Sterilization system and sterilization method

Abstract

A sterilization system for sterilizing a biofilm formed on an object, the sterilization system being provided with: a sterilization composition containing an organic compound having a standard oxidation-reduction potential (pH7) of-0.7 V to-0.2 V and water; a bactericidal composition supply unit for holding the bactericidal composition; a nozzle for supplying the bactericidal composition held by the bactericidal composition supply unit to the biofilm; an electrode provided on the nozzle so as to be in contact with the bactericidal composition; and a control unit which is connected to the electrode and which applies, to the electrode, a potential that is greater than the lower limit value of the potential window and that is-0.4 V or less based on the silver / silver chloride electrode.

Description

Technical Field

[0001] This invention relates to sterilization systems and sterilization methods. Background Technology

[0002] Currently, the management of infectious diseases in healthcare settings has become a global problem due to biofilms formed by bacterial colonies. One known method for killing bacteria is the use of chemical methods such as antibiotics. However, antibiotics pose a risk of antibiotic resistance due to improper use.

[0003] Therefore, sterilization techniques other than chemical methods have been studied for many years. For example, non-patent literature 1-4 reported methods for removing biofilms formed on the surface of conductors (electrical conductors) such as stainless steel and titanium by applying a high potential (e.g., around 7V) to the conductors and utilizing the generation of H2 and negative-negative surface repulsion.

[0004] Existing technical documents

[0005] Non-patent literature

[0006] Non-patent literature 1: Current Opinion in Solid State and Materials Science, Vol.25, Issue 4, August 2021, 100926.

[0007] Non-patent literature 2: Applied Sciences, 2022, Vol.12, Issue13, 6320.

[0008] Non-patent literature 3: Bioelectrochemistry 121 (2018) pp. 84-94.

[0009] Non-patent literature 4: Colloids and Surfaces B: Biointerfaces, Vol.117, 1, May 2014, p.152-157. Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] However, while the technologies disclosed in Non-Patent Literature 1-4 do not produce drug-resistant bacteria, they require implementation under harsh conditions involving electrolysis in solvents. Sterilization under such harsh conditions is costly due to the large energy consumption required, and sometimes, side reactions can produce reactive chemical species that have adverse effects on human health.

[0012] Therefore, the objective of this invention is to provide a sterilization system and method for sterilizing biofilms that can be carried out more efficiently under milder conditions.

[0013] Methods for solving problems

[0014] In order to achieve the above-mentioned problem, the inventors conducted in-depth research and found that the above-mentioned problem can be achieved through the following configuration.

[0015] [1] A sterilization system for sterilizing biofilms formed on an object, wherein the sterilization system comprises: The bactericidal composition comprises an organic compound with a standard oxidation-reduction potential (pH 7) of -0.7V to -0.2V and water; The sterilizing composition supply unit maintains the sterilizing composition. The nozzle supplies the bactericidal composition held by the bactericidal composition supply section to the biofilm; An electrode is disposed on the nozzle in such a manner as to contact the bactericidal composition; and The control unit is connected to the electrode and applies a potential to the electrode that is greater than the lower limit of the potential window and less than -0.4V based on the silver / silver chloride electrode.

[0016] [2] The sterilization system as described in [1], wherein the electrode is formed by at least a portion of the nozzle.

[0017] [3] The sterilization system as described in [1], wherein the electrode is composed entirely of the nozzle.

[0018] [4] The sterilization system as described in any one of [1] to [3], wherein the electrode has a ring shape.

[0019] [5] The sterilization system as described in any one of [1] to [4], wherein the electrode is a working electrode and further comprises a counter electrode and a reference electrode disposed in such a manner as to contact the sterilization composition.

[0020] [6] The sterilization system as described in [5], wherein the counter electrode and the reference electrode are disposed in the sterilization composition supply section.

[0021] [7] The sterilization system as described in any one of [1] to [6], wherein the organic compound contained in the sterilization composition is methyl violarin.

[0022] [8] The sterilization system as described in any one of [1] to [7], wherein the sterilization composition further comprises a cationic component.

[0023] [9] The sterilization system as described in any one of [1] to [8], wherein the sterilization composition further comprises silver ions.

[0024]

[10] The sterilization system as described in any one of [1] to [9], wherein the object is a medical device.

[0025]

[11] The sterilization system as described in any one of [1] to

[10] , wherein the object is a tubular body and the biofilm is formed on the inner surface of the tubular body.

[0026]

[12] The sterilization system as described in any one of [1] to

[11] , wherein the object is the insertion tube of an endoscope.

[0027]

[13] The sterilization system as described in any one of [1] to

[12] , wherein the control unit applies a potential of -1.2V to -0.8V to the electrode.

[0028]

[14] A sterilization method, which is a sterilization method for forming a biofilm on an object using any one of the sterilization systems described in [1] to

[13] , wherein the sterilization method includes: The bactericidal composition is brought into contact with an electrode to which a potential greater than the lower limit of the potential window and less than -0.4V based on a silver / silver chloride electrode is applied; and The bactericidal composition that has come into contact with the electrode is brought into contact with the biofilm.

[0029] The effects of the invention

[0030] This invention provides a sterilization system and method for sterilizing biofilms that can be carried out efficiently under mild conditions. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the sterilization system of this embodiment.

[0032] Figure 2 This is a schematic diagram illustrating the hypothetical sterilization mechanism in the sterilization system of this embodiment.

[0033] Figure 3 This is a schematic diagram illustrating the proposed sterilization mechanism in the sterilization system of this embodiment, and is an enlarged view of the vicinity of the bacteria.

[0034] Figure 4 This is a flowchart illustrating the sterilization method of this embodiment.

