WO2017017810A1

WO2017017810A1 - Method for killing spores

Info

Publication number: WO2017017810A1
Application number: PCT/JP2015/071499
Authority: WO
Inventors: 知美阪井
Original assignee: 花王株式会社
Priority date: 2015-07-29
Filing date: 2015-07-29
Publication date: 2017-02-02
Also published as: JP6537611B2; TWI696427B; JPWO2017017810A1; TW201707574A; SG11201800022VA

Abstract

A method for killing spores according to the present invention comprises carrying out step 1, step 2 and step 3. Specifically, step 1 comprises bringing a spore-forming bacterium into contact with dipicolinic acid or a salt thereof, step 2 comprises bringing the spore-forming bacterium into contact with a cationic surfactant, and step 3 comprises warming the spore-forming bacterium to 50ºC or higher. There may be a period during which step 1, step 2 and step 3 are carried out simultaneously.

Description

Sprouting method

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

Spore-forming bacteria such as Bacillus and Clostridium form a strong shell structure and form spores with extremely high resistance to heat and drugs. Certain spore-forming bacteria 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 sterilized by heating, but spore-forming bacteria that are heat resistant cannot be sterilized by heat sterilization. As a result, damage to the dead due to nosocomial infection of spore-forming bacteria via linen products has also occurred.

In order to sterilize spore-forming bacteria, in many cases, it is sterilized with high-pressure steam, or a strong chemical sterilizing agent 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.

US2014 / 0308162 discloses a disinfecting and rinsing composition used in laundry detergents. US2014 / 0238445 discloses a cleaning, disinfecting and rinsing method including a cleaning step using a composition containing phosphinosuccinic acid and a disinfecting and rinsing step using a composition containing a percarboxylic acid. ing. Furthermore, WO2013 / 079308 discloses disinfecting bacteria containing spores. US 2004/0058878 discloses improving the germicidal effect by combining a germinating agent with a quaternary ammonium salt.

The present invention relates to a germination method for performing the following step 1, step 2, and step 3.
Step 1: A step of bringing a spore-forming bacterium into contact with dipicolinic acid or a salt thereof;
Step 2: A step of bringing a spore-forming bacterium into contact with a cationic surfactant; and Step 3: A step of heating the spore-forming bacterium to 50 ° C. or higher.
(However, after step 1 and step 2 are started, step 3 is ended).

The present invention relates to a germination aid composition comprising dipicolinic acid or a salt thereof and a cationic surfactant.

Detailed Description of the Invention

The techniques disclosed so far have not been sufficient for bactericidal effects on spores, that is, dormant spore-forming bacteria, and have been insufficient in reducing the number of spore bacteria.

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.

In food processing, harsh heat treatment or low-temperature distribution methods are used 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 efficiently and effectively sterilizing spore-forming bacteria that are harmful in the medical and food fields. Furthermore, this invention relates to the germination adjuvant composition used in order to disinfect a spore formation microbe. In the present invention, “sprouting” mainly intends to germinate and kill bacteria having a spore.

As a result of diligent investigations in view of the above problems, the present inventor has found that dipicolinic acid (2,6-pyridinedicarboxylic acid) or a salt thereof and a cationic surfactant are brought into contact with a spore-forming bacterium so as to be mild. It discovered that it could kill effectively by heating, and came to this invention.

According to the present invention, a sprouting method capable of sterilizing spore-forming bacteria is provided. 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 that has 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. When these spore-forming bacteria form spores, the spore-forming bacteria have heat resistance. Here, the heat resistance is a state in which a spore having an initial bacterial count of 10 ⁷ to 10 ⁹ CFU / mL is formed at 80 ° C. for 30 minutes, as shown by the spore-forming bacteria used in the examples of the present invention. When heated above, 10 ⁷ CFU / mL or more of spore-forming bacteria can survive.

