JP2005131213A - Method for externimation of various bacteria using ozone for organism-growing apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sterilization method using ozone for an organism growing apparatus, eliminating bacteria which affect growth of useful organism stored in the growing apparatus easily and completely. <P>SOLUTION: Ozonic gas is brought into the organism growing apparatus 10 first prior to the transfer of useful organism into the apparatus 10, in order not only to maintain the appropriate temperature and gas composition including steam for their growth in the apparatus 10 where the useful organism is cultured, but also to eliminate bacteria which affect the growth, and bacteria will be eliminated when each part of the apparatus 10 is exposed to the ozonic gas. Then, a gas for adjusting the gas composition is brought into the apparatus 10, with which the gas composition in the apparatus 10 will include no ozone more and will become appropriate for the growth of the organism through at least three stages of process, ozone injection for eliminating bacteria included in the apparatus, degradation and removal of the ozone and dust removal by a HEPA filter F. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention eliminates various germs that become obstacles to the growth of useful organisms housed in various organism growth rooms, such as cell culture, fermentation of fungi, and cultivation of mushrooms. The present invention relates to a method for eliminating germs in adult rooms.

  Conventionally, for the elimination of various germs that hinder the growth of various useful organisms (culture, fermentation, cultivation) housed in organism growth rooms (eg, incubators) such as cells, fungi, and mushrooms In addition, hot water (boiling), heating with high temperature steam (high pressure, dry heat sterilization, etc.), irradiation with electromagnetic waves (ultraviolet rays, etc.), application of liquid disinfectants (alcohol, formalin, chlorine compounds, etc.), or immersion in it, gas Exposure to fungicides (such as ozone) has been carried out.

  Both have advantages and disadvantages, and are effective for removable members and parts of the organism growing room, but for the main body and non-removable or difficult members, heating and immersion in a liquid disinfectant are generally not possible. First of all, electromagnetic radiation has no effect on the shadowed parts, the disinfection is incomplete, and the application of liquid disinfectant generally depends on manual work and is extremely troublesome There is. On the other hand, exposure to gaseous sterilizers (such as ozone) can be carried out automatically if the equipment is in place because the gaseous sterilizer penetrates into narrow gaps, and even leakage is completely prevented. If there is no influence on the work environment, it is considered to be the most effective means.

In one conventional example, the inside of the culture apparatus for solid (eg, steamed defatted soybean) or the air conditioning path is pre-washed with air or water containing ozone, and ozone is used to quench the heat-treated solid raw material. It has been proposed that air containing ozone is used, and that gas containing ozone is supplied to the air taken into the air-conditioning path of the solid culture device in the culture (see, for example, Patent Document 1). ).

  However, for example, germs that adhere to the surface of a solid, such as steamed defatted soybeans, are exposed to ozone-containing air and are only removed. If it is short, it may not have a significant effect on its growth, but this is not the case for the growth of organisms that are sensitive to ozone and whose growth can be impaired by exposure (eg in the case of cell culture). Inapplicable or difficult. In culturing the cells, generally, detachable members, autoclaving for parts, and the main body and non-removable or difficult members are applied multiple times with liquid disinfectant, washing by it, The introduced gas is irradiated with ultraviolet rays.

Japanese Patent Laid-Open No. 05-131184 (Claims and FIG. 1)

  From the above, in order to eliminate the disadvantages of the prior art described above, the present invention easily and completely eliminates germs that become obstacles to the growth of useful organisms contained in the organism growth room. An object of the present invention is to provide a method for eliminating germs in an organism growing room by ozone.

In order to achieve the above object, according to the method for eliminating germs in an organism growth chamber with ozone according to the invention of claim 1, the gas composition including temperature and water vapor in the organism growth chamber 10 in which the useful organism is accommodated is used. Prior to the introduction of the useful organism into the organism growth chamber 10, the organism growth chamber is first maintained so that not only is it maintained suitable for the growth of the organism but also the germs that impair its growth are eliminated. The ozone-containing gas is introduced into the chamber 10, and various parts in the chamber 10 are exposed to the ozone-containing gas to eliminate germs. Then, the gas composition in the chamber 10 does not contain ozone and is useful Gas that has undergone at least three treatments: injection of ozone to eliminate germs contained in it, decomposition and removal of ozone, and dust removal by HEPA filter F so as to be suitable for the growth of organisms Forming adjustment of the gas is configured to be introduced.

In addition to the configuration of the invention of claim 1, the invention of claim 2 is included in at least the chamber 10 during the growth period of the useful organism housed in the organism growth chamber 10. A gas for adjusting the gas composition that has undergone the three processes of ozone injection for eliminating germs, decomposition and removal of ozone, and dust removal by the HEPA filter F is continuously introduced.

