Composition for inhibiting or removing bacterial biofilm and application thereof

Abstract

The present invention provides a composition for inhibiting and removing bacterial biofilm and its application. The composition comprises a ginger magnolia bark extract and a grape skin residue extract; wherein the bacteria are selected from at least one of Fusobacterium nucleatum and Porphyromonas gingivalis; the partial inhibition concentration index of the composition for Porphyromonas gingivalis is 0.32-0.50; the partial inhibition concentration index of the composition for Fusobacterium nucleatum is 0.28-0.48; the ginger magnolia bark and the grape skin residue extract are mainly used to inhibit the biofilm of Porphyromonas gingivalis and Fusobacterium nucleatum, and at the same time, the combination of ginger magnolia bark and the grape skin residue extract also produces a synergistic effect on inhibiting and removing the formation of biofilm, solving the problems of drug resistance and pathogenicity of Porphyromonas gingivalis and Fusobacterium nucleatum.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a composition for inhibiting and removing bacterial biofilm and an application thereof. Background Art

[0002] Bad breath, commonly known as halitosis, is a physiological or pathological phenomenon caused by multiple factors. It has an adverse effect on people's daily life and communication, and has become one of the reasons that affect social interaction and cause psychological disorders.

[0003] At present, the prevention and treatment of related pathogenic microorganisms in the treatment of halitosis face the following problems: Fusobacterium nucleatum and Porphyromonas gingivalis easily adhere to the surface of teeth and tongue coating to form dental plaque biofilms with stronger drug resistance, and are difficult to eradicate through mechanical therapy. Therefore, removing the biofilm of halitosis-causing bacteria is one of the keys to treating halitosis. At present, the main means of treating halitosis is to use mouthwashes containing chemical antibacterial agents to inhibit or kill halitosis-causing bacteria, such as chlorhexidine, triclosan, cetylpyridinium chloride and other antibacterial agents, but long-term use of such mouthwashes may lead to adverse consequences such as changes in taste, mucosal staining, burning sensation and parotid swelling, dysbiosis of oral flora, and increased bacterial resistance.

[0004] Many studies at home and abroad have shown that Chinese medicine or natural plant extracts have therapeutic or auxiliary therapeutic effects on halitosis. The application of Chinese medicine extracts in halitosis treatment can maintain the original microecological environment in the oral cavity, directly inhibit pathogens without producing drug resistance, and is relatively simple to prepare, with little toxicity and side effects, and the effect lasts for a long time without producing drug resistance. However, there are no reports on the inhibitory or elimination effects of ginger magnolia bark and grape skin residue extracts on oral flora biofilm.

[0005] Based on this, the present invention provides a composition for inhibiting and removing bacterial biofilm and an application technical means to solve the shortcomings of the above technical solutions. Summary of the invention

[0006] In view of the above technical problems, the present invention discloses a composition for inhibiting and removing bacterial biofilm and its application. The composition comprises a ginger magnolia bark extract and a grape peel residue extract. The ginger magnolia bark and the grape peel residue extract are mainly used to inhibit the biofilm and quorum sensing of Porphyromonas gingivalis and Fusobacterium nucleatum, thereby hindering the regulatory effect of quorum sensing on biofilm formation. At the same time, the combination of ginger magnolia bark and grape peel residue extract also has a synergistic effect on inhibiting and removing the formation of biofilm, solving the problems of drug resistance and pathogenicity of Porphyromonas gingivalis and Fusobacterium nucleatum, thereby effectively inhibiting or killing oral odor-causing bacteria.

[0007] The present invention aims to provide a composition for inhibiting and removing bacterial biofilm, which is achieved through the following technical solution.

[0008] A composition for inhibiting and removing bacterial biofilm, comprising a Zingiber officinale extract and a grape skin residue extract;

[0009] Wherein, the bacteria is selected from at least one of Fusobacterium nucleatum and Porphyromonas gingivalis;

[0010] The composition comprises the following components in mass fractions:

[0011] Ginger Magnolia Bark Extract 0.075-0.9%

[0012] Grape pomace extract 0.15-8%

[0013] Solvent Residue.

[0014] Furthermore, the total magnolia bark phenol content in the ginger magnolia bark extract is ≥90wt%.