In the present invention, “sprouting” mainly intends to germinate and kill bacteria having a spore. Germination refers to the phenomenon that spores of spore-forming bacteria germinate, thereby producing cells having normal growth and metabolic ability. Sprouting also includes reducing the total number of bacteria with spores present in the population. The spore killing effect is not particularly limited. For example, as shown in the Examples, the number of viable bacteria having spores was 10 ⁷ CFU / mL in which the initial number of bacteria was 10 ⁸ CFU / mL. Indicates a state of less than

This invention has the outstanding germination effect by performing the following process 1, the process 2, and the process 3.
Step 1: A step of bringing a spore-forming bacterium into contact with dipicolinic acid or a salt thereof;
Step 2: A step of bringing a spore-forming bacterium into contact with a cationic surfactant; and Step 3: A step of heating the spore-forming bacterium to 50 ° C. or higher.
(However, after step 1 and step 2 are started, step 3 is ended).

In the present invention, the order of the steps 1, 2, and 3 is not particularly limited. However, after starting Step 1 and Step 2, Step 3 is finished. That is, in a series of operations including Step 1, Step 2, and Step 3, starting Step 1 or Step 2 after the end of Step 3 or any of them is not included.

The reason why the germination method of the present invention has an excellent germination effect is not clear, but is considered as follows. By performing step 1, step 2 and step 3, the spore germinates. It is estimated that the cationic surfactant increases the affinity between dipicolinic acid or its salt and spore-forming bacteria, and that dipicolinic acid or its salt easily enters the spore-forming bacteria and effectively promotes germination when heated. The The germinated spore-forming bacteria can be easily sterilized in step 2 and step 3.

In addition, since the action of dipicolinic acid or a salt thereof in the spore-forming bacterium is not an action mediated by a specific receptor specific to the microbial species, it is considered to have a spore-killing effect regardless of the type of spore-forming bacterium.

In addition, it can confirm that it germinated here by confirming the heat resistant fall of a microbe. The decrease in heat resistance can be confirmed by treating spore-forming bacteria under an environment of, for example, 80 ° C. and examining the number of living bacteria.

In the present invention, when dipicolinate is used, the counter ion of dipicolinic acid is not particularly limited, but alkali metal ions such as sodium ion and potassium ion; alkaline earth metal ions such as calcium ion and magnesium ion are exemplified. The

In the present invention, the term “cationic surfactant” refers to a surfactant in which a hydrophilic portion is charged to a cation when dissolved in a solution, and is not limited, but includes a primary ammonium salt and a secondary ammonium salt. And tertiary ammonium salts and quaternary ammonium salts. More specifically, the cationic surfactant of the present invention is an alkyl trimethyl ammonium salt, an alkyl triethyl ammonium salt, an alkyl dimethyl ethyl ammonium salt, an alkyl methyl diethyl ammonium salt, a dialkyl dimethyl ammonium salt, a dialkyl diethyl ammonium salt, a dialkyl ethyl methyl. Examples include ammonium salts, benzalkonium salts, alkylpyridinium salts, quaternary ammonium salts such as alkylbenzetonium salts, and primary ammonium salts such as alkylamine salts.

Here, the alkyltrimethylammonium salt, alkyltriethylammonium salt, alkyldimethylethylammonium salt and alkylmethyldiethylammonium salt refer to a compound represented by the following general formula (1).

^Wherein any one of R 11 ^{~ R 14,} hydroxyl group, an ester group, an amide group hydrocarbon group having 3 or more carbon atoms which may have a ^remaining three of R 11 ^{~ R 14} Represents a methyl group or an ethyl group, and X ⁻ represents an inorganic or organic anionic compound. ]

Here, the number of carbon atoms of the hydrocarbon group having 3 or more carbon atoms is preferably 6 or more, more preferably 10 or more, more preferably 12 or more, more preferably from the viewpoint of further improving the germination effect. 14 or more, and preferably 22 or less, more preferably 20 or less, more preferably 18 or less. The hydrocarbon group having 3 or more carbon atoms may be a linear or branched alkyl group or alkenyl group, and is preferably a linear alkyl group. X ^- includes halogen ions such as chloride ions and bromide ions, sulfate ions, phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, nitrate ions, carbonate ions, bicarbonate ions, acetate ions and the like. Inorganic ions are preferred, halogen ions are more preferred, and chloride ions are more preferred.

Here, dialkyldimethylammonium, dialkyldiethylammonium salt and dialkylethylmethylammonium salt refer to compounds represented by the following general formula (2).

