CN109908185B - Method for inhibiting streptococcus mutans and candida albicans double-bacteria biofilm - Google Patents
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Abstract
The invention discloses a method for inhibiting a streptococcus mutans and candida albicans double-bacteria biofilm, belonging to the technical field of microorganisms. In different stages of the formation of the double-bacteria biomembrane, the lactobacillus plantarum CCFM8724 has the capacity of inhibiting the double-bacteria biomembrane of streptococcus mutans and candida albicans in vitro, the double-bacteria biomembrane can obviously reduce the exopolysaccharide production amount of the biomembrane and the number of pathogenic bacteria in the biomembrane under different time actions, can tolerate 1.6mg/mL of lysozyme, and has poor film-forming properties which are all lower than 0.80 under different sucrose concentrations, which indicates that the lactobacillus plantarum does not have the possibility of replacing cariogenic bacteria to form the cariogenic biomembrane. The method provided by the invention has the effects of reducing dental caries and dental caries, and is expected to open a new functional market for probiotic bacteria to prevent and treat dental caries.
Description
Technical Field
The invention relates to a method for inhibiting a streptococcus mutans and candida albicans double-bacteria biofilm, belonging to the technical field of microorganisms.
Background
Caries is a bacterial infectious disease that occurs in both enamel and dentin, and the formation of plaque biofilm is a prerequisite for caries development. The mature dental plaque biomembrane is a three-dimensional ecological environment formed by embedding oral bacteria in a matrix mainly containing insoluble polysaccharide, is rich in gaps and pipeline systems, and has a certain thickness. Once this ecological imbalance is caused by changes in host, diet, microbial growth, etc., cariogenic microbes in the plaque biofilm become dominant microflora, which rapidly metabolise breakdown of carbohydrates, especially sucrose, producing organic acids, lowering the pH below 5.5, leading to demineralization of the enamel and destruction of the hard tissue structure of the tooth to form caries cavities.
Mutans streptococci are recognized as the major cariogenic bacteria. Although S.mutans is not the most abundant bacterium in the oral cavity, it rapidly coordinates the formation of cariogenic biofilms and, in combination with other cariogenic bacteria, forms a more virulent cariogenic biofilm. Unlike planktonic bacteria in the oral cavity, bacteria in biofilms are often nutritionally restricted, bacteria in this near dormant state are more resistant to antibiotics and bacteriostatic agents than planktonic metabolically active bacteria, and in addition, many therapeutic agents bind to exopolysaccharides in biofilms before they act on the bacteria, thereby becoming ineffective. It is also one of the reasons why the antibacterial agent and antibiotic have poor effect of preventing and treating dental caries clinically. Thus, "probiotic therapy" is a hot spot in the development of current technology.
It has been shown that Lactobacillus plantarum inhibits the growth of Streptococcus mutans and thus the extent of caries (CN108486022A, published: 2018.09.04). However, clinical investigation has found that Candida albicans and Streptococcus mutans can be detected in the oral cavity of carious patients (Hajishengalis E et al, Molecular biology,2017,32(1):24-34.), so that it is increasingly important to study the inhibitory effect of probiotics on the double biofilms of Streptococcus mutans and Candida albicans, and to prevent and treat dental caries.
The association of candida albicans with streptococcus mutans enhances the pathogenicity of streptococcus mutans and enhances the formation of cariogenic biofilms. Candida albicans has a weak ability to adhere to the tooth surface, but after binding to Streptococcus mutans, the oral colonization ability is greatly enhanced. Glucosyltransferase (GTF) produced by S.mutans binds to mannan on the cell wall of Candida albicans, recruiting it to the early plaque biofilm (Ellepola K et al, Journal of dental research,2017,96(10): 1129-1135.). Candida albicans having a larger surface area may provide binding sites for more S.mutans, which in turn increases the number of Candida albicans. The two biofilms have higher biomass and cell numbers than the single biofilm, and studies have demonstrated that biofilms of candida albicans and streptococcus oralis are more resistant to antibiotics than single species (Shirtliff M E et al, FEMS microbiology letters,2009,299(1):1-8.), and these properties of the two biofilms undoubtedly increase the difficulty of treatment of the caries they cause.
Disclosure of Invention
The first purpose of the invention is to provide a method for inhibiting the biofilm of streptococcus mutans and candida albicans, which utilizes lactobacillus plantarum to inhibit the formation of the biofilm of streptococcus mutans and candida albicans.