[0015] Furthermore, the resveratrol content in the grape pomace extract is ≥10wt%.

[0016] Furthermore, the partial inhibitory concentration index of the composition against Porphyromonas gingivalis is 0.32-0.50;

[0017] Furthermore, the partial inhibitory concentration index of the composition against Fusobacterium nucleatum is 0.28-0.48.

[0018] Furthermore, the solvent is selected from at least one of water and alcohols.

[0019] Preferably, the solvent is water.

[0020] The present invention further provides use of the composition in regulating bacterial quorum sensing.

[0021] The present invention further provides an antibacterial agent for inhibiting and removing bacterial biofilm, comprising a Zingiber officinale extract and a grape skin residue extract.

[0022] Furthermore, the antibacterial agent is used in preparing toothpaste or mouthwash for removing bad odor in the oral cavity.

[0023] The composition for inhibiting and removing bacterial biofilm provided by the present invention has the following beneficial effects:

[0024] 1. The present invention uses ginger magnolia bark and grape peel residue extract as antibacterial or bactericidal agents. Both plant extracts are non-toxic, non-irritating natural antibacterial agents. They can effectively inhibit or kill oral odor-causing bacteria and remove dental plaque without affecting the ecological balance of oral microorganisms.

[0025] 2. The extracts of Magnolia officinalis and grape peel residue are mainly used to inhibit the biofilm and quorum sensing of Porphyromonas gingivalis and Fusobacterium nucleatum, thereby hindering the regulatory effect of quorum sensing on the formation of biofilm. At the same time, the combination of Magnolia officinalis and grape peel residue extracts also has a synergistic effect in inhibiting and removing the formation of biofilm, and solving the problems of drug resistance and pathogenicity of Porphyromonas gingivalis and Fusobacterium nucleatum. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a statistical diagram of the metabolic activity of each group of drugs on biofilm bacteria at each stage of Pg detected by CCK-8 method;

[0027] Figure 2 This is a statistical chart of the total amount of biofilm of Pg at each stage detected by each group of drugs using the crystal violet method;

[0028] Figure 3 The morphological structure diagram of the biofilm of Porphyromonas gingivalis at different stages observed by SEM.

[0029] Figure 4 This is a statistical diagram of the metabolic activity of each group of drugs on biofilm bacteria at each stage of Fn detected by CCK-8 method;

[0030] Figure 5 This is a statistical chart of the total amount of biofilm at each stage of Fn detected by each group of drugs using the crystal violet method;

[0031] Figure 6 This is a SEM observation of the morphological structure of the biofilm of Fusobacterium nucleatum at different stages after drug treatment. DETAILED DESCRIPTION

[0032] In order to more clearly illustrate the technical solution of the present invention, the following examples are listed. Unless otherwise stated, the raw materials, reactions and post-treatment methods shown in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0033] The bacteria described in the embodiments of the present invention are Porphyromonas gingivalis (standard strain, ATCC 33277) and Fusobacterium nucleatum (standard strain, ATCC 25586);

[0034] The culture medium described in the embodiment of the present invention adopts brain heart infusion agar medium (BHI-S, pH 7.4) and brain heart infusion liquid medium (BHI, pH 7.4).

[0035] The ginger magnolia bark extract described in the embodiment of the present invention was purchased from Beijing Bencao Fangyuan Pharmaceutical Group Co., Ltd., batch number: 20210109, wherein the total magnolia bark phenol content is 90wt%.

[0036] The grape skin extract described in the embodiment of the present invention was purchased from Shaanxi Baichuan Biotechnology Co., Ltd., batch number: BCSW221028-1, wherein the resveratrol content is 10wt%.

[0037] The components and concentrations of the brain heart infusion agar medium and the brain heart infusion liquid medium of the present invention are shown in Table 1 below;

[0038] Element Brain Heart Infusion Agar Brain Heart Infusion Broth Culture medium particles (g) 5.2 7.7 Hemin (μg / mL) 5.0 5.0 Vitamin K1 (μg / mL) 10.0 10.0 Sterile defibrinated sheep blood (%) 1％ / Deionized water (mL) 100 200

[0039] The Zingiber officinale extract stock solution is a lyophilized powder of Zingiber officinale extract, which is dissolved in sterile water to prepare a drug stock solution with a concentration of 2560 mg / mL;

[0040] The grape skin extract stock solution is grape skin extract powder, which is dissolved in sterile water to prepare a drug stock solution with a concentration of 2560 mg / mL.