[Wherein any two of R ²¹ to R ²⁴ are hydrocarbon groups having 3 or more carbon atoms which may have a hydroxyl group, an ester group or an amide group, and the remaining two of R ²¹ to R ^24] Represents a methyl group or an ethyl group, and X ⁻ represents an inorganic or organic anionic compound. Here, any two hydroxyl groups of R ²¹ to R ²⁴ , an ester group, and an amide group that may have a hydrocarbon group having 3 or more carbon atoms may not be the same. ]

Here, the carbon number of the hydrocarbon group having 3 or more carbon atoms is preferably 6 or more, more preferably 10 or more, and preferably 22 or less, more preferably 20 from the viewpoint of further improving the germination effect. Below, it is more preferably 18 or less, more preferably 14 or less. The hydrocarbon group having 3 or more carbon atoms may be a linear or branched alkyl group or alkenyl group, and is preferably a linear alkyl group. X ^- includes halogen ions such as chloride ions and bromide ions, sulfate ions, phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, nitrate ions, carbonate ions, bicarbonate ions, acetate ions and the like. Inorganic ions are preferred, halogen ions are more preferred, and chloride ions are more preferred.

The benzalkonium salt refers to a compound represented by the following general formula (3).

Wherein, R ³¹ is a hydrocarbon group, X ^- represents an inorganic or organic anionic compounds. ]

The carbon number of R ³¹ is preferably 6 or more, more preferably 10 or more, more preferably 12 or more, more preferably 14 or more, and preferably 22 or less, more preferably 20 or less, more Preferably it is 18 or less. R ³¹ may be a linear or branched alkyl group or alkenyl group, and is preferably a linear alkyl group. X ⁻ is preferably a halogen ion such as chloride ion or bromide ion, sulfate ion, phosphate ion, hydrogen phosphate ion, dihydrogen phosphate ion, nitrate ion, carbonate ion, hydrogen carbonate ion, acetate ion and the like. It is done. Inorganic ions are preferred, halogen ions are more preferred, and chloride ions are more preferred.

Here, “alkyl” in the case of alkylpyridinium salt, alkylbenzetonium salt, or alkylamine salt preferably refers to a hydrocarbon group having 3 or more carbon atoms. The carbon number of the hydrocarbon group having 3 or more carbon atoms is preferably 6 or more, more preferably 10 or more, and is preferably 22 or less, more preferably 20 or less, more preferably 18 or less. The hydrocarbon group having 3 or more carbon atoms may be a linear or branched alkyl group or alkenyl group, and is preferably a linear alkyl group. *

As the cationic surfactant, dialkyldimethylammonium salt, alkyltrimethylammonium salt or benzalkonium salt is preferable, dialkyldimethylammonium salt and alkyltrimethylammonium salt are more preferable, and alkyltrimethylammonium salt is further preferable.

Here, the salt is not limited, but halides such as chloride and bromide, sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, nitrate, carbonate, bicarbonate, acetate, etc. Is mentioned. Inorganic salts are preferred, halides are more preferred, and chlorides are more preferred.

As the cationic surfactant, dialkyldimethylammonium chloride, alkyltrimethylammonium chloride or benzalkonium chloride is preferable, dialkyldimethylammonium chloride and alkyltrimethylammonium chloride are more preferable, and alkyltrimethylammonium chloride is more preferable.

The molar ratio of dipicolinic acid to the cationic surfactant ([molar concentration of dipicolinic acid] / [molar concentration of cationic surfactant]) is preferably 1/1000 or more, more preferably 1/100 or more, More preferably 1/50 or more, more preferably 1/20 or more, more preferably 1/10 or more, more preferably 1/5 or more, more preferably 1/2 or more, And it is preferably 1000 or less, more preferably 100 or less, more preferably 50 or less, more preferably 20 or less, more preferably 10 or less, more preferably 5 or less, more Preferably it is 2 or less.

