JP2013094468A - Device and method of killing microorganisms by atmospheric pressure plasma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a device and a method of killing microorganisms at a high killing speed and inexpensively.SOLUTION: A microorganism killing device including an atmospheric pressure plasma generation means for introducing plasma into a container is provided, wherein the atmospheric pressure plasma generation means generates the plasma using a gas whose COcontent is 5% or higher as a process gas.

Description

  The present invention relates to an apparatus and method for killing by atmospheric pressure plasma.

  Conventionally, treatment to sterilize or sterilize microorganisms (fungi, fungi, viruses, etc.) present on the surface of packaging materials and molded products in medicine, medical equipment and food manufacturing processes, from ensuring medical safety and preventing food corruption. Has been done.

  Known techniques for sterilizing or sterilizing microorganisms include high-pressure steam sterilization (autoclave), ethylene oxide gas (EOG) sterilization, electron beam sterilization, and gamma ray sterilization.

  However, the high-pressure steam sterilization method requires high-temperature treatment at 121 ° C. or higher, so that it is difficult to treat plastic products that are vulnerable to heat. Further, the ethylene oxide gas (EOG) sterilization method has been pointed out to be toxic and carcinogenic to the human body, and there is a problem in the safety problem for the user. And the electron beam sterilization method and the gamma ray sterilization method have the subject that the apparatus to be used is very large and expensive.

  In recent years, as a means for solving such a problem, a sterilization method using plasma such as low-pressure discharge (Non-Patent Document 1) has been proposed. The plasma sterilization method is attracting attention as a new sterilization method because it can be sterilized at low temperatures without using harmful substances.

  The term “sterilization” refers to the act of reducing the initial number of bacteria of a specific microorganism, and the term “sterilization” refers to the act of achieving sterility, that is, removing all microorganisms. In the present specification, the act itself of reducing the number of bacteria is referred to as “killing”.

Masaaki Nagatsu: “3. Plasma Sterilization”, J. Am. Plasma Fusion Res. Vol. 83, no. 7, pp. 601-606 (2007)

  However, the conventional method of killing microorganisms using plasma has a drawback that the killing rate of microorganisms is slow because the density of active oxygen species in the plasma is low. In addition, the microorganism killing method using low-pressure discharge plasma has a drawback in that equipment such as a vacuum vessel and a vacuum exhaust pump is required to perform a decompression process, resulting in an increase in apparatus cost. Therefore, there is a need for a microorganism killing apparatus and method that has a high killing speed and low cost.

Therefore, in the present invention, carbonic acid (CO ₂ ) gas is used as the process gas supplied to the plasma generating means in the microorganism killing method and apparatus using atmospheric pressure plasma that does not require equipment for pressure reduction treatment. Accordingly, it is an object of the present invention to provide a microorganism killing apparatus and method having a high killing speed and low cost.

A first aspect of the present invention is a microorganism killing device provided with an atmospheric pressure plasma generating means for introducing plasma into a container, wherein the atmospheric pressure plasma generating means has a CO ₂ content of 5% or more. A microorganism killing device configured to generate plasma using a gas as a process gas.

According to a second aspect of the present invention, in the first aspect, gas replacement means for replacing the atmosphere in the container with a process gas containing CO ₂ is provided.

According to a third aspect of the present invention, there is provided a method for killing microorganisms by atmospheric pressure plasma generation means for introducing plasma into a container, wherein the atmospheric pressure plasma generation means contains a gas having a CO ₂ content of 5% or more. A method for killing microorganisms comprising the step of generating plasma as a process gas.

According to a fourth aspect of the present invention, in the third aspect, the atmosphere in the container is further replaced with a process gas containing CO ₂ .