[0035] Figure 5 This is a diagram illustrating the polytetrafluoroethylene (PTFE) tube (the object on which a biofilm is formed) used in the embodiments.

[0036] Figure 6 This is a graph showing the evaluation results (number of colonies) of the bactericidal effect in the examples.

[0037] Figure 7 This is a graph showing the evaluation results (bacterial reduction) of the bactericidal effect in the examples.

[0038] Figure 8 This is a graph showing the evaluation results (number of colonies) of the bactericidal effect in the examples.

[0039] Figure 9 This is a graph showing the evaluation results (bacterial reduction) of the bactericidal effect in the examples.

[0040] Figure 10 This is a graph showing the current density of the electrodes in the embodiment. Detailed Implementation

[0041] The following description of the constituent elements is sometimes based on representative embodiments of the present invention, but the present invention is not limited to such embodiments.

[0042] It should be noted that, in this specification, the numerical range represented by “~” refers to the range of values ​​recorded before and after “~” as the lower and upper limits.

[0043] [Sterilization System]

[0044] Figure 1 This is a schematic diagram of the sterilization system 100 according to this embodiment. The sterilization system 100 of this embodiment is a system for sterilizing biofilms formed on the target object (sterilization target object) 6. Figure 1 As shown, the sterilization system 100 of this embodiment includes, for example, a sterilization composition 10 for sterilizing biofilm, a nozzle 20 for supplying the sterilization composition 10 to the biofilm, a sterilization composition supply unit 30 for maintaining the sterilization composition 10, an electrode 40 provided on the nozzle 20, and a control unit 70 connected to the electrode 40 and applying a potential to the electrode 40 that is greater than the lower limit of the potential window and less than -0.4V based on a silver / silver chloride electrode.

[0045] Bactericidal composition 10 is a composition for sterilizing biofilms. Bactericidal composition 10 comprises an organic compound (hereinafter, appropriately referred to as "specific organic compound") with a standard redox potential (pH 7) of -0.7V to -0.2V and water. Details of bactericidal composition 10 will be described later.

[0046] The disinfectant composition supply unit 30 includes a container 31 for holding the disinfectant composition 10. The disinfectant composition 10 is held (stored) in the container 31. The disinfectant composition 10 is supplied from the container 31 via a nozzle 20 to the biofilm formed on the object 6. Figure 1 The diagram illustrates the structure in which object 6 is connected to nozzle 20.

[0047] Figure 2 This is a cross-sectional view focusing on the portion where the nozzle 20 connects to the object 6 in this embodiment. (Example) Figure 2 As shown, nozzle 20 is a tubular structure for supplying the bactericidal composition 10 to the biofilm 2, through which the bactericidal composition 10 passes. The bactericidal composition 10, passing through the interior of nozzle 20, is then ejected from nozzle 20 and comes into contact with the biofilm 2. Nozzle 20 can also be described as an element for bringing the bactericidal composition 10 into contact with the biofilm 2.

[0048] In this embodiment, an example is shown where the object 6 is a tubular body, and a biofilm 2 is formed on the inner surface of the object 6. The tubular object 6 is, for example, the insertion tube of an endoscope. Figure 1 and Figure 2 The diagram illustrates a structure in which the nozzle 20 also functions as an electrode 40.

[0049] The inventors have surprisingly discovered that by applying a potential to the electrode 40, and bringing the bactericidal composition 10, which passes through the nozzle 20 provided with the electrode 40, into contact with the biofilm 2 formed on the object 6, the activity of bacteria (bacterial cells) 1 in the biofilm 2 can be reduced, ultimately achieving sterilization, thus completing the present invention. Furthermore, the negative potential applied to the electrode 40 only needs to be a weak potential within the potential window range. Therefore, electrolysis of the solvent (water) does not occur, and sterilization can be performed under mild conditions.

[0050] The mechanism of this invention is speculated as follows. Figure 3 This is a schematic diagram illustrating the mechanism hypothesized in the sterilization method using sterilization system 100. For example... Figure 3 As shown, during the process by which bacteria 1 obtains energy by breaking down organic compounds (especially under anaerobic conditions), NAD+ is used to break down organic compounds. + Oxidized nicotinamide adenine dinucleotide (NADH) produces reduced nicotinamide adenine dinucleotide (NADH). NADH releases electrons (arrow 3) via the electron transport system and returns to NAD. + It can then be used to obtain energy.

[0051] Figure 2 The potential applied to electrode 40 and the movement of electrons are schematically illustrated. For example... Figure 2As shown, when a negative potential is applied to the electrode 40 disposed in the nozzle 20 (arrow 8), a specific organic compound 5 receives electrons from the electrode 40 (nozzle 20) (arrow 9), and in a state of increased reducing power, passes through the nozzle 20, coming into contact with or approaching the biofilm 2. Then, as... Figure 3 As shown, the release of electrons from bacteria 1 (arrow 3) is restricted by the proximity or contact of a specific organic compound 5 with enhanced reducing properties. Specifically, by applying a potential below -0.4V (based on a silver / silver chloride electrode), electrons move from the specific organic compound 5 to bacteria 1 (arrow 4), hindering the cell's energy acquisition, further reducing its activity, and ultimately killing bacteria 1 (bactericidal). Thus, it is hypothesized that the specific organic compound 5 acts as an electron medium, maximizing the movement of electrons from electrode 40 to bacteria 1. Therefore, even if electrode 40 is not in contact with bacteria 1, electron movement from electrode 40 to bacteria 1 occurs via the specific organic compound 5, enabling highly efficient bactericidal action even with a weak applied potential. This principle of thermodynamically inhibiting the metabolism of bacteria 1 is a revolutionary principle that promises a sustained effect against drug-resistant bacteria latent in the biofilm 2. It should be noted that the mechanisms described above are speculative and do not affect the scope of the invention.