Step 1 of the present invention is a step of bringing a spore-forming bacterium into contact with dipicolinic acid or a salt thereof. Step 2 of the present invention is a step in which spore-forming bacteria and a cationic surfactant are brought into contact with each other. Here, Step 1 and Step 2 include any of the following aspects.
(1) An embodiment in which Step 1 and Step 2 are performed simultaneously, that is, an embodiment in which contact is simultaneously made between a spore-forming bacterium, dipicolinic acid or a salt thereof and a cationic surfactant, and contact is ended simultaneously.
(2) A mode in which step 1 is performed first and step 2 is performed after step 1 is completed. For example, after performing Step 1 in which spore-forming bacteria are brought into contact with dipicolinic acid or a salt thereof, dipicolinic acid or a salt thereof is removed, and then a cationic surfactant is added to spore-forming bacteria and a cationic surfactant. Can be kept in contact with each other.
(3) A mode in which step 2 is performed first and step 1 is performed after step 2 is completed. For example, after performing Step 2 in which the spore-forming bacteria are brought into contact with the cationic surfactant, the cationic surfactant is removed, and then dipicolinic acid or a salt thereof is added to the spore-forming bacteria and dipicolinic acid or the The salt can be kept in contact.
(4) A mode in which step 1 is performed first and step 2 is started without ending step 1. Here, a spore-forming bacterium and dipicolinic acid or a salt thereof can be contacted first, and then a cationic surfactant can be contacted without removing dipicolinic acid or a salt thereof.
(5) A mode in which step 2 is performed first and step 1 is started without ending step 2. Here, spore-forming bacteria and a cationic surfactant can be contacted first, and then dipicolinic acid can be contacted without removing the cationic surfactant.

Dipicolinic acid or a salt thereof and a cationic surfactant can be provided in a liquid or solid state, respectively. In addition, dipicolinic acid or a salt thereof and a cationic surfactant are preferably provided as a germination aid composition from the viewpoint of simplicity.

The term “contact” as used herein refers to a method in which contact is made in a solution, or in the case where spore-forming bacteria are present on the surface of a solid, killing a liquid described later, such as a solution containing dipicolinic acid or a salt thereof and a cationic surfactant. The method of apply | coating a bud adjuvant composition to the solid substance surface is mentioned. Examples of the method for applying to the solid surface include a spraying method and a method using a tool such as a brush or a sponge. Here, the solid surface is not particularly limited, but refers to a hard surface or the like.

When the spore-forming bacterium and dipicolinic acid or a salt thereof are kept in contact with each other, the content of dipicolinic acid or a salt thereof in the solution at the time of contact is preferably from the viewpoint of further improving the germicidal effect. 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 the stability of the solution containing dipicolinic acid From the viewpoint, it is preferably 1 M or less, more preferably 200 mM or less, more preferably 100 mM or less, more preferably 50 mM or less, more preferably 20 mM or less. Further, when the spore-forming bacterium and dipicolinic acid or a salt thereof are contacted in a solution, the content of dipicolinic acid or a salt thereof in the solution is preferably 0.05 mM to 1 M, taking the above viewpoints together. More preferably, it is 0.05 to 200 mM, more preferably 3 to 100 mM, and more preferably 3 to 25 mM. Here, in the case of dipicolinate, the contents are all converted to acid (hereinafter, the same meaning unless otherwise specified).

In the contact, the relationship between the initial number of bacteria and the concentration of dipicolinic acid or a salt thereof is not particularly limited. For example, from the viewpoint of further improving the germicidal effect, the concentration of dipicolinic acid or a salt thereof with respect to the initial bacterial count of 10 ⁸ CFU / mL 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 1 M or less, more preferably 200 mM or less, more preferably 100 mM or less, more preferably 50 mM or less, more preferably Is 20 mM or less.

When the spore-forming bacteria and the cationic surfactant are kept in contact with each other in the solution, the content of the cationic surfactant in the solution at the time of the contact is preferably from the viewpoint of further improving the germicidal effect. 01 mM or more, more preferably 0.05 mM or more, more preferably 0.1 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 1000 mM or less, more preferably 500 mM or less, still more preferably 300 mM or less, more preferably 100 mM or less. In addition, when the spore-forming bacterium and the cationic surfactant are kept in contact with each other in the solution, the content of the cationic surfactant in the solution is preferably 0.01 to 1000 mM, taking the above viewpoints together, Preferably 0.05 to 1000 mM, more preferably 0.1 to 1000 mM, more preferably 0.5 to 500 mM, more preferably 3 to 300 mM, more preferably 4 to 100 mM, more preferably 6 to 100 mM, more preferably 8-100 mM.

