CN109908185A - A method of inhibiting Streptococcus mutans and the double bacterium biomembranes of Candida albicans - Google Patents
A method of inhibiting Streptococcus mutans and the double bacterium biomembranes of Candida albicans Download PDFInfo
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Abstract
The invention discloses a kind of methods of inhibition Streptococcus mutans and the double bacterium biomembranes of Candida albicans, belong to microorganisms technical field.In the different phase of double bacterium biofilm formations, lactobacillus plantarum CCFM8724 has the external ability for inhibiting Streptococcus mutans and the double bacterium biomembranes of Candida albicans, bacterium number of causing a disease in the extracellular polysaccharide amount and biomembrane that double bacterium biomembranes can substantially reduce biomembrane is acted in different time, and it is resistant to the lysozyme of 1.6mg/mL, film forming is all poor under different sucrose simultaneously, be below 0.80 hereinafter, illustrate the lactobacillus plantarum will not substituted cariogenic bacteria form the possibility of cariogenic biomembrane.Method provided by the invention has effects that reduce the cariogenic plaque in oral cavity, prevention and treatment saprodontia, is expected to prevent and treat one new function market of saprodontia opening for probiotics.
Description
Technical field
The present invention relates to a kind of methods of inhibition Streptococcus mutans and the double bacterium biomembranes of Candida albicans, belong to microorganism skill
Art field.
Background technique
Dental caries are the bacterial infection diseases betided on enamel and dentine, and the formation of Dental plaque biofilm is dental caries
Pathogenetic prerequisite.The Medium Culture formation that mature Dental plaque biofilm is oral bacteria based on the insoluble polysaccharide
A kind of ecological environment of 3 D stereo is rich in gap and pipe-line system, has certain thickness.Once due to host, diet, micro- life
The variation of the factors such as the growth of object and when leading to the disruption of ecological balance, the cariogenic microorganism in Dental plaque biofilm becomes dominant bacteria
Group, their tachymetabolisms decompose carbohydrate, especially sucrose, generate organic acid, pH is made to be reduced to 5.5 hereinafter, leading to tooth
Enamel demineralization and hard tooth tissue structural failure are to form cavity.
Streptococcus mutans are generally acknowledged main cariogenic bacterias.Although Streptococcus mutans are not the most abundant bacteriums in oral cavity,
The formation of its energy rapid coordination cariogenicity biomembrane, and the stronger cariogenic biomembrane of virulence can be formed in conjunction with other cariogenic bacterias.With
In oral cavity unlike planktonic bacteria, the usual nutrient restriction of bacterium in biomembrane, the bacterium under this approximate dormant state
Than the metabolic activity bacterium that swims more resistant to antibiotic and bacteriostatic agent, in addition, many therapeutic agents before they act on bacteriums just with
Exocellular polysaccharide in biomembrane combines, to lose effectiveness.This is also that antibacterial agent and antibiotic clinically prevent and treat saprodontia effect
One of bad reason.Therefore, " probiotic therapy " is the hot spot of current techniques exploitation.
Existing research proves that lactobacillus plantarum can inhibit the growth of Streptococcus mutans to inhibit the degree (CN of dental caries
108486022A publication date: 2018.09.04).But clinical investigation is found, can usually be detected simultaneously in the oral cavity of dental caries patient white
Color candida albicans and Streptococcus mutans (Hajishengallis E et al., Molecular oral microbiology, 2017,32
(1): 24-34.), therefore probiotics is studied to the inhibition work of double kind of biomembranes of Streptococcus mutans and fungi candida albicans
With, for prevention and treatment saprodontia it is more and more important.