1 is a schematic diagram of plasma irradiation processing in Example 1. FIG. 6 is a schematic diagram of plasma irradiation processing of Example 3. FIG. It is a figure which shows the result of the bactericidal characteristic of Example 1. FIG. It is a figure which shows the result of the bactericidal characteristic of Example 2. It is a figure which shows the result of the bactericidal characteristic of the comparative example 1. It is a figure which shows the result of the bactericidal characteristic of Example 3. It is a figure which shows the result of the bactericidal characteristic of the comparative example 2. It is a figure which shows the result of the bactericidal characteristic of Example 4. It is a figure which shows the result of the density | concentration measurement of Example 5. FIG. It is a figure which shows the result of the bactericidal characteristic of Example 6.

  In the present invention, a plasma jet is generated by using a nozzle type plasma generation device as an atmospheric pressure plasma generation means. This generating apparatus has an ejection hole having a diameter of about 1 mm on the sealed end surface of a cylindrical metal tube, supplies a process gas into the metal tube, and supplies a predetermined voltage and power from the power supply line between the internal electrodes. Is generated to generate plasma, and a plasma jet is ejected from the ejection hole. In addition, in this invention, the plasma jet was produced | generated using the well-known plasma concept Tokyo make damage free multi-gas plasma jet (trademark).

As the process gas, carbon dioxide (CO ₂ ) gas may be supplied at a content rate of 100%, or it may be added to a carrier gas such as argon (Ar) or nitrogen (N ₂ ). The carbonic acid (CO ₂ ) gas content contained in the process gas can be selected while observing the effect of the above. As the carbon dioxide (CO ₂ ) gas, an industrial grade (purity 99.9%) cylinder gas can also be used.

The plasma generated under the above conditions includes active oxygen such as atomic oxygen (O.) dissociated from CO ₂ , excited singlet oxygen molecules ( ¹ O ₂ ) and ozone (O ₃ ) as reaction products. And a large amount of highly reactive gas such as carbon monoxide (CO). Here, active oxygen is literally a general term for activated oxygen species and has a very high oxidizing power, and it is a reaction process of atoms / molecules and electron collisions in the gas phase described in known literatures. considering, the atmospheric pressure plasma jet, in addition to these, hydroxy radical (OH ·) generated from atmospheric moisture (H 2 _O), superoxide anion radicals (O 2 _- ^·) contains etc. It is thought that it is acting on the object to be processed.

  From the electron micrograph of the microorganism treated with the plasma jet of the present invention, it is presumed that the active oxygen species are chemically oxidized and etched on the surface of the microorganism to kill (inactivate) the microorganism. In particular, it is expected that active oxygen species in plasma in a vessel in which microorganisms are present is generated at a very high density, and data suggesting the presence of these active species is obtained from measurements by emission spectroscopy. And is considered to be the dominant killing factor.

In the present invention, the effect of killing can be further enhanced by replacing the atmosphere of the processing space with the process gas atmosphere from the atmosphere (air) in advance.
For example, when processing the inner wall surface of a plastic bottle in an in-line killing process, an effective bottle is prepared by providing a process gas filling step inside the bottle in advance, and then processing with an atmospheric pressure plasma jet. Internal microbe killing process can be realized. At this time, the plasma jet may not directly contact the inside of the bottle. For example, even if a plasma jet is generated in a place with a distance of about 5 mm, the killing process is possible.

The reason why the processing efficiency is improved when the atmosphere of the processing space is replaced with a process gas using carbon dioxide (CO ₂ ) gas is that oxygen (O ₂ ) and moisture contained in the air in the case of processing in normal air (H ₂ O) reacts with active oxygen species released from the plasma jet, particularly atomic oxygen (O.), in the gas phase. On the other hand, since atomic oxygen (O.) generated in plasma hardly reacts with the process gas CO ₂ to form tetraatomic molecules (CO ₃ ), compared with the case where other process gases are used. Thus, it is presumed that the lifetime of atomic oxygen (O.) in the gas phase is prolonged and it is easy to reach the workpiece.