[0052] The following describes in detail each structure of the sterilization system 100 of this embodiment. Furthermore, in this specification, "sterilization" includes not only killing all bacteria in the biofilm, but also killing a portion of the bacteria in the biofilm (i.e., reducing the number of bacteria in the biofilm).

[0053] As described above, the bactericidal composition 10 contains a specific organic compound 5 with a standard oxidation-reduction potential (pH 7) of -0.7V to -0.2V and water.

[0054] The standard redox potential (pH 7) of the specific organic compound 5 is only required to be in the range of -0.7V to -0.2V, and there is no particular limitation. However, from the point of view of obtaining a higher bactericidal effect, it is preferred to be -0.6V to -0.3V or -0.6V to -0.4V.

[0055] The specific organic compound 5 is any organic compound whose standard redox potential is within the above-mentioned range, and is not particularly limited. For example, among compounds containing nitrogen-containing aromatic heterocycles, compounds having a dipyridine structure are preferred. Specific compounds include the compounds shown in the table below and their derivatives. From the viewpoint of obtaining higher bactericidal effects, methyl viologen and its derivatives are preferred, and methyl viologen is more preferred. The specific organic compound 5 may consist of a single compound or two or more compounds.

[0056] Table 1

[0057] The content of the specific organic compound 5 in the bactericidal composition 10 is not particularly limited, but from the viewpoint of obtaining a higher bactericidal effect, it may be, for example, 1 μM (M = mol / L) to 1000 μM or 10 μM to 600 μM.

[0058] The water contained in the bactericidal composition 10 is not particularly limited and can be pure water, distilled water, ion-exchanged water, etc.

[0059] In addition, the bactericidal composition 10 may also contain cations. By combining a specific organic compound 5 with cations, a synergistic effect is achieved, further enhancing the bactericidal effect. The mechanism is not yet certain, but is speculated as follows: In the bactericidal composition 10 containing the specific organic compound 5, applying a negative potential to electrode 40 promotes the movement of electrons towards bacteria 1. Figure 3 (Arrow 4). At this time, if the bactericidal composition 10 contains cations, the cations flow into the interior of the bacteria (bacterial cell) 1. As a result, an osmotic pressure anomaly is generated within the bacteria (bacterial cell) 1, reducing the activity of the bacteria 1 and ultimately achieving sterilization. In addition, if the cations have the property of damaging the DNA or enzymes within the bacteria 1, the bactericidal effect can be further improved. Furthermore, the mechanism described above is speculative and has no impact on the scope of the present invention.

[0060] There are no particular limitations on the cation; examples include metal ions, complex ions, and organic cations generated by pharmaceuticals. Examples of metal ions and metal-containing cations include silver (Ag), copper (Cu), cobalt (Co), aluminum (Al), nickel (Ni), zinc (Zn), molybdenum (Mo), vanadium (V), zirconium (Zr), tungsten (W), palladium (Pd), and platinum (Pt). From the viewpoint of further improving the bactericidal effect, silver ions (Ag) are preferred. ＋ ), copper ions (Cu) ＋ Cu 2+ ), more preferably silver (Ag) ＋ Additionally, cations can also be cations derived from so-called physiological electrolytes, such as sodium ions (Na+). ＋ ), potassium ions (K) ＋ ), calcium ions (Ca 2＋ ), magnesium ions (Mg 2＋ ), etc. As a cation, one type of cation or two or more cations can be used.

[0061] The bactericidal composition 10 may also contain anion as a counterion to the cation. The anion is not particularly limited; examples include halide ions such as fluorine, chloride, bromine, and iodine. The cation may also be an ion derived from a salt composed of a cation and anion. Preferred salts include, for example, metal halides such as silver chloride and copper chloride. By dispersing or dissolving (including partially dissolving) these salts in the bactericidal composition 10, cations can be introduced into the bactericidal composition 10.

[0062] The concentration of cations in the bactericidal composition 10 is not particularly limited and can be appropriately adjusted within the range that achieves the effect of this embodiment. From the viewpoint of obtaining a higher bactericidal effect, for example, the concentration of cations in the bactericidal composition can be 1 μM to 1000 μM or 10 μM to 200 μM.

[0063] The bactericidal composition 10 may consist solely of the specific organic compound 5 and water, or it may contain other components besides the specific organic compound 5 and water, within the scope of achieving the effects of this embodiment. Other components, besides the aforementioned cations, include, for example, electron mediators, electron source compounds, buffers, and coagulants other than the specific organic compound 5. Electron source compounds (electron donors) are compounds used for bacterial metabolism and are not particularly limited; examples include organic compounds (amino acids, sugars, and organic acids). Buffers are not particularly limited and include borates, bicarbonates, Tris-HCl, citrates, phosphates, succinates, phosphates, and acetates. Coagulants include agar, gelatin, and agar, with agar being preferred.

[0064] Common culture media (liquid culture media, solid culture media), buffer solutions, physiological saline, etc., contain water and the other components mentioned above. Therefore, the bactericidal composition 10 of this embodiment may also be a mixture of a specific organic compound 5 and a common culture medium, etc.

[0065] The bactericidal composition 10 can be prepared by uniformly mixing a specific organic compound 5, water, and other ingredients as needed using a common method. Alternatively, the bactericidal composition 10 can also be prepared by adding a specified amount of the specific organic compound 5 to common culture media, buffer solutions, physiological saline, etc. Commercially available products can also be used as culture media, buffer solutions, physiological saline, etc.