Being associated with for candida albicans and Streptococcus mutans can enhance the pathogenic of Streptococcus mutans and strengthen cariogenic life
The formation of object film.The ability of Candida albicans adherence facing is weaker, but in conjunction with Streptococcus mutans after, oral cavity colonization ability is significantly
Enhancing.The glucosyltransferase (GTF) that Streptococcus mutans generate can be adhered to the mannosan on albicans cell wall,
Raised onto early stage Dental plaque biofilm (Ellepola K et al., Journal of dental research, 2017,
96(10): 1129-1135.).Candida albicans with larger surface area can provide bound site for more Streptococcus mutans
Point, the Streptococcus mutans quantity increased increase the quantity of Candida albicans again in turn.Double kinds of biomembranes are than single biomembrane
With higher biomass and cell quantity, and the biomembrane of existing research confirmation Candida albicans and Streptococcus oralis is to antibiosis
The drug resistance of element be higher than single species (Shirtliff M E et al., FEMS microbiology letters, 2009,299
(1): 1-8.), these characteristics of double bacterium biomembranes undoubtedly increase the difficulty to Caries therapy caused by it.
Summary of the invention
The first purpose of the invention is to provide the sides of a kind of inhibition Streptococcus mutans and the double bacterium biomembranes of Candida albicans
Method, the method are to inhibit the formation of Streptococcus mutans and the double bacterium biomembranes of Candida albicans using lactobacillus plantarum.
In one embodiment of the invention, the lactobacillus plantarum is lactobacillus plantarum CCFM8724.
In one embodiment of the invention, the Streptococcus mutans are Streptococcus mutans ATCC 25175.
It in one embodiment of the invention, is with lactobacillus plantarum CCFM8724 fermentation supernatant or bacterium mud described double
The different phase of bacterium biofilm formation is mediated.
In one embodiment of the invention, the lactobacillus plantarum CCFM8724 fermentation supernatant or bacterium mud are by plant
Lactobacillus CCFM8724 is cultivated in MRS fluid nutrient medium, and supernatant bacterium mud is respectively obtained after centrifugation.
In one embodiment of the invention, the mediation is 0h, 6h, 12h and for 24 hours in double bacterium biofilm formations
It is mediated respectively.
In one embodiment of the invention, the specific steps that described 0h, 6h, 12h are mediated are as follows: Streptococcus mutans are added
With Candida albicans bacteria suspension, it is separately added on lactobacillus plantarum CCFM8724 in double bacterium biofilm developments to 0h, 6h, 12h
Clearly, culture is then proceeded to for 24 hours.
In one embodiment of the invention, the specific steps mediated for 24 hours are as follows: Streptococcus mutans and white is added
Candida albicans bacteria suspension, double bacterium biofilm developments to for 24 hours when, after cleaning biomembrane with PBS, be added lactobacillus plantarum
CCFM8724 supernatant then proceedes to culture for 24 hours.
In one embodiment of the invention, the Streptococcus mutans and Candida albicans bacteria concentration be 1 ×
106cfu/mL。
The present invention also provides application of the lactobacillus plantarum CCFM8724 in terms of preparing anticaries drug.
Beneficial effects of the present invention:
(1) in the different phase of double bacterium biofilm formations, the inhibition that lactobacillus plantarum CCFM8724 supernatant can be apparent
The forming amount of Streptococcus mutans and the double bacterium biomembranes of Candida albicans, and make biomembrane reduction amount all ten in the different mediation time
Divide significantly, is above 65%, 0h mediated effects close to 90%, illustrates that lactobacillus plantarum CCFM8724 can inhibit pathogenic bacteria to form life
Object film.
(2) 0h be added after lactobacillus plantarum CCFM8724 supernatant mediates in double bacterium biomembranes pathogenic bacteria Streptococcus mutans and
Candida albicans quantitatively all has dropped 2~3 orders of magnitude, it is seen then that lactobacillus plantarum CCFM8724 supernatant can make viable bacteria
Number is reduced.
(3) lactobacillus plantarum CCFM8724 is when 0h is mediated, so that the reduction amount of exocellular polysaccharide amount is up to 80% or so,
He mediates the reduction amount of time exocellular polysaccharide to be also both greater than 40%, and effect is very significant.
(4) double bacterium biomembranes have preferable film forming under different sucrose, but lactobacillus plantarum CCFM8724 is not
It is all poor with film forming under sucrose concentration, be below 0.80 hereinafter, illustrate the lactobacillus plantarum will not substituted cariogenic bacteria formed
The possibility of cariogenic biomembrane.