Examples of the object to be treated on the surface of which microorganisms are present include, for example, inorganic materials such as glass and ceramics, and molded articles of plastics such as polyethylene, polyethylene terephthalate, polycarbonate, polypropylene, polyimide, polytetrafluoroethylene, and polyacetal. Although it is mentioned, it is not limited to the solid surface of these materials, For example, it is applicable also to the killing process of the microorganisms which exist in a liquid or a liquid surface.
When killing the microorganisms present on the liquid surface, the gas component is infiltrated into the liquid by blowing the process gas on the liquid surface in advance, so that the killing effect can be further enhanced. Furthermore, by providing a mechanism for convection of the liquid, it is possible to carry out the treatment by sending microorganisms present at the bottom to the vicinity of the surface.

  Hereinafter, examples and comparative examples of the present invention will be described with reference to the drawings. The examples and comparative examples described below are the most preferred examples of the present invention, and various modifications can be made within the scope of the gist of the present invention.

Example 1.
In Example 1, a nozzle type plasma generation apparatus 1 as shown in FIG. 1 was used as the atmospheric pressure plasma generation means. In addition, the plasma jet was produced | generated using the plasma concept Tokyo make damage free multi-gas plasma jet (trademark). In order to generate atmospheric pressure plasma, carbon dioxide (CO ₂ ) gas was supplied as a process gas between the electrodes inside the cylindrical metal tube of the plasma generation apparatus 1 at a flow rate of 5 liters / minute (hereinafter referred to as LM). . Further, a cover 4 for replacing the process gas is provided around the area where the atmospheric pressure plasma is generated, and the state in which the process gas is circulated from the ejection hole of the plasma generating apparatus is maintained for 30 seconds, and the processing atmosphere is changed to the process gas (CO ₂ ).

As shown in FIG. 1, a membrane filter 3 spotted in an area of about 105 Bacillus subtilis spores and a diameter of about ⁵ mm is located at a position 10 mm below the plasma ejection hole 2, and the central axis of the ejection hole 2. It was placed above and irradiated with a plasma jet for a predetermined time.

  After the plasma jet irradiation treatment, the membrane filter 3 on which the bacteria are spotted is put into sterilized water, the bacteria are sufficiently eluted, serially diluted with sterilized water, smeared on an agar medium, and then in an incubator at 36 ° C. for 24 hours. After culturing, the number of colonies formed on the agar medium was counted, and the bactericidal properties were evaluated.

Example 2
In Example 2, argon (Ar) gas and carbonic acid (CO ₂ ) gas as a process gas are mixed at a total flow rate of 5 LM and a flow rate ratio of 9: 1 between the electrodes inside the cylindrical metal tube of the plasma generator 1. Except supplying, it processed by the structure and conditions similar to Example 1, and evaluated bactericidal characteristics.

Comparative Example 1
In Comparative Example 1, treatment is performed under the same configuration and conditions as in Example 1 except that air (AIR) gas is supplied as a process gas at a flow rate of 5 LM between the electrodes inside the cylindrical metal tube of the plasma generation apparatus 1. The bactericidal properties were evaluated.

Example 3
In the third embodiment, the same plasma generation apparatus 11 as in the first embodiment is used, and a process gas replacement cover 14 is provided around the atmosphere where atmospheric pressure plasma is generated, and the gas flow state is maintained for 30 seconds. The treatment atmosphere was replaced with process gas.
As shown in FIG. 2, a test sample is prepared in which spore-forming bacteria (Geobacillus stearothermophilus) spores and Bacillus subtilis spores are smeared on the agar medium 13 at a predetermined concentration in advance to generate atmospheric pressure plasma. Therefore, argon (Ar) gas and carbonic acid (CO ₂ ) gas are mixed and supplied as a process gas at a total flow rate of 5 LM and a flow rate ratio of 9: 1 into the cylindrical metal tube of the plasma generation device 11. Time plasma jet was irradiated.

  The agar medium after the plasma jet irradiation treatment was cultured as it was in an incubator at a predetermined temperature, and colonies generated on the medium were visually observed and photographed to evaluate the bactericidal properties.

