JP2020109064A - Method and oral composition for reducing activity of pathogenic bacteria present inside oral biofilm - Google Patents

Abstract

To provide novel methods and oral compositions that can inhibit the onset and progression of oral diseases, such as caries and periodontal disease, by reducing the activity of pathogenic bacteria present inside the oral biofilm.SOLUTION: The method for reducing the activity of pathogenic bacteria present inside the oral biofilm according to one embodiment of the present invention reduces the activity of the pathogenic bacteria by controlling the extracellular electron transfer mechanism of the pathogenic bacteria. The oral composition according to one embodiment of the present invention contains a production inhibitor of electron transmitters produced by bacteria, which are present inside the oral biofilm, having the electron transmitter-producing ability.SELECTED DRAWING: Figure 3

Description

The present invention relates to a method and oral composition for reducing the activity of pathogenic bacteria present inside an oral biofilm.

BACKGROUND ART A biofilm is formed by attaching bacteria and an adherent extracellular polysaccharide (EPS) (also referred to as glycocalyx) produced on the solid phase surface. Dental plaque (plaque) in the oral cavity is a typical example of a biofilm produced through saliva.

The oral biofilm is composed of 500 to 700 kinds or more of bacteria, and bacteria of the genus Streptococcus are typical bacteria. Among them, Streptococcus mutans (S. mutans) is one of the bacteria that cause caries (cariogenic bacterium), which produces glycocalyx using sugar as a raw material and forms dental cartilage along with other oral bacteria on the tooth surface. Forming plaques. Bacteria such as S. mutans existing inside the oral biofilm are anaerobic bacteria that can grow even in the absence of oxygen, and many of these bacteria are pathogenic factors related to oral diseases such as caries and periodontal disease. It is a sex bacterium. Therefore, removal of the oral biofilm or sterilization of pathogenic bacteria present inside the oral biofilm (hereinafter also referred to as “pathogenic bacteria in oral biofilm”) is associated with the risk of the onset and progression of oral diseases. It is said to be important for reduction. Further, in recent years, it has been revealed that pathogenic bacteria in the oral biofilm are involved in various diseases of the whole body, and interest in oral care is further increasing.

In biofilms formed by oral indigenous bacteria and cariogenic bacteria on the surface of teeth, and biofilms formed by periodontopathic bacteria in the periodontal pocket, multiple microorganisms containing pathogenic bacteria interact with each other. Communities are formed by influencing each other. As the formation of the oral biofilm progresses, the structure of the biofilm changes more complicatedly, so that it becomes difficult for the antibacterial agent to penetrate into the oral biofilm, and it becomes difficult to effectively kill pathogenic bacteria.

For example, in Patent Document 1, by using a monoglyceride of a medium-chain fatty acid having 6 to 12 carbon atoms and an antibacterial component in combination, a bactericidal effect is exhibited against an oral biofilm that is difficult to be treated by a conventional antibacterial agent, and an oral biofilm. Is proposed to be removed.

In Patent Document 2, focusing on quorum sensing, which is a mechanism of information transmission between bacteria, a search was conducted based on the structure of a compound reported to have an inhibitory effect on oral biofilm formation by S. mutans. It has been proposed to suppress oral biofilm formation by using cinnamic acid analogs.

Patent No. 5953608 Publication Japanese Patent No. 6389212

In the approach using an antibacterial agent as disclosed in Patent Document 1, the resistance of the target pathogenic bacterium to the antibacterial agent may be increased, or the antibacterial agent may increase the metabolic activity of non-target bacteria. In addition, the possibility that new resistant bacteria may appear cannot be ruled out, and there is a problem that it is difficult to maintain and continue the bactericidal effect. In fact, the increase of resistant bacteria due to the use of agents such as antibacterial agents is one of the biggest problems with pathogenic bacteria.

In recent years, as it has become clear that quorum sensing is involved in the control of various functions of bacteria, active research is being conducted on quorum sensing control methods. The approach of Patent Document 2 is expected to be effective in that it is expected that the expression of bacterial pathogenicity is suppressed by controlling quorum sensing. In addition, chemical substances used in the control of quorum sensing may also be advantageous in that the risk of emergence of resistant bacteria is low, but this means that the effect of controlling quorum sensing is limited, and pathogenicity is limited. It means that bacteria cannot be killed. In addition, quorum sensing is involved in the formation of the three-dimensional structure of biofilm, such as the control of glycocalyx production by bacteria, but is not involved in the initial adsorption on the solid phase surface such as teeth. Therefore, it is difficult to prevent bacterial attachment only by controlling quorum sensing.

As described above, the conventional biochemical approach has a problem in that it may not exhibit sufficient preventive/therapeutic effect for oral diseases. Therefore, despite various attempts so far, there is still a demand for the development of a method that can contribute to the prevention and treatment of oral diseases by a completely different approach.

In view of the above circumstances, the present inventors have found that the energy production mechanism (metabolic reaction) of pathogenic bacteria represented by S. mutans present inside the oral biofilm (metabolic reaction), especially fermentation under anaerobic conditions (fermentation). Focusing on the mechanism, we have attempted to develop a novel method and oral composition that can suppress the onset and progression of oral diseases by reducing the activity of the pathogenic bacterium using a physicochemical approach.

For a pathogenic bacterium existing in an anaerobic environment in an oral biofilm, maintaining a redox balance is very important in maintaining metabolism (fermentation). For example, when S. mutans metabolizes sugar, organic acids such as lactic acid, acetic acid, and formic acid, hydrogen, carbon dioxide, and the like are produced, and the pH is lowered, so that the inside of the oral biofilm becomes a highly reducing environment. Under such reducing conditions, the regeneration of NAD+ due to the oxidation of NADH is unlikely to occur, and thus the maximization of energy production by fermentation is hindered. Therefore, for pathogenic bacteria in the oral biofilm, smooth regeneration of NAD+ from NADH is an important factor for maintaining redox balance and maintaining life activity. It is also believed that hydrogen consumption by the symbiotic bacteria forming a community within the oral biofilm, as well as individual bacterial levels, contributes to maintaining the intracellular energy balance of pathogenic bacteria. ..

It is known that some bacteria existing in nature (environmental bacteria) regenerate NAD+ by extracellular electron transfer (EET). These bacteria use an electron-accepting enzyme (cytochrome c) expressed on the extracellular membrane to generate an electron generated by the decomposition (metabolism) of an electron donor (organic substance) inside the bacterial cell, which is an extracellular electron acceptor. Since it has the ability to transmit to metals (metals, etc.), it is also called "current-producing bacteria". In recent years, as a sustainable energy production technology, research in the fields of microbial fuel cells that generate electricity using biomass etc. Is being actively conducted.

However, many pathogenic bacteria in oral biofilms such as S. mutans do not have cytochrome c localized on the cell surface such as current-producing bacteria or electron transport system associated therewith. Since it is known, whether these bacteria have the ability to carry out extracellular electron transfer (extracellular electron transfer ability) as described above has not been elucidated, and they have not been studied. Of course, as far as the present inventors can know, there is no such research report example.

On the other hand, the inventors of the present invention carry out research on the extracellular electron transfer mechanism of iron-reducing bacteria represented by Schwanella bacteria such as Shewanella oneidensis (S. oneidensis) and the industrial use of these bacteria as current-producing bacteria. Among them, focusing on the fact that the pathogenic bacteria in the oral biofilm are anaerobic bacteria as well as the iron-reducing bacteria, it is necessary to maintain the redox balance in the fermentation mechanism in the oral biofilm. The idea was that the transmission mechanism might be involved.

