CN111686243A - Composition for inhibiting oral pathogenic bacteria and application thereof - Google Patents
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Abstract
The invention relates to the field of prevention and treatment of oral diseases, and particularly relates to a composition for inhibiting oral pathogenic bacteria and application thereof. The composition for inhibiting the oral pathogenic bacteria comprises lysozyme and montmorillonite. The composition consists of the following medicines in parts by weight: 4 parts of lysozyme and 1 part of montmorillonite. The composition has antibacterial effect on oral pathogenic bacteria such as Escherichia coli, Streptococcus mutans, Staphylococcus aureus, Porphyromonas gingivalis, and Practilla intermedia, has wide antibacterial spectrum and high antibacterial rate, can efficiently and briefly inhibit the growth of the oral pathogenic bacteria, has low minimum antibacterial concentration, does not generate drug-resistant strains of the oral pathogenic bacteria after long-term use, can effectively play roles in preventing caries and periodontitis, and has unique advantages and wide application prospect.
Description
Technical Field
The invention relates to the field of prevention and treatment of oral diseases, and particularly relates to a composition for inhibiting oral pathogenic bacteria and application thereof.
Background
Lysozyme (also called as cell wall lytic enzyme) can selectively decompose microbial cell walls to achieve the bactericidal effect, and the principle is that beta-1, 4 glycosidic bonds between C-1 of N-acetylmuramic acid and C-4 of N-acetylglucosamine are catalyzed to destroy the cell walls of bacteria, and the bacteria can be broken under the action of intracellular pressure. And the lysozyme does not damage other biological tissues. Meanwhile, the bio-enzyme preparation is a natural protein, can be completely digested and absorbed in gastrointestinal tracts, has no toxicity and irritation to human bodies, and is a bio-enzyme preparation with high safety. Meanwhile, research finds that bacterial infection occurs when Recurrent Aphthous Ulcers (RAU) occur, and the infection range is not limited to the ulcer surface. The lysozyme helps to eliminate infection of a lesion part and promote ulcer healing by killing and inhibiting microorganisms. At present, some patents have applied lysozyme to the control of oral pathogenic bacteria, for example, an oral care product (CN201710922542.8) containing biological lysozyme discloses an oral care product containing biological lysozyme, the oral care product contains 0.1-15% (wt) of a biological lysozyme additive composition, and the biological lysozyme additive composition consists of the following components in percentage by weight: 10-40% of biological lysozyme, 0.01-2.0% of modified vegetable gum, 0.01-0.3% of EDTA disodium and the balance of deionized water. The oral care product can effectively inhibit and kill harmful bacteria causing oral problems such as oral ulcer, gingivitis, halitosis and the like. The lysozyme is used alone, the bacteriostatic spectrum is narrow, the bacteriostatic effect is general, and technicians in the field perform numerous compatibility experiments on the lysozyme so as to obtain a bacteriostatic composition with wide bacteriostatic spectrum and good bacteriostatic effect, but at present, no composition capable of achieving the purpose is obtained.
Natural montmorillonite is a dioctahedral phyllosilicate clay mineral composed of silicon-oxygen tetrahedron and aluminum-oxygen octahedron, and the random substitution of aluminum and magnesium for silicon and aluminum in the structure causes charge imbalance, so that the natural montmorillonite has the characteristics of swelling, adsorption, electrification and ion exchange when meeting water. Is clinically used as a medicament for treating diarrhea and digestive tract ulcer. Montmorillonite has no direct antibacterial effect of antibacterial drugs.
The invention unexpectedly discovers that the composition of lysozyme and montmorillonite has better antibacterial effect, wider antibacterial spectrum and lower minimum antibacterial concentration, can be used as a main component to be applied to preventing oral infectious diseases, prevents oral drug-resistant pathogenic bacteria caused by abuse of antibiotics and chemical disinfectants, can effectively play roles in preventing caries and periodontitis, and has unique advantages and wide application prospect.
Disclosure of Invention
In order to solve the technical problems, the invention provides a composition for inhibiting oral pathogenic bacteria, which comprises lysozyme and montmorillonite.
Preferably, the composition consists of the following medicines in parts by weight: 4 parts of lysozyme and 1 part of montmorillonite.
The lysozyme composition is applied to the preparation of medicines for inhibiting the growth of oral pathogenic bacteria.