[0041] The chlorhexidine stock solution is prepared by dissolving chlorhexidine powder in dimethyl sulfoxide to prepare a drug stock solution with a concentration of 2560 mg / mL.

[0042] The preparation methods of Porphyromonas gingivalis and Fusobacterium nucleatum in the test examples of the present invention are as follows:

[0043] In a sterile workbench, glycerol-frozen strains of Porphyromonas gingivalis and Fusobacterium nucleatum were inoculated on BHI-S agar plates, respectively, and placed in a 37°C constant temperature incubator for anaerobically cultured for 60 h. Sterile physiological saline was added to the culture medium cultured with mature test strains, and the colonies were shaken and washed to obtain suspensions of Porphyromonas gingivalis and Fusobacterium nucleatum.

[0044] By turbidimetry, the bacterial suspension was diluted with sterile saline to adjust the turbidity of the suspension to be consistent with that of the No. 0.5 turbidimetric tube, that is, the concentration of the bacterial suspension was 1.5×10 8 CFU / mL. Then adjust the concentration of the bacterial suspension to 1.0×10 6 CFU / mL, as the experimental bacterial suspension.

[0045] Example 1

[0046] A composition for inhibiting and removing bacterial biofilm, comprising a Zingiber officinale extract and a grape skin residue extract;

[0047] The concentration of the ginger magnolia bark extract is 2560-1.25 mg / mL;

[0048] The concentration of the grape pomace extract is 2560-1.25 mg / mL;

[0049] The Zingiber officinale extract and the grape pomace extract were diluted to the concentrations using a BHI liquid medium by a two-fold microdilution method.

[0050] Example 2

[0051] A composition for inhibiting and removing bacterial biofilm, comprising a Zingiber officinale extract and a grape skin residue extract;

[0052] The concentration of the ginger magnolia bark extract is 0.15-18 mg / mL;

[0053] The concentration of the grape pomace extract is 5.46-700 mg / mL;

[0054] The Zingiber officinale extract and the grape pomace extract were diluted to the concentrations using a BHI liquid medium by a two-fold microdilution method.

[0055] Example 3

[0056] A composition for inhibiting and removing bacterial biofilm, comprising a Zingiber officinale extract and a grape skin residue extract;

[0057] The concentration of the ginger magnolia bark extract is 0.75-1.13 mg / mL;

[0058] The concentration of the grape pomace extract is 70-80 mg / mL;

[0059] The Zingiber officinale extract and the grape pomace extract were diluted to the concentrations using a BHI liquid medium by a two-fold microdilution method.

[0060] Test Example 1

[0061] Determination of the minimum inhibitory concentration of the composition against Porphyromonas gingivalis and Fusobacterium nucleatum

[0062] 200 μL of 1×10 6 CFU / mL suspensions of Porphyromonas gingivalis and Fusobacterium nucleatum were inoculated into sterile 96-well microplates, respectively. Porphyromonas gingivalis was cultured anaerobically in a 37°C constant temperature incubator for 72 h, and Fusobacterium nucleatum was cultured under the same conditions for 48 h.

[0063] Use a sterile pipette to remove the culture medium and floating bacteria from each well, and gently rinse three times with 200 μL of sterile PBS;

[0064] The chlorhexidine drug stock solution was diluted to a concentration range of 2560-10 μg / mL using BHI liquid culture medium by the micro-two-fold dilution method, and 200 μL of the ginger magnolia bark extract, the grape peel extract and the diluted chlorhexidine drug stock solution described in Example 1 were respectively added to a 96-well plate inoculated with Porphyromonas gingivalis and Fusobacterium nucleatum, and a growth control well (containing only culture medium and Porphyromonas gingivalis or Fusobacterium nucleatum) and a negative control well (containing only liquid culture medium) were set, and the plates were placed in a 37°C constant temperature incubator for anaerobic culture for 24 hours and the results were observed.