In one embodiment of the present invention, the lactobacillus plantarum is lactobacillus plantarum CCFM 8724.
In one embodiment of the invention, the streptococcus mutans is streptococcus mutans ATCC 25175.
In one embodiment of the invention, the fermentation supernatant or sludge is mediated by lactobacillus plantarum CCFM8724 at different stages of the formation of the two-strain biofilm.
In an embodiment of the present invention, the lactobacillus plantarum CCFM8724 fermentation supernatant or bacterial sludge is obtained by culturing lactobacillus plantarum CCFM8724 in MRS broth and centrifuging.
In one embodiment of the invention, the mediation is performed at 0h, 6h, 12h and 24h of the formation of the two-strain biofilm, respectively.
In an embodiment of the present invention, the specific steps mediated by 0h, 6h and 12h are: adding the streptococcus mutans and candida albicans suspension, adding the supernatant of lactobacillus plantarum CCFM8724 when the double-bacterium biofilm grows for 0h, 6h and 12h respectively, and then continuing to culture for 24 h.
In an embodiment of the present invention, the specific steps mediated by 24h are: adding a streptococcus mutans and candida albicans suspension, washing the biomembrane with PBS when the double-bacteria biomembrane grows to 24h, adding lactobacillus plantarum CCFM8724 supernatant, and continuing to culture for 24 h.
In one embodiment of the present invention, the concentration of Streptococcus mutans and Candida albicans is 1 × 106cfu/mL。
The invention also provides application of the lactobacillus plantarum CCFM8724 in preparation of drugs for preventing caries.
The invention has the beneficial effects that:
(1) at different stages of the double-bacterium biofilm formation, the supernatant of the lactobacillus plantarum CCFM8724 can obviously inhibit the formation amount of the double-bacterium biofilm of streptococcus mutans and candida albicans, the reduction amount of the biofilm is very obvious in different mediation time, is higher than 65%, and the mediation effect is close to 90% in 0h, which shows that the lactobacillus plantarum CCFM8724 can inhibit the formation of the biofilm of pathogenic bacteria.
(2) After the lactobacillus plantarum CCFM8724 supernatant is added for mediating in 0h, pathogenic bacteria streptococcus mutans and candida albicans in the double-bacteria biomembrane are reduced by 2-3 orders of magnitude, and therefore the viable count can be reduced by the lactobacillus plantarum CCFM8724 supernatant.
(3) When the lactobacillus plantarum CCFM8724 is mediated for 0h, the reduction of the extracellular polysaccharide amount can reach about 80%, the reduction of the extracellular polysaccharide amount in other mediation time is also more than 40%, and the effect is very obvious.
(4) The two-bacterium biomembrane has better film-forming property under different sucrose concentrations, but the film-forming property of the lactobacillus plantarum CCFM8724 is poor under different sucrose concentrations, and the film-forming property is lower than 0.80, which indicates that the lactobacillus plantarum does not have the possibility of replacing cariogenic bacteria to form the cariogenic biomembrane.
(5) The highest tolerance concentration of the lactobacillus plantarum CCFM8724 to lysozyme is 1.6mg/mL, and the result is far higher than the concentration (0-57 mu g/mL) of lysozyme in saliva of a human oral cavity, which indicates that the lactobacillus plantarum CCFM8724 has the capability of surviving in the oral environment.
Biological material
The Lactobacillus plantarum CCFM8724 of the invention has been disclosed in the patent "A Lactobacillus plantarum CCFM8724 and its use" (CN201210046430.8) (published: 2012.07.04).
The streptococcus mutans ATCC25175 is purchased from China general microbiological culture Collection center (CGMCC).
Drawings
FIG. 1: the inhibition effect of the supernatant of lactobacillus plantarum CCFM8724 on the formation amount of double-bacterium biofilms is mediated at different times; indicates significant differences between the treated and untreated groups (P < 0.05).
FIG. 2: the lactobacillus plantarum CCFM8724 supernatant is 0h mediates the influence on the number of pathogenic bacteria in the streptococcus mutans and candida albicans biomembrane; indicates significant differences between the treated and untreated groups (P < 0.001).
FIG. 3: a laser confocal three-dimensional model diagram of live and dead bacteria staining in a double-bacteria biomembrane mediated by lactobacillus plantarum CCFM8724 supernatant at different times; wherein A: 24h untreated control; b: 6h mediation group; c: 12h mediation group; d: 24h mediation group.