When the spore-forming bacteria and the cationic surfactant are kept in contact with each other in the solution, the content of the cationic surfactant in the solution at the time of the contact is preferably 0.01 from the viewpoint of further improving the germicidal effect. It is at least mass%, more preferably at least 0.05 mass%, and preferably at most 10 mass%, more preferably at most 5 mass%, still more preferably at most 1 mass%. In addition, when the spore-forming bacterium, dipicolinic acid or a salt thereof and a cationic surfactant are kept in contact with each other in the solution, the content of the cationic surfactant in the solution is preferably 0. The content is 01 to 10% by mass, more preferably 0.05 to 5% by mass, and more preferably 0.05 to 1% by mass.

In the contact, the relationship between the initial number of bacteria and the concentration of the cationic surfactant is not particularly limited. For example, from the viewpoint of further improving the germicidal effect, the concentration of the cationic surfactant with respect to the initial bacterial count of 10 ⁸ CFU / mL is preferably 0.01 mM or more, more preferably 0.05 mM or more, more preferably 0.1 mM. Or more, more preferably 0.5 mM or more, more preferably 1 mM or more, more preferably 10 mM or more, more preferably 100 mM or more, and preferably 1000 mM or less, more preferably 500 mM or less, more preferably 300 mM or less, more Preferably it is 250 mM or less.

These preferable concentrations are the same regardless of whether dipicolinic acid or a salt thereof and a cationic surfactant are simultaneously contacted with spore-forming bacteria or when they are separately contacted.

Before starting Step 3 of the present invention, the temperature of Step 1 or Step 2 performed independently of Step 3 is preferably independently from the viewpoint of further improving the germicidal effect, preferably 0 ° C. or higher. Is 10 ° C. or more, more preferably 15 ° C. or more, more preferably 20 ° C. or more, and preferably less than 50 ° C., more preferably 49 ° C. or less, more preferably 45 ° C. or less, more preferably 40 ° C. or less, More preferably, it is 30 degrees C or less. In addition, the contact temperature between the spore-forming bacterium and dipicolinic acid, a salt thereof, or a solution containing a cationic surfactant in Step 1 and Step 2 performed independently of Step 3 is preferably 15 ° C. to It is less than 50 ° C., more preferably 15 ° C. to 40 ° C., more preferably 15 to 30 ° C.

These preferred temperatures are the same whether step 1 and step 2 are performed simultaneously or separately, but if step 1 and step 2 are performed separately, step 1 and step 2 The temperatures of 2 may be different or the same.

From the viewpoint of further improving the germicidal effect, the time of Step 1 or Step 2 performed independently of Step 3 of the present invention is preferably 1 second or more, more preferably 5 seconds or more, more preferably from the viewpoint of further improving the germicidal effect. Is 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 or more, more preferably 20 minutes or more, more preferably 25 minutes or more, and preferably 24 hours or less, more preferably 12 hours or less, more preferably 6 hours or less, more preferably 3 hours or less, more preferably 60 minutes or less, more Preferably it is 50 minutes or less, More preferably, it is 40 minutes or less, More preferably, it is 35 minutes or less. The contact time between dipicolinic acid or a salt thereof and the spore-forming bacterium in step 1 is preferably 1 second to 24 hours, more preferably 1 second to 6 hours, more preferably 1 second to It is 3 hours, more preferably 1 second to 60 minutes, more preferably 5 seconds to 60 minutes, and more preferably 30 seconds to 60 minutes.

<Step 3>
Step 3 of the present invention is a step of heating the spore-forming bacteria to 50 ° C. or higher.

Step 3 of the present invention is preferably a step of heating spore-forming bacteria at 50 ° C. or higher and 250 ° C. or lower.

The temperature for heating the spore-forming bacteria is 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., from the viewpoint of further improving the spore killing effect. More preferably, it is 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. From the above viewpoint, the temperature during heating in step 3 of the present invention is preferably 50 ° C. to 250 ° C., more preferably 50 ° C. to 100 ° C., more preferably 55 ° C. to 100 ° C., more preferably 60 ° C. It is -100 ° C, more preferably 65 ° C-90 ° C, more preferably 70 ° C-90 ° C, and more preferably 75 ° C-85 ° C.

Usually, 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 and Step 2, so that the germination effect can be obtained even at 100 ° C. or lower in Step 3. In the present invention, it is also possible to kill at a temperature higher than 100 ° C.