Preferably, the oral pathogenic bacteria comprise one or more of Streptococcus mutans (Streptococcus mutans), Escherichia coli (Escherichia coli), Porphyromonas gingivalis (Porphyromonas gingivalis), Staphylococcus aureus (Staphylococcus aureus), and Prevotella intermedia (Prevotella intermedia).
The application of the lysozyme composition in preparing an oral care agent.
The lysozyme composition is applied to the preparation of an oral care agent for inhibiting the growth of oral pathogenic bacteria.
Preferably, the oral pathogenic bacteria comprise one or more of Streptococcus mutans (Streptococcus mutans), Escherichia coli (Escherichia coli), Porphyromonas gingivalis (Porphyromonas gingivalis), Staphylococcus aureus (Staphylococcus aureus), and Prevotella intermedia (Prevotella intermedia).
Preferably, the lysozyme composition further comprises an orally acceptable carrier.
Preferably, the carrier is a toothpaste, tooth powder, mouthwash, spray, dental paste or cream formulation, dental gel, and chewing gum.
The invention has the beneficial effects that:
compared with a single-component lysozyme product, the lysozyme composition has more remarkable bacteriostatic effect on oral pathogenic bacteria such as Escherichia coli, streptococcus mutans, staphylococcus aureus, porphyromonas gingivalis, prevotella intermedia and other bacteria, has wide bacteriostatic spectrum and high bacteriostatic rate, can efficiently and briefly inhibit the growth of the oral pathogenic bacteria, has lower minimum bacteriostatic concentration, cannot generate drug-resistant strains of the oral pathogenic bacteria after long-term use, can effectively play roles in preventing caries and periodontitis, and has unique advantages and wide application prospect.
Drawings
FIG. 1 shows the bacteriostatic effect of Streptococcus mutans measured by Oxford cup method.
A is lysozyme treatment group, B is lysozyme and montmorillonite co-treatment group, and D is montmorillonite treatment group
FIG. 2 is a graph showing the bacteriostatic effect of Escherichia coli measured by Oxford cup method.
FIG. 3 is a graph showing the inhibition effect of Porphyromonas gingivalis by Oxford cup method.
FIG. 4 shows the bacteriostatic effect of Staphylococcus aureus measured by Oxford cup method.
FIG. 5 shows the bacteriostatic effect of Prevotella intermedia measured by the Oxford cup method.
Detailed Description
The technical solutions of the present invention are described in detail below by using specific examples, but the scope of the present invention is not limited by the following examples, and any technical solutions obtained by modifying the technical solutions of the present invention by those skilled in the art belong to the scope of the present invention.
The sources of the strains used in the experiment of the invention are as follows:
streptococcus mutans (Streptococcus mutans), Escherichia coli (Escherichia coli), Porphyromonas gingivalis (Porphyromonas gingivalis), Staphylococcus aureus (Staphyloccocusareus) and Prevotella intermedia (Prevotella intermedia) used in the present invention were purchased from China general microbiological culture Collection center.
The sources of the drugs used in the experiment of the invention are as follows:
lysozyme (Shanghai Zhonghua pharmaceutical Co., Ltd., China pharmacopoeia, batch number: 190202);
montmorillonite (Beijing Solibao, cat # YZ-100860);
an experimental instrument:
anaerobic incubators (GeneScience, E500, usa), bacteria thermostatted incubators (shanghai xin instrument, XY-DR-360), biosafety cabinets (shanghai liking, HFsafe-1200a2), full-wavelength plate readers (TECAN, Infinite 200Pro, switzerland), fluorescence microscopes (olympus, BX53), adjustable pipetting guns (mettler, XLS +), electronic balances (BT 25S, germany seudoli); bovine heart-brain broth medium (Beijing Huayuyo, GU9-2814), Brucella broth medium (Beijing Soilebao, LA3560), LB medium (Beijing Soilebao, L1010), Columbia blood agar medium (Jiangmen Kailin trade Co., Ltd.), hemin (Beijing Soilebao, H8130), WST-8 kit (Japanese colleague, M439)
Example one inhibition of oral bacteria by a Lysozyme composition for inhibiting oral pathogenic bacteria
In this experiment, an in vitro method was used to examine the inhibitory effect of the lysozyme oral care agent on the common oral bacteria Streptococcus mutans (Streptococcus mutans), Escherichia coli (Escherichia coli), Porphyromonas gingivalis (Porphyromonas gingivalis), Staphylococcus aureus (Staphylococcus aureus) and Prevotella intermedia (Prevotella intermedia). Because the common colony counting method has strong subjectivity, and a sample to be detected cannot be completely dispersed into single bacteria, the result accuracy of the plate colony counting is low, and a grown single colony can come from 2-3 or more cells in the sample, so that accurate counting is not easy to obtain. Therefore, the oxford cup method is adopted in the experiment, and the bacteriostatic effect of each experimental group is directly observed. The Oxford cup method is a common method for detecting the antibacterial effect, and the grouped antibacterial effect is judged by measuring the diameter of the antibacterial zone.
