WO2015152328A1

WO2015152328A1 - Method for killing spores

Info

Publication number: WO2015152328A1
Application number: PCT/JP2015/060338
Authority: WO
Inventors: 知美阪井
Original assignee: 花王株式会社
Priority date: 2014-04-02
Filing date: 2015-04-01
Publication date: 2015-10-08
Also published as: TW201540197A; JP6607688B2; TWI675624B; JP2015199723A; SG11201608232QA

Abstract

Provided is a method for killing spores that can efficiently and effectively kill spore-forming bacteria regardless of the bacterial strain. Provided is a method for killing spores in which step 2 described below is performed after completing step 1 described below. That is, after completing step 1 that is a step of bringing spore-forming bacteria and dipicolinic acid or a salt thereof into contact in conditions of pH 6 or lower, step 2 that is a step of killing the spore-forming bacteria is performed. Also provided is a method for killing spores that has a time period in which step 1 described below and step 2A described below are performed simultaneously. That is, step 1 is a step of bringing spore-forming bacteria and dipicolinic acid or a salt thereof into contact in conditions of pH 6 or lower, and step 2A is a step of physically killing the spore-forming bacteria.

Description

Sprouting method

The present invention relates to a method for killing spore-forming bacteria having spores.

Bacteria such as Bacillus spp. And Clostridium spp. Form a strong shell structure and form spores with extremely high resistance to heat and drugs. Bacteria that can form certain types of spores are known to produce toxins when they enter the human body. For example, in medical practice, linen products such as sheets and pillowcases are mainly disinfected by heat, but heat disinfection cannot sterilize bacteria having heat-resistant spores. For this reason, there are also cases where the death to the dead occurs due to nosocomial infection of bacteria that can form spores via linen products.

In order to sterilize bacteria having spore spores, in many cases, sterilization is performed with high-pressure steam, or a strong chemical disinfectant such as sodium hypochlorite is used at a high concentration. In food processing, severe heat treatment or low-temperature distribution method is taken as a measure against spores.

In WO 01/66471, a germination agent is brought into contact with a water-closure system for a sufficient time and under suitable conditions for germinable bacteria to germinate into vegetative cells. Disclosed is a method for controlling spores in a water-closed system, comprising sterilizing cells. JP 2009-118775 A discloses a germination method in which a bacterium having a spore contained in a liquid egg or egg processed product is subjected to pressure treatment at a predetermined temperature and pressure, and after germination by the method. A method for disinfecting germinated bacteria is disclosed.

This invention relates to the germination method which performs the following process 2 after completion | finish of the following process 1. FIG.
Step 1: A step of bringing a spore-forming bacterium into contact with dipicolinic acid or a salt thereof under a condition of pH 6 or lower; and Step 2: a step of sterilizing the spore-forming bacterium.

Moreover, this invention relates to the germination method which has a period which performs the following process 1 and the following process 2A simultaneously.
Step 1: A step of bringing a spore-forming bacterium into contact with dipicolinic acid or a salt thereof under a condition of pH 6 or lower; and Step 2A: a step of physically sterilizing the spore-forming bacterium.

Further, the present invention relates to a germination aid composition containing dipicolinic acid or a salt thereof and having a pH of 6 or less.

Detailed Description of the Invention

The technique specifically disclosed in Patent Document 1 for treating bacteria having spores with a germination inducer such as L-alanine does not have a sufficient bactericidal effect.

Germination methods using high-concentration disinfectants and high-pressure steam sterilization are not suitable for disinfecting a wide range of environments such as hospitals, and may be damaging to equipment or toxic to the human body. The usage environment is limited. For example, in the technique disclosed in Patent Document 2, it is necessary to apply a high pressure of 10 MPa or more to germinate bacteria having spores, and there is a problem that a special device is required. There is also concern about quality deterioration due to application to foods.

In food processing, severe heat treatment or low-temperature distribution methods are taken as measures against spores, but problems such as flavor and quality deterioration, and power costs also arise.

The present invention relates to a germination method capable of effectively and effectively sterilizing bacteria having spores that are harmful in the medical and food fields, regardless of the species. Furthermore, this invention relates to the germination adjuvant composition used in order to disinfect a spore formation microbe.

When the present inventors contact dipicolinic acid (2,6-pyridinedicarboxylic acid) or a salt thereof with a spore-forming bacterium at a pH of 6 or less, the present inventor can effectively use a fungicide having a concentration lower than that normally used or effectively at a lower temperature. The inventors have found that sprouting is possible and have reached the present invention.

According to the present invention, there is provided a spore killing method capable of sterilizing spore-forming bacteria having spores. Furthermore, in this invention, the germination adjuvant composition used in order to disinfect a spore formation microbe is provided.

A spore-forming bacterium refers to a bacterium having resistance to certain heat treatment and drying by forming spores in the absence of nutrients. The spore indicates a strong shell structure formed by bacteria formed by bacteria, and is distinguished from the bacteria themselves. The “spore-forming bacterium” that is a target for sprouting according to the present invention is a general spore-forming bacterium that exists in medical sites, foods, and beverage products. For example, bacteria belonging to the genus Bacillus, such as Bacillus cereus and Bacillus subtilis, bacteria belonging to the genus Clostridium bil, such as Clostridium phicil, And bacteria of the genus Sporosarcina, bacteria of the genus Geobacillus, bacteria of the genus Aerobacillus, bacteria of the genus Alicyclobacillus and the like. Since the “spore-forming bacterium” that is the subject of the present invention is a bacterium having a spore, it has heat resistance. Here heat resistance, as shown in the embodiment of the present invention, a bacterium having spores is an initial bacteria number ^{^{10 7 ~ 10 9 CFU / mL}} , when heated for 30 minutes or more at 80 ° C., 10 ⁷ It means that bacteria of CFU / mL or more can survive.

において In the present invention, “sprouting” mainly intends to germinate and kill bacteria having a spore. Germination means that a spore-forming bacterium having a spore germinates, and thereby refers to a phenomenon of becoming a normal microbial cell having proliferation and metabolic ability. Here, sprouting includes killing and sterilizing bacteria having spores. Germination also includes reducing the overall number of bacteria present in the population. The sprouting is not particularly limited, but when expressed as the sprouting effect index shown in the examples, it indicates a state of 0.21 or more, more preferably 0.3 or more, and further preferably 0.5 or more. In the present invention, the germicidal effect refers to the difference in the germicidal effect from the control represented by the germicidal effect index.

The reason why the germination method of the present invention has an excellent germination effect is not clear, but is considered as follows. In step 1, germination is promoted by allowing dipicolinic acid (hereinafter also referred to as DPA) or a salt thereof to act on spore-forming bacteria having spore under pH 6 or less, and sterilization in step 2 is easily performed. . Moreover, since dipicolinic acid under the conditions of pH 6 or less is affinity with bacteria, it is estimated that it is easy to enter into bacteria and germination is effectively promoted.

In addition, since the action of dipicolinic acid or its salt in the spore-forming bacteria is not an action through a specific receptor specific to the bacterial species, it is considered to have a spore-killing or bactericidal effect regardless of the type of spore-forming bacteria. It is done.

Note that germination can be confirmed, for example, by examining the presence or absence of spore darkening with a phase contrast microscope. That is, the larger the proportion of darkened spores, the more germination is induced.

工程 Step 1 of the present invention is a step of bringing a spore-forming bacterium into contact with dipicolinic acid or a salt thereof under a pH of 6 or less. In step 1, the spore-forming bacterium is preferably a bacterium having a spore. It may contain bacteria that do not have spores and may have spores.

Dipicolinic acid or a salt thereof is provided in a liquid or solid state. Dipicolinic acid or a salt thereof is preferably a liquid from the viewpoint of convenience and further improvement of the germicidal effect. In addition, dipicolinic acid or a salt thereof is preferably provided as a germination aid composition described below from the viewpoint of convenience.

In the method of contacting dipicolinic acid or a salt thereof and a spore-forming bacterium in Step 1 of the present invention, when the spore-forming bacterium is present in the liquid, a high concentration solution of dipicolinic acid or a salt thereof or dipicolinic acid or a salt thereof Examples thereof include a method in which the powder is mixed with a liquid containing spore-forming bacteria and the mixed liquid is always brought into contact with stirring, a method in which the mixed liquid is stirred, a method in which the mixed liquid is stirred, or a method in which the mixed liquid is allowed to stand. In the case where spore-forming bacteria are present on the solid, a method of applying a liquid germination aid composition described later such as dipicolinic acid or a salt solution thereof onto the solid may be mentioned. Examples of the method for applying a solution of dipicolinic acid or a salt thereof on a solid include a spraying method and a method using a tool such as a brush or a sponge. Moreover, the method of spraying water etc. by installing solid sprouting aid composition, such as a powder, in a solid is mentioned. Here, the term “on a solid” refers to a hard surface, although not particularly limited.