Therefore, the present inventors have actually used S. mutans (UA159 strain), which is known as a typical cariogenic bacterium, by the method described below to actually provide S. mutans with an extracellular electron transfer mechanism. I found that. Furthermore, according to an experiment using S. mutans, S. mutans regenerates NAD+ by transmitting the excessive reduction energy generated inside the cells due to fermentation etc. to the outside of the cells as an electron to maintain the lactic acid fermentation activity. I found that. In other words, it was clarified that for S. mutans, discharging electrons from the inside of the cell to the outside of the cell plays an important role in maintaining redox balance and maintaining life activity.

Based on these novel findings, the present inventors have conducted further research, and by controlling the extracellular electron transfer mechanism of pathogenic bacteria in the oral biofilm, the activity of the pathogenic bacteria can be reduced. As a result, they have found that the onset and progression of oral diseases can be suppressed, and completed the present invention.

That is, the present invention includes the following aspects.
(1) A method of reducing the activity of the pathogenic bacterium by controlling the extracellular electron transfer mechanism of the pathogenic bacterium existing inside the oral biofilm.
(2) The method according to (1), wherein the extracellular electron transfer mechanism of the pathogenic bacterium is controlled by suppressing electron transfer from the inside of the pathogenic bacterium to the outside of the cell.
(3) The method according to (2), wherein the electron transfer from the inside to the outside of the pathogenic bacterium is suppressed by suppressing the electron transfer from the inside to the outside of the oral biofilm.
(4) Suppressing electron transfer from intracellular to extracellular of the pathogenic bacterium by inhibiting the production of an electron mediator by a bacterium having the ability to generate an electron mediator present in the oral biofilm. The method according to (2) or (3).
(5) The method according to (2), wherein the electron transfer from the intracellular to the extracellular of the pathogenic bacterium is suppressed by inhibiting the activity of an electron transfer enzyme present in the extracellular membrane of the pathogenic bacterium. ..
(6) The method according to (4), wherein the electron mediator is riboflavin.
(7) The method according to any one of (1) to (6), wherein the pathogenic bacterium is a bacterium that causes oral diseases.
(8) The method according to (7), wherein the pathogenic bacterium is a cariogenic bacterium or a periodontopathic bacterium.
(9) The method according to (8), wherein the pathogenic bacterium is Streptococcus mutans, Streptococcus sanguinis, Porphyromonas gingivalis, or Aggregatibacter actinomycetemcomitans.
(10) A composition for oral cavity containing an inhibitor of the production of an electron mediator produced by a bacterium having the ability to produce an electron mediator, which is present inside an oral biofilm.
(11) The composition for oral cavity according to (10), wherein the electron mediator is riboflavin.
(12) A composition for oral cavity containing an activity inhibitor against an electron transfer enzyme present in the extracellular membrane of a pathogenic bacterium present in the oral biofilm.

According to the present invention, by controlling the extracellular electron transfer mechanism of pathogenic bacteria existing inside the oral biofilm, the activity of the pathogenic bacteria can be reduced, so that caries and periodontal disease, etc. The onset and progression of oral diseases can be suppressed. Further, according to the present invention, it is possible to suppress the onset and progression of systemic diseases other than oral diseases related to pathogenic bacteria in the oral biofilm.

Schematic diagram of NADH-dependent metabolic mechanism mediated by Rex protein in S. mutans. Chronoamperogram (current-time response) results obtained with S. mutans. Results of electrochemical measurement of glucose metabolism by S. mutans. (A) Current density (nA/cm ² ) change with time, (b) Glucose concentration (mM) change with time, (c) Lactate concentration (mM) change with time, (d) Ethanol concentration (mM) change with time .. Results of electrochemical measurement of glucose metabolism by S. mutans. (A) Time-dependent change of voltage (mV) vs. Ag/AgCl value under OCV conditions, (b) Time-dependent change of acetic acid concentration (mM), (c) Time-dependent change of formic acid concentration (mM). The result of the emission spectrum analysis showing the reducing ability of riboflavin by S. mutans in the presence of glucose. Results of electrochemical analysis performed using ¹⁵ NH ₄ Cl. Results of calculating the N isotope ratio (%) of each cell of S. mutans. (A) OCV conditions in the absence of riboflavin, (b) EET conditions in the presence of riboflavin, (c) OCV conditions in the presence of riboflavin, (d) EET conditions in the absence of riboflavin. FIG. 8 is a diagram summarizing the results obtained under each condition of FIG. 7. Schematic diagram showing the electron transfer mechanism from pathogenic bacteria such as S. mutans to the electrode surface. (I) Electron transfer mechanism via protein present on cell surface, (ii) Electron transfer mechanism via electron transfer substance (for example, riboflavin (RF)). Experimental results showing the existence of a direct electron transfer mechanism by S. mutans. (A) Current density (nA/cm ² ) measurement results before and after replacement of the culture medium, (b) DPV analysis result, (c) Redox using DAB in the presence of hydrogen peroxide (H ₂ O ₂ ). Transmission electron microscope (TEM) observation results of S. mutans cells subjected to dependency staining, (d) Transmission type of S. mutans cells subjected to DAB staining in the absence of hydrogen peroxide (H ₂ O ₂ ). Electron microscope (TEM) observation results. The result of observing the ITO electrode after the electrochemical measurement with a scanning electron microscope (SEM). (A) Time course of current generation value (μA) by S. mutans measured at ITO electrode, (b) Redox peak current at potential of about 20 mV with respect to standard hydrogen electrode (SHE) shown in FIG. 10(b). The graph which plotted the electric current generation value (μA) by S. mutans against the value (μA). Results of transmission electron microscope (TEM) observation of S. mutans cells stained with DAB. (A) Positive control conditions (DAB-positive), (b) Negative control conditions (DAB-negative). Results of TEM-LINE profile analysis of S. mutans cells treated with DAB in the presence of hydrogen peroxide. Results of TEM-LINE profile analysis of S. mutans cells treated with DAB in the absence of hydrogen peroxide.

A method of reducing the activity of a pathogenic bacterium present inside an oral biofilm according to an embodiment of the present invention is to control the activity of the pathogenic bacterium by controlling the extracellular electron transfer mechanism of the pathogenic bacterium. Reduce.

In the present specification, “pathogenic bacteria present inside the oral biofilm” causes oral diseases or systemic diseases other than oral diseases among bacteria existing inside the oral biofilm. Refers to bacteria. Examples of oral diseases include dental caries and periodontal disease. Examples of systemic diseases include heart disease (angina, myocardial infarction, valvular disease, etc.), brain disease (cerebral infarction, etc.), diabetes (type I diabetes, type II diabetes), renal disease (glomerulonephritis, nephrosclerosis). Diseases, etc.), gastric diseases (gastritis, gastric ulcer, etc.), skin diseases, respiratory infections, bacteremia, sepsis, bacterial endocarditis, arteriosclerosis, arthritis, osteoporosis, metabolic syndrome and the like. Unless otherwise specified in the present specification, when simply described as “pathogenic bacterium”, the pathogenic bacterium is intended to be a pathogenic bacterium existing inside the oral biofilm.

In one embodiment of the present invention, the pathogenic bacterium is a bacterium that causes oral diseases, and is typically a cariogenic bacterium and a periodontal pathogenic bacterium. Examples of the cariogenic bacteria and the periodontopathic bacteria include bacteria of the genus Streptococcus, the genus Porphyromonas, and the genus Aggregatibacter. More specific examples include, but are not limited to, Streptococcus mutans, Streptococcus sanguinis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and the like.