1. Preparation of culture Medium
Preparing a BHI culture medium: 3.7 percent of bovine heart brain infusion medium powder is placed in an ultra-clean bench to be cooled for standby after being sterilized at high temperature and high pressure for 25 minutes. Adding 1.5% agar, preparing agar plate, cooling to 50 deg.C, and pouring. Used for culturing the streptococcus mutans.
Preparing an LB culture medium: and (3) sterilizing the LB culture medium with the concentration of 2.5% at high temperature and high pressure for 25 minutes, and then placing the LB culture medium on a clean bench for cooling for later use. Adding 1.5% agar, preparing agar plate, cooling to 50 deg.C, and pouring. Used for culturing Escherichia coli.
Preparing a BHI blood agar culture medium: 3.7% bovine heart brain infusion medium powder, autoclaving at high temperature for 25 minutes, and adding hemin to a final concentration of 5 μ g/mL. Adding 1.5% agar, preparing agar plate, cooling to 50 deg.C, and pouring. Is used for culturing the porphyromonas gingivalis.
Preparing MH culture medium: weighing 21.0g of culture medium powder, heating and dissolving in 1000ml of distilled water, sterilizing at high temperature and high pressure for 25 minutes, and then placing in an ultra-clean bench for cooling for later use. Adding 1.5% agar, preparing agar plate, cooling to 50 deg.C, and pouring. The culture medium is used for culturing staphylococcus aureus.
Preparing a Columbia culture medium: weighing 39 g of culture medium powder, heating and dissolving in 1000ml of distilled water, subpackaging, sterilizing at high temperature and high pressure for 25 minutes, cooling to about 45-50 ℃, adding sterile gentamicin sulfate containing 20mg of gentamicin, and mixing uniformly for later use. Adding 1.5% agar, preparing agar plate, cooling to 50 deg.C, and pouring. The culture medium is used for culturing the prevotella intermedia.
2. Recovery and culture of strain
And (3) recovering the streptococcus mutans: streptococcus mutans lyophilized strains were inoculated on BHI agar plates and incubated in a 37 ℃ bacterial incubator (containing 5% CO)2) The culture was carried out for 24 hours.
Culturing streptococcus mutans: selecting recovered Streptococcus mutans strain, inoculating to BHICulturing in liquid culture medium for 24 hr, and preparing bacterial suspension (10) with PBS by McLeod's turbidimetry8CFU/mL), ready for use.
And (3) escherichia coli recovery: the lyophilized strain of Escherichia coli was inoculated on an LB agar plate and incubated at 37 ℃ in a bacteria incubator (containing 5% CO)2) The culture was carried out for 24 hours.
And E, culturing escherichia coli: selecting recovered Escherichia coli strain, inoculating into LB liquid culture medium, culturing for 24 hr, and preparing into bacterial suspension (10) with PBS by McLeod8CFU/mL), ready for use.
And (3) recovering the porphyromonas gingivalis: the lyophilized strain of Porphyromonas gingivalis was inoculated on BHI blood agar plates and anaerobically (volume ratio: 80% N) at 37 deg.C2，10％H2，10％CO2) The culture was carried out for 48 hours.
Culturing porphyromonas gingivalis: selecting recovered Porphyromonas gingivalis strain, inoculating into blood-containing BHI liquid culture medium, anaerobically culturing for 48 hr, and preparing into bacterial suspension (10) with PBS by McLeod turbidimetry8CFU/mL), ready for use.
And (3) recovering staphylococcus aureus: inoculating lyophilized Staphylococcus aureus strain on MH agar plate, and culturing in 37 deg.C bacteria incubator (containing 5% CO)2) The culture was carried out for 24 hours.
Culturing staphylococcus aureus: selecting recovered Staphylococcus aureus strain, inoculating to MH liquid culture medium, culturing for 24 hr, and preparing into bacterial suspension (10) with PBS by McLeod turbidimetry8CFU/mL), ready for use.