The contact method between dipicolinic acid or a salt thereof and spore-forming bacteria in Step 1 of the present invention is preferably a liquid composition containing dipicolinic acid or a salt thereof, more preferably dipicolinic acid or a salt thereof, from the viewpoint of simplicity. It is a method of contacting a solution, more preferably an aqueous solution of dipicolinic acid or a salt thereof.

In Step 1, when the spore-forming bacterium and dipicolinic acid or a salt thereof are contacted in a solution, the pH of the solution at 24 ° C. is preferably 5 or less, more preferably 4 from the viewpoint of further improving the germicidal effect. Hereinafter, it is more preferably 3 or less, more preferably 2.5 or less, and more preferably 1.8 or less. From the viewpoint of safety, the pH of the solution at 24 ° C. is preferably 1 or more, more preferably 1.2 or more. The pH at 24 ° C. of the solution is preferably 1 to 6, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, more preferably 1 to 3, taking the above viewpoints together. 2.5, more preferably 1.2 to 1.8. The pH can be measured at 24 ° C. using a desktop pH meter Model 9611 (manufactured by HORIBA).

The pH adjustment can be performed when a dipicolinic acid or salt solution is prepared in advance. Or it can also carry out after adding dipicolinic acid or its salt in the solution containing a spore formation microbe, when making it contact with a spore formation microbe. Examples of pH adjusters include commonly used acids and bases, for example, inorganic acids such as hydrochloric acid and sulfuric acid, organic acids such as lactic acid and citric acid or their salts, inorganic bases such as sodium hydroxide and potassium hydroxide, and triisopropanol. And organic bases such as amines. As the pH adjuster, from the viewpoint of versatility, the acid is preferably an inorganic acid such as hydrochloric acid or sulfuric acid, and the base is preferably an inorganic base such as sodium hydroxide or potassium hydroxide.

When the spore-forming bacterium and dipicolinic acid or a salt thereof are contacted in a solution, the content of dipicolinic acid in the solution is preferably 0.05 mM or more, more preferably 0. 5 mM or more, more preferably 3 mM or more, more preferably 4 mM or more, more preferably 6 mM or more, more preferably 8 mM or more, and preferably from the viewpoint of stability and economics of a solution of dipicolinic acid or a salt thereof. It is 30 mM or less, more preferably 25 mM or less, more preferably 20 mM or less, more preferably 15 mM or less, more preferably 12 mM, more preferably 10 mM or less. When the spore-forming bacteria and dipicolinic acid are brought into contact with each other in the solution, the content of dipicolinic acid in the solution is preferably 0.05 to 30 mM, more preferably 0.5 to 30 mM, taking the above viewpoints together. More preferably 3-30 mM, more preferably 3-25 mM, more preferably 3-20 mM, more preferably 3-15 mM, more preferably 4-15 mM, more preferably 6-12, more preferably 8-10 mM. is there. Here, the content of dipicolinic acid is a numerical value in terms of dipicolinic acid when a dipicolinic acid salt is used.

In the contact, the relationship between the initial number of bacteria and the concentration of dipicolinic acid is not particularly limited. For example, from the viewpoint of further improving the germicidal effect, the concentration of dipicolinic acid relative to the initial cell count of 10 ⁸ CFU / mL is more preferably 0.1 mM or more, more preferably 1 mM or more, more preferably 10 mM or more, more preferably From the economical viewpoint, the concentration of dipicolinic acid relative to the initial bacterial count of 1 CFU / mL is preferably 1 M or less, more preferably 100 mM or less, more preferably 30 mM or less, more preferably 10 mM or less. Preferably it is 1 mM or less.

The contact temperature between the spore-forming bacterium and dipicolinic acid or a salt thereof in step 1 before the start of step 2 of the present invention is preferably 15 ° C. or higher, more preferably from the viewpoint of further improving the germicidal effect and economy. From the viewpoint of economy, it is preferably less than 50 ° C, more preferably 49 ° C or less, more preferably 45 ° C or less, more preferably 40 ° C or less, and more preferably 30 ° C or less. Further, from the same viewpoint, the contact temperature between the spore-forming bacterium and the dipicolinic acid or a salt thereof in step 1 is preferably 15 ° C. to less than 50 ° C., more preferably 15 ° C. to 49 ° C., more preferably 15 ° C. -40 ° C, more preferably 15-30 ° C.

The contact time for contacting the spore-forming bacterium with dipicolinic acid or a salt thereof at pH 6 or less in Step 1 before the start of Step 2 of the present invention is preferably 30 seconds or more, more preferably from the viewpoint of further improving the germination effect. 1 minute or more, more preferably 3 minutes or more, more preferably 5 minutes or more, more preferably 8 minutes or more, more preferably 10 minutes or more, more preferably 15 minutes or more, more preferably 20 minutes or more, more preferably Is 25 minutes or longer, and from the viewpoint of economy, it is 60 minutes or shorter, more preferably 50 minutes or shorter, more preferably 40 minutes or shorter, more preferably 35 minutes or shorter. The contact time between the solution of dipicolinic acid or a salt thereof and the spore-forming bacterium in Step 1 before the start of Step 2 is preferably 30 seconds to 60 minutes, more preferably 1 minute to 60 minutes, considering the above viewpoints. More preferably 3 minutes to 60 minutes, more preferably 5 minutes to 60 minutes, more preferably 5 minutes to 50 minutes, more preferably 5 minutes to 40 minutes, more preferably 8 minutes to 40 minutes, more preferably 8 minutes. Minutes to 35 minutes, more preferably 10 minutes to 35 minutes, more preferably 15 minutes to 35 minutes, more preferably 20 minutes to 35 minutes, more preferably 25 minutes to 35 minutes.

The total contact time of the spore-forming bacterium and dipicolinic acid or a salt thereof in step 1 of the present invention is preferably 5 minutes or more, more preferably 10 minutes or more, more preferably 15 from the viewpoint of further improving the germicidal effect. Min. Or more, more preferably 20 min. Or more, more preferably 30 min. Or more, more preferably 45 min. Or more, and from the viewpoint of economy, it is preferably 90 minutes or less, more preferably 80 minutes or less, more preferably 70 minutes or less.
Here, when step 2 ends before step 1, the time of step 1 from the end of step 2 to the end of step 1 is not included in the total contact time.

Although the process 2 is basically performed after the process 1, the process 2 may be performed after the process 1 is finished. As will be described later, the process 1 and the process 2 are performed simultaneously without completing the process 1. You may go. Moreover, the process 1 and the process 2 may be started simultaneously, and the process 1 may be started after starting the process 2.

Step 1 can be completed by removing dipicolinic acid or a salt thereof. As a method of removing dipicolinic acid or a salt thereof at the end of step 1, when spore-forming bacteria and dipicolinic acid or a salt thereof are present in the liquid, the supernatant is discarded by centrifugation and dipicolinic acid or a salt thereof Is excluded.
Step 1 can also be terminated by adjusting the pH to above 6. As a method for adjusting the pH when step 1 is completed, a pH adjuster can be used. Examples of the pH adjuster include those described above, and from the viewpoint of versatility, an inorganic acid or an inorganic base is preferable, and hydrochloric acid, sulfuric acid, sodium hydroxide, or potassium hydroxide is more preferable.

<Step 2>
Step 2 of the present invention is a step of sterilizing spore-forming bacteria. Here, the spore-forming bacterium in Step 2 refers to a spore-forming bacterium in a state where germination is promoted by the treatment in Step 1. Depending on the conditions described below, bacteria that are not germinated are also included.

Examples of the means for sterilization include a means for physical sterilization and a means for chemical sterilization.
In the step 2, the means for physically sterilizing and the means for chemically sterilizing may be used alone or in combination of two or more of the physical means and two or more of the chemical means. Also good. Further, a combination of two or more of physical means and chemical means may be used.

[Physical sterilization]
<Process 2A>
Step 2A of the present invention is a step of physically sterilizing spore-forming bacteria, for example, spore-forming bacteria whose germination has been promoted.

Examples of the means for physically sterilizing include heat, pressure, and electromagnetic waves. Examples of electromagnetic waves include radiation such as X-rays and γ rays, and ultraviolet rays. The means for physically sterilizing is preferably heat from the viewpoint of economy and convenience.
Step 2A can be initiated by applying physical means to the spore-forming bacterium that has performed Step 1. Also, step 2A can be stopped by stopping the physical means.