FIG. 4: the lactobacillus plantarum CCFM8724 bacterial sludge mediates the influence on the structure of the biological membrane of the streptococcus mutans and the candida albicans by 0 h; A. c, D: double-bacterium biofilm control; B. e, F: CCFM8724 mediated two bacterial biofilm.
FIG. 5: the influence of different time mediation of the lactobacillus plantarum CCFM8724 supernatant on the extracellular polysaccharide production amount of the double-bacterium biofilm.
FIG. 6: the lactobacillus plantarum CCFM8724 has the self-polymerization and pathogenic bacterium copolymerization capacity.
FIG. 7: the ability of lactobacillus plantarum CCFM8724 to form biofilms at different sucrose concentrations.
FIG. 8: the tolerance of lactobacillus plantarum CCFM8724 to various concentrations of lysozyme.
FIG. 9: comparing the effects of different lactobacilli on a single-bacterium biomembrane and a double-bacterium biomembrane of streptococcus mutans and candida albicans; a: the 23 strains of lactobacillus mediate the reduction of the single bacterial biomembrane of the streptococcus mutans for 12 h; b, reducing the amount of a single candida albicans biofilm mediated by 23 strains of lactobacillus for 12 h; and c, the 23 strains of lactobacillus are 12h to mediate the reduction of the double-bacterial biofilm.
Detailed Description
MRS culture medium: the beef extract contains 5.0g/L yeast powder, 10.0g/L beef extract, 10.0g/L peptone, 20.0g/L glucose, 2.0g/L anhydrous sodium acetate, 2.0g/L hydrogencitrate diamine, 2.6g/L dipotassium hydrogen phosphate, 0.25g/L manganese sulfate monohydrate, 0.5g/L magnesium sulfate heptahydrate and Tween-801 mL, and the pH value is 6.2-6.4.
TSBY medium: is tryptone soy broth medium supplemented with 0.6% yeast powder. Contains 6.0g/L yeast powder, 3.0g/L soybean protein, 17.0g/L tryptone, 2.5g/L glucose, 5g/L sodium chloride and 2.5g/L dipotassium hydrogen phosphate, and has the pH value of 7.0-7.4. The TSBY medium used to form the two-strain biofilm was supplemented with 5% additional sucrose.
YPD medium: 1.0g/L of yeast powder, 2.0g/L of glucose and 2.0g/L of peptone. YPD medium for biofilm formation was supplemented with 5% additional sucrose.
Lactobacillus plantarum CCFM8724 supernatant: inoculating Lactobacillus plantarum CCFM8724 in MRS liquid culture medium at an inoculum size of 2%, culturing at 37 deg.C for 24 hr, centrifuging at 4 deg.C for 5min at 8000r/min, and filtering with 0.22 μm sterile membrane to obtain supernatant.
Lactobacillus plantarum CCFM8724 bacterial sludge: inoculating lactobacillus plantarum CCFM8724 in MRS liquid culture medium at an inoculum size of 2%, culturing at 37 ℃ in an incubator for 24h, then performing 8000r/min, centrifuging at 4 ℃ for 5min, and removing supernatant to obtain bacterial sludge.
Streptococcus mutans suspension is prepared by culturing Streptococcus mutans strain ATCC25175 in TSBY culture medium containing 0.6% yeast powder at 37 deg.C for 12 hr, and adjusting the concentration of the bacterial suspension to 1 × 106cfu/mL。
Candida albicans suspension is prepared by culturing Candida albicans in YPD medium at 37 deg.C for 12 hr, and adjusting the concentration of the suspension to 1 × 106cfu/mL。
The formation of dental plaque biomembranes mainly comprises four stages of an attachment stage (0-6 h), an initial microcolonic stage (6-12 h), a biomembrane maturation stage (12-24 h) and a spreading stage (24-48 h), so that the research is mainly carried out from several time points of 0h, 6h, 12h and 24h when the inhibition effect of the lactobacillus plantarum CCFM8724 on the double-bacterium biomembrane of streptococcus mutans and candida albicans is verified.