[0066] As described above, when the object to be sterilized 6 is a tubular body, and the biofilm 2 formed on the inner surface of the tubular body is sterilized, such as... Figure 1 and Figure 2As shown, a connector 21 (e.g., a silicone tube) can be used to connect the nozzle 20 and the object (tubular body) 6, allowing the bactericidal composition 10 to flow from the nozzle 20 into the interior of the object (tubular body) 6. The nozzle 20 and the object 6 are connected to each other using the connector 21, such that the interior (flow path) of the nozzle 20 is connected to the interior (flow path) of the object 6. However, the shape of the object 6 is not limited to a tubular body. Alternatively, for example, when the object 6 is a human or a tooth of an organism other than a human, and when bactericidalizing a biofilm formed within the pores of the tooth, the nozzle 20 can be inserted into the pores, allowing the bactericidal composition 10 to flow into the pores.

[0067] The material of the nozzle 20 is not particularly limited; metal materials and various plastic materials can be used. As a metal material, the same material used in the electrode 40 described later can be used, and the same method is preferred. When the nozzle 20 is made of metal, it is preferable that the entire nozzle 20 or a portion thereof can function as the electrode 40. Various plastics can be used, for example, polyvinyl chloride, polycarbonate, ABS resin, polypropylene, polyethylene, polystyrene, fluoropolymers, polyethylene terephthalate, methyl methacrylate, polyamide, polyethersulfone, polysulfone, polyurethane, ethylene-vinyl acetate copolymer resin, silicone resin, thermoplastic elastomer, liquid silicone rubber (LSR), etc. The nozzle 20 can be made of a single material or a composite of multiple materials.

[0068] The size (thickness) of the nozzle 20 is not particularly limited and can be appropriately designed according to the object 6 to be sterilized. For example, when the object 6 is the insertion tube of an endoscope, it is preferable that the diameter (outer diameter) of the nozzle 20 is not much different from the diameter of the insertion tube, for example, it can be about 1mm to 100mm.

[0069] Electrode 40 is disposed on nozzle 20 to contact the bactericidal composition 10 passing through nozzle 20. By disposing of electrode 40 on nozzle 20, electrode 40 can be brought close to the biofilm 2, which is the target of sterilization. As a result, more of the specific organic compound 5, which has increased its reducing power by accepting electrons from electrode 40, can be delivered to biofilm 2, further improving the sterilization effect.

[0070] like Figure 1 and Figure 2 As shown, electrode 40 can be formed from part or all of nozzle 20. If part or all of nozzle 20 is used as electrode 40, it becomes easier to enlarge electrode 40, increasing the contact area between electrode 40 and bactericidal composition 10. As a result, the amount (concentration) of the specific organic compound 5 that enhances reducing properties increases, thereby improving the bactericidal effect.

[0071] Alternatively, electrode 40 can be provided in nozzle 20 as a component different from nozzle 20. When electrode 40 is provided as a component different from nozzle 20, for example, a structure in which a tubular electrode 40 is provided at the front end of nozzle 20, a structure in which electrode 40 is provided inside nozzle 20, or a structure in which a linear electrode 40 extends from the inside of the tubular nozzle 20. Electrode 40 can be annular, allowing the bactericidal composition 10 to flow inside. In this case, the size (thickness, diameter) and length (length along the flow direction of bactericidal composition 10) of electrode 40 are not particularly limited and can be appropriately designed according to the object to be sterilized, such as the object 6. The size (thickness, diameter) of electrode 40 can be the same as that of nozzle 20 described above. The length of electrode 40 can be, for example, 0.1 cm to 30 cm, 0.5 cm to 10 cm, etc.

[0072] The material constituting electrode 40 can be any conductor capable of applying a potential, and there are no particular limitations. Examples of materials include metallic materials such as silver, copper, aluminum, nickel, iron, and alloys containing these metals (e.g., stainless steel (SUS)), as well as carbon materials such as amorphous carbon, graphite, and carbon nanotubes.

[0073] In this embodiment, electrode 40 is used as the working electrode in the three-electrode method, and it may also have two electrodes (counter electrode 50 and reference electrode 60). By using the three-electrode method, the potential applied to the electrode (working electrode) 40 can be controlled more accurately and easily. The counter electrode 50 and reference electrode 60 only need to be in contact with the bactericidal composition 10, and their placement is not particularly limited, but they are preferably placed in the bactericidal composition supply section 30. By placing the counter electrode 50 and reference electrode 60 in the bactericidal composition supply section 30, and only placing the electrode (working electrode) 40 in the nozzle 20, the nozzle 20 can be miniaturized compared to a structure in which the counter electrode 50 and reference electrode 60 are also placed in the nozzle 20 in addition to the electrode 40. However, a structure in which the counter electrode 50 and reference electrode 60 are also placed in the nozzle 20 in addition to the electrode 40 is also adopted. The material of the counter electrode 50 and reference electrode 60 is not particularly limited, and known counter electrodes and reference electrodes used for electrochemical measurements can be used. In addition, the reference electrode 60 is preferably a silver / silver chloride electrode.

[0074] The preferred current density of the current flowing in electrode 40 during sterilization, which is related to the sterilization reaction, is -10 μA / cm. 2 ~ -0.01μA / cm 2 More preferably -10 μA / cm 2 ~ -0.1μA / cm 2By keeping the current density in electrode 40 within the aforementioned range, sterilization can be performed with a small current value. Therefore, it has the advantages of suppressing side reactions on the electrode, minimizing damage to the object 6 during washing, and having minimal impact on tissues even when used in vivo.