Step 2A of the present invention is preferably a step of sterilizing spore-forming bacteria with heat from the viewpoint of further improving the germicidal effect and economical efficiency, and more preferably spore-forming bacteria at 50 ° C to 250 ° C. It is a process of sterilizing.

The temperature at which the spore-forming bacteria are sterilized by heat is preferably 50 ° C. or higher, more preferably 55 ° C. or higher, more preferably 60 ° C. or higher, more preferably 65 ° C. or higher, from the viewpoint of further improving the germicidal effect. More preferably, it is 70 ° C. or more, more preferably 75 ° C. or more, and from the viewpoint of further improving the germicidal effect, economy and versatility, preferably 250 ° C. or less, more preferably 200 ° C. or less, more Preferably it is 150 degrees C or less, More preferably, it is 120 degrees C or less, More preferably, it is 100 degrees C or less, More preferably, it is 90 degrees C or less, More preferably, it is 85 degrees C or less.

The temperature at the time of sterilization by heat in Step 2 of the present invention is preferably 50 ° C. to 250 ° C., more preferably 50 ° C. to 200 ° C., more preferably 50 ° C. to 150 ° C., more preferably 50 ° C. from the above viewpoint. ° C to 100 ° C, more preferably 55 ° C to 100 ° C, more preferably 60 ° C to 100 ° C, more preferably 65 ° C to 90 ° C, more preferably 70 ° C to 90 ° C, more preferably 75 ° C to 85 ° C. ° C.

Normally, spore-forming bacteria are difficult to sterilize at 100 ° C or below unless they germinate. However, in the present invention, germination is promoted by step 1, so that a bactericidal effect can be obtained even at 100 ° C. or lower in step 2. In the present invention, it is possible to sterilize at a temperature higher than 100 ° C., and in some cases, sterilization at a temperature higher than 100 ° C. is also possible.

The time for sterilization by heat in step 2A of the present invention is preferably 3 minutes or more, more preferably 5 minutes or more, more preferably 8 minutes or more, more preferably 10 minutes or more, from the viewpoint of further improving the germination effect. More preferably, it is 20 minutes or more, more preferably 25 minutes or more, and from the viewpoint of economy and versatility, it is preferably 90 minutes or less, more preferably 70 minutes or less, more preferably 50 minutes or less, more preferably 40 minutes or less, more preferably 35 minutes or less.

In view of the above, the time for sterilization by the heat in step 2A of the present invention is preferably 3 minutes to 90 minutes, more preferably 5 minutes to 70 minutes, more preferably 8 minutes to 50 minutes, more preferably 10 minutes to 50 minutes, more preferably 20 to 40 minutes, and more preferably 25 to 35 minutes.

方法 As a method of sterilizing by heat of step 2A of the present invention, a method of applying dry heat, wet heat, boiling, hot water, steam heat, etc. to spore-forming bacteria can be mentioned. For example, by immersing spore-forming bacteria in hot water, by placing a solid surface such as a hard surface where spore-forming bacteria are present or a liquid containing spore-forming bacteria in a thermostatic bath, by heat treatment such as cooking in food processing The method etc. are mentioned.

The pH conditions for sterilization by heat in the cocoon step 2A are not particularly limited, and can be performed under any conditions such as pH 1-9. The same pH 6 or lower conditions as in step 1 may be used.

In the germination method of the present invention, the step 2A can be completed by rapidly cooling the spore-forming bacteria sterilized by heat in the step 2A with water containing ice or a cooled container. Alternatively, the spore-forming bacteria sterilized by heat in the step 2A can be completed by cooling the heat naturally after a while. Here, the sterilized spore-forming bacteria may be present in the liquid.

When spore-forming bacteria are present in the liquid in step 1 or step 2A, the germination method of the present invention may have a step of stirring the spore-forming bacteria before or after step 1 or step 2A. . Step 1 and step 2 may be repeated.

[Chemical sterilization]
<Process 2B>
Step 2B of the present invention is a step of chemically sterilizing spore-forming bacteria.

The means for chemically sterilizing is preferably using a sterilizing agent from the viewpoint of economy.

As the bactericidal agent used in the method of sterilizing with the bactericidal agent in step 2 of the present invention, as a bactericidal agent used as a liquid substance, hypochlorous acid or a salt thereof, chlorous acid or a salt thereof, dichloroisocyanuric acid or a salt thereof, Disinfectants containing halides such as chlorine-based disinfectants that are aqueous solutions of trichloroisocyanuric acid or salts thereof, chlorine dioxide, and iodine-based disinfectants such as popidone iodine and iodoform; ethanol, 1-propanol, isopropyl alcohol, methanol, etc. Examples include alcohols; aldehydes such as formaldehyde and glutaraldehyde; phenols such as lime acid, cresol, xylenol, isopropylmethylphenol, and triclosan; and peroxides such as peracetic acid, hydrogen peroxide, and ozone. Moreover, ethylene oxide, formaldehyde, hydrogen peroxide, ozone, chlorine dioxide etc. are mentioned as bactericides used in gaseous form.

In the present invention, germination of spore-forming bacteria having spores is promoted in step 1, so that the spore-forming bacteria treated in step 1 can be sterilized with a smaller amount of bactericide than usual. For example, a chlorinated disinfectant, preferably a hypochlorous acid and its salt, chlorous acid and its salt, dichloroisocyanuric acid and its salt, trichloroisocyanuric acid and its salt, and a chlorine-based disinfectant selected from chlorine dioxide More preferably, when spore-forming bacteria having spores are sterilized with sodium hypochlorite, an effective chlorine concentration of 1000 ppm or more is usually required if step 1 of the present invention is not performed. On the other hand, when performing the process 1 of this invention, the effective chlorine concentration required for germination may be less than 1000 ppm. Specifically, in the present invention, the effective chlorine concentration is preferably 10 ppm or more, more preferably 50 ppm or more, more preferably 80 ppm or more from the viewpoint of further improving the germicidal effect, and 1000 ppm from the viewpoint of economy. Below, more preferably 800 ppm or less, more preferably 500 ppm or less, more preferably 300 ppm or less, more preferably 150 ppm or less. In addition, the temperature at which spore-forming bacteria are sterilized by chemical sterilization means such as chlorinated antiseptics such as sodium hypochlorite is preferably 5 ° C. or more, more preferably from the viewpoint of further improving the germicidal effect. Is 10 ° C. or more, more preferably 15 ° C. or more, more preferably 20 ° C. or more, more preferably 23 ° C. or more, and from the viewpoint of economy, it is preferably 50 ° C. or less, more preferably 40 ° C. or less, more Preferably it is 30 degrees C or less, More preferably, it is 25 degrees C or less.

In addition, also in this invention, it can disinfect with the usage-amount of a normal disinfectant. For example, a chlorine-based disinfectant having an effective chlorine concentration of 1000 ppm or more, preferably selected from hypochlorous acid and its salt, chlorous acid and its salt, dichloroisocyanuric acid and its salt, trichloroisocyanuric acid and its salt, and chlorine dioxide It can be sterilized with a chlorinated disinfectant, more preferably sodium hypochlorite.

Step 2 can be performed after step 1 is completed (Aspect 1).
When step 2 is step 2A in which spore-forming bacteria are physically sterilized, a period in which step 1 and step 2A are performed simultaneously may be included (aspect 2). Aspect 2 is divided as follows.
Aspect 2-1 Start Step 1 first Aspect 2-2 Start Step 1 and Step 2A at the same time Aspect 2-3 Start Step 2A first In Aspect 2, from the viewpoint of further improving the germination effect, Aspect 2-1 is preferred.
Moreover, it is preferable that process 2A of aspect 2 is a process sterilized with heat.

[Aspect 1: Aspect where Step 2 is performed after Step 1 is completed]
In the present invention, Step 2 can be performed after Step 1 is completed. Here, the end of step 1 can be ended when the germination of spore-forming bacteria is promoted. Whether the process 2 is the process 2A (physical sterilization) or the process 2B (chemical sterilization), in any case, the preferable conditions of the process 1 and the process 2 of this embodiment 1 are as described above. Street. Step 1 can be terminated by bringing the pH above 6 or removing dipicolinic acid. The aspect is as described above.
Here, “perform step 2 after the end of step 1” means that step 2 is performed at the end of step 1, preferably within one hour after completion of step 1 at the latest, more preferably 30. It means to move to step 2 within minutes, more preferably within 15 minutes, more preferably within 5 minutes, and even more preferably within 1 minute.