Comparative Example 2
In Comparative Example 2, argon (Ar) gas and oxygen (O ₂ ) gas are mixed and supplied as a process gas at a total flow rate of 5 LM and a flow rate ratio of 9: 1 into the cylindrical metal tube of the plasma generator 11. Except for the above, the treatment and conditions were the same as in Example 3, and the bactericidal properties were evaluated.

Example 4
In Example 4, plasma jet irradiation treatment was performed under the same configuration and conditions as in Example 3. The irradiation time of the plasma jet is 5 minutes, and B. subtilis spores and spore-forming bacteria (G. stearo.) Spores before and after the plasma jet irradiation treatment are respectively scanned by an electron microscope (JSM-6510, JEOL). Observed at.

Example 5 FIG.
In the fifth embodiment, the atmosphere in which atmospheric pressure plasma is generated is surrounded by a process gas replacement cover, and the atmosphere of the processing space is replaced in advance from the atmosphere (air) to the process gas atmosphere, and the process is not surrounded by the cover. In the case where the gas atmosphere was not replaced, the production concentrations of ozone (O ₃ ) and carbon monoxide (CO) in the space of the plasma jet irradiation treatment were measured with a Kitagawa gas detector tube.

Example 6
In Example 6, the process gas argon (Ar) gas and carbon dioxide (CO ₂₎ gas, a total flow rate 5 lm, carbonate to the argon (Ar) gas (CO ₂₎ by changing the addition flow rate ratio of the gas to 0-50% The same treatment and evaluation as in Example 2 were performed except that the irradiation time was fixed for 3 minutes.

FIG. 3 shows the results of treatment of B. subtilis spores with atmospheric pressure plasma (CO ₂ gas) in Example 1. The vertical and horizontal axes in FIG. 3 represent the ratio of B. subtilis spores remaining and the plasma jet irradiation time, respectively.

FIG. 4 shows the results of treatment of B. subtilis spores with atmospheric pressure plasma (Ar + CO ₂ gas) in Example 2. The vertical axis and the horizontal axis in FIG. 4 represent the ratio of remaining B. subtilis spores and the plasma jet irradiation time, respectively.

  FIG. 5 shows the results of treatment of B. subtilis spores with atmospheric pressure plasma (AIR gas) in Comparative Example 1. The vertical axis and the horizontal axis in FIG. 5 represent the ratio of remaining B. subtilis spores and the plasma jet irradiation time, respectively.

From the results shown in FIG. 3 to FIG. 5, the treatment with atmospheric pressure plasma to which carbon dioxide (CO ₂ ) gas was added linearly decreased the number of fungi, and a fungicidal effect was recognized. On the other hand, with air (AIR) gas, there was almost no decrease in the number of germs, and no killing effect was observed.

FIG. 6 shows a photograph of an agar medium after irradiation treatment with atmospheric pressure plasma (Ar + CO ₂ gas) in Example 3. Also, it is shown in Comparative Example 2, the agar photograph after irradiation of atmospheric pressure plasma (Ar + O ₂ gas) in FIG. 6 and 7, the plasma jet irradiation time from the left represents 10 seconds, 1 minute, and 5 minutes, respectively.

From the results shown in FIG. 6, in any of the indicator bacteria, suppression of colony formation was observed in a radial manner from the center of the agar medium in the atmospheric pressure plasma to which CO ₂ gas was added. On the other hand, from the results shown in FIG. 7, in the atmospheric pressure plasma to which O ₂ was added, no significant change was observed after the plasma jet irradiation treatment.

  An electron microscope observation image of Example 4 is shown in FIG. From the results shown in FIG. 8, in any of the indicator bacteria, a change in physical form was observed such that the spores contracted or adhered to each other after the plasma jet irradiation.

