CN111588838A - Composition for inhibiting streptococcus mutans and application thereof - Google Patents
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Abstract
The invention discloses a composition for inhibiting streptococcus mutans and application of the composition in preparation of a medicament for inhibiting streptococcus mutans. Lactic acid continuously secreted by lactobacillus plantarum is continuously converted into hydrogen peroxide under the chain catalytic action of lactate oxidase and ferroferric oxide nanoenzyme, and hydroxyl radicals with bactericidal activity better than that of hydrogen peroxide are further generated, so that the effects of sterilizing and inhibiting biofilm formation are achieved. The composition exhibits mild and sustained germicidal action. The lactobacillus plantarum-lactate oxidase-ferroferric oxide nanoenzyme composition provided by the invention can generate better inhibition effect on streptococcus mutans, which is a main pathogenic bacterium of oral caries, and a formed biological membrane thereof, and can become a new means for preventing and treating dental caries.
Description
Technical Field
The invention belongs to the technical field of streptococcus mutans inhibitors, and particularly relates to a composition for inhibiting streptococcus mutans and application thereof.
Background
The biomembrane is a bacterial aggregate membrane formed by coating bacterial colonies with polysaccharide matrixes secreted by bacteria, and many diseases of human beings are caused by the biomembrane, wherein one of the diseases is dental caries. Studies have shown that the main pathogenic bacteria of dental caries are streptococcus mutans(s), which adheres relatively easily to our tooth surfaces, produces acid using carbohydrates in the oral cavity and forms a dense biofilm. The biofilm tightly attached to the tooth surface is difficult to remove, and provides a barrier for bacteria in the membrane, so that the sterilization difficulty is greatly increased; the acidic substances generated by bacteria can cause tooth demineralization, and as substances such as calcium, phosphorus and the like are lost, firm teeth become brittle gradually, and caries are formed. Biofilm bacteria have strong resistance to antibiotics and host immune defense mechanisms, and the inhibition effect of some existing anti-caries means such as conventional antibacterial agents chlorhexidine on oral biofilms is very limited.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the technical problems, the invention provides a novel composition which takes lactobacillus plantarum as an auxiliary and jointly applies natural enzymes, namely lactate oxidase and ferroferric oxide nanoenzyme, and hydrogen peroxide and hydroxyl radicals with the functions of sterilizing and removing biological membranes are continuously generated through the chain catalytic reaction of the two enzymes. The mode is applied to the prevention and treatment of dental caries, and has better sterilization and removal effects on oral streptococcus mutans and a biological membrane thereof.
The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a composition for inhibiting streptococcus mutans, wherein the composition comprises lactobacillus plantarum, lactate oxidase and ferroferric oxide nanoenzyme.
Preferably, the method comprises the following steps:
the ferroferric oxide nanoenzyme is synthesized into the ferroferric oxide nanoenzyme with enzyme-like activity by a hydrothermal method.
The synthetic method of the ferroferric oxide nanoenzyme comprises the following steps:
fully dissolving anhydrous ferric chloride and polyethylene glycol in ethylene glycol, adding sodium acetate, uniformly stirring, carrying out hydrothermal reaction at the temperature of 180 ℃ and 220 ℃ for 10-14h, and washing off impurities to obtain the iron-based catalyst.
The concentration of Lactobacillus plantarum is (1-10) × 104CFU/mL, preferably 5 x 104CFU/mL。
The lactate oxidase related by the invention can catalytically convert lactic acid continuously generated by bacteria into hydrogen peroxide, and the generated hydrogen peroxide can play a certain sterilization role, and the hydrogen peroxide generated in the process has low concentration and small irritation, and the sterilization process is mild and durable. The lactate oxidase concentration should be 0.1U/mL or more, preferably 0.1-5U/mL, and more preferably 1U/mL.