[0075] The current flowing in electrode 40 during sterilization includes not only the current related to the sterilization reaction but also a system-specific background current unrelated to the sterilization reaction. The aforementioned "current density of the current related to the sterilization reaction" is obtained by subtracting the system-specific background current from the current measured at electrode 40 and dividing the result by the contact area between electrode 40 and sterilization composition 10. The method for calculating the "current density of the current related to the sterilization reaction" is not particularly limited; for example, it can be calculated using the method described in the examples.

[0076] like Figure 1 As shown, the container 31 of the sterilization composition supply unit 30 is configured to be connected to the nozzle 20 via, for example, a tube 32 and a connector 33, to supply the sterilization composition 10 to the nozzle 20. However, the container 31 and the nozzle 20 may also be directly connected. The materials of the container 31, tube 32, and connector 33 are not particularly limited and can be appropriately selected from common materials. In addition, as described above, the sterilization composition supply unit 30 may also include a counter electrode 50 and a reference electrode 60 as needed.

[0077] The sterilization composition 10 is supplied from the sterilization composition supply unit 30 to the nozzle 20, for example, via a liquid delivery mechanism (not shown). The sterilization system 100 of this embodiment may or may not include a liquid delivery mechanism as a component. If not included, a liquid delivery mechanism (liquid delivery device) separate from the sterilization system can be used. As such a liquid delivery mechanism, a general-purpose liquid delivery pump such as a tubular roller pump or a syringe pump can be used.

[0078] The sterilizing composition 10 supplied from the sterilizing composition supply unit 30 to the nozzle 20 further flows through the nozzle 20 to the object 6, contacting and sterilizing the biofilm 2. The sterilizing composition 10 after contact with the object 6 can be directly discarded (drained) or returned to the sterilizing composition supply unit 30. For example, when the object 6 is a tubular body such as the insertion tube of an endoscope, the outlet (the opening on the side opposite to the nozzle 20) of the object 6 (tubular body) can be directly or connected to the sterilizing composition supply unit 30 using a tube or the like. This allows the sterilizing composition 10 to circulate between the sterilizing composition supply unit 30, the nozzle 20 (electrode 40), and the object 6 (tubular body), enabling more efficient sterilization of the biofilm.

[0079] The control unit 70 is connected to the electrode 40, for example, via wiring 71, and applies a predetermined potential to the electrode 40. Specifically, the control unit 70 applies a potential to the electrode 40 that is greater than the lower limit of the potential window and less than -0.4V based on a silver / silver chloride electrode. The control unit 70 uses any known device capable of applying a potential. In the case of using the three-electrode method, the control unit 70 can be a potentiostat that is connected to and controls the three electrodes (electrode 40, counter electrode 50, and reference electrode 60) via wiring 71.

[0080] The inventors have discovered that, regardless of the type of bacteria, by using the bactericidal composition of this embodiment and applying a weak potential of -0.4V or less, electrons can be promoted to move from electrode 40 to bacteria 1 via a specific organic compound 5. Figure 2 and Figure 3 Arrow 4) hinders the cell's energy acquisition, ultimately killing bacteria. From the viewpoint of obtaining a higher sterilization effect, it is preferable to apply a potential of -0.6V or less, -0.8V or less, or -0.9V or less to electrode 40. In addition, the lower limit of the negative potential applied to electrode 40 is not particularly limited as long as it is greater than the lower limit of the potential window. The lower limit of the potential window is a value determined by the composition of the sterilization composition 10, pH, and the material of electrode 40, which is obvious to those skilled in the art. By applying a potential greater than the lower limit of the potential window, sterilization can be carried out under mild conditions where the solvent (water) does not undergo electrolysis. The sterilization method of this embodiment is low in cost because it can suppress energy consumption, and it can also suppress side reactions of active chemical species that may have adverse effects on the human body. Furthermore, if a high potential is applied and water electrolysis occurs, reactions such as hydrogen production are preferentially promoted, and electrons move from the sterilization composition 10 to the bacteria 1 ( Figure 2 and Figure 3 Arrow 4) can sometimes be suppressed. In this embodiment, since the applied potential is weak, no hydrogen or the like is generated, and the metabolism of bacteria 1 can be effectively inhibited to kill bacteria. The lower limit of the potential, for example, based on a silver / silver chloride electrode, is preferably -1.4V or higher, -1.2V or higher, or -1.1V or higher.

[0081] [Target for sterilization]

[0082] The object 6, which is the target of sterilization, is not particularly limited. For example, medical devices can be included. Examples of medical devices include those used through the mucous membranes of an organism or in direct contact (e.g., forceps and endoscopes), or those implanted in an organism (e.g., dental implants and various other implants). These medical devices sometimes generate biofilms in minute areas that are not thoroughly cleaned mechanically. Bacteria in such biofilms can be killed (sterilized) by the sterilization method of this embodiment, and the effect of preventing bacterial infection is expected. In particular, the sterilization system 100 of this embodiment allows the sterilization composition 10 to flow from the tip of the nozzle 20 into the tubular body that is the object 6, enabling efficient sterilization of biofilms formed on the inner surface (inner wall) of the tubular body (e.g., the insertion tube of an endoscope). Alternatively, the object 6 can also be, for example, the teeth of a human or an organism other than a human. When sterilizing the biofilm formed in the pores of teeth, the nozzle 20 is inserted into the pores, allowing the sterilizing composition 10 to flow into the pores, thus achieving efficient sterilization.

[0083] If the object 6 is not a tubular body (e.g., forceps or implant), sterilization can be performed, for example, by spraying the sterilizing composition 10 onto the object 6 using the nozzle 20. It should be noted that if the object 6 is inside the body (e.g., an implant), the sterilization system 100 preferably has a discharge section for inhaling and expelling the used sterilizing composition 10 sprayed onto the object 6.