[Aspect 2: A mode having a period in which Step 1 and Step 2A are performed simultaneously]
In the present invention, a period in which Step 1 and Step 2A are performed simultaneously can be provided.
The time and temperature conditions in which Step 1 and Step 2A overlap in the period in which Step 1 and Step 2A are performed simultaneously are the same as those in Step 2A when Step 2A is a step of sterilization by heat. When Step 2A is a step of physical sterilization other than heat, the temperature condition when performing Step 1 and Step 2A at the same time is the same as that of Step 1 above.
The conditions other than time and temperature, for example, the content of dipicolinic acid and the pH conditions during the period in which Step 1 and Step 2A are performed simultaneously are the same as those in Step 1 above.
Preferred conditions when performing only step 1 other than the above are as described above.

[Aspect 2-1: Aspect in which step 1 is started first]
The germination method which has the period which performs the process 1 and the process 2 of this invention simultaneously can start the process 1 previously. That is, the sterilization of the step 2A can be started by applying physical means in the middle of the step 1. For example, when step 2A is a step of sterilizing spore-forming bacteria by heat, when the step of contacting dipicolinic acid or a salt thereof with spore-forming bacteria in step 1 is performed in a solution, the step 2A is directly subjected to step 2A. The temperature of the solution can be shifted to the temperature zone of step 2A.

終了 Each step may be terminated by either ending step 1 first, ending step 1 and step 2A simultaneously, or ending step 2A first. In addition, the completion method of the process 1 and the process 2A is as having mentioned above. In addition, when step 2A ends before step 1, the time of step 1 from the end of step 2A to the end of step 1 is not included in the “total contact time” in this specification.

In embodiment 2-1, the contact time between dipicolinic acid or a salt thereof and the spore-forming bacterium in step 1 before starting step 2A is preferably 30 seconds or more, more preferably from the viewpoint of further improving the germicidal effect. 1 minute or more, more preferably 3 minutes or more, more preferably 5 minutes or more, more preferably 8 minutes or more, more preferably 10 minutes or more, more preferably 15 minutes or more, more preferably 20 minutes or more, more preferably 25 From the viewpoint of economy, it is preferably 60 minutes or less, more preferably 50 minutes or less, more preferably 40 minutes or less, more preferably 35 minutes or less, more preferably 30 minutes or less, more Preferably it is 25 minutes or less, More preferably, it is 20 minutes or less, More preferably, it is 15 minutes or less, More preferably, it is 10 minutes or less. The contact time is preferably 30 seconds to 60 minutes, more preferably 1 minute to 60 minutes, more preferably 3 minutes to 60 minutes, and more preferably 5 minutes to 60 minutes from the viewpoint of further improving the germicidal effect. Minutes, more preferably 5-50 minutes, more preferably 5-40 minutes, more preferably 8-40 minutes, more preferably 10-40 minutes, more preferably 15-40 minutes, more preferably 20 minutes to 40 minutes, more preferably 25 minutes to 40 minutes. The contact time is preferably 25 to 35 minutes from the viewpoint of further improving the germicidal effect and economical efficiency.

In aspect 2-1, the total contact time of the spore-forming bacterium and dipicolinic acid or a salt thereof in step 1 is preferably 30 minutes or more, more preferably 35 minutes or more, from the viewpoint of further improving the germination effect. More preferably 40 minutes or more, more preferably 45 minutes or more, more preferably 50 minutes or more, more preferably 55 minutes or more, more preferably 60 minutes or more, and from the viewpoint of economy, preferably 90 minutes or less. More preferably, it is 80 minutes or less, More preferably, it is 70 minutes or less, More preferably, it is 65 minutes or less.
Here, when step 2A ends before step 1, the time of step 1 from the end of step 2A to the end of step 1 is not included in the total contact time.

条件 Other conditions such as temperature and pH during the period in which Step 1 or Step 2A is carried out independently are the same as those in the embodiment in which Step 2 is carried out after Step 1 is completed.

[Aspect 2-2: Start step 1 and step 2A simultaneously]
The germination method having a period in which Step 1 and Step 2A of the present invention are performed simultaneously can start Step 1 and Step 2A simultaneously. That is, the contact between the spore-forming bacteria and dipicolinic acid or a salt thereof at pH 6 or less can be started simultaneously with the physical means. When heat is used as the physical means, the temperature when performing Step 1 and Step 2A at the same time is the same as the temperature of the step of sterilization by the heat of Step 2A. That is, from the beginning, dipicolinic acid or a salt thereof and a spore-forming bacterium are brought into contact with each other under the condition of pH 6 or less in the temperature range of the step of sterilization by the heat of step 2A.

[Aspect 2-3: Start step 2A first]
The germination method having a period in which Step 1 and Step 2A of the present invention are performed simultaneously can start Step 2A first. That is, the physical means of step 2A is first applied to the spore-forming bacteria, and contact between the spore-forming bacteria and dipicolinic acid or a salt thereof is started while the physical means is being applied. When heat is used as a physical means, the temperature of the spore-forming bacterium is set in the temperature range of step 2A in advance, and dipicolinic acid or a salt thereof is brought into contact with the spore-forming bacterium at a pH of 6 or less while the temperature is maintained.

<Sprouting aid composition>
The germination aid composition of the present invention is a composition containing dipicolinic acid or a salt thereof. The germination aid composition of the present invention is used for a treatment performed for the purpose of germination. Further, it is distinguished from a bactericide composition intended to kill at normal temperature (around 20 ° C. to 38 ° C.). In particular, it refers to a composition that can exert a germicidal effect or a bactericidal effect by sterilization by physical or chemical means after Step 1 of the present invention or simultaneously with Step 1. In the present invention, the germination aid composition may be used in step 1.

The sprouting aid composition includes a liquid composition or a solid composition.

The liquid sprouting aid composition comprises dipicolinic acid or a salt thereof and a solvent.

When the sprouting aid composition is a liquid, examples of the solvent contained include water and a hydrophilic solvent. Examples of the hydrophilic solvent include alcohols such as ethanol, methanol, and isopropanol, polyhydric alcohols such as glycerin, ethylene glycol, and propylene glycol, and carbitols such as methyl carbitol and ethyl carbitol. The solvent is preferably water or a mixture of water and a hydrophilic solvent, more preferably water, from the viewpoint of further improving the germicidal effect and economical efficiency.

From the viewpoint of convenience, the liquid germination aid composition is preferably a solution of dipicolinic acid or a salt thereof, more preferably an aqueous solution of dipicolinic acid or a salt thereof.

The liquid germination aid composition can further contain a pH adjuster. The pH adjuster contained in the liquid germination aid composition includes commonly used acids and bases, for example, inorganic acids such as hydrochloric acid and sulfuric acid, organic acids such as lactic acid and citric acid or their salts, sodium hydroxide And inorganic bases such as potassium hydroxide and organic bases such as triisopropanolamine. As the pH adjuster, from the viewpoint of versatility, the acid is preferably an inorganic acid such as hydrochloric acid or sulfuric acid, and the base is preferably an inorganic base such as sodium hydroxide or potassium hydroxide.

The pH at 24 ° C. of the liquid germination aid composition, preferably a solution of dipicolinic acid or a salt thereof, more preferably an aqueous solution of dipicolinic acid or a salt thereof, is preferably 1 or more, more preferably from the viewpoint of safety. 1.2 or more, and 6 or less, preferably 5 or less, more preferably 4 from the viewpoint of further improving the germination effect when the spore-forming bacterium is brought into contact with a solution of dipicolinic acid or a salt thereof. Hereinafter, it is more preferably 3 or less, more preferably 2.5 or less, and more preferably 1.8 or less. The pH at 24 ° C. of the solution of dipicolinic acid or a salt thereof is preferably 1 to 6, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, More preferably, it is 1 to 2.5, and more preferably 1.2 to 1.8.

The content of dipicolinic acid in the liquid germicidal aid composition, preferably in a solution of dipicolinic acid or a salt thereof, more preferably in an aqueous solution of dipicolinic acid or a salt thereof, is from the viewpoint of further improving the germicidal effect. Preferably it is 0.05 mM or more, More preferably, it is 0.5 mM or more, More preferably, it is 3 mM or more, More preferably, it is 5 mM or more, More preferably, it is 8 mM or more, and stability and economical efficiency of the solution of dipicolinic acid or its salt From this viewpoint, it is preferably 30 mM or less, more preferably 25 mM or less, more preferably 20 mM or less, more preferably 15 mM or less, and more preferably 12 mM or less. In addition, the content of dipicolinic acid in the solution of dipicolinic acid or a salt thereof is preferably 0.05 to 30 mM, more preferably 0.5 to 30 mM, more preferably 3 to 30 mM. It is preferably 3 to 25 mM, more preferably 3 to 20 mM, more preferably 3 to 15 mM, more preferably 5 to 15 mM, more preferably 8 to 12 mM.