The ferroferric oxide nanoenzyme is an enzyme-like material with good peroxidase activity, has certain pH dependency, shows high activity of the peroxidase in an acid environment formed by an oral mixed biomembrane, can efficiently catalyze hydrogen peroxide to generate hydroxyl radicals, further initiates oxidative stress imbalance in bacteria, and achieves better effects of sterilizing and inhibiting the formation of the biomembrane. The ferroferric oxide nanoenzyme still has good stability and high biological safety in a strong acid environment. The concentration of the ferroferric oxide nanoenzyme is more than 100ug/mL, preferably 100ug/mL and 500ug/mL, and more preferably 200 ug/mL.
The invention also provides application of the composition in preparing a medicament for inhibiting streptococcus mutans.
The streptococcus mutans is planktonic bacteria without forming a biological film, or is streptococcus mutans in a state of forming a biological film and reducing the sensitivity to drugs.
The invention finally provides the use of said composition for the preparation of a medicament for the prevention and/or treatment of dental caries. The composition has good bactericidal and cleaning effects on oral streptococcus mutans and a biological membrane thereof, so that the composition can be applied to the prevention and treatment of dental caries.
Has the advantages that: the composition of the invention combines the lactobacillus plantarum, the lactate oxidase and the ferroferric oxide nanoenzyme, and has better sterilization and removal effects on the cariogenic bacteria streptococcus mutans with or without a formed biological film. Under the condition provided by the invention, hydrogen peroxide generated by catalysis of lactic acid oxidase does not strongly stimulate normal cells in an oral cavity, and the acid environment of a biological membrane is very beneficial to ferroferric oxide nanoenzyme to exert the peroxidase activity, so that the hydrogen peroxide is catalyzed to generate hydroxyl free radicals, oxidative stress imbalance is caused in bacteria, the quantity of the bacteria is obviously reduced, the dry weight of the biological membrane is obviously reduced, streptococcus mutans is effectively inhibited, and further dental caries can be effectively prevented and treated.
Drawings
FIG. 1 is a transmission electron microscope image of the ferroferric oxide nanoenzyme described in example 1.
FIG. 2 is a morphological diagram of cells grown on LMW plates together with Streptococcus mutans and Lactobacillus plantarum.
FIG. 3 is a graph showing the effect of the composition of the present invention on oral biofilm (43h) inhibition.
FIG. 4 is a graph showing the results of the dry weight effect on oral biofilms (43h) after application of the composition according to the present invention.
FIG. 5 is a scanning electron microscope image showing the effect of the composition of the present invention on oral biofilms (43h) after application.
FIG. 6 is an image of the effect of the composition of the present invention on oral biofilms (43h, 67h, and 91h) after application, observed under a two-photon fluorescence electron microscope.
FIG. 7 is a graph showing the effect of the combination mode of the invention on bacteriostasis of oral biofilms cultured for a long time (29-91 h).
FIG. 8 is a graph showing the effect of the combination of the present invention on the dry weight of oral biofilms cultured over a long period of time (29-91 h).
Detailed Description
The technical solution of the present invention is further described in detail by the following specific examples.
Example 1: synthesis of ferroferric oxide nanoenzyme
The ferroferric oxide nanoenzyme is prepared by a hydrothermal synthesis method, which comprises the following steps:
10mL of ethylene glycol was mixed with 30mL of polyethylene glycol, 0.4g of anhydrous ferric chloride was added, and the mixture was stirred with a magnetic stirrer to dissolve the anhydrous ferric chloride sufficiently. 3.6g NaAC 3H was added2And O, continuously stirring and mixing, reacting the reaction solution at 200 ℃ for 12h, and washing with ethanol and water to remove impurities. The transmission electron microscope image of the ferroferric oxide nanoenzyme prepared by the method is shown in figure 1, and the result shows that the spherical nano-particles with the diameter of about 40nm are prepared.
Example 2: influence of the combination of lactobacillus plantarum, lactate oxidase and ferroferric oxide nanoenzyme on oral biomembrane (43h)
1. Culture method of mixed biological membrane
Selecting Streptococcus mutans and Lactobacillus plantarum colony, culturing in LMW + 1% glucose culture medium and MRS culture medium respectively, and culturing in medium containing 5% CO2Was activated overnight at 37 ℃. Transferring the cells the next day, culturing in incubator to OD600To reach 1.