And (3) recovering the intermediate prevotella: the lyophilized strain of Prevotella intermedia was inoculated on a Columbia blood agar plate and anaerobically incubated at 37 ℃ (volume: 85% N)2，5％H2，10％CO2) The culture was carried out for 48 hours.
Culturing intermediate prevotella: selecting recovered strain, inoculating into Columbia blood agar culture medium, placing in 37 deg.C anaerobic incubator, vacuumizing the incubator with vacuum pump, and charging oxygen-free mixed gas (5% H)2，10％CO2，85％N2). Culturing for 48 hr, and mixing with PBS by McLeod's turbidimetryAdult bacteria suspension (10)8CFU/mL), ready for use.
3. Drug configuration and experimental grouping
Experimental group 1: accurately weighing 0.4g of lysozyme, ultrasonically dispersing the lysozyme in 100mL of 5 bacterial culture media to be detected, taking 2mL of the solution, adding 2 mu L of bacterial suspension to be detected, wherein the final concentration of the medicine is as follows: 0.004g/ml lysozyme;
experimental group 2: 1.0g of montmorillonite is accurately weighed, the montmorillonite is ultrasonically dispersed in 100mL of 5 bacteria culture media to be detected, 2mL of the solution is taken, 2 mu L of bacteria suspension to be detected is added, and the final concentration of the medicine is as follows: 0.01g/ml montmorillonite;
experimental group 3: accurately weighing 0.4g of lysozyme and 1.0g of montmorillonite, ultrasonically dispersing in 100mL of 5 bacterial culture media to be detected, taking 2mL of the solution, adding 2 mu L of bacterial suspension to be detected, wherein the final concentration of the medicine is as follows: 0.004g/ml lysozyme and 0.01g/ml montmorillonite;
4. procedure of experiment
And (3) culturing each experimental group for 12h under corresponding culture conditions, and testing the antibacterial effect of each group of samples by adopting an Oxford cup method: and (3) uniformly coating corresponding bacteria on the surface of a corresponding agar culture plate by using a sterile coating rod, after the agar culture plate is basically dried in an ultra-clean bench, coating the plate on the surface of the culture plate according to regions, placing a plurality of sterile oxford cups, and injecting corresponding samples in groups into the sterile oxford cups. After culturing for 12h under corresponding bacteria culture conditions, observing and evaluating the diameter of the bacteriostatic zone, and judging the reference standard of the antibacterial effect: the National Committee for Clinical Laboratory Standardization (NCCLS).
SPSS 21.0 software is used for data processing, single-factor variance analysis is adopted, and statistical significance is achieved when P is less than 0.05.
5. Results of the experiment
Each group 108Diluting the CFU/mL bacterial solution by 1000 times by using a corresponding drug-containing culture medium, uniformly coating a culture dish, adding drugs, and culturing for 12 h. The bacteriostatic effect of different oral pathogenic bacteria of each experimental group is shown in figures 1-5, and the measurement data is shown in table 1:
TABLE 1 inhibition effect of each experimental group on oral pathogenic bacteria
As can be seen from FIGS. 1 to 5 and Table 1, montmorillonite itself has no bacteriostatic ability; the lysozyme has antibacterial effect on streptococcus mutans, escherichia coli, porphyromonas gingivalis, staphylococcus aureus and prevotella intermedia; the montmorillonite and lysozyme composition can also inhibit streptococcus mutans, escherichia coli, porphyromonas gingivalis, staphylococcus aureus and prevotella intermedia, and compared with lysozyme and montmorillonite, the antibacterial effect of the montmorillonite and lysozyme composition is obviously improved.
EXAMPLE two WST-8 test of bacteriostatic Effect of each group
Compared with the common colony counting method, the WST-8 method has the advantages of high sensitivity, reliable data, good repeatability, simple and convenient operation and the like, and can quantify the antibacterial effect more accurately.
1. Experiment grouping
The experimental groups were the same as in example one.