[0065] Results judgment criteria: The lowest drug concentration at which no turbidity is observed by the naked eye is the minimum membrane inhibitory concentration (MBIC), and the results are shown in Table 2;

[0066] In order to ensure that the drug sensitivity test operation is carried out within acceptable standards and the test results are credible, quality control should be performed during each measurement. The quality control QC strain is Bacteroides fragilis (ATCC 25285) and the quality control drug is metronidazole. Under parallel operation conditions, the minimum inhibitory concentration (MIC) is in the range of 0.25-2μg / mL. In this way, the test results are considered valid and credible.

[0067] Table 2 MBIC values ​​of drugs against Porphyromonas gingivalis and Fusobacterium nucleatum (n=3)

[0068]

[0069] As shown in Table 2, the extract of Magnolia officinalis bark has a certain clearing effect on the biofilm of Porphyromonas gingivalis and Fusobacterium nucleatum, among which the extract of Magnolia officinalis bark has the best clearing effect on the biofilm of Porphyromonas gingivalis.

[0070] Test Example 2

[0071] Determination of drug sensitivity concentration of ginger magnolia bark extract and grape skin extract

[0072] 200 μL of 1×10 6 CFU / mL Porphyromonas gingivalis suspension and Fusobacterium nucleatum suspension were inoculated into sterile 96-well microplates, respectively. Porphyromonas gingivalis was cultured anaerobically in a 37°C constant temperature incubator for 72 hours, and Fusobacterium nucleatum was cultured under the same conditions for 48 hours. Use a sterile pipette to aspirate and remove the culture medium and floating bacteria in each well, and take 200 μL of sterile PBS to gently rinse three times.

[0073] According to the results of test example 1, the MBIC value of the ginger magnolia bark extract was selected, and its 2MIC value was taken as the first concentration. 100 μL of ginger magnolia bark extract was added to the A1-G1 columns of a sterile 96-well microplate respectively;

[0074] Add 50 μL of BHI liquid medium to rows A - G and columns 2 - 10 of a sterile 96 - well microplate. Use a sterile pipette to aspirate 50 μL from the first column and transfer it to the second column for two - fold dilution, and then perform serial two - fold dilutions horizontally to the eighth column (1 / 64 MIC). Discard the 50 μL in the eighth column.

[0075] Dilute the grape skin extract to 7 concentrations (1 MIC - 1 / 64 MIC), and add 50 μL of each concentration to rows A - G of the sterile 96 - well microplate.

[0076] In addition, set up a growth control well (containing Porphyromonas gingivalis suspension or Fusobacterium nucleatum suspension and medium) and a negative control well (containing only liquid medium). Except for the negative control well, add 100 μL of bacterial suspension (1.0×10 6 CFU / mL) to each well, and adjust the volume of each well to 200 μL with BHI liquid medium.

[0077] After culturing under anaerobic conditions at 37 °C for 24 h, observe the results. By visual inspection, the concentration corresponding to the drug well without colony growth is its MBIC combination. The results are shown in Table 3.

[0078] Result evaluation criteria: Use the fractional inhibitory concentration index (FICI) as the judgment basis for the combined drug sensitivity test: FICI ≤ 0.5 indicates a synergistic effect; 0.5 < FICI ≤ 1 indicates an additive effect; 1 < FICI ≤ 2 indicates an irrelevant effect.

[0079] FICI = MBICA 药联用 / MBICA 药单用 +MBICB 药联用 / MBICB 药单用

[0080] Table 3 Results of the combined drug sensitivity test of Magnolia officinalis extract and grape skin extract (n = 3)

[0081]

[0082] As can be seen from the results, the FICI values of the combined use of Magnolia officinalis extract and grape skin extract against Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) are both < 0.5, indicating that the combination of the two has a synergistic effect and a better antibacterial effect (FICI ≤ 0.5 indicates a synergistic effect). For Porphyromonas gingivalis, the MIC of Magnolia officinalis extract after combination is 1 / 8 of that when used alone, and the MIC of grape skin extract after combination is 1 / 4 of that when used alone; for Fusobacterium nucleatum, the MIC of Magnolia officinalis extract after combination is 1 / 8 of that when used alone, and the MIC of grape skin extract after combination is 1 / 4 of that when used alone, both indicating that the combination can reduce the dosage of any single drug.