The germination aid composition of the present invention is a composition containing dipicolinic acid or a salt thereof and a pH adjuster, water or other solvent, and does not contain any of peracetic acid, sulfoperoxycarboxylic acid, or dicarboxylic acid diester. obtain. Not included here indicates that the germination aid composition does not have a germicidal effect, preferably 1000 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less, more preferably 1 ppm or less, more preferably It is substantially 0 ppm. Here, “substantially” is below the detection limit.

液体 The liquid germicidal aid composition of the present invention is preferably a solution of dipicolinic acid or a salt thereof, more preferably an aqueous solution of dipicolinic acid or a salt thereof. In this case, the germination aid composition can be prepared by mixing, preferably dissolving, powdered dipicolinic acid or a salt thereof with water or another solvent.

Examples of the solid germination aid composition include dipicolinic acid or a salt thereof or dipicolinic acid or a salt thereof and a solidifying agent.

The solidifying agent contained in the solid germination aid composition is not limited to this, but polyethylene glycol having a number average molecular weight of 1,000 to 100,000; carnauba wax, candelilla wax, jojoba oil Waxes such as beeswax and lanolin; hydrocarbons having 15 or more carbon atoms such as paraffin, petrolatum, ceresin and microcrystalline wax; higher fatty acids having 12 to 22 carbon atoms such as lauric acid, myristic acid and stearic acid; cetyl alcohol; Examples thereof include higher alcohols having 14 to 22 carbon atoms such as stearyl alcohol.

The solid sprouting aid composition can further contain a pH adjuster. The pH adjuster contained in the solid germination aid composition includes commonly used acids and bases, for example, inorganic acids such as hydrochloric acid and sulfuric acid, organic acids such as lactic acid and citric acid or their salts, sodium hydroxide And inorganic bases such as potassium hydroxide and organic bases such as triisopropanolamine. As the pH adjuster, from the viewpoint of versatility, the acid is preferably an inorganic acid such as hydrochloric acid or sulfuric acid, and the base is preferably an inorganic base such as sodium hydroxide or potassium hydroxide.

The content of dipicolinic acid in the solid germination aid composition is preferably 0.1% by mass or more, more preferably 1% by mass or more, more preferably 5% by mass from the viewpoint of further improving the germination effect. Or more, more preferably 10% by mass or more, more preferably 20% by mass or more, more preferably 30% by mass or more, more preferably 40% by mass or more, more preferably 50% by mass or more, and from the viewpoint of economy Therefore, it is preferably 100% by mass or less, more preferably 90% by mass or less, more preferably 80% by mass or less, more preferably 70% by mass or less, more preferably 60% by mass or less, more preferably 50% by mass or less. It is.

The content of the solidifying agent in the solid germination aid composition is preferably 0% by mass or more, more preferably 10% by mass or more, more preferably 20% by mass or more, more preferably from the viewpoint of simplicity. 30% by mass or more, more preferably 40% by mass or more, more preferably 50% by mass or more, and from the viewpoint of further improving the germicidal effect, preferably from 99.9% by mass or less, More preferably 99% by mass or less, more preferably 95% by mass or less, more preferably 90% by mass or less, more preferably 80% by mass or less, more preferably 70% by mass or less, more preferably 60% by mass or less, More preferably, it is 50 mass% or less.

When the germination aid composition of the present invention is used in step 1 of the germination method of the present invention, the pH at 24 ° C. of the solution containing the spore-forming bacteria and the germination aid composition is a safety point of view. From the viewpoint of further improving the germicidal effect, preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or more. It is preferable to use it so that it may become 0.5 or less, more preferably 1.8 or less. Moreover, when the germination aid composition of the present invention is used in step 1 of the germination method of the present invention, the pH at 24 ° C. of the solution containing the spore-forming bacteria and the germination aid composition is as described above. From a comprehensive viewpoint, it is preferably 1 to 6, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, more preferably 1 to 2.5, more preferably 1.2 to 1.. It is preferably used so that it is 8.

When the sprouting aid composition of the present invention is used in step 1 of the sprouting method of the present invention, the content of dipicolinic acid in the solution containing the spore-forming bacteria and the sprouting aid composition is From the viewpoint of further improving the effect, it is preferably 0.05 mM or more, more preferably 0.5 mM or more, more preferably 3 mM or more, more preferably 4 mM or more, more preferably 6 mM or more, more preferably 8 mM or more, and From the viewpoint of stability and economics of a solution of dipicolinic acid or a salt thereof, preferably 30 mM or less, more preferably 25 mM or less, more preferably 20 mM or less, more preferably 15 mM or less, more preferably 12 mM, more preferably 10 mM. It is preferably used as follows. Moreover, when the germination aid composition of the present invention is used in step 1 of the germination method of the present invention, the content of dipicolinic acid in the solution containing the spore-forming bacteria and the germination aid composition is Taking the above viewpoints together, it is preferably 0.05 to 30 mM, more preferably 0.5 to 30 mM, more preferably 3 to 30 mM, more preferably 3 to 25 mM, more preferably 3 to 20 mM, more preferably 3 to 3 mM. It is preferable to use 15 mM, more preferably 4 to 15 mM, more preferably 6 to 12, and more preferably 8 to 10 mM.

ジ Dipicolinic acid or a salt thereof of the present invention can be used either synthetically or by fermentation using a method of extraction from microorganisms.

に関し Regarding the embodiment described above, the present invention discloses the following germination method and germination aid composition.

<Section 1>
The germination method of performing the following step 2 after completion of the following step 1:
Step 1: A step of bringing a spore-forming bacterium into contact with dipicolinic acid or a salt thereof under a condition of pH 6 or lower; and Step 2: a step of sterilizing the spore-forming bacterium.

<Section 2>
The germination method according to <Item 1>, wherein the step 2 is the following step 2A.
Step 2A: Step of physically sterilizing spore-forming bacteria.

<Section 3>
<2> The germination method according to <2>, wherein Step 2A is a step of sterilizing spore-forming bacteria with heat, pressure, or electromagnetic waves.

<Section 4>
The germination method according to <Item 2> or <Item 3>, wherein the spore-forming bacteria are sterilized at 50 ° C. or higher and 250 ° C. or lower in the step 2A.

<Section 5>
In the step 2A, the spore-forming bacterium is preferably 50 ° C. or higher, more preferably 55 ° C. or higher, more preferably 60 ° C. or higher, more preferably 65 ° C. or higher, more preferably 70 ° C. or higher, more preferably 75 ° C. or higher. And preferably 250 ° C. or lower, more preferably 200 ° C. or lower, more preferably 150 ° C. or lower, more preferably 120 ° C. or lower, more preferably 100 ° C. or lower, more preferably 90 ° C. or lower, more preferably 85 ° C. or lower. Further, it is preferably sterilized with 50 ° C to 250 ° C, more preferably 50 ° C to 200 ° C, more preferably 50 ° C to 150 ° C, more preferably 50 ° C to 100 ° C, more preferably 55 ° C to 100 ° C, More preferably 60 ° C to 100 ° C, more preferably 65 ° C to 90 ° C, more preferably 70 ° C to 90 ° C, more preferably Sterilized at 75 ° C. - 85 ° C., <claim 2> ~ quit method according to any one of <claim 4>.

<Section 6>
The time for sterilizing the spore-forming bacteria in the step 2A is preferably 3 minutes or more, more preferably 5 minutes or more, more preferably 8 minutes or more, more preferably 10 minutes or more, more preferably 20 minutes or more, more preferably 25 minutes or more, and preferably 90 minutes or less, more preferably 70 minutes or less, more preferably 50 minutes or less, more preferably 40 minutes or less, more preferably 35 minutes or less. Item 5. The germination method according to any one of items 5>.

<Section 7>
The germination method according to any one of <Item 1> to <Item 6>, wherein the step 2 is the following step 2B.
Step 2B: A step of chemically sterilizing spore-forming bacteria.

<Section 8>
The sprouting method according to <Item 7>, wherein Step 2B is a step of sterilizing spore-forming bacteria with a bactericide.

<Section 9>
The germination method according to <Item 8>, wherein the disinfectant is used as a liquid.

<Section 10>
The disinfectant is at least one selected from halides, alcohols, aldehydes, phenols and peroxides, hypochlorous acid or a salt thereof, chlorous acid and a salt thereof, dichloroisocyanuric acid and a component thereof Salt, trichloroisocyanuric acid and its salts, chlorine dioxide, popidone iodine, iodoform, ethanol, 1-propanol, isopropyl alcohol, methanol, formaldehyde, glutaraldehyde, lime, cresol, xylenol, isopropyl methylphenol, triclosan, peracetic acid, peroxy The germination method according to any one of <Item 8> to <Item 9>, wherein the method is one or more selected from hydrogen oxide and ozone.