[0084] Biofilm 2 is a higher-order structure formed by bacteria 1 attached to the surface of the solid phase (object 6), for example, covered by polysaccharides produced by bacteria 1. The bacteria 1 contained in the biofilm 2, i.e., the bacteria 1 targeted for sterilization in this embodiment, are not particularly limited and can be any type of Gram-positive or Gram-negative bacteria. For example, as bacteria that significantly impact endoscope contamination, the sterilization method of this embodiment is also effective against Klebsiella pneumoniae (Gram-negative bacteria), Pseudomonas aeruginosa (Gram-negative bacteria), and Staphylococcus epidermidis (Gram-positive bacteria) as defined by the U.S. Food and Drug Administration (FDA).

[0085] Furthermore, regarding the sterilization method of this embodiment, if the bacteria contained in the biofilm are Gram-negative bacilli, especially Gram-negative bacilli belonging to the Enterobacteriaceae family, then electron movement ( Figure 2 and Figure 3 Arrow 4) indicates greater efficiency and superior sterilization effect. Examples of bacteria belonging to the Enterobacteriaceae family include, for example, Klebsiella pneumoniae, Enterobacter, Escherichia, Salmonella, Serratia, Shigella, and Yersinia.

[0086] [Sterilization method]

[0087] For use Figure 1 The method for sterilizing the biofilm in the sterilization system 100 shown will be described. The sterilization method of this embodiment includes, for example, the following steps ( Figure 4 ).

[0088] Step S1: Pass the sterilization composition 10 through the nozzle 20 (electrode 40).

[0089] Step S2: The electrode 40, which has been subjected to a potential greater than the lower limit of the potential window and less than -0.4V based on the silver / silver chloride electrode, is brought into contact with the bactericidal composition 10.

[0090] Step S3: The bactericidal composition 10 passing through the nozzle 20 (i.e., in contact with the electrode 40) comes into contact with the biofilm 2.

[0091] Step S1: The sterilization composition 10 passes through nozzle 20

[0092] The sterilizing composition 10 is passed through the nozzle 20, for example, using a liquid delivery mechanism (not shown). The speed at which the sterilizing composition 10 passes through the nozzle 20 (liquid delivery speed) is not particularly limited, and for example, it can be set to 0.1 mL / min to 10 mL / min.

[0093] Step S2: Bring the sterilizing composition 10 into contact with the electrode 40 to which a potential has been applied.

[0094] A potential is applied to the electrode 40 via the control unit 70. In the sterilization system 100 of this embodiment, a three-electrode method is preferably used, and the control unit 70 is preferably a potentiostat. The magnitude of the applied potential is as described above. The application of the potential can be continuous or intermittent, but from the viewpoint of efficient sterilization, continuous application is preferred. The specific organic compound 5 in the sterilization composition 10 receives electrons from the electrode 40 when passing through the nozzle 20, thereby increasing its reducing power.

[0095] It should be noted that, in Figure 1 In the structure (where the nozzle 20 also functions as an electrode 40), the bactericidal composition 10 passes through the nozzle 20 and comes into contact with the electrode 40 to which a potential is applied. That is, steps S1 and S2 are performed in parallel on the time axis. On the other hand, for example, in a structure where an electrode 40 different from the nozzle 20 is provided at the front end of the nozzle 20, step S2 is performed after step S1. As can be understood from the above description, the order of steps S1 and S2 is not particularly limited.

[0096] Step S3: Contact between the sterilization composition 10 and the biofilm 2

[0097] The method for contacting the bactericidal composition 10 (i.e., the bactericidal composition 10 containing a specific organic compound 5 with enhanced reducing properties) with the biofilm 2 is not particularly limited. For example, when sterilizing the biofilm formed on the inner surface of the tubular body, such as... Figure 1 and Figure 2 As shown, the nozzle 20 is connected to the target object (tubular body) 6, allowing the bactericidal composition 10 to flow from the nozzle 20 into the interior of the target object (tubular body) 6. Alternatively, for example, when sterilizing a biofilm formed within pores, the nozzle 20 can be inserted into the pores, allowing the bactericidal composition 10 to flow into the pores. Alternatively, the bactericidal composition 10 can be sprayed onto the biofilm from the nozzle 20. The bactericidal composition 10 passing through the nozzle 20 contains a specific organic compound 5 with enhanced reducing properties. By bringing it into contact with or close to the biofilm 2, bacteria 1 in the biofilm 2 can be sterilized.

[0098] The contact time between the bactericidal composition 10 and the biofilm 2 (sterilization time) is not particularly limited and can be adjusted appropriately according to the type of specific organic compound, the type of bacteria, the liquid delivery rate, etc. For example, the sterilization time can be 30 minutes to 24 hours. Furthermore, the temperature of the bactericidal composition 10 in contact with the biofilm 2 (sterilization temperature) is not particularly limited. For example, the sterilization temperature can be 0°C to 100°C or room temperature.

[0099] The contact between the bactericidal composition 10 and the biofilm 2 can be carried out under anaerobic conditions (e.g., a nitrogen environment) or under aerobic conditions (e.g., in the atmosphere). Since the environment is not limited to anaerobic conditions, the bactericidal method of this embodiment can be implemented with simpler equipment.

[0100] According to the above description of the sterilization system 100 of this embodiment and the sterilization method using the sterilization system 100, by applying a weak potential to the electrode 40, the activity of bacteria 1 in the biofilm 2 can be reduced, ultimately killing all or part of the bacteria in the biofilm 2 (sterilization). Therefore, a sustained effect against drug-resistant bacteria latent in the biofilm 2 can be expected. In addition, in the sterilization system 100, by placing the electrode 40 at the nozzle 20, a specific organic compound 5 with enhanced reducing properties can be easily delivered to the biofilm 2, further improving the sterilization effect. By using the sterilization system 100 of this embodiment, antibacterial agents are not required, or the amount of antibacterial agents used can be reduced, thus suppressing the emergence of new drug-resistant bacteria.