In the present specification, the term "extracellular electron transfer mechanism" used for bacteria means that the electrons generated by the decomposition (metabolism) of an electron donor (organic substance) in the bacterial cell of the bacterium are extracellular electron acceptors. Means a mechanism that is transmitted to (metal, etc.). That is, a bacterium having an extracellular electron transfer mechanism means a bacterium having an ability to perform extracellular electron transfer (EET) (extracellular electron transfer ability).

Therefore, in a preferred embodiment of the present invention, the extracellular electron transfer mechanism of the pathogenic bacterium is controlled by suppressing electron transfer from intracellular to extracellular of the pathogenic bacterium present inside the oral biofilm. ..

In addition, as described above, since the oral biofilm is composed of a plurality of microorganisms containing pathogenic bacteria and their products, electron transfer from the inside of the oral biofilm to the outside also occurs in the oral biofilm. It is considered to be an important factor in maintaining the redox balance of the pathogenic bacteria. Therefore, in a preferred embodiment of the present invention, the electron transfer from the inside to the outside of the pathogenic bacterium is suppressed by suppressing the electron transfer from the inside to the outside of the oral biofilm.

As detailed below, intracellular to extracellular electron transfer of pathogenic bacteria within oral biofilms can be mediated by electron mediators such as riboflavin. The electron mediator may also mediate electron transfer from the interior of the oral biofilm to the exterior. This means that there is a possibility that there is a bacterium having an electron mediator-producing ability inside the oral biofilm as well as a habitat of bacteria existing in nature and a community formed between bacteria. , By inhibiting the production of electron mediators by bacteria having the ability to produce electron mediators, electron transfer from the intracellular to extracellular of pathogenic bacteria in the oral biofilm and from the inside to the outside of the oral biofilm. It means that the electron transfer of can be suppressed. Therefore, in a preferred embodiment of the present invention, by inhibiting the production of an electron mediator by a bacterium capable of producing an electron mediator, which is present inside the oral biofilm, from inside the cell to outside the cell of the pathogenic bacterium. Suppresses electron transfer.

The bacterium having the ability to generate an electron mediator may be the same as the pathogenic bacterium in the oral biofilm, or may be a bacterium different from the pathogenic bacterium in the oral biofilm.

In addition, as described above, the current-producing bacteria existing in nature, through the electron transfer enzyme (cytochrome c) expressed on the extracellular membrane, breathes solids such as iron oxide existing outside the cells into the final electrons. It is known that it can be used as a receptor. This means that the pathogenic bacteria in the oral biofilm may have cytochrome c or an electron transfer enzyme having a function equivalent to that in the extracellular membrane, and the activity of the electron transfer enzyme. Means that the electron transfer from the intracellular to the extracellular of the pathogenic bacterium can be suppressed. Therefore, in a preferred embodiment of the present invention, by inhibiting the activity of the electron transfer enzyme present in the extracellular membrane of the pathogenic bacterium present inside the oral biofilm, the activity of the pathogenic bacterium from inside the cell to outside the cell is reduced. Suppresses electron transfer.

An oral composition according to an embodiment of the present invention contains an electron mediator production inhibitor produced by a bacterium having an electron mediator-producing ability, which is present inside an oral biofilm. Examples of electron mediators include, but are not limited to, riboflavin. In the present invention, a substance produced by bacteria present inside the oral biofilm, wherein electron transfer of pathogenic bacteria from the inside of the oral biofilm to the outside of the cell and/or inside of the oral biofilm. It is possible to target an electron mediator confirmed to be involved in the electron transfer from the outside to the outside, and a production inhibitor of the electron mediator can be appropriately selected. Such a production inhibitor may be an agent that inhibits the biosynthetic pathway of the electron mediator by bacteria.

An oral composition according to an embodiment of the present invention contains an activity inhibitor against an electron transfer enzyme present in the extracellular membrane of a pathogenic bacterium present inside an oral biofilm. Examples of the electron transfer enzyme include, but are not limited to, cytochrome c. In the present invention, an electron transfer enzyme (electron transfer protein) confirmed to be present in the extracellular membrane of a pathogenic bacterium in the oral biofilm can be targeted, and an activity inhibitor of the electron transfer enzyme can be used. It can be appropriately selected.

In the present specification, the terms “inhibit activity” and “inhibition of activity” used with respect to an electron transfer enzyme mean that the enzyme of interest has an electron transfer from intracellular to extracellular of a pathogenic bacterium. The term “activity inhibitor” means an agent that inhibits the electron transfer mediated effect.

The form of the oral composition of the present invention is not particularly limited, and examples thereof include liquids, solids, gels, semisolids such as pastes, and the like, and specifically, mouthwash, liquid toothpaste, mouth freshener. It may be in the form of an agent, a mouthwash (gargle), a toothpaste such as a liquid toothpaste and a toothpaste, a mouthwash (gargle), a troche, and a chewing gum. These can be made into a formulation by a known means.

Hereinafter, specific embodiments of the present invention will be described by taking as an example the case where the pathogenic bacterium existing inside the oral biofilm is S. mutans.

<Evaluation of extracellular electron transfer ability of pathogenic bacteria in oral biofilm>
S. mutans (UA159 strain) is a typical pathogenic bacterium present in the oral biofilm, and is known to have a Rex protein. The Rex protein is a redox sensor, and acts as a DNA transcriptional repressor by binding to a gene involved in the early regulation mechanism of energy metabolism. As shown in FIG. 1, Rex protein suppresses the transcription of pyruvate dehydrogenase (pdhAB) and promotes the lactic acid fermentation pathway when the ratio of cytoplasmic NADH/NAD+ is low. On the other hand, when the ratio of NADH/NAD+ is high, the binding of Rex protein to the target gene is inhibited, so that the ethanol production pathway is promoted. Therefore, the electron transfer in the experimental electrochemical system can be quantitatively evaluated by using the amount of ethanol produced by glucose oxidation as an indicator of whether NADH is in excess of NAD+. ..

In addition, in the previous research on bacteria existing in nature, substances that function as electron mediators (also called “electron shuttles”) that carry intracellular reduction energy to the outside of cells and reduce metals are found. It is known to exist, and a typical example thereof is riboflavin. Riboflavin, also called vitamin B ₂ (Vitamin B ₂ ), is a physiologically active substance classified as a water-soluble vitamin among vitamins, and is known to be biosynthesized by microorganisms in the human body. There is.

Therefore, by evaluating the extracellular electron transfer ability in combination with an electron transfer substance such as riboflavin, it is possible to obtain more detailed knowledge regarding the extracellular electron transfer ability of the target pathogenic bacterium and regulate its activity ( It is possible to efficiently select an effective method. In the following, S. mutans (strain UA159) was used as a pathogenic bacterium, and a method for evaluating the extracellular electron transfer ability of S. mutans and the utilization ability of riboflavin as an electron shuttle by S. mutans and the results thereof are described in detail. Explained.

<Preparation of cell culture>
S. mutans (strain UA159) obtained from ATCC (American Type Culture Collection) was precultured at 37° C. in 20 mL of Brain Heart Infusion (BHI) medium in a vial having a butyl rubber stopper. Headspace in the vial is filled with until a late logarithmic growth phase _{CO 2 / N 2 (20:80 v} / v) was anoxic. At this time, the absorbance (OD ₆₀₀ ) at a wavelength of 600 nm was 1.0, and the pH of the medium was lowered to 4.6±0.2. The obtained culture was centrifuged at 7800 rpm and 37° C. for 10 minutes, and the obtained cell pellet was washed twice with synthetic medium (DM). The synthetic medium was prepared by partially improving a commonly known method. The composition of the synthetic medium (per liter) is as follows: NaHCO ₃ 2.5 g; CaCl ₂ .2H ₂ O 0.09 g; NH ₄ Cl 1.0 g; MgCl ₂ .6H ₂ O 0.2 g. NaCl 10 g; HEPES 7.2 g; yeast extract 0.5 g.