Adding phosphate buffer solution with the same volume into saliva with empty stomach, adding PMSF (1mM), mixing, filtering and sterilizing. The hydroxyapatite disk or human tooth slice used in the experiment is soaked in the obtained sterile saliva, incubated for 1h in an incubator at 37 ℃, and then washed with sterile water.
Taking brain heart infusion Broth (BHI) added with 1% glucose as culture medium for culturing mixed biological membrane, adding two bacterial solutions 280uL into 24-well plate respectively, supplementing culture medium until total volume is 2.8mL, and controlling initial bacterial count to be 10 respectively5CFU/mL and 5 x 104CFU/m, only adding the streptococcus mutans bacterial liquid to the single streptococcus mutans biofilm group, and the other conditions are the same. The culture process is carried out at 37 deg.C and 5% CO2Is carried out in an incubator.
2. Experiment of administration modes for respective groups
Experimental setup 5 groups, respectively:
s.m (Streptococcus mutans isolated culture group)
Mix (two-bacterium mixed culture group)
3.mix+LOD
4.mix+LOD+MNPs
5.mix+MNPs
After culturing for 19h, a biofilm was initially formed and the solution was changed. The hydroxyapatite tablets or dental sections were rinsed in sterile water and placed in fresh medium, with no other treatment being performed on the two control groups (s.m group and mix group). Lactate oxidase is added during liquid changing, the final concentration is 1U/mL, ferroferric oxide nanoenzyme is added one hour before liquid changing (not added during 18 hours), the final concentration is 200ug/mL, and the total action time is 1 hour.
The total culture time of the oral cavity mixed biological membrane is 43h, the liquid changing time points (the same time point of adding lactate oxidase) are 19h and 29h, and the time point of adding ferroferric oxide nanoenzyme is 18h and 42 h.
3. Experimental effect evaluation mode
Two indicators of the effect were evaluated, one being the biofilm dry weight and the other being the bacteriostatic effect.
After the biofilm was cultured for 43h, each experimental group was treated. Carefully scraping off a biological film on the surface of the hydroxyapatite or the tooth slice, performing ultrasonic treatment, taking liquid, diluting, coating, and performing inverted culture in a carbon dioxide incubator at 37 ℃ for more than 24 h. On the LMW plate, the two bacteria can be clearly distinguished according to the colors and the sizes of the streptococcus mutans and the lactobacillus plantarum, the colony number is counted, and the growth forms of the two bacteria on the LMW plate are shown in figure 2. Drying the biological membrane suspension in an oven at 60 ℃ for 5-7h, and weighing the dry weight, wherein the experimental results are shown in figures 3 and 4.
As can be seen from FIG. 2, Streptococcus mutans and Lactobacillus plantarum can be clearly distinguished on the LMW plate, the colony of the Streptococcus mutans is obviously smaller than that of the Lactobacillus plantarum, the color of the Streptococcus mutans is whitish, and the Lactobacillus plantarum is light yellow. Fig. 3 and 4 are statistics of bacteriostatic and dry weight data, respectively, showing that: compared with an independent streptococcus mutans group, the number of bacteria of streptococcus mutans of the combined lactate oxidase and ferroferric oxide nanoenzyme group can be reduced by about 4lg, and the dry weight is reduced by 57%; compared with a mixed culture group, the number of bacteria of streptococcus mutans of the combined lactate oxidase and the ferroferric oxide nanoenzyme group can be reduced by 3-4lg, the dry weight is reduced by about 48%, and the bacteriostatic and dry weight effects of the enzyme or the ferroferric oxide nanoenzyme are not combined.
Example 3: the effect of the combined mode of the invention on the oral biomembrane (43h) under the observation of an electron microscope
1. The effect of the combined mode of the invention on the oral biomembrane (43h) under the biological scanning electron microscope
Four groups were set up for the experiment: 1, S.m group 2.mix group 3.mix + LOD group 4.mix + LOD + MNPs group
Activating Streptococcus mutans and Lactobacillus plantarum overnight, transferring next day, culturing in 5% carbon dioxide incubator at 37 deg.C to OD600To 1. The hydroxyapatite tablets were soaked in sterile saliva and transferred to an oven at 37 ℃ for 1 h.