[0101] Example

[0102] The present invention will be described in more detail below based on embodiments. The materials, amounts, proportions, processing contents, processing steps, etc., shown in the following embodiments can be appropriately modified without departing from the spirit of the invention. Therefore, the scope of the present invention should not be interpreted as limited by the embodiments shown below.

[0103] [Experiment 1]

[0104] In this experiment, the following was used Figure 1 The sterilization system 100 shown sterilizes *Pseudomonas aeruginosa* (PA) in a biofilm formed within a PTFE tube (object 6). In this experiment, a three-electrode method was used, where a potentiostat (control unit 70) controls the working electrode 40, the counter electrode 50, and the reference electrode 60. The nozzle 20 is entirely composed of electrode 40 (SUS tube, diameter: 0.41 cm, length: 1 cm). Methyl viologen (MV) was used as the specific organic compound.

[0105] (1) Formation of biofilms

[0106] First, prepare 15 mL of LB (Luria-Bertani) medium in a 50 mL tube, add frozen PA bacteria (-80℃) sample, and incubate overnight at 37℃ with shaking under aerobic conditions. Then, autoclave the PTFE tubes (diameter: 0.5 cm, length: 20 cm) at 120℃ for 20 minutes. Fill the sterilized PTFE tubes with cultured PA bacteria (OD). 600 A TSB (tryptic soy broth) medium solution of 0.01 g / L was used, and both ends of the tube were sealed with stoppers. The tube was then incubated at 37°C for 24 hours. As a result, a biofilm formed on the inner surface (inner wall) of the PTFE tube.

[0107] (2) Preparation of bactericidal composition

[0108] Add methyl viologen (MV) to DM (defined medium, liquid medium) to a concentration of 500 μM and mix thoroughly. It should be noted that DM medium uses an electrolyte solution containing the following electrolytes: CaCl2. 2H2O: 0.08g / L; NH4Cl: 1.0g / L; NaHCO3: 2.5g / L; NaCl: 10.0g / L; MgCl2 6H2O: 0.2 g / L; and HEPES (4-hydroxyethylpiperazine ethanesulfonic acid): 7.2 g / L.

[0109] (3) Sterilization of biofilms

[0110] A platinum (Pt) wire is used as the counter electrode 50, and a silver / silver chloride electrode is used as the reference electrode 60. The working electrode 40 (nozzle 20) is electrically connected to a potentiostat (control unit 70). The prepared bactericidal composition 10 is injected into the bactericidal composition supply unit 30 (glass beaker), and the counter electrode 50 and the reference electrode 60 are positioned in contact with the bactericidal composition 10. The bactericidal composition supply unit 30 is connected to the nozzle 20 (SUS tube) using a PTFE tube 32 and a connector 33 (silicon tube). The nozzle 20 (SUS tube) is connected to one end of a PTFE tube (object 6) on which the biofilm 2 is formed using a connector 21 (silicon tube). Furthermore, a drain pipe (not shown) is connected to the other end of the PTFE tube (object 6). As a liquid delivery device, an inlet pump (not shown) is connected to the bactericidal composition supply unit 30, and a tubular roller pump (not shown) is connected to the drain pipe (not shown).

[0111] The liquid delivery device (injection pump and tubing pump) was used to allow the disinfectant composition 10 to flow sequentially from the disinfectant composition supply section 30 through the nozzle 20, the PTFE tubing (object 6), and the drain pipe (not shown). The liquid delivery rate was set to 2 mL / min. A potential of -1.0V (with Ag / AgCl as the reference electrode) was applied to the working electrode 40. The disinfectant composition was allowed to flow for 1 hour under the applied potential. It should be noted that this experiment was conducted under aerobic conditions (atmospheric).

[0112] [Evaluation of bactericidal effect]

[0113] Cut out from the PTFE tubing (object 6) after the experiment. Figure 5 The diagram shows three parts: A (1cm), B (1cm), and C (1cm). Part A is the portion 2.5cm to 3.5cm from the upstream end (one end) of the PTFE tube, and Part C is the portion 2.5cm to 3.5cm from the downstream end (the other end) of the PTFE tube. Part B is approximately the central part of the PTFE tube.

[0114] Parts A, B, and C were treated as follows: First, each part was washed three times with PBS (pH = 7.2). After washing, each part was placed in a container, and 1 mL of PBS was added. The mixture was then subjected to strong vortexing (30 seconds), sonication (7 minutes), and a second strong vortexing (30 seconds) to detach the biofilm from each part. The PBS containing the detached biofilm was diluted to 10. 7To prepare a sample, 10 μL of diluted PBS containing the biofilm was spread onto a TSB-agar plate and incubated overnight at 37°C. After incubation, the number of colonies formed on the sample was counted, and the colony count (Colony Forming Units (CFU / mL)) was determined. This experiment was repeated three times, and the average value was calculated. The results are shown below. Figure 6 As shown.

[0115] For comparison, as a control, the bacterial colony counts were also calculated for PTFE tubes that had formed biofilm 2 but were not subsequently sterilized, in parts A, B, and C. The results are presented together. Figure 6 .

[0116] In addition, to make the experimental results clearer, Figure 7 This represents the individual bacterial reduction amounts (log) in parts A, B, and C. 10 (Bacterial reduction). Bacterial reduction is from... Figure 6 The colony counts (log) of the control group (before sterilization treatment) shown for each part are as follows. 10 Subtract the number of colonies in each part (log) 10 The difference obtained is...

