofilm structure is complete.In experimental group, three mediations time point, i.e. 0h, 6h, 12h before 24h, biofilm structure is destroyed completely, and thalline is substantially with loose some formal distribution, and bacteria total amount obviously declines.And after 24h with 48h after mediate time, although the amplitude that antibacterial area declines, not as the effect of mediation before 24h, still has remaining biofilm structure to exist, be essentially dead bacterium.In all experimental grouies, biomembrane activity all has significant difference (P < 0.05) compared with matched group.
Embodiment 4: Lactobacillus plantarum FB-T9 metabolite is on the impact of extracellular polysaccharide in Streptococcus mutans biomembrane
The preparation of biomembrane specimen is as embodiment 3, but fluorescent dye used is Calcofluor
tM, concentration is 10 μ g/ml, and it can make extracellular polysaccharide send blue-fluorescence.The exciting light used when observing with CLSM is 400 ~ 490nm, and other condition is with embodiment 3.Its result of the test as shown in Figure 3.
As can be seen from accompanying drawing 3, after the biomembrane of the metabolite effect Streptococcus mutans of Lactobacillus plantarum FB-T9, sparse and sparse along with Multiple drug resistance of the distribution of extracellular polysaccharide in biomembrane, lumps, streak structure reduce, the area of extracellular polysaccharide also reduces accordingly, but slip reduces along with the postponement of mediation time.
Embodiment 5: the impact of biofilm states lower variation streptococcus being synthesized to insoluble extracellular polysaccharide
First, in 24 hole polystyrene Tissue Culture Dishs, it is 1 × 10 that every hole adds bacteria concentration
5the Streptococcus mutans bacteria suspension 250 μ l of cfu/ml and the TSB culture medium 750 μ l containing 0.25% sucrose, N
295%, CO
25%, 37 DEG C of Anaerobic culturel.1ml lactic acid bacteria fermentation supernatant is added during 0h, 6h, 12h in Streptococcus mutans 24h biofilm formation.Remove culture medium in hole after 24h, PBS hole flushing 2 times, remove the antibacterial swum in surface.Or every hole adds the TSB culture medium that 500 μ l Streptococcus mutans bacteria suspensions and 1500 μ l contain 0.25% sucrose, Anaerobic culturel, form 24h or 48h biomembrane, remove the culture medium in hole, PBS hole flushing 2 times, remove the antibacterial swum in surface, every hole adds lactic acid bacteria fermentation supernatant 2000 μ L, Anaerobic culturel 24h, removes the liquid in hole, PBS hole flushing 2 times, removes the antibacterial swum in surface.Then every hole adds 1.5ml, the NaOH of 0.4M, firmly the resuspended antibacterial be close at the bottom of hole, collects thalline, 4 DEG C, 1500g, 20min, collect supernatant, precipitation uses 1.5ml again, and the NaOH of 0.4M washs 4 times, merges supernatant, as the sample of water-insoluble extracellular polysaccharide.The content of extracellular polysaccharide is finally surveyed with Anthrone-sulfuricacid method.Its result of the test as shown in Figure 4.
As can be seen from accompanying drawing 4, after the biomembrane of the metabolite effect Streptococcus mutans of Lactobacillus plantarum FB-T9, the content of the insoluble extracellular polysaccharide in each experimental group biomembrane significantly declines (P < 0.05), but slip also reduces along with the postponement of mediation time.
It is to be noted: * represents that processed group and untreated fish group have significant difference (P < 0.05).
The present invention is by above-mentioned analysis, the metabolite of Lactobacillus plantarum FB-T9 is applied to treatment and caries prevention aspect, and prove by experiment, the metabolite of Lactobacillus plantarum FB-T9 has better effect to treatment and caries prevention, the further function excavating probiotic bacteria, for safeguarding oral health, caries prevention provides new approach and solution.