The time for heating in step 3 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 germicidal effect. Preferably it is 20 minutes or more, more preferably 25 minutes or more, and preferably 3 hours or less, more 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, it is 35 minutes or less. From the above viewpoint, the heating time in step 3 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. Minutes, more preferably 20 to 40 minutes, more preferably 25 to 35 minutes. This time may include a period simultaneously with Step 1 and / or Step 2.

The heating in step 3 of the present invention is not particularly limited as long as it results in an increase in the temperature of the system containing spore-forming bacteria. Examples of the heating include a method of applying dry heat, wet heat, boiling, hot water, steam heat, etc. to the spore-forming bacteria. Here, for example, a method of immersing spore-forming bacteria in hot water, a method of installing a solid surface such as a hard surface where spore-forming bacteria are present, a liquid containing spore-forming bacteria, etc., a cooking process in food processing, etc. Examples thereof include a method by heat treatment and a method in which the temperature of the spore increases as a result of laser irradiation or the like.

In Step 1, Step 2, and Step 3, the pH of the solution at 24 ° C. is not particularly limited, but is preferably 3 or more, more preferably 4 or more, and even more preferably 6 or more, from the viewpoint of safety. Therefore, it is preferably 12 or less, more preferably 9 or less, and more preferably 8.5 or less. The pH of the solution at 24 ° C. is preferably 3 to 12, more preferably 3 to 9, and more preferably 4 to 8.5, taking the above viewpoints together.

PH adjustment can be performed when dipicolinic acid or a salt thereof or a cationic surfactant solution is prepared in advance. Or it can also carry out after adding dipicolinic acid, its salt, or a cationic surfactant 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, 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.

In the germination method of the present invention, step 3 can be completed by rapidly cooling the spore-forming bacteria heated in step 3 with water containing ice or a cooled container. Alternatively, the spore-forming bacteria heated in step 3 can be cooled for a while and then step 3 can be completed. Here, the heated spore-forming bacterium may be in a state of being present in the liquid.

When spore-forming bacteria are present in the liquid, the spore killing method of the present invention may have a step of stirring the spore-forming bacteria before or after step 1, step 2, and step 3 or those steps. Step 1, step 2 and step 3 can be repeated.

Process 3 can be performed after completing Process 1 and Process 2 (Aspect 1).

In aspect 1, “perform step 3 after step 1 and step 2” means to perform step 3 after removing dipicolinic acid or a salt thereof and a cationic surfactant. It means that with the end. In aspect 1, after completing step 1 and step 2, preferably within 24 hours, more preferably within 12 hours, more preferably within 6 hours, more preferably within 1 hour, more preferably within 30 minutes, still more preferably The process proceeds to step 3 within 15 minutes, more preferably within 5 minutes, and even more preferably within 1 minute.

It may have a period of performing Step 1 or Step 2 and Step 3 at the same time (Aspect 2).

In aspect 2, “having a period in which step 1 or step 2 and step 3 are performed simultaneously” means performing step 3 after removing dipicolinic acid, a salt thereof, or a cationic surfactant. It means that, with the end of 2. In aspect 2, after step 1 or step 2, it is preferably within 24 hours, more preferably within 12 hours, more preferably within 6 hours, more preferably within 1 hour, more preferably within 30 minutes, even more preferably The process proceeds to step 3 within 15 minutes, more preferably within 5 minutes, and even more preferably within 1 minute.

You may have the period which performs the process 1, the process 2, and the process 3 simultaneously (aspect 3). Aspect 3 is divided as follows.
Aspect 3-1 Start Step 1 and / or Step 2 before Step 3 Aspect 3-2 Start Step 1, Step 2 and Step 3 at the same time Aspect 3-3 Step 3 before Step 1 and Step 2 Embodiment 3-4 Starting Step 1 or Step 2 and Step 3 at the same time Among Embodiment 3, from the viewpoint of further improving the germicidal effect, Embodiment 3-1 is preferable, but not limited thereto.

The aspect (mode 3) which has the period which performs the process 1, the process 2, and the process 3 simultaneously from the viewpoint of improving the germination method more is preferable.