<Section 11>
The disinfectant is at least one aqueous solution selected from hypochlorous acid and salts thereof, chlorous acid and salts thereof, dichloroisocyanuric acid and salts thereof, trichloroisocyanuric acid and salts thereof, and chlorine dioxide. 8>-<Item 10> The germination method according to any one of items 10.

<Section 12>
The effective chlorine concentration of the disinfectant is preferably 10 ppm or more, more preferably 50 ppm or more, more preferably 80 ppm or more, and 1000 ppm or less, more preferably 800 ppm or less, more preferably 500 ppm or less, more preferably 300 ppm or less, The germination method according to any one of <Item 8> to <Item 11>, more preferably 150 ppm or less.

<Section 13>
The germicidal method according to any one of <8> to <12>, wherein the disinfectant is gaseous and is at least one selected from ethylene oxide, formaldehyde, hydrogen peroxide, ozone, and chlorine dioxide.

<Section 14>
The pH at 24 ° C. upon contact in Step 1 is preferably 1 or more, more preferably 1.2 or more, and is preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2.5 or less, more preferably 1.8 or less, and preferably 1 to 6, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, more preferably 1 to 2. The sprouting method according to any one of <Item 1> to <Item 13>, which is 0.5, more preferably 1.2 to 1.8.

<Section 15>
The germination method according to any one of <Item 1> to <Item 14>, wherein in the step 1, the spore-forming bacterium is contacted with dipicolinic acid or a salt thereof in a liquid.

<Section 16>
The germination method according to any one of <Item 1> to <Item 15>, wherein the content of dipicolinic acid in the liquid is 0.05 mM or more and 30 mM or less.

<Section 17>
The content of dipicolinic acid in the liquid is preferably 0.05 mM or more, more preferably 0.5 mM or more, more preferably 3 mM or more, more preferably 4 mM or more, more preferably 6 mM or more, more preferably 8 mM or more. And preferably 30 mM or less, more preferably 25 mM or less, more preferably 20 mM or less, more preferably 15 mM or less, more preferably 12 mM, more preferably 10 mM or less, and preferably 0.05 to 30 mM, more Preferably 0.5-30 mM, more preferably 3-30 mM, more preferably 3-25 mM, more preferably 3-20 mM, more preferably 3-15 mM, more preferably 4-15 mM, more preferably 6-12, Any one of <Item 1> to <Item 16>, more preferably 8 to 10 mM. Stop method claim wherein.

<Section 18>
The germination method according to any one of <Item 1> to <Item 17>, wherein Step 1 is performed at 15 ° C. or more and less than 50 ° C.

<Section 19>
Step 1 is preferably 15 ° C. or higher, more preferably 20 ° C. or higher, and preferably less than 50 ° C., more preferably 49 ° C. or lower, more preferably 45 ° C. or lower, more preferably 40 ° C. or lower, more Preferably, the reaction is carried out at 30 ° C. or less, preferably 15 ° C. to less than 50 ° C., more preferably 15 ° C. to 49 ° C., more preferably 15 ° C. to 40 ° C., more preferably 15 to 30 ° C. <Item 1 The sprouting method according to any one of> to <18>.

<Section 20>
The germination method according to any one of <Item 1> to <Item 19>, wherein the step 1 is performed for 5 minutes to 60 minutes.

<Section 21>
The step 1 is preferably performed for 30 seconds or more, more preferably 1 minute or more, more preferably 3 minutes or more, more preferably 5 minutes or more, more preferably 8 minutes or more, more preferably 10 minutes or more, more preferably 15 Minutes, more preferably 20 minutes or more, more preferably 25 minutes or more, and 60 minutes or less, more preferably 50 minutes or less, more preferably 40 minutes or less, more preferably 35 minutes or less, and preferably 30 Second to 60 minutes, more preferably 1 minute to 60 minutes, more preferably 3 minutes to 60 minutes, more preferably 5 minutes to 60 minutes, more preferably 5 minutes to 50 minutes, more preferably 5 minutes to 40 minutes, More preferably 8 minutes to 40 minutes, more preferably 8 minutes to 35 minutes, more preferably 10 minutes to 35 minutes, more preferably 15 minutes to 35 minutes, more preferably 20 minutes to 35 minutes, more preferably. Stop method of the performing 25 minutes to 35 minutes, <claim 1> to any one of claims <claim 20>.

<Section 22>
The germination method which has the period which performs the following process 1 and the following process 2A simultaneously:
Step 1: A step of bringing a spore-forming bacterium into contact with dipicolinic acid or a salt thereof under a condition of pH 6 or lower; and Step 2A: a step of physically sterilizing the spore-forming bacterium.

<Section 23>
The germination method according to <Item 22>, wherein Step 2A is a step of sterilizing spore-forming bacteria with heat, pressure, or electromagnetic waves.

<Section 24>
The germination method according to <Item 22> or <Item 23>, wherein the spore-forming bacteria are sterilized at 50 ° C. or more and 250 ° C. or less in the step 2A.

<Section 25>
In the step 2A, the spore-forming bacterium is preferably 50 ° C. or higher, more preferably 55 ° C. or higher, more preferably 60 ° C. or higher, more preferably 65 ° C. or higher, more preferably 70 ° C. or higher, more preferably 75 ° C. or higher. And preferably 250 ° C. or lower, more preferably 200 ° C. or lower, more preferably 150 ° C. or lower, more preferably 120 ° C. or lower, more preferably 100 ° C. or lower, more preferably 90 ° C. or lower, more preferably 85 ° C. or lower. Further, it is preferably sterilized with 50 ° C to 250 ° C, more preferably 50 ° C to 200 ° C, more preferably 50 ° C to 150 ° C, more preferably 50 ° C to 100 ° C, more preferably 55 ° C to 100 ° C, More preferably 60 ° C to 100 ° C, more preferably 65 ° C to 90 ° C, more preferably 70 ° C to 90 ° C, more preferably Sterilized at 75 ° C. - 85 ° C., <claim 22> ~ quit method according to any one of <claim 24>.

<Section 26>
The germination method according to any one of <Item 22> to <Item 25>, wherein the time for sterilizing the spore-forming bacteria at 50 ° C. or higher and 250 ° C. or lower in Step 2A is 3 minutes or longer and 90 minutes or shorter.

<Section 27>
The time for sterilizing the spore-forming bacteria in the step 2A is preferably 3 minutes or more, more preferably 5 minutes or more, more preferably 8 minutes or more, more preferably 10 minutes or more, more preferably 20 minutes or more, more preferably 25 minutes or more, and preferably 90 minutes or less, more preferably 70 minutes or less, more preferably 50 minutes or less, more preferably 40 minutes or less, more preferably 35 minutes or less. Item 26> The germination method according to any one of items 26>.

<Section 28>
The germination method according to any one of <Item 22> to <Item 27>, wherein in the step 1, spore-forming bacteria are contacted with dipicolinic acid or a salt thereof in a liquid.

<Section 29>
The pH at 24 ° C. upon contact in Step 1 is preferably 1 or more, more preferably 1.2 or more, and is preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2.5 or less, more preferably 1.8 or less, and preferably 1 to 6, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, more preferably 1 to 2. The germination method according to any one of <Item 22> to <Item 28>, which is 0.5, more preferably 1.2 to 1.8.

<Section 30>
The germination method according to any one of <Item 22> to <Item 29>, wherein the concentration of dipicolinic acid in the solution is 0.05 mM or more and 30 mM or less.

<Section 31>
The content of dipicolinic acid in the liquid is preferably 0.05 mM or more, more preferably 0.5 mM or more, more preferably 3 mM or more, more preferably 4 mM or more, more preferably 6 mM or more, more preferably 8 mM or more. And preferably 30 mM or less, more preferably 25 mM or less, more preferably 20 mM or less, more preferably 15 mM or less, more preferably 12 mM, more preferably 10 mM or less, and preferably 0.05 to 30 mM, more Preferably 0.5-30 mM, more preferably 3-30 mM, more preferably 3-25 mM, more preferably 3-20 mM, more preferably 3-15 mM, more preferably 4-15 mM, more preferably 6-12, Any one of <Item 22> to <Item 30>, more preferably 8 to 10 mM. Quit The method according (1).

<Section 32>
The sprout killing method according to any one of <Item 22> to <Item 31>, wherein the step 1 is performed at a temperature of 15 ° C. or higher and lower than 50 ° C. before the start of the step 2A.