[0117] like Figure 6 and Figure 7 As shown, in all parts of sections A, B, and C, the number of bacterial colonies decreased after sterilization treatment compared to the control (before sterilization treatment), confirming the effectiveness of the sterilization method using sterilization system 100.

[0118] It should be noted that, in Figure 7 In comparison to sections A and C (near both ends of the PTFE tubing), the bacterial reduction in section B (the center of the PTFE tubing) was slightly lower. This is because biofilm formation is difficult in the center of the PTFE tubing (object 6). Therefore, this result does not imply a bias in bactericidal effect due to the location of the PTFE tubing. On the other hand, as... Figure 7 As shown, the same level of sterilization effect is obtained at both ends of the PTFE tube (part A near the upstream end and part C near the downstream end). This indicates that the same effect can be obtained even at a distance, demonstrating the practicality of the invention.

[0119] [Experiment 2]

[0120] Except that the length of electrode 40 (nozzle 20, SUS tube) was set to 5 cm, the sterilization experiment was conducted under the same conditions as in Experiment 1, and the evaluation was performed in the same manner. The results are as follows: Figure 8 and Figure 9 As shown. Figure 8 and Figure 9 As shown, Experiment 2 also achieved the same bactericidal effect as Experiment 1.

[0121] [Calculation of current density]

[0122] As electrodes, the current values ​​in the 5cm and 10cm SUS tubes used in Experiment 2 were measured, and the current density was calculated in the following order based on the results.

[0123] First, the current flowing through a 5cm SUS tube was measured using a potentiostat (control unit 70). Additionally, the sterilization experiment was conducted under the same conditions as in Experiment 2, except that the length of electrode 40 (nozzle 20, SUS tube) was set to 10cm, and the current was measured similarly for a 10cm SUS tube. Since the background current specific to the system is independent of the electrode size, it was approximately the same in both the 5cm and 10cm SUS tubes.

[0124] On the other hand, the magnitude of the current associated with the sterilization reaction depends on the size of the electrode (i.e., the length of the SUS tube). Therefore, the current associated with the sterilization reaction flowing through the 5cm SUS tube is obtained by subtracting the current value of the 5cm SUS tube from the current value of the 10cm SUS tube (length difference: 5cm). Dividing this by the area of ​​the inner side of the 5cm SUS tube (the contact area between the electrode and the sterilization composition) yields the current density. The results are shown in... Figure 10 .like Figure 10 As shown, the current density is approximately -0.7 μA / cm². 2 .

[0125] Industrial applicability

[0126] The sterilization method of the present invention can be used for washing and cleaning to prevent infections caused by medical devices or medical implants.

[0127] Explanation of reference numerals in the attached figures

[0128] 1: Bacterial cells (bacteria).

[0129] 2: Biomembrane.

[0130] 5: Specific organic compounds.

[0131] 6: Object.

[0132] 10: Bactericidal composition.

[0133] 20: Nozzle.

[0134] 21: Connector.

[0135] 30: Sterilization composition supply unit.

[0136] 31: Container.

[0137] 32: tube.

[0138] 33: Connector.

[0139] 40: Electrode (working electrode).

[0140] 50: Counter electrode.

[0141] 60: Reference electrode.

[0142] 70: Control Department.

[0143] 71: Wiring.

[0144] 100: Sterilization system.

Claims

1. A sterilization system for sterilizing a biofilm formed on an object, wherein The sterilization system includes: a sterilization composition including an organic compound having a standard oxidation-reduction potential of -0.7 V to -0.2 V at pH 7 and water; a sterilization composition supply section that holds the sterilization composition; a nozzle that supplies the sterilization composition held by the sterilization composition supply section to the biofilm; an electrode disposed in contact with the sterilization composition in the nozzle; and a control section connected to the electrode and applying a potential of -0.4 V or less on a silver / silver chloride electrode basis that is greater than a lower limit value of a potential window to the electrode. The electrode is composed of at least a part of the nozzle.

2. The germicidal system of claim 1, wherein, The electrode is composed of the entire nozzle.

3. The germicidal system of claim 1, wherein, The electrode has a ring shape.

4. The germicidal system of any of claims 1-3, wherein, 5. The sterilization system according to any one of claims 1 to 4, wherein the electrode is a working electrode, the sterilization system further includes a counter electrode and a reference electrode disposed in contact with the sterilization composition. The counter electrode and the reference electrode are disposed in the sterilization composition supply section.

6. The germicidal system of claim 5, wherein, The organic compound contained in the sterilization composition is methyl viologen.

7. The germicidal system of any of claims 1-6, wherein, The sterilization composition further includes a cation.

8. The germicidal system of any of claims 1-7, wherein, The sterilization composition further includes silver ions.

9. The germicidal system of any of claims 1-8, wherein, The object is a medical instrument.

10. The germicidal system of any of claims 1-9, wherein, The object is a tubular body, and the biofilm is formed on an inner surface of the tubular body.

11. The germicidal system of any of claims 1-10, wherein, The object is an insertion tube of an endoscope.

12. The germicidal system of any of claims 1-11, wherein, The control section applies a potential of -1.2 V to -0.8 V to the electrode.

13. The germicidal system of any of claims 1-12, wherein, The sterilization method includes:

14. A sterilization method of a biofilm formed on an object using the sterilization system according to any one of claims 1 to 13, wherein contacting the sterilization composition with the electrode to which a potential of -0.4 V or less on a silver / silver chloride electrode basis that is greater than a lower limit value of a potential window is applied; and contacting the biofilm with the sterilization composition contacted with the electrode. ​

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