(5) lactobacillus plantarum CCFM8724 is 1.6mg/mL to the highest tolerable concentration of lysozyme, as a result significantly larger than people
The concentration (0~57 μ g/mL) of lysozyme illustrates that lactobacillus plantarum CCFM8724 has and survives in oral environment in saliva of buccal cavity
Ability.
Biomaterial
Lactobacillus plantarum CCFM8724 of the invention is in patent " a kind of lactobacillus plantarum CCFM8724 and application thereof "
(CN201210046430.8) it is disclosed in (publication date: 2012.07.04).
Streptococcus mutans ATCC 25175 of the invention is bought in China General Microbiological Culture Collection Center CGMCC.
Detailed description of the invention
Fig. 1: lactobacillus plantarum CCFM8724 supernatant different time mediates the inhibiting effect to double bacterium biofilm formation amounts；*
Indicate that processing group and untreated fish group have significant difference (P < 0.05).
Fig. 2: lactobacillus plantarum CCFM8724 supernatant 0h mediates in Streptococcus mutans and the double bacterium biomembranes of Candida albicans
The influence for bacterium number of causing a disease；* * indicates that processing group and untreated fish group have significant difference (P < 0.001).
Fig. 3: lactobacillus plantarum CCFM8724 supernatant different time mediates total to dead bacterium dyeing laser living in double bacterium biomembranes
Focus three-dimensional model diagram；Wherein A:24h untreated control；B:6h mediation group；C:12h mediation group；D:24h mediation group.
Fig. 4: lactobacillus plantarum CCFM8724 bacterium mud 0h mediates to Streptococcus mutans and the double bacterium biomembrane knots of Candida albicans
The influence of structure；A, C, D: double bacterium biomembrane controls；B, double bacterium biomembranes that E, F:CCFM8724 are mediated.
Fig. 5: lactobacillus plantarum CCFM8724 supernatant different time mediates the influence to the extracellular polysaccharide amount of double bacterium biomembranes.
Fig. 6: lactobacillus plantarum CCFM8724 autohemagglutination and with pathogenic bacteria copolymerized ability.
Fig. 7: lactobacillus plantarum CCFM8724 forms the ability of biomembrane under different sucrose.
Fig. 8: the lactobacillus plantarum CCFM8724 tolerance to the lysozyme of various concentration.
Fig. 9: different lactobacillus compare Streptococcus mutans and Candida albicans single bacterium biomembrane and double bacterium biomembrane effects；
A:23 plants of lactobacillus 12h mediate Streptococcus mutans single bacterium biomembrane reduction amount；B:23 plants of lactobacillus 12h mediate Candida albicans
Single bacterium biomembrane reduction amount；C:23 plants of lactobacillus 12h mediate double bacterium biomembrane reduction amounts.
Specific embodiment
MRS culture medium: containing yeast powder 5.0g/L, beef extract 10.0g/L, peptone 10.0g/L, glucose 20.0g/L,
Anhydrous sodium acetate 2.0g/L, citric acid hydrogen diamine 2.0g/L, dipotassium hydrogen phosphate 2.6g/L, Manganous sulfate monohydrate 0.25g/L, seven water
Close magnesium sulfate 0.5g/L, Tween-80 1mL, pH6.2~6.4.
TSBY culture medium: for the pancreas peptone soybean broth culture medium for being added to 0.6% yeast powder.Contain yeast powder
6.0g/L, soybean protein 3.0g/L, tryptone 17.0g/L, glucose 2.5g/L, sodium chloride 5g/L and dipotassium hydrogen phosphate
2.5g/L, pH 7.0~7.4.The TSBY culture medium for being used to form double bacterium biomembranes separately adds 5% sucrose.
YPD culture medium: yeast powder 1.0g/L, glucose 2.0g/L and peptone 2.0g/L.It is used to form double bacterium biomembranes
YPD culture medium separately add 5% sucrose.