[0083] Test Example 3

[0084] Effects of drugs on adhesion, aggregation and mature biofilm of tested bacteria

[0085] 1×10 6 200 μL of the test bacterial solution with a CFU / mL was inoculated into a flat-bottom 96-well plate, and Porphyromonas gingivalis was cultured anaerobically at 37°C for 36 h, 60 h, and 84 h, respectively; Fusobacterium nucleatum was cultured for 24 h, 42 h, and 60 h, respectively, and then the culture was terminated (the culture medium was replaced every 24 h), and the biofilms of Porphyromonas gingivalis in the adhesion, aggregation, and maturity stages and Fusobacterium nucleatum in the adhesion, aggregation, and maturity stages were obtained, respectively;

[0086] The adhesion period of Porphyromonas gingivalis is 36 hours of culture, the aggregation period is 60 hours of culture, and the maturity period is 84 hours of culture;

[0087] The adhesion period of the cultured Fusobacterium nucleatum is 24 hours, the aggregation period is 42 hours, and the maturity period is 60 hours.

[0088] After establishing biofilms at different stages according to the above method, the culture medium was discarded and the biofilms were rinsed with PBS;

[0089] 200 μL of drug test solution was added respectively, and the Porphyromonas gingivalis was divided into the following groups: 0.75 mg / mL ginger magnolia bark extract group, 80 mg / mL grape skin extract group, 0.75 mg / mL ginger magnolia bark extract + 80 mg / mL grape skin extract combined group, 10 μg / mL chlorhexidine group as positive control, and 200 μL of culture medium was added as growth control group;

[0090] Fusobacterium nucleatum was divided into the following groups: 1.13 mg / mL ginger magnolia bark extract group, 70 mg / mL grape skin extract group, 1.13 mg / mL ginger magnolia bark extract + 70 mg / mL grape skin extract combined group, and 5.62 μg / mL chlorhexidine group as positive controls, and 200 μL of culture medium was added as the growth control group. All of them were placed in a 37°C constant temperature incubator for anaerobically cultured for 24 hours, the culture medium and drug solution were discarded, and PBS was rinsed for determination. The test results are shown in the figure. Figure 1-Figure 6 shown.

[0091] See also Figure 1The drugs were used to intervene in the biofilm of Porphyromonas gingivalis in the adhesion stage (A1), aggregation stage (B1), and maturity stage (C1). The results showed that the combined group could effectively reduce the metabolic activity of bacteria in the biofilm (P < 0.0001), and its metabolic activity decreased by 88.23%, 96.10%, and 90.85% respectively compared with the growth control group. In the maturity stage (84h), the effect of the drug combination group was better than that of each single drug group (P < 0.0001), and its effect was 8.21 times that of the single use of ginger magnolia bark extract and 10.50 times that of the single use of grape skin extract. Compared with the positive control group of chlorhexidine, the combined group had a better effect (P < 0.0001).

[0092] During the adhesion period (36h) and aggregation period (60h), the effect of the drug combination group was also better than that of each single drug group (P < 0.01). The effects were 3.92 times and 13.12 times that of the single use of Magnolia officinalis extract, and 8.53 times and 23.07 times that of the single use of grape skin extract. Compared with the positive control group of chlorhexidine, there was no significant difference in the effect of the combination group (P > 0.05).

[0093] It can be seen that the combination of ginger magnolia bark extract and grape skin extract can effectively inhibit the metabolic activity of bacteria in the biofilm at all stages of Porphyromonas gingivalis biofilm formation, and the effect is better than using either drug alone.

[0094] See also Figure 2 The drugs intervened in the biofilms of Porphyromonas gingivalis in the adhesion stage (A2), aggregation stage (B2), and maturity stage (C2). The results showed that the combined group could effectively reduce the total amount of biofilm matrix (P < 0.0001), and its metabolic activity decreased by 64.23%, 78.34%, and 88.23% respectively compared with the growth control group.

[0095] During the aggregation period (60h), the effects of the drug combination group were better than those of the single drug groups (P<0.05), and the effects were 3.53 times that of the single use of Magnolia officinalis extract and 3.37 times that of the single use of grape skin extract.