<Electrochemical analysis (whole cell method)>
Electrochemical measurements were performed using a measuring device (single chamber) equipped with a three-electrode system composed of a working electrode, a counter electrode, and a reference electrode. The tin-doped indium oxide (ITO) of the working electrode was formed on a glass substrate using a thin film forming apparatus (made by SPD Laboratory) by a spray pyrolysis method, and was placed at the bottom of the measuring device (resistance value 8Ω/□; Thickness 1.1 mm; surface area 3.14 cm ² ). A platinum wire and Ag/AgCl (KCl _sat ) were used as the counter electrode and the reference electrode, respectively.
10 μM riboflavin as an electron acceptor was added to a synthetic medium containing glucose (10 mM) as an electrolyte (electron donor) added to this electrochemical cell (4.8 mL). A medium (4.8 mL) supplemented with (RF) was injected, and the solution in the cell was purged with N ₂ gas for at least 15 minutes to remove dissolved oxygen. Then, 0.2 mL of cell suspension (optical density (OD) at 600 nm: 2.5) newly prepared using S. mutans prewashed with synthetic medium was injected, and a standard hydrogen electrode (SHE) was applied. As a reference, the final OD ₆₀₀ was set to 0.1 under the constant potential condition where the potential difference of the ITO electrode was +0.4V. During the electrochemical measurement, the temperature of the measuring device was maintained at 37° C., and the device was not shaken or the solution was not stirred.

As a control, the same electrochemical analysis was carried out using a sterile medium containing no glucose and riboflavin (sterile medium) and a sterile medium supplemented with 10 μM riboflavin.
In addition, as a comparative test, the same electrochemical analysis was performed under an open circuit voltage (OCV; Open Circuit Voltage) condition in which the electrodes did not receive electrons.

Chronoamperometry measurement and differential pulse voltammetry (DPV) measurement were performed using an automatic polarization system (VMP3, manufactured by Bio Logic Company). The measurement conditions of DPV are as follows: potential difference based on SHE: −0.6 V to +0.7 V, pulse height 50 mV, pulse width 0.3 s, potential step 5 mV/s, step time 5 s. The open source software SOAS software was used for data analysis in the differential pulse voltammogram.

<Determination of glucose and metabolite concentrations>
Approximately 200 μL aliquots were collected from the measuring device every 8 hours, and the filtrate from which cells were removed through a filter having a pore size of 0.22 μm was stored at −20° C. and used for analysis. The glucose concentration was measured using a glucose assay kit (GAGO-20, manufactured by Sigma-Aldrich). In the measurement of metabolites, the above filtrate was diluted 100 times with distilled water and the pH was adjusted to 8.0, which was used as a sample. The concentrations of lactic acid, acetic acid, and formic acid were measured using an ion chromatography system (HIC-20Asuper, manufactured by Shimadzu). The injection amount of the sample was set to 50 μL, and the anion measurement was performed by the non-suppressor method. As the analysis column and the guard column, Shim-pack IC-A3 and Shim-pack IC-GA3 (all manufactured by Shimadzu) were used. The mobile phase contained 8 mM p-hydroxybenzoic acid, 3.2 mM Bis-Tris and 50 mM boronic acid, and the flow rate was 1.2 mL/min. The column temperature was maintained at 40° C. and the parameters of the detector (CDD-10A _SP ) were set as described in the operating instructions for the system. Analysis of peak areas was performed using analytical workstation software (LabSolutions) provided by Shimadzu. The elution times of acetic acid, lactic acid, and formic acid were about 2.4 minutes, about 3.25 minutes, and about 3.4 minutes, respectively, and the calibration curve showed sufficiently high linearity (R ² =0.999). It was By ion chromatography analysis, metabolites other than lactic acid, acetic acid and formic acid were not detected.

<Analysis of ethanol production by titration using an acidic solution of dichromic acid>
The standard titration method using an acidic solution of dichromic acid was partially modified to measure the ethanol concentration in the sample. The solution used for titration was prepared as follows.
(1) Dichromic acid solution (0.01 mol/L in 5.0 mol/L sulfuric acid): 125 mL of water is put into a 500 mL conical flask, and 70 mL of concentrated sulfuric acid is slowly added dropwise while swirling at a constant speed. .. After cooling the flask, 0.75 g of potassium dichromate (K ₂ Cr ₂ O ₇ ) is added and the mixture is diluted with distilled water to 250 mL.
(2) Starch solution (indicator solution; 1.0%): Add 100 g of soluble starch to 100 mL of boiling water and stir until dissolved.
(3) Sodium thiosulfate solution (Na ₂ S ₂ O ₃ .5H ₂ O; 0.03 mol/L): 7.44 g of Na ₂ S ₂ O ₃ .5H ₂ O was added to a 1 L volumetric flask, and distilled water was added. Dissolve into 1 L solution.
(4) Potassium iodide solution (KI; 1.2 mol/L): Dissolve 5 g of KI in 25 mL of water.

10 mL of dichromic acid solution was transferred to a 50 mL Falcon tube. Dilute the sample 1:50 with distilled water, pipette 1 mL from the diluted sample solution into an Eppendorf tube, place this sample tube in the sample holder provided in the Falcon tube containing the dichromic acid solution, It was stored overnight in an incubator set at 37°C. Then, the Falcon tube was cooled to room temperature, the cap was slowly loosened, and the sample holder was taken out. The wall surface of the Falcon tube was rinsed with distilled water, about 100 mL of distilled water and 1 mL of potassium iodide solution were added, and the tube was swirled to mix the contents. The sample solution in each tube was titrated using a burette filled with a sodium thiosulfate solution. When the brown color of iodine turned yellow to yellow, 1 mL of starch solution was added and the titration was continued until the blue color disappeared. A blank test was performed to measure the amount of dichromic acid in the absence of added alcohol. Calculate the average volume of the sodium thiosulfate solution required for the titration of the sample solution and the blank test solution (blank solution), and use the value obtained by dividing the value in the titration of the sample solution by the value in the titration of the sample solution. , The molar number of sodium thiosulfate was calculated, and the alcohol concentration was determined. The relationship between the number of moles of sodium thiosulfate and the number of moles of ethanol was calculated as follows: 6 mol S ₂ O ₃ ²⁻ is equivalent to 1 mol Cr ₂ O ₇ ²⁻ and ² mol Cr. ₂ O ₇ ²⁻ is equivalent to 3 mol of C ₂ H ₅ OH. Therefore, 1 mol of S ₂ O ₃ ²⁻ is equivalent to 0.25 mol of C ₂ H ₅ OH.

FIG. 2 is a result of the chronoamperogram (current-time response). In FIG. 2, the description “SM added” means that the cell suspension of S. mutans was injected at the time point indicated by the arrow. Further, the description “10 mM Glucose” means that 10 mM glucose was injected at the time indicated by the arrow. In this experimental system, glucose and other electrolytes were not contained in the synthetic medium before S. mutans injection, but 10 μM riboflavin was added.

After the injection of S. mutans, the current value was stable at about 0.06 μA, and immediately after the injection of glucose, the current value rapidly increased to a value exceeding 0.10 μA. This indicates that S. mutans has the ability to generate an electric current by oxidizing glucose.