Lactobacillus plantarum CCFM8724 supernatant: by lactobacillus plantarum CCFM8724 with 2% inoculation in MRS fluid nutrient medium
Amount inoculation, the 8000r/min after 37 DEG C of incubator cultures for 24 hours, are centrifuged 5min, are filtered to take with 0.22 μm of sterilised membrane filter by 4 DEG C
Supernatant.
Lactobacillus plantarum CCFM8724 bacterium mud: by lactobacillus plantarum CCFM8724 with 2% inoculation in MRS fluid nutrient medium
Amount inoculation, the 8000r/min after 37 DEG C of incubator cultures for 24 hours, are centrifuged 5min by 4 DEG C, abandon supernatant and obtain bacterium mud.
Suspension of S. mutans: Streptococcus mutans strain ATCC 25175 is cultivated in the TSBY for being added to 0.6% yeast powder
In base at 37 DEG C, 12h is cultivated, adjusting bacteria suspension concentration is 1 × 106cfu/mL。
Candida albicans bacteria suspension: by Candida albicans 37 DEG C in YPD culture medium, 12h is cultivated, its bacteria suspension concentration is adjusted
It is 1 × 106cfu/mL。
The formation of Dental plaque biofilm mainly includes attachment stage (0~6h), initial microcolony stage (6~12h) and life
Object film maturation (12~for 24 hours) and (24~48h) this four-stage is disseminated the stage, therefore in verifying lactobacillus plantarum CCFM8724
Main from 0h, 6h, 12h and for 24 hours these times when to the inhibiting effect of the double bacterium biomembranes of Streptococcus mutans and Candida albicans
Point is studied.
Embodiment 1: influence of the lactobacillus plantarum CCFM8724 to double bacterium biofilm formation amounts
Streptococcus mutans and each 50 μ L of Candida albicans bacteria suspension are added in 96 orifice plates, in 0h, 6h, 12h is separately added into
100 μ L of lactobacillus plantarum CCFM8724 supernatant, 37 DEG C of cultures are for 24 hours.And the method mediated for 24 hours be double bacterium biofilm developments extremely
It is carefully cleaned with PBS biomembrane 2 times when for 24 hours, 100 μ L of lactobacillus plantarum CCFM8724 supernatant is added, 37 DEG C of cultures are for 24 hours.Double bacterium
Biomembrane control replaces lactobacillus plantarum supernatant with blank MRS fluid nutrient medium, and every group setting 6 parallel.It is used after culture
PBS is carefully cleaned biomembrane 2 times, is stored at room temperature after drying biomembrane so that 100 μ L, 0.1% crystal violet solution is added to every hole, will
Biomembrane dyes 30min, is cleaned 2 times after dyeing with PBS, every hole is dissolved with 95% ethyl alcohol, under microplate reader OD600nm
Read absorbance value.
As seen from Figure 1 in the different phase of double bacterium biofilm formations, lactobacillus plantarum CCFM8724 supernatant can be apparent
Inhibit the forming amount of the double bacterium biomembranes of Streptococcus mutans and Candida albicans, and reduce biomembrane in the different mediation time
Amount is all very significant, is above 65%, 0h mediated effects close to 90%, illustrates that lactobacillus plantarum CCFM8724 supernatant has and inhibit
The effect of pathogenic bacteria formation biomembrane.