[0096] The effect in the adhesion period (36h) and maturity period (84h) was also better than that of each single drug group, and its effect was 1.54 times and 2.49 times that of the single use of ginger magnolia bark extract, and 2.09 times and 2.97 times that of the single use of grape skin extract. Compared with the positive control group of chlorhexidine, there was no significant difference in the effect of the combined group (P>0.05).

[0097] It can be seen that the combined use of ginger magnolia bark extract and grape skin extract can effectively reduce the total amount of matrix in each stage of Porphyromonas gingivalis biofilm formation, and the effect is better than the use of either drug alone.

[0098] See also Figure 3, the drug effects on the microstructure of Porphyromonas gingivalis biofilm during the adhesion, aggregation and mature stages;

[0099] During the adhesion period (36h), compared with the growth control group, the number of bacteria decreased relatively under the action of each drug group, and the bacterial structure changed to varying degrees. Under the treatment of ginger magnolia bark extract and grape skin extract, the number of bacteria decreased and the structure was slightly deformed, but normal cell division and differentiation could still be carried out. In the combined group, in addition to the significant reduction in the number of bacteria, there were almost no formed bacteria. Under the action of the positive control chlorhexidine, the number of bacteria decreased relatively and some bacteria were severely deformed.

[0100] During the aggregation period (60h), the bacteria in the growth control group gradually aggregated into clumps, and extracellular matrix adhesion was visible on the outside of the bacteria. Under the action of the drug, the number of bacteria decreased relatively, and the bacterial bodies were deformed and ruptured. Among them, under the treatment of ginger magnolia extract and grape skin extract, the number of bacteria decreased relatively, but some bacterial clumps that aggregated into clumps were still visible. In the combined group, the number of bacteria decreased significantly and the bacterial clumps disappeared, and the bacterial bodies showed obvious deformation and rupture, with traces of content outflow. Under the action of the positive control chlorhexidine, the number of bacteria decreased significantly and obvious ruptures were visible on the surface of the bacteria.

[0101] At the mature stage (84h), the bacteria in the growth control group formed a dense biofilm adhered to the extracellular matrix and had a three-dimensional structure. Under the action of the drug, the number of bacteria and the bacteria were reduced and deformed to varying degrees. Under the treatment of ginger magnolia extract and grape skin extract, the surface of the bacteria was slightly ruptured and deformed, but the dense biofilm structure still existed. In the combined group, the number of bacteria was greatly reduced and the bacterial clumps were broken, the three-dimensional structure of the biofilm was severely damaged, and traces of a large number of bacterial deaths were left. Under the action of the positive control chlorhexidine, the number of bacteria was greatly reduced, the biofilm structure was destroyed, only a small number of bacterial clumps remained, and the bacterial morphology was obviously ruptured and deformed.

[0102] See also Figure 4 The drugs were used to intervene in the biofilm of Fusobacterium nucleatum in the adhesion stage (D1), aggregation stage (E1), and maturity stage (F1). The results showed that the combined group could effectively reduce the metabolic activity of bacteria in the biofilm (P < 0.0001), and its metabolic activity decreased by 93.27%, 90.25%, and 93.32% respectively compared with the growth control group. In the maturity stage (60h), the effect of the drug combination group was better than that of each single drug group (P < 0.001), and its effect was 7.33 times that of the single use of ginger magnolia extract and 9.71 times that of the single use of grape skin extract.

[0103] In the adhesion period (24h) and aggregation period (42h), the effect of the drug combination group was also better than that of each single drug group (P < 0.01), and its effect was 5.26 times and 3.82 times that of the single use of ginger magnolia bark extract, and 10.18 times and 4.84 times that of the single use of grape skin extract. Compared with the positive control group of chlorhexidine, there was no significant difference in the effect of the combination group (P > 0.05).

[0104] It can be seen that the combination of ginger magnolia bark extract and grape skin extract can effectively inhibit the metabolic activity of bacteria in the biofilm at all stages of Fusobacterium nucleatum biofilm formation, and the effect is better than using the two drugs alone.

[0105] See also Figure 5 The drugs intervened in the biofilm of Porphyromonas gingivalis in the adhesion stage (D2), aggregation stage (E2), and maturity stage (F2). The results showed that the combined group could effectively reduce the total amount of biofilm matrix (P < 0.0001), and its metabolic activity decreased by 95.69%, 78.27% and 79.06% respectively compared with the growth control group.