FIG. 3A is a time-dependent change in current density (nA/cm ² ). In FIG. 3( a ), the description “SM added” means that the cell suspension of S. mutans was injected at the time indicated by the arrow.
When the control sterile medium was used, no change in the current density due to the addition of S. mutans was observed in the presence (Sterile medium w/ RF) and the absence (Sterile medium) of riboflavin. On the other hand, when a synthetic medium supplemented with 10 mM glucose as an electron donor was used (EET), a current density of about 30 nA/cm ² was shown in the absence of riboflavin. Further, in the presence of riboflavin (RF EET), the current density increased at an earlier timing than in the absence of riboflavin, and the maximum value was about 40 nA/cm ² .
Further, as shown in FIGS. 3(b) to 3(d), when a synthetic medium containing glucose (EET), a synthetic medium containing glucose and riboflavin (RF EET) is used, OCV In both cases of using a synthetic medium containing glucose under the conditions (OCV) and using a synthetic medium containing glucose and riboflavin under the OCV conditions (RF OCV), the glucose concentration decreased with the generation of current. (FIG. 3(b)), production of a significant amount of lactic acid was confirmed (FIG. 3(c)), but production of ethanol was not confirmed (FIG. 3(d)).
3(b) and 3(c), the correspondence between each condition and the graph is indicated by using the symbols (1) to (4). The same applies to FIG. 4B, FIG. 4C, and FIG. 8 described later.

These results mean that after the addition of S. mutans, lactic acid was produced by the oxidation of glucose in the medium, and an electric current was generated by the transfer of electrons to the electrode surface. Therefore, it was demonstrated that S. mutans has extracellular electron transfer ability.

Thus, it can be said that the environment in which glucose is present as an electron donor is an environment in which S. mutans can perform extracellular electron transfer (EET). Therefore, hereinafter, the conditions using the medium containing glucose are also referred to as “EET conditions” for convenience.

From the studies so far, it has been confirmed that current-producing bacteria existing in nature can obtain current densities of 10 to 100 μA/cm ² in the same experimental system as described above. Compared with this value, the current value that can be generated by the extracellular electron transfer mechanism of S. mutans is about 100 times smaller than the current value by the current-producing bacteria used in microbial fuel cells and the like. I can say.
That is, the pathogenic bacterium whose activity is reduced by the present invention is intracellular by an extracellular electron transfer mechanism as compared with Shewanella genus (Shewanella) bacterium and Geobacter genus (Geobacter) bacterium which are generally known as current-producing bacteria. Note that the amount of electrons transferred from the cell to the outside of the cell may be small.
However, the generation of minute electric currents due to such small amount of electron transfer is significantly related to the promotion of lactic acid fermentation by S. mutans and the inhibition of ethanol production. The fact that it significantly contributes to the relaxation of the reduction energy is a new finding by the present inventors and is a technical idea that forms the basis of the present invention.

<Evaluation of availability of electron mediator by pathogenic bacteria in oral biofilm>
Hereinafter, the results of the electrochemical analysis by the above experimental system will be described in more detail.
After 5 hours from the start of the electrochemical measurement, the value of the current density under the EET condition reached saturation (FIG. 3(a)), and as the current density subsequently decreased, the glucose consumption rate and the lactate production rate also increased. It decreased (Fig. 3(b), Fig. 3(c)).
On the other hand, even after 24 hours from the start of the electrochemical measurement, almost no ethanol production was observed under EET conditions (FIG. 3(d)), and the production amounts of acetic acid and formic acid were very small (FIG. 4). (B), FIG. 4(c)).
These results suggest that the extracellular electron transfer mechanism of S. mutans has a correlation with lactic acid fermentation and glucose oxidation.

In contrast, under OCV conditions, when the current generation was maintained at zero, 6 mM ethanol production was confirmed 8 hours after the start of the electrochemical measurement (FIG. 3(d)). The tendency of the change in glucose concentration with time is almost the same as the result under the EET condition (FIG. 3(b)), and the lactate concentration increases from the start of the electrochemical measurement to 8 hours and then decreases. It was shown (FIG.3(c)).
Considering that more NAD+ is regenerated by the production of ethanol as compared with lactic acid production, under OCV conditions, the Rex gene detects that the amount of NADH is high, and promotes the production of NAD+ by promoting the production of ethanol. It is thought to be promoting the generation of.

Therefore, these results indicate that the generation of current confirmed in this experimental system is associated with the regeneration of NAD+, and that the extracellular electron transfer mechanism contributes to lactate production in S. mutans.

On the other hand, under EET conditions, the current density in the presence of riboflavin was maintained at 60% or more of the maximum value even after 20 hours from the start of the electrochemical measurement (FIG. 3(a)), and the precursor formic acid was used. The production rate was less than 5% of the initial glucose concentration (10 mM), that is, less than 0.5 mM, 8 hours after the start of the electrochemical measurement (FIG. 4(c)).
This indicates that the generation of electric current confirmed in the present experimental system is related to the electron shuttling process, not controlled by the oxidation of hydrogen produced by fermentation. There is.

FIG. 5 shows the results of emission spectrum analysis showing the ability of S. mutans to reduce riboflavin in the presence of glucose. The emission spectrum was measured in a cuvette under anaerobic conditions, and the excitation wavelength was 450 nm.
Based on the fluorescence intensity of 10 μM riboflavin at 530 nm as a standard (A), S. mutans was added so that OD ₆₀₀ =0.1 (B), and then 10 mM glucose was added (C), so that riboflavin The fluorescence intensity at 530 nm was significantly reduced.
This result shows that riboflavin is oxidized (oxidized riboflavin is produced), and that the oxidation of riboflavin is due to S. mutans, and glucose due to S. mutans It has been shown to be associated with oxidation.
In FIG. 5, “DM” is the emission spectrum of the synthetic medium.

Here, focusing on the results obtained under the OCV condition in the absence of riboflavin (FIG. 3(c), FIG. 3(d)), even under the OCV condition, the reduction of riboflavin is constant with respect to the metabolic pathway. You can see that it has an influence. This indicates that a small amount of electron transfer has a significant effect on the cytoplasmic NADH/NAD+ ratio.

Comparing the time course of the measurement results of metabolites under OCV conditions in the presence and absence of riboflavin, the ethanol production rate increased by 50% in the presence of riboflavin at 8 hours after the start of the electrochemical measurement (Fig. 3(d)), lactic acid production was significantly reduced more than 8 hours after the start of electrochemical measurement (FIG. 3(c)). On the other hand, the glucose consumption rate was not significantly different in the presence and absence of riboflavin (Fig. 3(b)). Considering that NADH is produced by the consumption of lactic acid, S. mutans is considered to accumulate reducing energy in cells. In addition, as compared with the case of the absence of riboflavin, the electrode potential required to maintain the current value at zero was rapidly shifted to the negative potential with the addition of S. mutans in the presence of riboflavin ( FIG. 4(a)). This indicates that riboflavin promotes the excretion of reducing energy from inside the cell of S. mutans.

Riboflavin is considered to be an essential nutrient in the growth medium, but under the experimental conditions of the present inventors, the electrolyte itself contains a sufficient amount of yeast extract (the content of riboflavin is less than 3 nM). It can be said that the effect of the presence or absence of riboflavin is due to the ability of riboflavin to accept electrons.