It should be noted that, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (3)
1. an analytical method for Lactobacillus plantarum metabolite inhibition, is characterized in that: comprise,
Lactobacillus plantarum FB-T9 metabolite is to the impact analysis of Streptococcus mutans biofilm formation, and it comprises:
A () is on the impact of Streptococcus mutans biofilm formation amount:
Prepare Streptococcus mutans bacteria suspension;
When 0h, add Streptococcus mutans bacteria suspension in culture medium, these three time points of the 0h in cultivation, 6h, 12h, add Lactobacillus plantarum FB-T9 fermented supernatant fluid respectively, cultivate 24h together; Or Streptococcus mutans single culture 24h or 48h, the antibacterial of removing culture medium and swimming, then add Lactobacillus plantarum FB-T9 fermented supernatant fluid;
Negative control group and positive controls add normal saline and the chlorhexidine acetate solution of equivalent respectively;
After cultivation terminates, abandon free bacteria, deionized water wash, natural drying;
Add crystal violet solution, dye under room temperature, make the antibacterial of combination painted;
Incline after dyeing liquor, deionized water wash, add the ethanol/acetone mixed liquor colour developing of mixing after drying, microplate reader 600nm measures absorbance;
B () is on Streptococcus mutans biofilm structure and active impact:
Prepared by biomembrane specimen: in culture dish, add Streptococcus mutans bacteria suspension and the TSB culture medium containing 0.25% sucrose, Lactobacillus plantarum FB-T9 fermented supernatant fluid is added respectively when incubation time is 0h, 6h, 12h, each time point cultivates 3 samples, Anaerobic culturel 24h, PBS rinses, remove surperficial planktonic bacteria, dye in camera bellows under room temperature immediately; Or first cultivate Streptococcus mutans, after forming 24h or 48h biomembrane, remove culture medium, PBS washs, and removes surperficial planktonic bacteria, adds Lactobacillus plantarum FB-T9 fermented supernatant fluid, Anaerobic culturel 24h, removes flcating germ, fluorescence staining;
The preparation of fluorescent dye and dyeing: respectively viable bacteria, dead bacterium are dyeed with fluorescein based dye CFSE, PI, make antibacterial alive, extremely antibacterial send green fluorescence respectively, red fluorescence, thus can biofilm structure be observed, dyeing terminates rear PBS and washs, removing residual dye;
Confocal laser scanning microscope: observe under the above-mentioned biomembrane specimen having completed fluorescence staining is placed on CLSM, object lens × 20, eyepiece × 10, the exciting light observing biofilm structure is 510/480.With sample peak signal point for focal plane, with this plane for reference point, carrying out 2um along Z axis is that step pitch carries out tomoscan, finally carries out three-dimensional reconstruction, obtain biomembrane stereochemical structure, process record biofilm thickness, the antibacterial gross area, viable bacteria area, dead bacterium area and calculating viable bacteria percentage ratio;
Lactobacillus plantarum FB-T9 metabolite is to the impact analysis of extracellular polysaccharide in Streptococcus mutans biomembrane: adopt as the method in step (b) prepares biomembrane specimen, make extracellular polysaccharide send blue-fluorescence with fluorescent dye, the exciting light used when observing with CLSM is 400 ~ 490nm;
Biofilm states lower variation streptococcus is synthesized to the impact analysis of insoluble extracellular polysaccharide, it comprises:
In polystyrene cell culture dish, add Streptococcus mutans bacteria suspension and the TSB culture medium containing 0.25% sucrose, Anaerobic culturel;
Add Lactobacillus plantarum FB-T9 fermented supernatant fluid during 0h, 6h, 12h in Streptococcus mutans 24h biofilm formation, remove culture medium after 24h, PBS washs, and removes the antibacterial swum in surface; Or add Streptococcus mutans bacteria suspension and the TSB culture medium containing 0.25% sucrose, Anaerobic culturel, form 24h or 48h biomembrane, remove culture medium, PBS washs, remove the antibacterial swum in surface, add Lactobacillus plantarum FB-T9 fermented supernatant fluid, Anaerobic culturel 24h, remove the liquid in hole, PBS washs, and removes the antibacterial swum in surface;
Add NaOH, collect thalline, collect supernatant, precipitation with NaOH washing, merges supernatant, as the sample of water-insoluble extracellular polysaccharide again;
The content of extracellular polysaccharide is surveyed with Anthrone-sulfuricacid method.