Example 1: effect of Lactobacillus plantarum CCFM8724 on the amount of biofilm formation by the double bacteria
Adding 50 μ L of each of Streptococcus mutans and Candida albicans suspension into a 96-well plate, adding 100 μ L of Lactobacillus plantarum CCFM8724 supernatant at 0h, 6h and 12h, and culturing at 37 deg.C for 24 h. And the 24h mediated method comprises the steps of carefully cleaning the biological membrane for 2 times by PBS when the double-bacterium biological membrane grows to 24h, adding 100 mu L of supernatant of lactobacillus plantarum CCFM8724, and culturing for 24h at 37 ℃. The double-bacterial biofilm control replaces lactobacillus plantarum supernatant with blank MRS liquid medium, and 6 replicates were set per group. After the culture, the biological membrane is carefully cleaned by PBS for 2 times, the biological membrane is kept stand at room temperature and dried in the air, then 100 mu L of 0.1% crystal violet solution is added into each hole, the biological membrane is dyed for 30min, the biological membrane is cleaned by PBS for 2 times after the dyeing is finished, each hole is dissolved by 95% ethanol, and the absorbance value is read under an enzyme-labeling instrument OD600 nm.
As can be seen from the figure 1, at different stages of the double-bacterium biofilm formation, the supernatant of the lactobacillus plantarum CCFM8724 can obviously inhibit the formation amount of the double-bacterium biofilm of streptococcus mutans and candida albicans, the reduction amount of the biofilm is very obvious in different mediation time, the reduction amount is higher than 65%, and the mediation effect of 0h is close to 90%, which indicates that the supernatant of the lactobacillus plantarum CCFM8724 has the effect of inhibiting the formation of the biofilm of pathogenic bacteria.
TABLE 1 biofilm reduction Rate
Example 2: effect of Lactobacillus plantarum CCFM8724 on the number of pathogenic bacteria in a double-bacterial biofilm
A sterile 18mm × 18mm coverslip was placed in a 6-well plate, 2mL each of Streptococcus mutans and Candida albicans suspensions was added, 250 μ L of Lactobacillus plantarum CCFM8724 supernatant was added at 0h, and the control was replaced with blank MRS liquid medium at 37 ℃ for 24 h. After the culture is finished, the cover glass with the biomembrane is clamped into a 50mL centrifuge tube by using sterile forceps, the centrifuge tube is immersed by 10mL of 0.89% sterile normal saline, and the centrifuge tube is placed into an ultrasonic cleaning device for cleaning for 2h to ensure that the biomembrane is eluted from the cover glass and is suspended in the normal saline at the same time. The biofilm suspension was diluted in gradients, 100. mu.L of each dilution was taken, plated with TSB medium and YPD medium, incubated at 37 ℃ for 24h and counted in triplicate, and the experiment was repeated three times. The result is shown in fig. 2, and it can be seen that the number of pathogenic bacteria in the double-bacteria biomembrane is reduced by 2-3 orders of magnitude after the lactobacillus plantarum CCFM8724 supernatant is added for 0h for mediation.
TABLE 2 biofilm pathogen counts (logCFU/mL)
Example 3: effect of Lactobacillus plantarum CCFM8724 on Activity of double-bacterial biofilms
CFSE dye: 8 μm, CFSE dye stains live bacteria in biofilms.
PI dye: 4 μ M, PI dye can stain dead bacteria in the biofilm.
A sterile cover glass with the diameter of 24mm multiplied by 50mm is placed in a disposable plate with the diameter of 90mm, 8mL of each of streptococcus mutans and candida albicans suspension is added, the culture is conducted by adding lactobacillus plantarum CCFM8724 supernatant at 6h, 12h and 24h, the biomembrane is taken out and placed in a fresh culture medium containing 2mL of lactobacillus plantarum CCFM8724 supernatant at 24h mediation, a blank MRS liquid culture medium is used as a double-bacterium biomembrane control to replace the supernatant, and all the mediation group and the control group are cultured for 24h at 37 ℃. Carefully cleaning the biological membrane after the culture is finished, dyeing the biological membrane in a dark room, firstly adding 300 mu L of CFSE dye to cover the whole cover glass, incubating for 30min at 37 ℃ in a dark place, then cleaning the biological membrane twice by using sterile water, covering the whole cover glass by using 300 mu L of PI dye, incubating for 30min at 37 ℃ in a dark place, cleaning the biological membrane twice by using the sterile water after the culture is finished, placing the biological membrane under a laser confocal microscope for observation after the biological membrane is dried, setting the multiple of an objective lens to be 20 x, setting the multiple of 488nm, moving the objective lens downwards and upwards to adjust the layer which is focused to the brightest focal plane and then is respectively set to be the bottommost layer and the topmost layer, setting the scanning step distance to be 2 mu, calculating the layer number according to the distance between the topmost layer and the bottommost layer of the biological membrane for scanning and shooting, previewing the 3D effect after the shooting. As shown in fig. 3, it was found that the biofilm coverage formed after 6h mediation (fig. 3B) was the least, the biofilm reduction effect was worse the later the mediation was, and the biofilm was formed even after 24h mediation (fig. 3D), but the thickness of the biofilm was significantly reduced, the ratio of dead bacteria was greatly increased, and the biofilm activity was significantly reduced. The result shows that the addition of the supernatant of the lactobacillus plantarum CCFM8724 for mediating at 24h can greatly reduce the activity of the biomembrane although the reduction of the biomembrane amount is not significant.