These results suggest that under OCV conditions in the absence of riboflavin, the ratio of NADH/NAD+ is high, and therefore under these conditions S. mutans may lose its metabolic activity. In addition, the function of riboflavin as an electronic shuttle, which was previously clarified only in studies of naturally occurring bacteria, was specifically confirmed for S. mutans.
That is, regarding fermentative bacteria (anaerobic bacteria), there are electron mediators (electron shuttles) that help maintain the intracellular potential balance, and bacteria actually use such electron shuttles to achieve redox balance. It was revealed for the first time by the present inventors that It was also shown that among the microorganisms constituting the oral biofilm, there may be a bacterium that produces an electron transfer substance that assists the transfer of electrons from the inside of the pathogenic bacterium to the outside of the cell.

<Quantitative evaluation of metabolic activity of pathogenic bacteria in oral biofilm>
Next, a method for quantitatively evaluating the metabolic activity of S. mutans by performing secondary ion mass spectrometry (NanoSIMS analysis) using a cell-specific stable isotope and the result thereof will be described.

A anaerobic reactor equipped with an ITO electrode (surface area 3.14 cm ² ) was injected with a synthetic medium containing 10 mM glucose or a synthetic medium containing 10 mM glucose and 10 μM riboflavin, and the standard hydrogen electrode (SHE (4) as a reference, the S. mutans was incubated under a constant potential condition where the potential difference of the ITO electrode was +0.4 V, with ¹⁵ NH ₄ Cl as the sole nitrogen source being supplied to the ITO electrode surface.

Results are shown in FIG. In FIG. 6, the description “SM added” means that the cell suspension of S. mutans was injected at the time point indicated by the arrow.
Although a change likely to be due to the isotope effect due to ¹⁵ N was observed, generation of a current similar to that using unlabeled HN ₄ Cl (not shown) was confirmed. Further, in the presence of riboflavin (W/riboflavin), the maximum value of the current value obtained by the electrochemical measurement for 15 hours was 22% higher than that in the absence of riboflavin (W/o riboflavin).

<Sample preparation for secondary ion mass spectrometry>
After performing the electrochemical measurement, the ITO electrode to which the biofilm containing S. mutans was attached was collected from the reaction device and fixed in 0.1 M phosphate buffer containing 2.5% glutaraldehyde for 10 minutes. The electrodes were washed by immersing them in fresh 0.1 M phosphate buffer. The washed electrode sample was dehydrated with a gradient of ethanol (25%, 50%, 75%, 90%) in phosphate buffer and 100% ethanol, solvent exchange with t-butanol, vacuum drying, and then Quick Coater. SC-701 (manufactured by Sanyu Electronics Co., Ltd.) was used for coating with platinum. The surface of the sample was irradiated with a Cs ⁺ beam using a CAMECA NanoSIMS 50 L system (manufactured by CAMECA), and the amount of secondary ions ( ¹² C ¹⁴ N ⁻ and ¹² C ¹⁵ N ⁻ ) was measured. A region of interest (ROI) is marked for each cell, the total value of ionic strength in each ROI is calculated, and the ratio of ¹⁵ N to _total nitrogen (N _total ) ( ¹⁵ N/N _total ) The N isotope ratio (%) of each cell was calculated. For each sample, 200 or more cells were measured.

The results are shown in FIGS. 7 and 8. 7(a) to 7(d), the numbers shown in the upper right represent the number (n) of cells for which measurement was performed.
Under OCV conditions in the absence of riboflavin (FIG. 7(a); OCV), 42% of the cells (n=231) showed relatively low activity ( ¹⁵ N/N _total <10%). On the other hand, under OCV conditions in the presence of riboflavin (FIG. 7(c); RF OCV) and EET conditions in the presence of riboflavin (FIG. 7(b); RF EET), the value of ¹⁵ N/N _total was less than 10%. The number of cells was 14% and 9%, respectively, which was significantly lower than that in the OCV condition in the absence of riboflavin shown in FIG. 7(a). The number of cells having a ¹⁵ N/N _total value of less than 10% under EET conditions in the absence of riboflavin (FIG. 7(d); EET) was 4%.
As can be understood from FIG. 8 which summarizes the results obtained under each condition, these results indicate that riboflavin mediates the electron transfer of S. mutans from the inside to the outside of the cell, and that riboflavin is present. It was suggested that by doing so, the metabolic activity of cells with low activity was enhanced.

Further, in the OCV condition in the absence of riboflavin (FIG. 7(a)), 50% or more of the analyzed cells had the same activity as the cells under the other conditions, and the average value of ¹⁵ N/N _total was determined. Values were similar to the other three conditions. This indicates that in S. mutans, the influence on the efficiency of energy production due to the change in metabolic pathway is small.

From the results of NanoSIMS analysis on the activity of these single cells, the following matters are derived.
The transfer of electrons from the intracellular to the extracellular of S. mutans, which is confirmed as a small current value, plays a major role in controlling the intracellular NADH/NAD+ ratio.
・Riboflavin mediates electron transfer inside and outside of S. mutans and inside and outside of biofilm in oral biofilm.
-NAD+ regeneration occurs by discharging electrons from the intracellular reducing environment to the extracellular oxidative environment, thereby promoting lactic acid fermentation by S. mutans.

Such electron transfer is a phenomenon that has already been confirmed in bacteria existing in nature including bacteria called current-producing bacteria, but the present inventors have confirmed that pathogenic bacteria present in the oral biofilm are It was demonstrated for the first time using S. mutans that it has an extracellular electron transfer mechanism and that a small amount of electron transfer is largely involved in maintaining the redox balance of the pathogenic bacterium.

Although there are reports that a Coulombic efficiency of more than 80% is obtained in bacteria such as Schwanella spp. and Diobacter spp., which are called current-producing bacteria, compared with those bacteria, S. mutans The coulombic efficiency 24 hours after the start of the electrochemical measurement in the experimental system using was estimated to be less than 0.05% from the total coulombic value and the amount of glucose consumed. These results indicate that the role of the extracellular electron transfer mechanism in S. mutans is not to drive the electron flow produced by metabolism as seen in naturally occurring bacteria, but rather from intracellular to extracellular. It is to assist the movement of electrons.

Considering the above findings comprehensively, in the bacterial community inside the oral biofilm, especially in the oral biofilm containing S. mutans, the homeostasis of the redox homeostasis (redox homeostasis) is maintained. It is strongly suggested that the electron transfer is deeply involved in. This means that S. mutans inherently has an extracellular electron transfer mechanism, that is, whether or not there is a substance (electron transfer substance) that mediates electron transfer as an external factor, as described below. , It is also supported by having a mechanism to transfer electrons from inside the cell to the outside of the cell.

As shown in the results obtained in the above experimental system, even when riboflavin as an electron acceptor was not added, a significant amount of current was generated with the improvement of lactic acid fermentation and the assimilation of ¹⁵ N. On the other hand, the production of ethanol was confirmed to a non-negligible level compared with the case of the presence of riboflavin (Fig. 3, Fig. 7(d)).

In addition, as shown in FIG. 3( a ), the tendency of change over time in current generation was similar in the presence and absence of riboflavin, and the glucose consumption rates were almost the same (FIG. 3( b )). ), production of acetic acid and formic acid was confirmed to a non-negligible level (FIGS. 4(b) and 4(c)). The reason why the amount of current generated in the absence of riboflavin is smaller than that in the presence of riboflavin is because riboflavin transfers electrons not only from bacteria attached to the electrode but also from plankton cells present in the experimental system. It is possible that

These results indicate that S. mutans transfers electrons to the electrode surface not only by electron transfer (shutling) via riboflavin but also by a direct electron transfer mechanism not through electron mediators such as riboflavin. Suggests that you have the ability. That is, bacteria such as S. mutans existing inside the oral biofilm have an electron transfer mechanism mediated by an electron transfer substance (for example, riboflavin (RF)) as schematically shown in FIG. 9(ii). It is considered to have an electron transfer mechanism mediated by a protein existing on the cell surface, as schematically shown in 9(i).