[0106] In the adhesion period (24h) and the maturity period (60h), the effect of the drug combination group was better than that of each single drug group (P < 0.001), and its effect was 7.93 times and 2.48 times that of the single use of ginger magnolia bark extract, and 9.62 times and 2.94 times that of the single use of grape skin extract. The effect in the maturity period (84h) was also better than that of each single drug group (P < 0.05), and its effect was 2.50 times that of the single use of ginger magnolia bark extract, and 2.86 times that of the single use of grape skin extract. Compared with the positive control group of chlorhexidine, there was no significant difference in the effect of the combination group (P > 0.05).

[0107] It can be seen that the combined use of ginger magnolia bark extract and grape skin extract can effectively reduce the total amount of matrix in each stage of Fusobacterium nucleatum biofilm formation, and the effect is better than the use of either drug alone.

[0108] See also Figure 6 , the drug affects the microstructure of biofilm of Fusobacterium nucleatum during adhesion, aggregation and maturity stages;

[0109] During the adhesion period (24h), long rod-shaped bacteria were scattered in the growth control group. Compared with the growth control group, the number of bacteria decreased relatively under the action of each group of drugs. Under the treatment of ginger magnolia bark extract and grape skin extract, the number of bacteria decreased, but the morphological structure did not change, and normal cell division and differentiation could still be carried out. In the combined group, in addition to the significant reduction in the number of bacteria, there were no formed bacteria, and only traces of bacterial rupture remained. Under the action of the positive control chlorhexidine, the number of bacteria decreased and the bacteria were severely ruptured.

[0110] During the aggregation period (42h), the bacteria in the growth control group had extracellular matrix adhesion and aggregated into clumps. Under the action of the drug, the number of bacteria decreased relatively, and the bacterial cells showed different degrees of deformation and rupture. Among them, under the treatment of ginger magnolia extract and grape skin extract, the number of bacteria decreased relatively, but some bacterial clumps were still visible. In the combined group, the bacterial clumps disappeared, and only single bacteria remained, and the bacteria showed obvious deformation and rupture. Under the action of the positive control chlorhexidine, the number of bacteria decreased significantly, and the bacteria were ruptured, wrinkled and shapeless.

[0111] At the mature stage (60h), the bacteria in the growth control group formed a three-dimensional dense biofilm with a certain thickness. Under the action of the drug, the number of bacteria and the bacteria were reduced and deformed to varying degrees. Under the treatment of ginger magnolia extract and grape skin extract, the growth of bacteria was inhibited, the bacteria became shorter and the number decreased, but the dense biofilm structure still existed. In the combined group, the number of bacteria was greatly reduced, the three-dimensional structure of the biofilm was severely damaged and the thickness was reduced, and a large number of bacteria were ruptured. Under the action of the positive control chlorhexidine, the number of bacteria was greatly reduced, the growth was inhibited, the morphology changed from slender to short, and the biofilm structure was destroyed.

[0112] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0113] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A composition for inhibiting or removing bacterial biofilm, characterized in that: in, The bacteria is selected from at least one of Fusobacterium nucleatum and Porphyromonas gingivalis; The composition is composed of the following components by mass fraction: Ginger Magnolia Bark Extract 0.075-0.113% Grape pomace extract 7-8% Solvent residue; The content of total magnolia bark phenols in the ginger magnolia bark extract is ≥ 90wt%; The resveratrol content in the grape pomace extract is ≥10 wt %.

2. The composition for inhibiting or removing bacterial biofilm according to claim 1, characterized in that: The partial antibacterial concentration index of the composition to Porphyromonas gingivalis is 0.32-0.

50.

3. The composition for inhibiting or removing bacterial biofilm according to claim 1, characterized in that: The partial antibacterial concentration index of the composition against Fusobacterium nucleatum is 0.28-0.

48.

4. The composition for inhibiting or removing bacterial biofilm according to claim 1, characterized in that: The solvent is selected from at least one of water and alcohols.

5. An antibacterial agent for inhibiting or removing bacterial biofilm, characterized in that: Containing the composition according to claim 1.

6. Use of the antibacterial agent for inhibiting or removing bacterial biofilm as claimed in claim 5 in the preparation of toothpaste or mouthwash for removing bad odour in the mouth.

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