Example 4: effect of Lactobacillus plantarum CCFM8724 on the Structure of the double-bacterial biofilm
Placing 18mm × 18mm sterile cover glass in 6-well plate, adding 2mL each of Streptococcus mutans and Candida albicans suspension, and adding 20 μ L of Lactobacillus plantarum CCFM8724 bacterial mud at 0h (bacterial mud suspended in PBS to adjust OD0.6, CFU is 106～107) Incubate at 37 ℃ for 24h, and replace the control with blank MRS liquid medium. After the culture, the cover glass is washed by sterile water, fixed by 3% glutaraldehyde overnight at 4 ℃, then dehydrated continuously for 20min by 70%, 80%, 96% and 100% ethanol, sprayed with gold after air drying, and observed under 10kv voltage.
The results are shown in fig. 4, and as can be seen from fig. 4A, 4C, and 4D, the biofilms formed by the biofilm-biofilm contrast are connected into one piece and only have a few gaps as nutrient channels, and after amplification, the thickness of the biofilm-biofilm can be seen, and candida albicans hyphae as virulence factors are criss-cross at the bottom of the biofilm-biofilm, so that more streptococcus mutans and candida albicans are adhered to the biofilm, and the biofilm thickness is gradually accumulated. And fig. 4B, fig. 4E and fig. 4F are graphs of results of lactobacillus plantarum CCFM8724 bacterial sludge mediation, fig. 4B shows that a continuous biofilm is not formed obviously, and after amplification, single lactobacillus plantarum and hypha crosslinking of candida albicans are not seen basically, the thickness of the biofilm is reduced obviously, and the biofilm is thin and has no convex three-dimensional structure. According to the result of a scanning electron microscope, the CCFM8724 reduces the biofilm amount of the double-bacterium biofilm, the three-dimensional structure is damaged, the formation of candida albicans hyphae is inhibited, and the effect of the lactobacillus plantarum CCFM8724 is very obvious.
Example 5: influence of lactobacillus plantarum CCFM8724 on extracellular polysaccharide yield of double-bacterium biofilm
The determination method of the content of the extracellular polysaccharide comprises the following steps: 1mL of each of Streptococcus mutans and Candida albicans suspension was added to each well of a 24-well plate, 150. mu.L of the supernatant of Lactobacillus plantarum CCFM8724 was added at 0h, 6h, and 12h, the biofilm was washed twice with PBS after 24 h-mediated culture, 2mL of the supernatant of Lactobacillus plantarum CCFM8724 was added, and the culture was carried out at 37 ℃ for 24 h. After the culture is finished, the biomembrane at the bottom of the pore plate is eluted by 1.5mL of 0.4M NaOH and then is sucked into a 2mL centrifugal tube, the supernatant is sucked after centrifugation for 3min at 8000r/min, the residual precipitate is washed for 4 times by 0.4M NaOH, the supernatant after each centrifugation is collected, and the content of the extracellular polysaccharide is measured by an anthrone-sulfuric acid method.
The result is shown in fig. 5, the reduction amount of the extracellular polysaccharide can reach about 80% in 0h, the reduction amounts of the extracellular polysaccharide in other mediated times are all more than 40%, and the effect is very obvious. The extracellular polysaccharide is the most key substrate of the biological membrane, is a product of the growth and development of pathogenic bacteria and is a strong base of the pathogenic bacteria, and the reduction of the amount of the extracellular polysaccharide produced by the biological membrane indicates that the lactobacillus plantarum CCFM8724 plays a better role in inhibiting the double-bacteria biological membrane.