1 biofilm biomass slip of table
Embodiment 2: influence of the lactobacillus plantarum CCFM8724 to bacterium number of causing a disease in double bacterium biomembranes
It is put into 18mm × 18mm sterile cover slips in 6 orifice plates, Streptococcus mutans are added and Candida albicans bacteria suspension is each
250 μ L of lactobacillus plantarum CCFM8724 supernatant is added in 0h in 2mL, and 37 DEG C of cultures for 24 hours, are compareed with blank MRS fluid nutrient medium generation
It replaces.The coverslip with biomembrane is sandwiched in 50mL centrifuge tube with aseptic nipper after culture, with 0.89% nothing of 10mL
Bacterium physiological saline is submerged, under cleaning 2h guarantees that biomembrane is eluted from coverslip in centrifuge tube placement ultrasonic cleaning equipment
Come, while being suspended in physiological saline.Gradient dilution biomembrane suspension, each gradient take 100 μ L to dilute suspension, are cultivated with TSB
Base and YPD culture medium distinguish pouring plate, count after 37 DEG C of cultures for 24 hours, a formula three is parallel, tests in triplicate.As a result such as
Shown in Fig. 2, it is seen that pathogenic bacteria quantity all declines in double bacterium biomembranes after the mediation of lactobacillus plantarum CCFM8724 supernatant is added in 0h
2~3 orders of magnitude.
2 biomembrane pathogenic bacteria of table count (logCFU/mL)
Embodiment 3: influence of the lactobacillus plantarum CCFM8724 to double bacterium biomembrane activities
CFSE dyestuff: the viable bacteria in 8 Μ m, CFSE dye capable of dyeing colour biological films.
PI dyestuff: 4 μM, the dead bacterium in PI dye capable of dyeing colour biological film.
It is put into the sterile cover slips of 24mm × 50mm in the disposable plate of diameter 90mm, Streptococcus mutans and white is added
Lactobacillus plantarum CCFM8724 supernatant is added for 24 hours and is mediated, when mediating for 24 hours in 6h, 12h by each 8mL of color candida albicans bacteria suspension
Biomembrane taking-up is put into the fresh culture of the CCFM8724 supernatant of lactobacillus plantarum containing 2mL, double bacterium biomembrane controls are with sky
White MRS fluid nutrient medium replaces supernatant, and all mediation groups and control group are at 37 DEG C, and culture is for 24 hours.It is carefully cleaned after culture
Biomembrane dyes biomembrane under darkroom, and first plus 300 μ L of CFSE dyestuff covers entire coverslip, is protected from light 37 DEG C of incubations
30min, then with twice of sterile water wash biomembrane, entire coverslip is covered with 300 μ L of PI dyestuff, is protected from light 37 DEG C of incubations
30min, after use twice of sterile water wash biomembrane, be placed under laser confocal microscope and observe after dry, object lens times
Number 20 ×, be arranged exciting light 488nm, first focus on most bright focal plane move down and up respectively again object lens be adjusted to almost without
One layer of fluorescence signal is set as the bottom and top, after 2 μ of Scanning step is set, according to the biomembrane top of setting and
Bottom distance calculates the number of plies and carries out layer-by-layer scanning shoot, and preview 3D effect after shooting saves picture.As a result such as Fig. 3
It is shown, it is seen that the biomembrane overlay capacity that 6h mediates (Fig. 3 B) to be formed afterwards is minimum, and it is more late poorer to the reduction effect of biomembrane to mediate,
Mediate (Fig. 3 D) although still foring biomembrane for 24 hours, the thickness of biomembrane substantially reduces, and dead bacterium ratio substantially on
It rises, which significantly reduces.Although illustrating to be mediated biomembrane in addition lactobacillus plantarum CCFM8724 supernatant for 24 hours
Amount reduction is not significant, but can substantially reduce biomembrane activity.
Embodiment 4: influence of the lactobacillus plantarum CCFM8724 to double bacterium biofilm structures
It is put into 18mm × 18mm sterile cover slips in 6 orifice plates, Streptococcus mutans are added and Candida albicans bacteria suspension is each
2mL, 20 μ L of lactobacillus plantarum CCFM8724 bacterium mud is added in 0h, and (bacterium mud is suspended from PBS and adjusts OD0.6, CFU 106~107), 37
DEG C culture for 24 hours, control with blank MRS fluid nutrient medium replacement.With sterile water wash coverslip after culture, with 3% at 4 DEG C
Glutaraldehyde is fixed overnight, then with 70%, 80%, 96% and 100% ethyl alcohol continuous dehydration 20min, metal spraying after air-drying, in 10kv
It is observed under voltage.