<Section 33>
Prior to the start of step 2A, step 1 is preferably performed at 15 ° C or higher, more preferably 20 ° C or higher, and preferably less than 50 ° C, more preferably 49 ° C or lower, more preferably 45 ° C or lower, and more. It is preferably carried out at 40 ° C. or less, more preferably 30 ° C. or less, and preferably 15 ° C. to less than 50 ° C., more preferably 15 ° C. to 49 ° C., more preferably 15 ° C. to 40 ° C., more preferably 15 to 30 ° C. The germination method according to any one of <Item 22> to <Item 32>, wherein the method is carried out at ° C.

<Section 34>
The germination method according to any one of <22> to <33>, wherein the step 1 is performed for 30 seconds to 60 minutes before the start of the step 2A.

<Section 35>
Prior to the start of Step 2A, the Step 1 is preferably performed for 30 seconds or more, more preferably 1 minute or more, more preferably 3 minutes or more, more preferably 5 minutes or more, more preferably 8 minutes or more, more preferably Is 10 minutes or more, more preferably 15 minutes or more, more preferably 20 minutes or more, more preferably 25 minutes or more, and preferably 60 minutes or less, more preferably 50 minutes or less, more preferably 40 minutes or less, more preferably 35 minutes or less, preferably 30 seconds to 60 minutes, more preferably 1 minute to 60 minutes, more preferably 3 minutes to 60 minutes, more preferably 5 minutes to 60 minutes, more preferably 5 minutes to 50 minutes. Minutes, more preferably 5 minutes to 40 minutes, more preferably 8 minutes to 40 minutes, more preferably 10 minutes to 40 minutes, more preferably 15 minutes to 40 minutes, more preferably 20 minutes to 40 minutes, more 25 minutes to 40 minutes preferred, and more preferably is carried out 25 minutes to 35 minutes, <claim 22> ~ quit method according to any one of <claim 34>.

<Section 36>
The germination method according to any one of <Item 22> to <Item 35>, wherein the step 1 is performed for 30 minutes to 90 minutes in total.

<Section 37>
The total of step 1 is preferably 30 minutes or more, more preferably 40 minutes or more, more preferably 45 minutes or more, more preferably 50 minutes or more, more preferably 55 minutes or more, more preferably 60 minutes or more, and The germination method according to any one of <Item 22> to <Item 36>, which is preferably performed for 90 minutes or less, more preferably for 80 minutes or less, more preferably for 70 minutes or less, and even more preferably for 65 minutes or less.

<Section 38>
A germicidal aid composition comprising dipicolinic acid or a salt thereof and having a pH of 6 or less.

<Section 39>
The pH at 24 ° C. is preferably 1 or more, more preferably 1.2 or more, and
Preferably it is 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2.
The germination aid composition according to <Item 38>, which is 5 or less, more preferably 1.8 or less.

<Section 40>
The germination aid composition according to <Item 38> or <Item 39>, which is a liquid composition containing 0.05 mM or more and 30 mM or less of dipicolinic acid.

<Section 41>
The content of dipicolinic acid is preferably 0.05 mM or more, more preferably 0.5 mM or more, more preferably 3 mM or more, more preferably 5 mM or more, more preferably 8 mM or more, and preferably 30 mM or less, more Preferably it is 25 mM or less, more preferably 20 mM or less, more preferably 15 mM or less, more preferably 12 mM or less, preferably 0.05 to 30 mM, more preferably 0.5 to 30 mM, more preferably 3 to 30 mM. Any one of <Item 38> to <Item 40>, more preferably 3 to 25 mM, more preferably 3 to 20 mM, more preferably 3 to 15 mM, more preferably 5 to 15 mM, and more preferably 8 to 12 mM. A germination aid composition according to item 1.

<Section 42>
Any one of <Item 38> to <Item 41>, which is a liquid composition containing dipicolinic acid or a salt thereof and a solvent, or a liquid composition comprising dipicolinic acid or a salt thereof, a pH adjuster, and a solvent. The germination aid composition as described.

<Section 43>
43. The germination aid composition according to any one of <Item 38> to <Item 42>, wherein the solvent is preferably water or a mixture of water and a hydrophilic solvent, more preferably water.

<Item 44>
The pH adjuster is preferably an inorganic acid such as hydrochloric acid or sulfuric acid, an organic acid such as lactic acid or citric acid or a salt thereof, preferably an inorganic base such as sodium hydroxide or potassium hydroxide, and an organic such as triisopropanolamine. The sprouting aid composition according to any one of <Item 38> to <Item 43>, which is selected from bases.

<Section 45>
It contains dipicolinic acid or a salt thereof, a pH adjuster and water or other solvent, and the content of peracetic acid, sulfoperoxycarboxylic acid and dicarboxylic acid diester is preferably 1000 ppm or less, more preferably 100 ppm or less, more preferably 10 ppm or less. The sprouting aid composition according to any one of <Item 38> to <Item 44>, more preferably 1 ppm or less, and more preferably substantially 0 ppm.

<Section 46>
Use of a sprouting aid composition as a sprouting aid, comprising dipicolinic acid or a salt thereof and having a pH of 6 or less.

Test 1: Examples 1-1 to 1-4, Comparative Example 1-1:
Bactericidal test A 10 mM dipicolinic acid aqueous solution (hereinafter also referred to as a DPA solution) adjusted to the pH shown in Table 1 and B. cereus NBRC 13494 strain were used as test strains and tested according to the following procedure. The effect was evaluated. The evaluation results are shown in Table 1. In addition, spore-forming bacteria were used in the experiments after confirming that 95% or more of the spores were formed by microscopic observation in all the following tests.
In the following Step 1-1, Step 1-2, and Step 2A-1, the spore-forming bacteria are in contact with dipicolinic acid (hereinafter also referred to as DPA) at pH 6 or lower, and are included in Step 1. Step 2A is included in Step 2. This test corresponds to Embodiment 2-1, paragraph 0052.

Dipicolinic acid aqueous solution: Reagents (manufactured by Wako Pure Chemical Industries, Ltd., production code 165-05342) were used for dipicolinic acid, ion-exchanged water was used for water, and hydrochloric acid or sodium hydroxide was used for the pH adjuster. The dipicolinic acid aqueous solution was prepared at 24 ° C. While measuring the pH of the aqueous dipicolinic acid solution with a desktop pH meter model 9611 (manufactured by HORIBA), a pH adjuster was dropped into the aqueous dipicolinic acid solution to adjust the pH of the aqueous dipicolinic acid solution. In the examples, unless otherwise specified, the pH is a pH at 24 ° C.

-procedure-
(1) Step 1-1: 100 μL of an aqueous dispersion (hereinafter referred to as spore solution) containing 10 ⁸ CFU / mL of a spore-forming spore-forming bacterium (B. cereus NBRC 13494 strain) is listed in the table. Each of these was mixed with 900 μL of 10 mM DPA solution having the pH adjusted to obtain a test solution.
(2) Step 1-2: The test solution of (1) above was allowed to stand at 24 ° C. for 30 minutes.
(3) Step 2A: After the above (2), the test solution was heat-treated at 80 ° C. for 30 minutes.
An aluminum block thermostatic chamber MD-01N-110 manufactured by Major Science was used for the heating process.
(4) The test solution heat-treated in (3) was serially diluted with sterilized water, each diluted solution was smeared on LB agar medium (manufactured by BD), and cultured at 30 ° C. for 16 hours.
(5) After culturing in (4), the number of viable bacteria X (CFU / mL) was determined from the number of colonies that had grown.
(6) The germination effect was evaluated by the germination effect index obtained by the following formula.
Here, the larger the germination effect index, the higher the germination effect.

(Sprouting effect index) = Log 10 [(control viable cell count) / (viable cell count X)]

Here, the viable cell count of the control (CFU / mL) was determined by performing the above (1) to (6) using sterilized water adjusted to each pH instead of the DPA solution. is there. In the table, the number of viable bacteria X and the number of viable bacteria in the control are also shown in logarithm with 10 as the base. Since the viable cell count X of Example 1-1 was below the detection limit, the viable cell count X was calculated as 1 for convenience.

As shown in Table 1, Examples 1-1 to 1-4 are superior to Comparative Example 1-1 in that the germination method of the present invention has excellent germination and bactericidal effects.

Test 2: Examples 2-1 to 2-3:
A test was conducted under the same conditions as in Example 1-2, except that a DPA solution having the concentration shown in Table 2 was used instead of the 10 mM dipicolinic acid aqueous solution, and the germicidal effect was evaluated. The evaluation results are shown in Table 2.