TABLE 3 insoluble exopolysaccharide reduction (%)
Example 6: self-polymerization and pathogenic bacterium copolymerization capacity of lactobacillus plantarum CCFM8724
The self-polymerization of Lactobacillus plantarum CCFM8724 and its ability to copolymerize with Streptococcus mutans and Candida albicans were examined spectrophotometrically. The bacteria with strong self-polymerization ability have certain colonization ability in the oral cavity, and the bacteria with strong copolymerization with the pathogenic bacteria can carry the pathogenic bacteria away from the oral cavity along with drinking or gargling.
(1) Test for self-polymerization Capacity
Lactobacillus plantarum CCFM8724 suspended in PBS for OD modulation600Obtaining a lactobacillus plantarum CCFM8724 suspension at 0.6, standing the lactobacillus plantarum CCFM8724 suspension, sucking 100 mu L of upper-layer liquid every 2h, and measuring the absorbance at 600nm in a 96-well plate, wherein the formula is as follows:
(2) Copolymerization ability test
Mixing equal amount (1 mL each) of Lactobacillus plantarum with Streptococcus mutans and Candida albicans, standing at room temperature, sucking supernatant liquid every 2h, and measuring OD at different time600The value, the formula is:
The results are shown in fig. 6, the self-assembly ability of lactobacillus plantarum CCFM8724 reaches 20% at 4h, and then the self-assembly ability rises linearly, which indicates that the lactobacillus plantarum CCFM8724 has a certain permanent planting ability in the oral cavity; and the lactobacillus plantarum CCFM8724 can be copolymerized with streptococcus mutans and candida albicans, the copolymerization capacity is respectively close to 60% and 40% within 2h, the copolymerization effect is better along with the prolonging of time, and the CCFM8724 has the potential of bringing the pathogenic bacteria away from the oral cavity along with drinking water or gargling.
Example 7: capability of lactobacillus plantarum CCFM8724 in forming biological membrane under different sucrose concentrations
Adjusting the bacterial concentration of the lactobacillus plantarum CCFM8724 bacterial suspension to 10 by using a TSB culture medium containing 0%, 0.25%, 1%, 2% and 5% of sucrose6CFU/mL, inoculated in 96-well plates, each 100 u L, with two bacteria biofilm as control, 37 degrees C culture 24h, in 6 parallel. Washing the biological membrane twice with PBS, standing at room temperature, drying, performing 0.1% crystal violet staining and 95% ethanol dissolving and color development on the biological membrane, and measuring the light absorption value at 600nm with a microplate reader to judge the generation capacity of the biological membrane.
The results are shown in fig. 7, the two-bacterium biofilm is taken as a control, the two-bacterium biofilm has better film forming property under different sucrose concentrations, but the lactobacillus plantarum CCFM8724 has poor film forming property under different sucrose concentrations and has small difference, which indicates that the lactobacillus plantarum does not have the possibility of replacing cariogenic bacteria to form cariogenic biofilms.
TABLE 4 film Forming ability at different sucrose concentrations
Example 8: tolerance of lactobacillus plantarum CCFM8724 to different concentrations of lysozyme
A certain amount of 200. mu.L of MRS medium was added to a 96-well culture plate, and lysozyme solutions of different concentrations were added so that the final concentrations were 0.4, 0.8, 1.2, 1.6, 2.0, and 3.0(mg/mL), respectively. Inoculating Lactobacillus plantarum CCFM8724 culture in an inoculum size of 5% (V/V) into 96-well culture plate, culturing at 37 deg.C for 24h, and determining OD600Lower absorbance, i.e. the tolerance of Lactobacillus plantarum CCFM8724 to lysozyme is judged according to the growth of Lactobacillus, and the control group is added with 1 × 10 with the same volume8CFU/mL Lactobacillus, the amount of lysozyme solution was replaced with an equal volume of sterile water.
The results are shown in fig. 8, based on the OD value of the experimental group/OD value of the control group × 100% > 60%, the highest tolerance concentration of lactobacillus plantarum CCFM8724 to lysozyme is 1.6mg/mL, and the result is much higher than the concentration (0-57 μ g/mL) of lysozyme in saliva of human oral cavity, which indicates that lactobacillus plantarum CCFM8724 has the ability to survive in the oral environment.