As a result as shown in figure 4, from Fig. 4 A, Fig. 4 C, Fig. 4 D as it can be seen that double bacterium biomembranes compare the biomembrane to be formed joins together
And only several gaps can be seen that the thickness of double bacterium biomembranes, Candida albicans mycelia conduct as nutrition channel, after amplification
Virulence factor is criss-cross in double bacterium biomembranes bottom, makes more Streptococcus mutans and Candida albicans adherence thereon, by
Step builds up biofilm thickness.And Fig. 4 B, Fig. 4 E, Fig. 4 F figure are the result figure after lactobacillus plantarum CCFM8724 bacterium mud mediates,
Fig. 4 B can see single lactobacillus plantarum apparently without the sheet of biomembrane of the company of being formed, after amplification and not see substantially white
The mycelia of color candida albicans is crosslinked, and the thickness of biomembrane significantly reduces, several pieces only very thin, not raised stereochemical structure.From sweeping
It retouches Electronic Speculum result to see, CCFM8724 reduces the biofilm biomass of double bacterium biomembranes, and three-dimensional structure is destroyed, and Candida albicans mycelia
Formation be inhibited, lactobacillus plantarum CCFM8724 effect is very significant.
Embodiment 5: influence of the lactobacillus plantarum CCFM8724 to the extracellular polysaccharide amount of double bacterium biomembranes
The measuring method of exocellular polysaccharide content: Streptococcus mutans and Candida albicans bacteria suspension is added in every hole in 24 orifice plates
150 μ L of lactobacillus plantarum CCFM8724 supernatant is added in 0h, 6h, 12h in each 1mL, mediates clean for PBS after culture for 24 hours for 24 hours
Twice of biomembrane, add lactobacillus plantarum CCFM8724 supernatant 2mL, 37 DEG C of cultures are for 24 hours.The 0.4M of 1.5mL is used after culture
NaOH will be drawn after the biology membrane elution of orifice plate bottom to 2mL centrifuge tube, draw supernatant after 8000r/min centrifugation 3min, remaining
After precipitating washes 4 times with 0.4M NaOH again, the supernatant after each centrifugation is collected, is contained with Anthrone-sulfuricacid method measurement exocellular polysaccharide
Amount.
As a result as shown in figure 5, the reduction amount 0h of exocellular polysaccharide amount is up to 80% or so, other mediate time exocellular polysaccharide
Reduction amount is also both greater than 40%, and effect is very significant.Matrix of the exocellular polysaccharide as biomembrane most critical is pathogenic bacteria into long hair
The product educated is also the base that they grow, and the reduction of the extracellular polysaccharide amount of biomembrane illustrates that lactobacillus plantarum CCFM8724 is sent out
The preferable effect for inhibiting double bacterium biomembranes is shot.
The insoluble exocellular polysaccharide reduction amount (%) of table 3
Embodiment 6: the autohemagglutination of lactobacillus plantarum CCFM8724 and the ability with pathogenic bacteria copolymerization
The autohemagglutination and itself and Streptococcus mutans and Candida albicans of lactobacillus plantarum CCFM8724 are investigated with spectrophotometry
Copolymerized ability.The strong bacterium of autohemagglutination ability has certain colonization ability in oral cavity, and being copolymerized strong bacterium with pathogenic bacteria can incite somebody to action
Pathogenic bacteria are taken away oral cavity with drinking water or gargling by pathogenic bacteria.
(1) test of autohemagglutination ability
Lactobacillus plantarum CCFM8724 is suspended from PBS and adjusts OD600Lactobacillus plantarum CCFM8724 suspension is obtained for 0.6,
Lactobacillus plantarum CCFM8724 suspension is stood, 100 μ L of supernatant liquid is drawn every 2h and surveys extinction at 600nm in 96 orifice plates
Degree, formula are as follows:
(A0For initial OD values, AtFor different time OD value).