The total volume of each hole in the 24-hole plate is 2.8mL, streptococcus mutans and lactobacillus plantarum bacterial liquid is added according to experimental settings, and the initial bacterial count is controlled to be 10 respectively5CFU/mL and 5 x 104CFU/mL. Only the streptococcus mutans bacterial liquid is added to the single streptococcus mutans biofilm group, and other conditions are the same. Changing the culture medium when the culture is carried out for 19h at 37 ℃ in a 5% carbon dioxide incubator, adding 1U/mL of lactate oxidase for continuous culture, adding 200ug/mL of ferroferric oxide nanoenzyme when the culture is carried out for 28h, changing the fresh culture medium after 1h of action, and adding the lactate oxidase with the final concentration of 1U/mL into the new culture medium. The cultivation was continued in this manner until the cultivation time reached 43 h.
The biofilm was washed with water, soaked overnight in 2.5% glutaraldehyde solution in the dark, and pre-fixation of the sample was performed. Then, gradient dehydration is carried out by using 30 percent, 50 percent, 70 percent, 90 percent, 95 percent and 100 percent of ethanol in sequence, and finally, drying and spraying gold are carried out.
The imaging analysis was performed using a scanning electron microscope, resulting in fig. 5.
The results showed that significant biofilm was observed in both the group of S.mutans alone and the mixed group, and the biofilm densely packed the bacteria to form a globular colony. The treatment with lactate oxidase can inhibit the biofilm and bacteria, and as shown in FIG. 8, the biofilm in the visual field is obviously reduced, but a layer of fine and dense biofilm is still attached to the bottom. The combination method provided by the invention has more obvious effect, and compared with a control group, the number of the biomembrane spherical colonies is reduced, the size is obviously reduced, the surface of the hydroxyapatite sheet is smoother, and the inhibition effect of the biomembrane is obvious.
2. The effect of the combined mode of the invention on the oral biomembrane (43h) under the two-photon fluorescence electron microscope
Four groups were set up for the experiment: 1, S.m group 2.mix group 3.mix + LOD group 4.mix + LOD + MNPs group
Extracellular polymers of biofilm and bacteria were stain labeled with Alexa Fluor 647-dextran dye and SYTO9 fluorescent nucleic acid stain. The marking method comprises the following steps: AlexaFluor 647-dextran dye (final concentration 1mM) was added to the medium throughout the culturing of the biofilm; at 43h of culture, the biofilm was stained by immersion in SYTO9 fluorescent nucleic acid stain (final concentration 2.5mM), and finally the biofilm extracellular matrix was labeled red and the bacteria green.
SYTO9 and Alexa Fluor 647 have an excitation wavelength of 780nm and emission wavelengths of 498nm and 668nm, respectively. The image was taken with a 10 × L Plan objective two-photon fluorescence microscope to obtain a three-dimensional 3D image of the biofilm as shown in fig. 6.
The result of the diphosgene image is consistent with the dry weight result in the example 2, the biomembranes in the independent streptococcus mutans group and the mixed culture group are distributed spherically and densely, and the biomembrane formation is obviously reduced and the bacterial count is obviously reduced after the method for combining the lactate oxidase and the ferroferric oxide nanoenzyme is used.
Example 4: the effect of the combined mode on the oral biomembrane cultured for a long time
The experiment sets four time points of different culture times of 29h, 43h, 67h and 91h, and simultaneously sets four experimental groups at each time point as follows: 1, S.m group 2.mix group 3.mix + LOD group 4.mix + LOD + MNPs group.
Activating Streptococcus mutans and Lactobacillus plantarum overnight, transferring next day, culturing in 5% carbon dioxide incubator at 37 deg.C to OD600To 1. The hydroxyapatite tablets were soaked in sterile saliva and transferred to an oven at 37 ℃ for 1 h.