Test 3: Example 3-1:
A test similar to Example 1-2 was performed except that the standing time in step 1-2 was changed to the time described in Table 3, and the germicidal effect was evaluated. The evaluation results are shown in Table 3. Here, the standing time of Step 1-2 is 0, indicating that Steps 1 and 2 were started simultaneously.

Test 4: Examples 4-1 to 4-3:
A test similar to Example 1-2 was performed except that the temperature of the heat treatment in Step 2A was changed to the heating temperature described in Table 4, and the germicidal effect was evaluated. The evaluation results are shown in Table 4.

In Example 4-3, the germicidal effect was relatively high even in the control, and the bactericidal effect index was small.

Test 5: Examples 5-1 to 5-2:
A test similar to Example 1-2 was performed except that the heat treatment time in Step 2A was changed to the heat time described in Table 5, and the germicidal effect was evaluated. The evaluation results are shown in Table 5.

Test 6: Comparative examples 6-1 to 6-4:
Using the aqueous solution described in Table 6, the same test as in Test 1 was performed to evaluate the germicidal effect. Specifically, a test was conducted using each drug having a concentration shown in Table 6 instead of DPA, and the germicidal effect was evaluated. The evaluation results are shown in Table 6. The water used was ion exchange water, and the pH adjuster used hydrochloric acid or sodium hydroxide. Moreover, the chemical | medical agent of Table 6 is as follows.

DPA-Ca: prepared using dipicolinic acid Ca salt, DPA (manufactured by Wako Pure Chemical Industries, Ltd., production code 165-05342) and calcium chloride (manufactured by Wako Pure Chemical Industries, Ltd., production code 039-00475).
L-alanine: manufactured by Wako Pure Chemical Industries, Ltd., production code 010-01042
Inosine: manufactured by Wako Pure Chemical Industries, Ltd., production code 095-00233

As shown in Table 6, Examples 1-2 and 1-4 show an excellent germination effect as compared with Comparative Examples 6-1 to 6-4. That is, the germination method of the present invention using a DPA solution having a pH of 6 or less is compared to the case of using normal DPA-Ca at a normal pH or using L-alanine or L-alanine and inosine in combination. Has an excellent germination effect.

Test 7: Examples 7-1 and 7-2:
Bactericidal test Using a 10 mM dipicolinic acid aqueous solution (hereinafter also referred to as DPA solution) adjusted to pH 2.2 and B. cereus NBRC 13494 as a test strain, a spore-killing test was performed according to the following procedure. The effect was evaluated. Table 7 shows the evaluation results.

In addition, in the following Step 1-1 and Step 1-2, the spore-forming bacterium is in contact with dipicolinic acid (hereinafter also referred to as DPA) at a pH of 6 or less, and is included in Step 1. Step 2B is included in Step 2. This test corresponds to aspect 1 of the 0052 paragraph.

-procedure-
(1) Step 1-1: 100 μL of an aqueous dispersion (hereinafter referred to as spore solution) having a spore-forming spore-forming bacterium (B. cerus NBRC 13494 strain) content of 10 ⁸ CFU / mL and a pH of 2. 2 were mixed with 900 μL of 10 mM DPA solution to prepare a test solution.
(2) Step 1-2: The above test solution (1) was allowed to stand at 24 ° C. for the time shown in Table 7.
(3) Step 1-3: The test solution in Step 1-2 was centrifuged at 12 krpm for 10 minutes in a centrifuge (Tabletop Micro Cooling Centrifuge 3500 manufactured by Kubota Corporation), and the supernatant was removed.
(4) Step 2B: After Step 1-3, the supernatant was removed, and 1 mL of an aqueous sodium hypochlorite solution having the concentration shown in Table 7 was immediately added, and the mixture was allowed to stand at 24 ° C. for 10 minutes.
(5) Dilute the test solution of Step 2B 10 times by adding 4.5 mL of LP solution containing 0.1% sodium thiosulfate to 0.5 mL of the test solution of Step 2B, and leave it for 10 minutes. Then, the bactericidal action of sodium hypochlorite was stopped.
(6) The test solution of (5) was serially diluted with sterilized water, and 100 μL of each diluted solution was smeared on an LB agar medium (manufactured by BD) and cultured at 30 ° C. for 16 hours.
(7) The viable cell count X (CFU / mL) was determined from the number of colonies that had grown after the culture of (6).
(8) The germination effect was evaluated by the germination effect index obtained by the following formula.
Here, the larger the germination effect index, the higher the germination effect.

Here, the viable cell count (CFU / mL) of the control was determined by performing the above (1) to (8) using sterilized water adjusted to pH 2.2 instead of the DPA solution. Is a number. In the table, the number of viable bacteria X and the number of viable bacteria in the control are also shown in logarithm with 10 as the base.

The drugs used in Test 7 are as follows.
-Sodium hypochlorite: manufactured by Nankai Chemical Co., Ltd.-LP solution containing 0.1% sodium thiosulfate: LP diluent "DAIGO" manufactured by Wako Pure Chemical Industries, Ltd. and sodium thiosulfate manufactured by Wako Pure Chemical Industries, Ltd. Was prepared according to the manual for LP Diluent “DAIGO”.

Normally, effective chlorine concentration of about 1000 ppm is required for germination, but as shown in Table 7, even when a lower concentration of sodium hypochlorite is used, the germination effect index is high, and therefore, step 1 It was shown that the germination and bactericidal effects are excellent even with a low concentration of sodium hypochlorite.

<Confirmation of germination>
In the present specification, “germination” can be confirmed, for example, by confirming darkening of spores. As a method for confirmation, after step 1 and step 2, the presence or absence of darkened spores can be confirmed. That is, the presence or absence of darkening is confirmed with a phase contrast microscope. Here, when germination is not occurring, spores that appear white are darkened when germinated, and those that are darkened indicate germination.

The germination method of the present invention can be used in a wide range of applications such as linen cleaning, food spoilage prevention, and environmental purification.

Claims

The germination method of performing the following step 2 after completion of the following step 1:
Step 1: A step of bringing a spore-forming bacterium into contact with dipicolinic acid or a salt thereof under a condition of pH 6 or lower; and Step 2: a step of sterilizing the spore-forming bacterium.
The germination method according to claim 1, wherein the step 2 is the following step 2A.
Step 2A: Step of physically sterilizing spore-forming bacteria.
The germination method according to claim 1, wherein the step 2 is the following step 2B.
Step 2B: A step of chemically sterilizing spore-forming bacteria.
The germination method according to claim 2, wherein the step 2A is a step of sterilization by heat.
The germination method of Claim 3 whose said process 2B is a process of disinfecting a spore formation microbe with a disinfectant.
The spore killing method according to any one of claims 1 to 5, wherein in the step 1, spore-forming bacteria are contacted with dipicolinic acid or a salt thereof in a liquid.
The germination method according to claim 6, wherein the content of dipicolinic acid in the liquid is 0.05 mM or more and 30 mM or less.
The germination method according to any one of claims 1 to 7, wherein the step 1 is performed at 15 ° C or more and less than 50 ° C.
The germination method according to any one of claims 1 to 8, wherein the step 1 is performed for 5 minutes to 60 minutes.
The germination method which has the period which performs the following process 1 and the following process 2A simultaneously:
Step 1: A step of bringing a spore-forming bacterium into contact with dipicolinic acid or a salt thereof under a condition of pH 6 or lower; and Step 2A: a step of physically sterilizing the spore-forming bacterium.
The germination method according to claim 10, wherein the step 2A is a step of sterilization by heat.
The spore killing method of Claim 10 or 11 which sterilizes a spore formation microbe in 50 degreeC or more and 250 degrees C or less in the said process 2A.
The germination method according to claim 12, wherein the time for sterilizing the spore-forming bacteria at 50 ° C or higher and 250 ° C or lower in the step 2A is 3 minutes or longer and 90 minutes or shorter.
The spore killing method according to any one of claims 10 to 13, wherein, in the step 1, the spore-forming bacterium and dipicolinic acid or a salt thereof are contacted in a liquid.
The germination method according to claim 14, wherein the content of dipicolinic acid in the liquid is 0.05 mM or more and 30 mM or less.
The germination method according to any one of claims 10 to 15, which has a period of performing the step 1 at 15 ° C or higher and lower than 50 ° C before the start of the step 2A.
The germination method according to any one of claims 10 to 16, wherein the step 1 is performed for 30 seconds to 60 minutes before the start of the step 2A.
The germination method according to any one of claims 10 to 17, wherein the step 1 is carried out for 30 minutes or more and 90 minutes or less in total.
A germicidal aid composition containing dipicolinic acid and having a pH of 6 or less.
The germicidal aid composition according to claim 19, which is a liquid composition containing dipicolinic acid in an amount of 0.05 mM to 30 mM.