FIG. 9 shows measurement results of ozone (O ₃ ) and carbon monoxide (CO) production concentrations in the plasma jet irradiation space in Example 5. When the atmosphere where atmospheric pressure plasma is generated is surrounded by a process gas replacement cover and the atmosphere of the processing space is replaced from the atmosphere (air) to the process gas atmosphere in advance, ozone (O ₃ ) 35 ppm, carbon monoxide (CO ) More than 50 ppm. On the other hand, if the atmosphere in the processing space is not previously replaced with the process gas atmosphere from the atmosphere (air) without surrounding the atmosphere where the atmospheric pressure plasma is generated with the process gas replacement cover, the ozone (O ₃ ) generation concentration is The production concentration of 2.5 ppm and carbon monoxide (CO) was 3 ppm.

The result of the processing in Example 6 is shown in FIG. It was confirmed that if the carbon dioxide (CO ₂ ) gas content was 5% or more, a plasma jet could be generated and the killing effect could be sufficiently exhibited.
Here, although carbonate (CO ₂₎ gas content exemplified embodiments up to 50% is obtained similar killing effect in the range of 50-100%, acts as a scope of the present invention I have confirmed that.

Although an example is not shown here, it is remarkable when the atmosphere in which atmospheric pressure plasma is generated is surrounded by a process gas replacement cover and the atmosphere of the processing space is replaced in advance from the atmosphere (air) to the process gas atmosphere. Increased the killing effect. When the atmosphere in which the atmospheric pressure plasma is generated is not surrounded by the process gas replacement cover and the atmosphere in the processing space is not previously replaced with the process gas atmosphere from the atmosphere (air), oxygen (O ₂₎ contained in the surrounding atmosphere ) And moisture (H ₂ O), atomic oxygen (O.) is considered to be quenched (decayed).

From the results shown in FIGS. 3 to 10, electrons accelerated by the electric field in the plasma collide with the CO ₂ gas to dissociate the gas molecules, and a large amount of active oxygen species such as atomic oxygen (O.) It is considered that microorganisms have been killed (inactivated) by being generated without being chilled (attenuated), released to the outside of the plasma source, and etching the cell wall on the surface of the microorganism as a dominant factor.

  From these verification results, it has become clear that microorganisms can be effectively killed by the microorganism killing apparatus and method using atmospheric pressure plasma of the present invention. In the present invention, an example of processing using a plasma generation source having a plasma processing diameter of a few millimeters and a small direct processing area has been shown. However, the diameter of the plasma injection hole is enlarged, the gas flow rate, input power, etc. This increase makes it possible to easily expand this processing area. It is also possible to reduce the processing time.

Further, in the embodiment of the present invention, a method of generating a plasma by using a process gas blown from a plasma nozzle without first generating a plasma, replacing a processing atmosphere, and then performing a process is illustrated. For example, the gas replacement means (gas supply means) may be provided in a gas replacement cover portion or the like, and the killing process may be performed after the gas replacement. By adopting such a configuration, for example, microorganisms attached to the inner surface of a container such as plastic conveyed in-line can be surely killed.
Note that the plasma killing apparatus of the present invention is not limited to the first to sixth embodiments as long as it includes an atmospheric pressure plasma generating means for introducing plasma into the container. Various modifications are possible without departing from the scope of the invention. For example, the plasma source may have any configuration as long as it can be converted to plasma with a gas having a carbon dioxide content of 5% or more.

1,11. Plasma generator 2, 12. 2. Ejection hole Membrane filter 13. Agar medium 4,14. cover

Claims (4)

A microbe killing device provided with an atmospheric pressure plasma generating means for introducing plasma into a container,
The microorganism killing apparatus, wherein the atmospheric pressure plasma generation means is configured to generate plasma using a gas having a CO ₂ content of 5% or more as a process gas. A microorganism killing apparatus according to claim 1, further the atmosphere of the container, microbial killing device having a gas replacement means for replacing a process gas comprising CO _2. A method for killing microorganisms by means of atmospheric pressure plasma generation means for introducing plasma into a container,
A method for killing microorganisms, wherein the atmospheric pressure plasma generation means includes a step of generating plasma using a gas having a CO ₂ content of 5% or more as a process gas. A microorganism killing method of claim 3, the atmosphere in the container, further comprising a microorganism killing method the step of replacing a process gas comprising CO _2.