The total volume of each hole in the 24-hole plate is 2.8mL, streptococcus mutans and lactobacillus plantarum bacterial liquid is added according to experimental settings, and the initial bacterial count is controlled to be 10 respectively5CFU/mL and 5 x 104CFU/mL. Only the streptococcus mutans bacterial liquid is added to the single streptococcus mutans biofilm group, and other conditions are the same. Changing the culture medium when the culture is carried out for 19h at 37 ℃ in a 5% carbon dioxide incubator, adding 1U/mL of lactate oxidase for continuous culture, adding 200ug/mL of ferroferric oxide nanoenzyme when the culture is carried out for 28h, changing the fresh culture medium after 1h of action, and adding the lactate oxidase with the final concentration of 1U/mL into the new culture medium. The cultivation was continued in this manner until cultivation times of 29h, 43h, 67h and 91h were reached.
After the experimental culture time is reached, the biofilm of the corresponding group is scraped, homogenized by ultrasound, diluted in liquid and plated and weighed in dry weight. All manipulations were performed under sterile conditions.
The bacteriostatic and dry weight data obtained are shown in figures 7 and 8.
The results show that the number of the streptococcus mutans is reduced by more than 99.9% at the four time points of 29h, 43h, 67h and 91h, and the biomembrane clearance reaches 65% at 91 h. The combination mode provided by the invention can play a continuous and effective inhibiting role on the growth of the streptococcus mutans and the formation of a biological film under the experimental condition (29-91 h).
Claims (10)
1. The composition for inhibiting streptococcus mutans is characterized by consisting of lactobacillus plantarum, lactate oxidase and ferroferric oxide nanoenzyme.
2. The composition for inhibiting streptococcus mutans according to claim 1, wherein the ferroferric oxide nanoenzyme is an enzyme-like activity ferroferric oxide nanoenzyme synthesized by a hydrothermal method.
3. The composition for inhibiting streptococcus mutans according to claim 1, wherein the ferroferric oxide nanoenzyme is synthesized by the following method:
fully dissolving anhydrous ferric chloride and polyethylene glycol in ethylene glycol, adding sodium acetate, uniformly stirring, carrying out hydrothermal reaction at the temperature of 180 ℃ and 220 ℃ for 10-14h, and washing off impurities to obtain the iron-based catalyst.
4. The composition for inhibiting Streptococcus mutans according to claim 1, wherein the Lactobacillus plantarum concentration is (1-10) × 104CFU/mL。
5. The Streptococcus mutans inhibiting composition of claim 1, wherein the lactate oxidase concentration is 0.1U/mL or more, preferably 0.1-5U/mL.
6. The composition for inhibiting streptococcus mutans according to claim 1, wherein the concentration of the ferroferric oxide nanoenzyme is more than 100ug/mL, preferably 100ug/mL and 500 ug/mL.
7. Use of a composition according to any one of claims 1 to 6 in the manufacture of a medicament for inhibiting streptococcus mutans.
8. The use according to claim 7, wherein the Streptococcus mutans is a planktonic bacteria that does not form a biofilm.
9. The use according to claim 7, wherein the Streptococcus mutans is a biofilm-formed Streptococcus mutans having reduced susceptibility to a drug.
10. Use of a composition according to any one of claims 1 to 6 for the preparation of a medicament for the prevention and/or treatment of dental caries.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN114832831A (en) * | 2022-04-21 | 2022-08-02 | 中国科学院长春应用化学研究所 | Composite nano enzyme synergistic catalytic fiber material and preparation method and application thereof |
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| CN104561227A (en) * | 2014-10-17 | 2015-04-29 | 江南大学 | Analysis method of rejection capability of lactobacillus plantarum metabolite and application of lactobacillus plantarum metabolite |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN114832831A (en) * | 2022-04-21 | 2022-08-02 | 中国科学院长春应用化学研究所 | Composite nano enzyme synergistic catalytic fiber material and preparation method and application thereof |
| CN114832831B (en) * | 2022-04-21 | 2023-08-15 | 中国科学院长春应用化学研究所 | Composite nano enzyme synergistic catalytic fiber material and preparation method and application thereof |
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