Example 9: inhibition effect of single-bacterium biofilm and double-bacterium biofilm of different lactobacilli
Lactobacillus supernatant: respectively inoculating different lactobacillus (including 23 strains of Lactobacillus salivarius, Lactobacillus paracasei, Lactobacillus reuteri and Lactobacillus fermentum) in MRS liquid culture medium at an inoculation amount of 2%, culturing at 37 deg.C for 24 hr, then 8000r/min, centrifuging at 4 deg.C for 5min, and filtering with 0.22 μm sterile filter membrane to obtain supernatant.
(1) Inhibitory effect on biofilm of Streptococcus mutans
100 mu L of streptococcus mutans suspension is added into a 96-well plate, 100 mu L of supernatant of different lactobacilli (including 23 strains of lactobacillus salivarius, lactobacillus paracasei, lactobacillus reuteri and lactobacillus fermentum) is added when a streptococcus mutans single-bacterium biomembrane grows for 12h, and the culture is continued for 24h at 37 ℃.
(2) Inhibitory effect on Candida albicans single-bacterium biofilm
Adding 100 μ L of Candida albicans suspension into a 96-well plate, adding 100 μ L of different lactobacillus (including 23 strains of Lactobacillus salivarius, Lactobacillus paracasei, Lactobacillus reuteri and Lactobacillus fermentum) supernatants when the Candida albicans single bacterial biofilm grows to 12h, and culturing at 37 deg.C for 24 h.
(3) Inhibiting effect of biological membrane of single streptococcus mutans and candida albicans
100 mul of each of streptococcus mutans single-bacterium and candida albicans suspension is added into a 96-well plate, when a double-bacterium biomembrane grows to 12h, 100 mul of supernatant of different lactobacilli (including 23 strains of lactobacillus salivarius, lactobacillus paracasei, lactobacillus reuteri and lactobacillus fermentum) is added, and the culture is continued for 24h at 37 ℃.
The results show that the 23 strains of lactobacillus reduce the single bacterial biofilm of the streptococcus mutans by 44-72.3% (figure 9a) and reduce the single bacterial biofilm of the candida albicans by 9.4-62.4% (figure 9b), and the effect of the lactobacillus on the double bacterial biofilms is only 7.2-40% (figure 9c) and the effect is not more than 60%, which indicates that the lactobacillus does not necessarily have the inhibiting effect on the double bacterial biofilms of the streptococcus mutans and the candida albicans even though the lactobacillus has the inhibiting effect on the single bacterial biofilms.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. A method for inhibiting the biofilm of streptococcus mutans and candida albicans, wherein lactobacillus plantarum is used to inhibit the formation of the biofilm of streptococcus mutans and candida albicans; the lactobacillus plantarum is lactobacillus plantarum CCFM 8724.
2. The method according to claim 1, wherein the streptococcus mutans is streptococcus mutans ATCC 25175.
3. The method of claim 1, wherein the fermentation supernatant or puree is mediated by lactobacillus plantarum CCFM8724 at different stages of the formation of the two-strain biofilm.
4. The method according to claim 3, wherein the fermentation supernatant or bacterial sludge of Lactobacillus plantarum CCFM8724 is obtained by culturing Lactobacillus plantarum CCFM8724 in MRS liquid medium and centrifuging to obtain supernatant and bacterial sludge, respectively.
5. The method of claim 3, wherein the mediating is performed at 0h, 6h, 12h and 24h, respectively, of the formation of the double-fungal biofilm.
6. The method according to claim 5, wherein the specific steps mediated by 0h, 6h and 12h are as follows: adding the streptococcus mutans and candida albicans suspension, adding the supernatant of lactobacillus plantarum CCFM8724 when the double-bacterium biofilm grows for 0h, 6h and 12h respectively, and then continuing to culture for 24 h.
7. The method according to claim 5, wherein the specific steps mediated by 24h are: adding a streptococcus mutans and candida albicans suspension, washing the biomembrane with PBS when the double-bacteria biomembrane grows to 24h, adding lactobacillus plantarum CCFM8724 supernatant, and continuing to culture for 24 h.
8. The method of any one of claims 6 or 7, wherein the mutation is in the form of a mutationBoth Streptococcus and Candida albicans concentrations were 1 × 106cfu/mL。
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