(2) copolymerized ability is tested
Equivalent (each 1mL) lactobacillus plantarum is mixed with Streptococcus mutans, Candida albicans respectively, is stored at room temperature, every
2h draws the OD that supernatant liquid surveys different time600Value, formula are as follows:
(AxAnd AyFor lactobacillus and pathogenic bacteria initial OD values, AtWhen being different
Between OD value).
As a result as shown in fig. 6, lactobacillus plantarum CCFM8724 autohemagglutination ability in 4h up to 20%, linearly rise later
Trend illustrates that it has certain colonization ability in oral cavity；And lactobacillus plantarum CCFM8724 can be with Streptococcus mutans and white
Color candida albicans is copolymerized, interior in 2h, altogether agglutinability difference nearly 60% and 40%, extension at any time, co-agglomeration effect
Fruit is better, illustrate can with CCFM8724 have pathogenic bacteria are taken away into the potentiality in oral cavity with drinking water or gargling.
Embodiment 7: lactobacillus plantarum CCFM8724 forms the ability of biomembrane under different sucrose
Lactobacillus plantarum CCFM8724 bacteria suspension is adjusted with the TSB culture medium containing 0%, 0.25%, 1%, 2%, 5% sucrose
Dense middle bacterium is 106CFU/mL is inoculated in 96 orifice plates, and every 100 μ L of hole, using double bacterium biomembranes as control, 37 DEG C are cultivated for 24 hours, and one
Formula 6 parallel.Twice of biomembrane rear room temperature standing and drying is washed with PBS, 0.1% violet staining, 95% second are carried out to biomembrane
Alcohol dissolution colour developing, measures its light absorption value at 600nm with microplate reader and judges its biomembrane generative capacity.
As a result as shown in fig. 7, using double bacterium biomembranes as control, double bacterium biomembranes have preferably under different sucrose
Film forming, but lactobacillus plantarum CCFM8724 film forming under different sucrose is all poor, and difference is little, illustrates the plant
Lactobacillus will not substituted cariogenic bacteria form the possibility of cariogenic biomembrane.
Film forming ability under 4 different sucrose of table
Embodiment 8: tolerance of the lactobacillus plantarum CCFM8724 to the lysozyme of various concentration
A certain amount of 200 μ L MRS culture medium is added in 96 well culture plates, then adds the lysozyme of various concentration respectively
Solution makes final concentration be respectively 0.4,0.8,1.2,1.6,2.0,3.0 (mg/mL).By lactobacillus plantarum CCFM8724 culture
It is inoculated into 96 well culture plates with the inoculum concentration of 5% (V/V), 37 DEG C of cultures for 24 hours, measure OD600Lower absorbance, i.e., according to newborn bar
The growing state of bacterium judges lactobacillus plantarum CCFM8724 to the tolerance of lysozyme.Control group only adds isometric 1 ×
108The amount of CFU/mL lactobacillus, lysozyme soln is replaced with isometric sterile water.
As a result as shown in figure 8, using experimental group OD value/control group OD value × 100% > 60% be according to as bacterial strain to bacteriolyze
Enzyme has the standard of height endurability, and lactobacillus plantarum CCFM8724 is 1.6mg/mL to the highest tolerable concentration of lysozyme, as a result
The concentration (0~57 μ g/mL) of lysozyme significantly larger than in human mouth saliva, this illustrates that lactobacillus plantarum CCFM8724 has in mouth
The ability survived in chamber environment.
Embodiment 9: the single bacterium biomembrane of different lactobacillus and double bacterium biomembrane inhibitory effects
Lactobacillus supernatant: respectively by different lactobacillus (including Lactobacillus salivarius, lactobacillus paracasei, lactobacillus reuteri,
Totally 23 plants of lactobacillus fermenti) it is inoculated in MRS fluid nutrient medium with 2% inoculum concentration, after 37 DEG C of incubator cultures for 24 hours
8000r/min, is centrifuged 5min, the supernatant filtered to take with 0.22 μm of sterilised membrane filter by 4 DEG C.