In addition to the structure of the invention of claim 1 or 2, the invention of claim 3 is at least a time of carrying in a new organism during the growth period of the useful organism housed in the organism growth chamber 10, and Preferably, the ozone-containing gas is introduced into the chamber 10 each time there is a concern about contamination with germs during the check of the growth state and when a part of the organism is carried out, and preferably at a predetermined cycle. It is configured to be introduced temporarily.

  According to a fourth aspect of the present invention, in addition to the configuration of any one of the first to third aspects, the gas in the chamber 10 is drawn out and becomes at least part of the gas composition adjusting gas through the gas composition adjusting portion 20. It is configured to be circulated.

According to the invention of claim 1, each part in the organism growth chamber 10 is exposed to the ozone-containing gas, including a small gap, and the germs are exterminated, and at least for eliminating the germs contained therein. The gas composition in the chamber 10 does not contain ozone by introducing gas for adjusting the gas composition that has undergone the three processes of ozone injection, ozone decomposition and removal, and dust removal by the HEPA filter F. Relatively large spore-shaped bacteria that have entered the gas for gas composition adjustment for some reason are captured by the HEPA filter F, and relatively small bacteria contain ozone. Since it is extinguished by gas, an environment is secured that does not hinder the growth of organisms (eg, cell culture) that are sensitive to ozone and may lose their growth.

  According to the invention of claim 2, in addition to the effect of the invention of claim 1, during the growth period of the useful organism housed in the organism growth chamber 10, similarly, The gas for adjusting the gas composition after three treatments of ozone injection for eliminating germs contained therein, decomposition and removal of the ozone, and dust removal by the HEPA filter F of relatively large bacteria was continuously introduced. As a result, the gas in the chamber 10 is drawn out of the room, so that even if the gas in the chamber 10 is contaminated with germs for any reason, the germs are continuously removed from the chamber 10. . Therefore, an environment that is sensitive to ozone and that does not hinder the growth of organisms (for example, cell culture) that are likely to lose their growth is continuously secured.

According to the invention of claim 3, in addition to the effect of the invention of claim 1 or 2, at least when a new organism is carried in during the growth period of the useful organism housed in the organism growth chamber 10, Preferably, each time the growing state is checked and when a part of the organism is carried out, the gas containing the ozone is contained in the chamber 10 every time there is a concern about contamination with germs, and preferably at a predetermined cycle. It is necessary to set the exposure time to the organism and the ozone concentration to be sufficiently short and low according to the organism. The environment is more actively protected from the risk of contamination.

  According to the invention of claim 4, in addition to the effects of the inventions of claims 1 to 3, the gas in the chamber 10 is drawn out and becomes at least a part of the gas for adjusting the gas composition through the gas composition adjusting portion 20. Since it is circulated, the burden of introducing a new gas and adjusting the gas composition is alleviated accordingly.

  Referring to FIG. 1 as an example of Aspergillus culturing, a method for eliminating germs in an organism growing room by ozone, which relates to the best mode for carrying out the present invention, 10 is an organism in which an organism that is Aspergillus is contained and cultured. It is a body growing room (incubator), surrounded by a stainless steel door (not shown) and a wall provided with a heat insulating material (not shown), and by a partition wall having a HEPA filter F and two dampers D The filtering chamber 12 having the gas introduction port 12a on the side surface and the culture chamber 11 having the gas discharge port 11a on the side surface are separated vertically. In addition, inside the chamber 10, a culture rack 11b on which organisms that have entered a petri dish or the like are placed, and on the wall, sensors such as a thermometer T, a pressure gauge P, a CO2 meter C1, an ozone meter C2, and the chamber 10 Heating means (not shown) for keeping the temperature at a set value is also provided.

  Reference numeral 20 denotes a gas composition adjusting portion, and main equipment includes a circulation fan 21, an ozonizer 22, a gas humidifier 23, an ozone decomposer 24, a gas heater 25 built in the gas humidifier 23, and a gas cooling unit. A container 26 is also provided. 27 is a CO2 cylinder, 28 is an oxygen cylinder, and 30 is a control part for controlling the entire apparatus including an operation panel.

The piping will be described as one example. The gas discharged from the gas discharge port 11a from the inside of the chamber 10 is sucked by the circulation fan 21, supplied from the oxygen cylinder 28, and part thereof is converted to ozone by the ozonizer 22. The ozone-containing oxygen and the CO2 supplied from the CO2 cylinder 27 are entrained, sent to the gas humidifier 25, contacted with water, and a part of ozone and CO2 is dissolved in the water, and most of the most A line L1 is provided so that the gas is humidified and further led directly to the gas inlet 12a of the filtering chamber 12 via the gas cooler 26.