<Evaluation of direct electron transfer ability by pathogenic bacteria in oral biofilm>
Next, an experimental method for evaluating the direct electron transfer ability by the pathogenic bacteria in the oral biofilm as described above and the result thereof will be described.

To evaluate the electron transfer by environment-independent pathways in biofilms, we have developed an experimental system used to evaluate the electron transfer capacity of naturally occurring bacteria such as Schwanella bacteria and Diobacter bacteria. Using. Specifically, using an electrochemical cell having the same configuration as the electrochemical cell used in the above electrochemical analysis, the used culture medium was removed, and a sterile synthetic medium sparged with N ₂ (10 mM glucose was added). Was included), and the current density before and after the medium replacement was measured. The medium replacement degrees, the biofilm attached to the electrode, was rinsed twice with synthetic medium sparged with _{N 2 (N 2 -sparged DM)} . Further, while the medium was being replaced, the headspace in the cell was continuously sparged with N ₂ in order to avoid oxygen from entering the electrochemical cell. The spent culture medium was evaluated for the accumulation of soluble redox mediators in the medium. The culture filtrate was collected from around the biofilm, centrifuged to remove cell debris, placed in an electrochemical cell with the medium replaced, and sparged with N ₂ . The filter was not used in order to avoid the adsorption of organic molecules on the filter. The same differential pulse voltammetry (DPV) measurement as described above was performed to detect redox molecules.

FIG. 10A shows the measurement result of the current density. In FIG. 10( a ), the description “cells added” means that the cell suspension of S. mutans was injected at the time indicated by the arrow. The expression "exchange w/ fresh medium" means that the medium was replaced with a sterile synthetic medium containing 10 mM glucose at the time point indicated by the arrow. The description "exchange w/ spent medium" means that the used synthetic medium was replaced at the time indicated by the arrow.
As a result, even after the medium was exchanged, it was continuously confirmed that the electric current was generated at the same level as that before the exchange. That is, the change of the medium did not confirm the influence of S. mutans on the current generation, and the current generation by S. mutans was confirmed even when the medium did not contain plankton cells.

In addition, as a result of observing the ITO electrode after the electrochemical measurement with a scanning electron microscope (SEM), it was confirmed that S. mutans was directly attached to the surface of the electrode, as shown in FIG. The sample for SEM observation was prepared by the same procedure as the sample for secondary ion mass spectrometry, and the platinum-coated electrode sample was observed using a VE-9800 electron microscope (manufactured by Keyence). ..

The above results indicate that soluble redox mediators and hydrogen do not play a dominant role in current production by S. mutans.

In addition, from the results of the DPV analysis shown in FIG. 10(b), in the biofilm before replacement of the medium (Established biofilm) and the biofilm after replacement of the medium (biofilm-exchange w/ fresh DM), the redox peak was Detected at 20 mV and 250 mV against the hydrogen electrode (SHE), these peaks were not detected in spent synthetic medium (spent DM) and sterile synthetic medium (fresh DM). This indicates that the redox-active component is not affected by the replacement of the medium, and remains on the electrode surface as it is along with S. mutans.

FIG. 12A shows a constant potential condition using an ITO electrode (surface area: 3.14 cm ² ) in the presence of 10 mM glucose, with the potential difference of the ITO electrode being +0.4 V based on the standard hydrogen electrode (SHE). It is a graph which shows the time passage of the measured electric current generation value (microampere) by S. mutans. In FIG. 12( a ), the description “cells added” means that the cell suspension of S. mutans was injected at the time indicated by the arrow. Further, the arrows other than this indicate the timing of DPV measurement, indicating that the measurement was performed at intervals of 2 hours.
FIG. 12B shows a current generation value by S. mutans in contrast to the redox peak current value (μA) at a potential of about 20 mV with respect to the standard hydrogen electrode (SHE) in the DP voltammogram shown in FIG. 10B. 6 is a graph in which (μA) is plotted.
As shown in the graph inserted in the upper left of FIG. 12(b), except for the result 2 hours after the start of the electrochemical measurement, the phase was changed between the peak current value at 20 mV and the current generation value by S. mutans. A linear relationship is obtained with the square of the number of relations (R ² ) = 0.9489, and further, since the regression line shown in FIG. 12B passes through the origin, the redox molecule preferentially generates current. It has been shown that hydrogen and small amounts of riboflavin, which are mediated and contained in yeast extracts, are only slightly involved in this process.

Thus, due to the non-negligible effect of replacement of the electrolyte supernatant solution and the presence of redox molecules along with S. mutans, S. mutans mediated biomolecules present on the cell surface of the electrode surface. It was experimentally confirmed that the electrons are directly transferred to.

Furthermore, the existence of a direct electron transfer mechanism from the cell surface of S. mutans to the electrode surface is confirmed by the redox-dependent staining using 3,3′-diaminobenzidine (DAB), and It can also be confirmed by observation with a transmission electron microscope (TEM).

<Observation procedure using a transmission electron microscope (TEM)>
S. mutans cells were recovered by centrifuging 20 mL of cell culture at 7800 rpm for 10 minutes, and the recovered S. mutans cells were ice-cooled in a solution containing 2% paraformaldehyde and 2.5% glutaraldehyde. And fixed it. All operations after fixation were performed in a 2 mL Eppendorf tube. The cells were washed 5 times while being gently suspended in 1.5 mL of the washing solution, and centrifuged at 5000 xg for 4 minutes using 50 mM Na ⁺ -HEPES (pH 7.4, 35 g/L NaCl). Next, after treatment with 3,3′-diaminobenzidine (DAB) and hydrogen peroxide (H ₂ O ₂ ) and post-fixation with an osmium tetroxide (OsO ₄ ) solution, the cell membrane and peptidoglycan layer were stained, and then the resin encapsulation was performed. Buried. An 80 nm section was cut out from the obtained resin block, placed on a copper microgrid, and observed under an accelerating voltage condition of 80 kV using a JEM-1400 electron microscope (made by JEOL Ltd.). Quantitative analysis of electron beam absorption by the cell membrane of S. mutans was carried out by comparing the electron beam transmission (ET) amount of LR white resin, cytoplasm, and cell membrane region obtained from the LINE profile of DAB-positive stained cells. Deng, X., Dohmae, N., Nealson, KH, Hashimoto, K. & Okamoto, A. Multi-heme cytochromes provide a pathway for survival in energy-limited environments.Sci. Adv. 4, eaao5682 (2018) Please refer to.).

The TEM observation results are shown in FIG. 10(c), FIG. 10(d) and FIG.
In the positive control condition (DAB positive) in which DAB staining was performed in the presence of hydrogen peroxide, staining of the cell membrane and peptidoglycan layer was confirmed as shown in FIGS. 10(c) and 13(a). When hydrogen peroxide was not used as a negative control condition (DAB negative), little or no staining of the cell membrane or peptidoglycan layer was confirmed as shown in FIGS. 10(d) and 13(b). .. In FIG. 10(c), it can be confirmed that the redox activator is present on the cell surface or the peptidoglycan layer as indicated by the white arrow, but in FIG. 10(d), such a substance is present at the location indicated by the white arrow. Cannot be confirmed to exist. The scale bar in FIGS. 10C and 10D is 100 nm.