(1) Streptococcus mutans single bacterium biomembrane inhibitory effect
100 μ L of suspension of S. mutans is added in 96 orifice plates, when Streptococcus mutans single bacterium biofilm development is to 12h,
Different lactobacillus (including totally 23 plants of Lactobacillus salivarius, lactobacillus paracasei, lactobacillus reuteri, lactobacillus fermenti) supernatants are added
100 μ L continue culture for 24 hours at 37 DEG C.
(2) Candida albicans single bacterium biomembrane inhibitory effect
100 μ L of Candida albicans bacteria suspension is added in 96 orifice plates, when Candida albicans single bacterium biofilm development is to 12h,
Different lactobacillus (including totally 23 plants of Lactobacillus salivarius, lactobacillus paracasei, lactobacillus reuteri, lactobacillus fermenti) supernatants are added
100 μ L continue culture for 24 hours at 37 DEG C.
(3) Streptococcus mutans single bacterium and the double bacterium biomembrane inhibitory effects of Candida albicans
Streptococcus mutans single bacterium and each 100 μ L of Candida albicans bacteria suspension are added in 96 orifice plates, in double bacterium biofilm developments
When to 12h, different lactobacillus are added, and (including Lactobacillus salivarius, lactobacillus paracasei, lactobacillus reuteri, lactobacillus fermenti are total
23 plants) supernatant 100 μ L, continues culture for 24 hours at 37 DEG C.
The result shows that this 23 plants of lactobacillus make Streptococcus mutans single bacterium biomembrane reduction amount in 44%~72.3% (Fig. 9 a),
Make Candida albicans single bacterium biomembrane single bacterium biomembrane reduction amount in 9.4%~62.4% (Fig. 9 b), and same lactobacillus is to double
Bacterium biomembrane effect is only more than 60% without effect in 7.2%~40% (Fig. 9 c), even if illustrating lactobacillus to single bacterium biology
Film has inhibiting effect, not necessarily has inhibiting effect to Streptococcus mutans and the double bacterium biomembranes of Candida albicans.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of method for inhibiting Streptococcus mutans and the double bacterium biomembranes of Candida albicans, which is characterized in that the method is benefit
Inhibit the formation of Streptococcus mutans and the double bacterium biomembranes of Candida albicans with lactobacillus plantarum.
2. the method according to claim 1, wherein the lactobacillus plantarum is lactobacillus plantarum CCFM8724.
3. the method according to claim 1, wherein the Streptococcus mutans are Streptococcus mutans ATCC
25175。
4. the method according to claim 1, wherein being with lactobacillus plantarum CCFM8724 fermentation supernatant or bacterium mud
It is mediated in the different phase of double bacterium biofilm formations.
5. according to the method described in claim 4, it is characterized in that, the lactobacillus plantarum CCFM8724 fermentation supernatant or bacterium mud
It is to cultivate lactobacillus plantarum CCFM8724 in MRS fluid nutrient medium, supernatant bacterium mud is respectively obtained after centrifugation.
6. according to the method described in claim 4, it is characterized in that, it is described mediation be double bacterium biofilm formations 0h, 6h,
It 12h and is mediated respectively for 24 hours.
7. according to the method described in claim 6, it is characterized in that, the specific steps that described 0h, 6h, 12h are mediated are as follows: be added and become
Different streptococcus and Candida albicans bacteria suspension are separately added into lactobacillus plantarum in double bacterium biofilm developments to 0h, 6h, 12h
CCFM8724 supernatant then proceedes to culture for 24 hours.
8. according to the method described in claim 6, it is characterized in that, the specific steps mediated for 24 hours are as follows: variation hammer is added
Bacterium and Candida albicans bacteria suspension, double bacterium biofilm developments to for 24 hours when, after cleaning biomembrane with PBS, be added lactobacillus plantarum
CCFM8724 supernatant then proceedes to culture for 24 hours.
9. according to any method of claim 7 or 8, which is characterized in that the Streptococcus mutans and Candida albicans bacterium
Concentration is 1 × 106cfu/mL。
10. application of the lactobacillus plantarum CCFM8724 in terms of preparing anticaries drug described in claim 1.
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