  In addition, a bypass line L2 into which an ozonolysis device 24 is inserted is provided between the outlet of the gas heater 25 and the gas inlet 12a, and between the outlet of the circulation fan 21 and the gas inlet 12a. Is provided with a bypass line L3. V is a flow control valve, and RV is a pressure reducing valve.

The above-described operation is not only maintained so that the gas composition including the temperature and water vapor in the organism growth chamber 10 in which the useful organisms are accommodated is suitable for the growth of the useful organisms. The germs that impair its growth are eliminated. That is, first, an ozone-containing gas is introduced into the organism growth chamber 10 by the gas composition adjusting means 20, each part in the chamber 10 is exposed to the ozone-containing gas, and water in the gas humidifier 25 is Various bacteria are exterminated by contact with ozone dissolved in water. Note that the gas heater 25 and the gas cooler 26 adjust the humidity and temperature of the gas sent to the organism growth chamber 10 to values suitable for the growth of the organism.

  In detail, when the door (not shown) is closed, the valve V of the line L1 is opened, and the valves V of the lines L2 and L3 are closed, the air in the sealed organism growth chamber 10 is The gas withdrawn from the gas discharge port 11a by the circulation fan 21 is composed of ozone-containing oxygen and CO2 in which a part of oxygen supplied from the oxygen cylinder 28 is converted into ozone by the ozonizer 22 on the discharge side of the circulation fan 21. Along with CO2 supplied from the cylinder 27, it is sent to the gas humidifier 25 and comes into contact with water. Ozone and a part of CO2 are dissolved in the water, and most of the remaining gas is a temperature suitable for controlling germs. Adjusted to humidity.

The gas further becomes a gas for adjusting the gas composition (if only germs are eliminated, ozone addition and humidification are sufficient, and CO2 addition may be omitted), and the gas is introduced directly to the filtering chamber 12 via the gas inlet 12a. It is burned. The two dampers D are closed, and the gas guided to the filtering chamber 12 is removed by the HEPA filter F and then guided to the culture chamber 11. The gas is drawn out by the circulation fan 21 from the gas discharge port 11a, and most of the gas is repeatedly circulated while receiving the above-described operation. The temperature in the chamber 10, the ozone concentration, and the CO2 concentration are controlled to the respective set values by the control unit 30 based on signals from the sensors T, C1, and C2, respectively.

Thereby, while the gas circulates, relatively large spore-like bacteria entrained therein are captured by the HEPA filter F, and each part in the organism growth chamber 10 is exposed to the ozone-containing gas including a small gap. At the same time, when the gas comes into contact with ozone dissolved in the water in the gas humidifier 25, all germs are eliminated. Note that ozone dissolved in water has a significantly higher sterilizing power than ozone-containing gas. If the conditions such as the gas composition have reached the set values during the operation described in the above (0023), the valve V in the lines L1 and L2 is closed and the valve V in the line L3 is opened. Even if 10 gases are circulated through the line L3, germs are eliminated.

After completion of the germs in the organism growing room 10, the valve V of the line L2 is opened and the valves V of the lines L1 and L3 are closed so that the gas for adjusting the gas composition passes through the ozonolysis device 24. Then, the ozone contained therein is decomposed and removed, and introduced into the culture chamber 11 as a gas containing no ozone. For the time being, the same gas composition and temperature adjustment operations are performed, and when the gas is repeatedly circulated, the ozone-containing gas in the chamber 10 does not contain the gas having a composition suitable for growing organisms. Is completely replaced. Since the influence on the worker is thereby eliminated, it becomes possible to carry in the organism into the chamber 10. In addition, the ozone concentration before the ozonolysis removal of the gas composition adjustment gas during this period may be lower than that during the extermination of germs in the organism growing chamber 10 described in (0022) to (0024).

Moreover, of course, the above-mentioned operation is maintained during the growth period of useful organisms, so that relatively large spore-like bacteria are captured by the HEPA filter F even if there is an invasion of germs for any reason. In addition, since relatively small bacteria are exterminated by ozone-containing gas and ozone dissolved in the water in the gas humidifier 25, organisms that are sensitive to ozone and whose growth may be impaired (for example, cell culture) An environment that does not hinder the development of the plant will continue to be secured.

Finally, during the growth period of the useful organism housed in the organism growth chamber 10, at least when a new organism is carried in, and preferably when the growth state is checked, when a part of the organism is carried out The valve V of the lines L2 and L3 is closed, the valve V of the line L1 is opened, and gas containing ozone is supplied to the chamber every time there is a concern about contamination with various germs, and preferably at a predetermined cycle. 10 is temporarily introduced. Note that the humidity and CO2 concentration of the ozone-containing gas are maintained as before. Thus, of course, the exposure time and ozone concentration of the organism need to be set sufficiently short and low depending on the organism, but the exposure of the organism to the ozone-containing gas Because it is temporary, the environment for growing organisms is always more positively protected from the danger of contamination by bacteria without compromising its growth.