14A and 14B are TEM-LINE profile analysis of areas of the peptidoglycan layer that had been contrast-enhanced with DAB activators to normalize electron beam transmission and show previously reported current generation in nature. It is a result compared with the data regarding the extracellular membrane cytochrome c of the bacterium.
FIG. 14A is a result of DAB staining of S. mutans cells under a positive control condition (DAB positive), and FIG. 14B is a result of DAB staining of S. mutans cells under a negative control condition (DAB negatiev).
From the TEM images obtained under each condition, one cell was arbitrarily selected as shown in the upper left of FIGS. 14A and 14B, three sites were selected as analysis targets, and the LINE profile analysis of the electron beam transmission amount was performed. Regarding the LINE profile analysis results with reference numerals 1 to 3, the Y axis is the electron beam transmission amount, and the X axis is the length (nm) of the frame used for the analysis.
Each profiling result can be roughly divided into three areas. The region with the lowest value of electron beam transmission is the cell membrane as shown by the black arrow, and the regions on both sides of the part surrounded by the dotted line are LR white resin (resin) or inside the cell (cytoplasm). is there. White arrows in FIG. 14A indicate DAB activators localized on the cell surface or peptidoglycan layer. The normalized average value of the electron beam transmission amount obtained from the three profiling results shown in FIG. 14A is estimated to be 1.23, which is close to the value (1.88) reported for naturally occurring bacteria. The value was obtained.

These results suggest that the redox-active enzyme is localized on the surface of the peptidoglycan layer of S. mutans cells, which indicates that S. mutans has a direct extracellular electron transfer ability. Is consistent with being On the other hand, it has been reported that S. mutans lacks a gene encoding cytochrome c, which is an essential gene for the electron transfer mechanism in current-producing bacteria existing in nature. Therefore, in the above experimental system, it is possible that DAB was stained not at the heme center but at the transition metal ion, NADH or FAD cofactor. This suggests that in the direct electron transfer mechanism by S. mutans, there is an electron transfer mechanism involving a novel type of redox enzyme different from cytochrome c.

Thus, from the results of electrochemical analysis and transmission electron microscopy using S. mutans stained with a redox dye, direct cell mediated oxidoreductase binding to the cell surface of S. mutans was observed. It was confirmed that there is an external electron transfer mechanism.

The innate and direct extracellular electron transfer ability of S. mutans (not via electron transfer substances such as riboflavin) seems to enhance the rate of electron transfer. The new findings obtained also indicate that there is a wide range of alternative electron transfer mechanisms (ie, conductive matrices) in oral biofilms.

It is known that naturally occurring bacteria include bacteria that form a conductive biofilm along with the production of pili (for example, G. sulfurrenducens PCA). Further, it has been reported so far that a similar nanoscale structure exists in the pathogenic biofilm of the oral cavity, and it has been confirmed that it has conductivity by observation with an atomic force microscope. Given that these electrically conductive matrices are present in oral biofilms, S. mutans may be able to drain the excess reducing energy stored intracellularly into the oxidative environment outside the biofilm. To be This is also supported by the fact that the small amount of electric current (charge) confirmed in the above experimental example significantly affects metabolic activity. This is because even such low levels of electrical conduction should be sufficiently meaningful for the bacteria present in the biofilm.

Various electrochemical analysis results obtained using the above experimental system, bacteria existing inside the oral biofilm, the electron transfer mechanism to move electrons from the inside of the cell to the outside (excretion), In addition, electrons are transferred (exhausted) from the inside of the biofilm to the outside through the network between bacteria and electron mediators, which causes excessive reduction energy to be accumulated inside the bacteria and biofilm. Is eliminated and homeostasis of redox balance is maintained (homeostasis).

Thus, the extracellular electron transfer mechanism possessed by S. mutans can be confirmed as a minute current value in a given electrochemical system, and this small amount of electron transfer is anaerobic as in the oral biofilm. Since it is greatly involved in metabolic pathways and activities under conditions and greatly influences the homeostasis of redox balance during fermentation, it controls the extracellular electron transfer mechanism of pathogenic bacteria in oral biofilms. By doing so, it was specifically shown that the activity of the pathogenic bacterium can be reduced, and the onset and progression of oral diseases such as caries and periodontal disease can be suppressed.

Given that redox homeostasis and continuous NAD+ regeneration are universal challenges for a wide range of fermentative bacteria that form different types of biofilms, the phenomenon of excreting excess reducing energy (ie, microbial Survival strategies by) may exist everywhere. Since the utilization of electrons by microorganisms in the anaerobic respiratory chain is a matter of interest in the field of environmental microbiology, the novel findings by the present inventors elucidate the energy metabolism of fermentative bacteria in biofilms, and It should be the first step in controlling based on that understanding. In addition, since the extracellular electron transfer mechanism plays an important role in enhancing the activity of bacteria in biofilms, by identifying the enzyme that contributes to the extracellular electron transfer mechanism, it is possible to control oral and systemic diseases. It is expected that new approaches for new prevention and treatment will be possible. That is, the extracellular electron transfer mechanism can be a promising target for drug control or physicochemical control of pathogenic bacteria in the microbiome.

Although the embodiment of the present invention has been described in detail above by taking S. mutans as an example, the specific form is not limited to the above-mentioned embodiment, and the design within the scope not departing from the gist of the present invention The present invention includes modifications and the like.

The present invention provides novel methods and oral compositions that can contribute to the prevention and treatment of oral diseases by utilizing physicochemical techniques, which is completely different from conventional biochemical approaches. Further, according to the present invention, it is considered possible to suppress the onset and progression of systemic diseases other than oral diseases related to pathogenic bacteria in the oral biofilm. The present invention, like the conventional antibacterial agent, is not related to the problem of the permeability of the drug into the oral biofilm, the problem of the generation of new resistant bacteria, and the like. As described above, it is basically assumed that the application scene and effect of the drug are not limited, and various applications are expected.

Claims (12)

A method for reducing the activity of the pathogenic bacterium by controlling the extracellular electron transfer mechanism of the pathogenic bacterium existing inside the oral biofilm. The method according to claim 1, wherein the extracellular electron transfer mechanism of the pathogenic bacterium is controlled by suppressing electron transfer from the intracellular to the extracellular of the pathogenic bacterium. The method according to claim 2, wherein electron transfer from the inside to the outside of the pathogenic bacterium is suppressed by suppressing electron transfer from the inside to the outside of the oral biofilm. 3. The electron transfer from the intracellular to the extracellular of the pathogenic bacterium is suppressed by inhibiting the production of the electron mediator by the bacteria having the ability to generate the electron mediator existing inside the oral biofilm. Or the method described in 3. The method according to claim 2, wherein the electron transfer from the intracellular to the extracellular of the pathogenic bacterium is suppressed by inhibiting the activity of an electron transfer enzyme present in the extracellular membrane of the pathogenic bacterium. The method of claim 4, wherein the electron mediator is riboflavin. The method according to any one of claims 1 to 6, wherein the pathogenic bacterium is a bacterium that causes an oral disease. The method according to claim 7, wherein the pathogenic bacterium is a cariogenic bacterium or a periodontopathic bacterium. The method according to claim 8, wherein the pathogenic bacterium is Streptococcus mutans, Streptococcus sanguinis, Porphyromonas gingivalis, or Aggregatibacter actinomycetemcomitans. An oral composition comprising an inhibitor of the production of an electron mediator produced by a bacterium having the ability to produce an electron mediator, which is present inside an oral biofilm. The oral composition according to claim 10, wherein the electron transfer substance is riboflavin. An oral composition comprising an activity inhibitor against an electron transfer enzyme existing in the extracellular membrane of a pathogenic bacterium existing inside an oral biofilm.