2. Procedure of experiment
Each group of samples cultured for 6h and 12h was tested using the WST-8 kit. After each experimental group was cultured for a predetermined period of time under the corresponding bacterial culture conditions and left to stand for 30 minutes, 3 samples, each 0.15mL, were added to a 96-well plate from the supernatant of each group. Adding 15 μ L WST-8 reagent into each well of 96-well plate, mixing, incubating at 37 deg.C for 1h, and measuring OD with full-wavelength microplate reader450nm. The calculation formula of the antibacterial rate is as follows:
(OD) antibacterial ratePositive control-ODExperimental group)/(ODPositive control-ODNegative control)×100％
The OD positive control is the absorbance value of the liquid to be detected of the positive control group, the OD experimental group is the absorbance value corresponding to the liquid to be detected of each experimental group, and the OD negative control is the absorbance value corresponding to the liquid to be detected of the negative control group. The experiment takes the antibacterial rate of more than 95 percent according to the national standard to have the antibacterial effect, and the antibacterial rate of more than 80 percent has the antibacterial effect.
3. Bacteriostatic effect
TABLE 2 analysis of antibacterial rate of each experimental group against oral pathogens at different times
As can be seen from Table 2, the lysozyme can inhibit the growth of oral pathogenic bacteria, namely streptococcus mutans, escherichia coli, porphyromonas gingivalis, staphylococcus aureus and prevotella intermedia, the antibacterial rate is close to or reaches 80 percent, the antibacterial effect is achieved, and the antibacterial rate is obviously increased along with the increase of the culture time; montmorillonite has no inhibiting effect on oral pathogenic bacteria such as Streptococcus mutans, Escherichia coli, Porphyromonas gingivalis, Staphylococcus aureus and Prevotella intermedia. The antibacterial rate of the lysozyme and montmorillonite composition group to oral pathogenic bacteria streptococcus mutans, escherichia coli, porphyromonas gingivalis, staphylococcus aureus and prevotella intermedia reaches more than 85 percent, and the antibacterial rate is obviously increased along with the increase of the culture time. The antibacterial effect of the lysozyme and montmorillonite composition group is obviously better than that of the lysozyme group and the montmorillonite group.
In conclusion, the composition for inhibiting the oral pathogenic bacteria has an antibacterial effect on the oral pathogenic bacteria such as escherichia coli, streptococcus mutans, staphylococcus aureus, porphyromonas gingivalis, prevotella intermedia and other bacteria, has a wide antibacterial spectrum and a high antibacterial rate, can efficiently and temporarily inhibit the growth of the oral pathogenic bacteria, has a low minimum antibacterial concentration, does not generate drug-resistant strains of the oral pathogenic bacteria after long-term use, can effectively play roles in preventing caries and periodontitis, and has unique advantages and a wide application prospect.
Claims (9)
1. A lysozyme composition for inhibiting oral pathogenic bacteria, wherein the composition comprises lysozyme and montmorillonite.
2. The lysozyme composition of claim 1, wherein the composition comprises the following drugs in parts by weight: 4 parts of lysozyme and 1 part of montmorillonite.
3. Use of a lysozyme composition according to claim 1 or 2 for the preparation of a medicament for inhibiting the growth of pathogenic bacteria in the oral cavity.
4. The use according to claim 3, wherein the oral pathogenic bacteria comprise one or more of Streptococcus mutans (Streptococcus mutans), Escherichia coli (Escherichia coli), Porphyromonas gingivalis (Porphyromonas gingivalis), Staphylococcus aureus (Staphylococcus aureus), Prevotella intermedia (Prevotella intermedia).
5. Use of a lysozyme composition according to claim 1 or 2 in the preparation of an oral care agent.
6. Use of a lysozyme composition according to claim 1 or 2 for the preparation of an oral care agent for inhibiting the growth of oral pathogenic bacteria.
7. The use according to claim 6, wherein the oral pathogenic bacteria comprise one or more of Streptococcus mutans (Streptococcus mutans), Escherichia coli (Escherichia coli), Porphyromonas gingivalis (Porphyromonas gingivalis), Staphylococcus aureus (Staphylococcus aureus), Prevotella intermedia (Prevotella intermedia).
8. A lysozyme composition according to claim 1 or 2, further comprising an orally acceptable carrier.
9. Lysozyme composition according to claim 8, wherein the carrier is a toothpaste, a tooth powder, a mouthwash, a spray, a dental paste or cream formulation, a tooth gel, and a chewing gum.
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| CN112708654A (en) * | 2021-01-11 | 2021-04-27 | 苏州国辰生物科技股份有限公司 | Method for evaluating antibacterial effect of oral cleaning product |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112708654A (en) * | 2021-01-11 | 2021-04-27 | 苏州国辰生物科技股份有限公司 | Method for evaluating antibacterial effect of oral cleaning product |
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