  Moreover, since the gas in the chamber 10 is drawn and circulated through the gas composition adjusting portion 20 as at least a part, preferably a large part of the gas composition adjusting gas, the introduction of new gas and other gas composition adjustments are performed. The burden for is reduced accordingly.

The present invention will be described in more detail with reference to the following examples, which illustrate the control of various germs in the cultivation of Aspergillus oryzae for genetic manipulation. However, the scope of the present invention is not limited thereto. That is, first, the mildew that is the specimen is not accommodated, and a gas containing CO 2 with an ozone concentration of 20 ppm is introduced and circulated in the organism growth chamber 10 for 30 minutes (L1 line), and then passes through the ozonolysis device 24. A gas not containing ozone was introduced and circulated for 30 minutes (L2 line), whereby the gas in the organism growth chamber 10 did not contain ozone and was replaced with a gas suitable for Aspergillus.

Next, the specimen was placed on the culture rack 11b and cultured for 2 weeks. The culture state was observed once every two days during the culture. At that time, since there was concern about the invasion of germs by opening and closing the door of the organism growth chamber 10, a gas containing a concentration of 2.5 ppm was introduced into the organism growth chamber 10 and circulated for 30 minutes in accordance with the opening and closing timing of the door. As a result, various germs were removed. As a result, the culture was carried out in the medium without propagation of various bacteria in the two weeks.

FIG. 2 shows the time course of the survival rate when Aspergillus niger, Bacillus subtilis, baker's yeast, and Escherichia coli are exposed to an atmosphere with an ozone concentration of 2.5 ppm. This data shows that these bacteria and fungi differ in their resistance to ozone depending on the type, and by utilizing this characteristic, the targeted bacteria can be selectively sterilized. Also in this example, the idea of selective sterilization was applied to the removal of various germs that hindered the cultivation of Aspergillus, and the effect was confirmed.

It is an apparatus block diagram of the apparatus applied to the best example for implementing this invention. It is a graph which shows the relationship between the exposure time to ozone with respect to Aspergillus oryzae and other bacteria to be controlled for explaining Example 1 and the survival rate.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Organism growth room 11 Culture room 11a Gas discharge port 11b Culture rack 12 Filtering room 12a Gas introduction port 20 Gas composition adjustment part 21 Circulating fan 22 Ozonizer 23 Gas humidifier 24 Ozone decomposer 25 Gas heater 26 Gas cooler 27 CO2 Cylinder 28 Oxygen cylinder 30 Control part
C1 CO2 meter C2 Ozone meter D Damper L1, L2, L3 Piping line F HEPA filter P Pressure gauge T Thermometer
V Flow control valve VR Pressure reducing valve

Claims (4)

Not only is the gas composition containing the temperature and water vapor in the organism growth chamber (10) in which the useful organisms are accommodated maintained so as to be suitable for the growth of the useful organisms, but the germs that impair the growth are controlled. Prior to the introduction of the useful organism into the organism growth chamber (10), first, an ozone-containing gas is introduced into the organism growth chamber (10), and each part in the chamber (10) contains the ozone. The bacteria contained in the chamber (10) are contained at least so that the gas composition in the chamber (10) does not contain ozone and is suitable for the growth of the useful organism. Biomass by ozone, characterized by introducing gas for gas composition adjustment after three treatments of ozone injection, ozone decomposition and removal, and dust removal by HEPA filter (F) Bacteria method of combating growing chamber. During the growth period of the useful organism housed in the organism growth chamber (10), at least ozone injection for eliminating germs contained in the chamber (10) and decomposition of the ozone The method for eliminating germs in an organism-growing room with ozone according to claim 1, wherein the gas for gas composition adjustment that has undergone the three processes of removal and dust removal by the HEPA filter (F) is continuously introduced. . During the breeding period of the useful organism housed in the organism breeding room (10), at least when a new organism is carried in, and preferably at the time of checking the breeding state, when a part of the organism is carried out, The ozone-containing gas is temporarily introduced into the chamber (10) every time there is a concern about contamination of each of the microorganisms, and preferably at a predetermined cycle. 2. A method for eliminating germs in an organism-growing room with ozone according to 2. The gas in the chamber (10) is drawn out and circulated as at least part of a gas for gas composition adjustment through a gas composition adjustment portion (20). A method for eliminating germs in an organism-growing room with ozone as described in 1.

Images