2. the analytical method of Lactobacillus plantarum metabolite inhibition as claimed in claim 1, is characterized in that: to described Lactobacillus plantarum FB-T9 metabolite to the impact analysis of Streptococcus mutans biofilm formation before, also comprise:
With the fermented supernatant fluid for the treatment of of different temperature Lactobacillus plantarum FB-T9, then observed the situation of change of its biocidal property by bacteriostatic experiment, obtain effective antibacterial substance and there is heat stability, get rid of the possibility of macro-molecular protein as antibacterial substance;
The pH of the fermented supernatant fluid of Lactobacillus plantarum FB-T9 is transferred to different values, then observes the situation of change of its biocidal property by bacteriostatic experiment, obtain effective antibacterial substance and have pH dependency, namely pH is lower, and fungistatic effect is better;
With the fermented supernatant fluid of trypsin, papain and E.C. 3.4.21.64 process Lactobacillus plantarum FB-T9, the situation of change of its biocidal property is observed again by bacteriostatic experiment, obtain fungistatic effect significantly to decline, prove in its effective antibacterial substance main containing micromolecule polypeptide bacterioid element;
With the fermented supernatant fluid of hydrogen peroxide ferment treatment Lactobacillus plantarum FB-T9, then observed the situation of change of its biocidal property by bacteriostatic experiment, obtain fungistatic effect and decline, illustrate in its effective antibacterial substance containing hydrogen peroxide.
3. the application of metabolite in treatment and caries prevention of a Lactobacillus plantarum FB-T9.
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| CN107523526A (en) * | 2017-10-17 | 2017-12-29 | 无限极（中国）有限公司 | A kind of lactobacillus reuteri and application thereof |
| CN108486022A (en) * | 2018-07-02 | 2018-09-04 | 四川大学 | One plant of anti-caries disease lactobacillus plantarum and its application |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107523526A (en) * | 2017-10-17 | 2017-12-29 | 无限极（中国）有限公司 | A kind of lactobacillus reuteri and application thereof |
| CN107523526B (en) * | 2017-10-17 | 2020-05-12 | 无限极（中国）有限公司 | Lactobacillus reuteri and application thereof |
| US10968495B2 (en) | 2017-10-17 | 2021-04-06 | Infinitus (China) Company Ltd. | Lactobacillus reuteri and use thereof |
| CN108486022A (en) * | 2018-07-02 | 2018-09-04 | 四川大学 | One plant of anti-caries disease lactobacillus plantarum and its application |
| CN108486022B (en) * | 2018-07-02 | 2020-08-28 | 四川大学 | Anti-caries lactobacillus plantarum and application thereof |
| CN109908185A (en) * | 2019-03-29 | 2019-06-21 | 江南大学 | A method of inhibiting Streptococcus mutans and the double bacterium biomembranes of Candida albicans |
| CN109908185B (en) * | 2019-03-29 | 2020-10-09 | 江南大学 | Method for inhibiting streptococcus mutans and candida albicans double-bacteria biofilm |
| CN111588838A (en) * | 2020-05-29 | 2020-08-28 | 扬州大学 | Composition for inhibiting streptococcus mutans and application thereof |
| CN111588838B (en) * | 2020-05-29 | 2023-01-31 | 扬州大学 | Composition for inhibiting streptococcus mutans and application thereof |
| CN112852686A (en) * | 2021-04-14 | 2021-05-28 | 四川高福记生物科技有限公司 | Lactobacillus plantarum LP220 with caries prevention function and application thereof |
| CN113702559A (en) * | 2021-08-16 | 2021-11-26 | 江南大学 | Method for separating and identifying active substances of lactobacillus plantarum source inhibiting double-bacterium biological membrane |
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