<Sprouting aid composition>
The sprouting aid composition of the present invention is a composition comprising dipicolinic acid or a salt thereof and a cationic surfactant. The germination aid composition of the present invention is used for pre-heating treatment for the purpose of germination. Further, it is distinguished from a germicide composition intended to germinate using the composition itself at room temperature (about 20 ° C. to about 38 ° C.). In particular, it refers to a composition that can exert a germicidal effect by heating after Step 1 and Step 2 of the present invention or simultaneously with Step 1 and Step 2. In the present invention, the germination aid composition can be used in Step 1 and Step 2.

Examples of the germination aid composition include a liquid composition and a solid composition.

The liquid germination aid composition contains dipicolinic acid or a salt thereof, a cationic surfactant, and a solvent.

When the germination aid composition is a liquid, examples of the solvent included include water and a hydrophilic solvent. Examples of the hydrophilic solvent include monohydric 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. From the viewpoint of further improving the germicidal effect, the solvent is preferably water or a mixture of water and a hydrophilic solvent, more preferably water.

From the viewpoint of simplicity, the liquid germination aid composition is preferably a solution containing dipicolinic acid or a salt thereof and a cationic surfactant, more preferably an aqueous solution containing dipicolinic acid or a salt thereof and a cationic surfactant. is there.

The liquid germicidal 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 monoethanolamine, triethanolamine, and triisopropanolamine. As the pH adjuster, 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 containing dipicolinic acid or a salt thereof and a cationic surfactant, more preferably an aqueous solution containing dipicolinic acid or a salt thereof and a cationic surfactant is particularly limited. However, it is preferably 3 or more, more preferably 4 or more, and still more preferably 6 or more. From the viewpoint of safety, it is preferably 12 or less, more preferably 9 or less, more preferably 8.5. It is as follows. The pH of the solution at 24 ° C. is preferably 3 to 12, more preferably 3 to 9, and more preferably 4 to 8.5, taking the above viewpoints together.

The content of dipicolinic acid in a solution containing dipicolinic acid or a salt thereof and a cationic surfactant, and more preferably in an aqueous solution containing dipicolinic acid or a salt thereof and a cationic surfactant 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, from the viewpoint of further improving the germicidal effect. From the viewpoint of stability of a solution containing dipicolinic acid or a salt thereof and a cationic surfactant, it is preferably 1 M or less, more preferably 200 mM or less, more preferably 100 mM or less, more preferably 50 mM or less, More preferably, it is 20 mM or less. Further, the content of dipicolinic acid or a salt thereof in the liquid germination aid composition is preferably 0.05 mM to 1 M, more preferably 0.05 mM to 200 mM, more preferably, taking the above viewpoints into consideration. It is 3 mM to 100 mM, more preferably 3 mM to 15 mM, and more preferably 4 mM to 15 mM.

The liquid germination aid composition preferably comprises a cationic surfactant in a solution comprising dipicolinic acid or a salt thereof and a cationic surfactant, more preferably in an aqueous solution comprising dipicolinic acid or a salt thereof and a cationic surfactant. The content 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 from the viewpoint of further improving the germicidal effect. 8 mM or more, and preferably 1 M or less, more preferably 200 mM or less, more preferably 100 mM or less, more preferably 50 mM or less, more preferably 20 mM or less. The content of the cationic surfactant in the liquid germination aid composition is preferably 0.1 to 1 M, more preferably 0.5 to 500 mM, more preferably 3 to 3, in view of the above viewpoints. 300 mM, more preferably 4 to 100 mM, more preferably 6 to 100 mM, and 8 to 100 mM.

The content of the cationic surfactant in the liquid germination aid composition is preferably 0.01% by mass or more, more preferably 0.05% by mass or more, from the viewpoint of further improving the germination effect, and Preferably it is 10 mass% or less, More preferably, it is 5 mass% or less, More preferably, it is 1 mass% or less. Further, the content of the cationic surfactant in the liquid germination aid composition is preferably from 0.01 to 10% by mass, more preferably from 0.05 to 5% by mass, more preferably from the above viewpoints. Is 0.05 to 1% by mass.

The germination aid composition of the present invention comprises dipicolinic acid or a salt thereof and a cationic surfactant, water or other solvent, and does not contain any of peracetic acid, sulfoperoxycarboxylic acid, or dicarboxylic acid diester. possible. 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.

The liquid germination aid composition of the present invention, preferably a solution containing dipicolinic acid or a salt thereof and a cationic surfactant, more preferably an aqueous solution containing dipicolinic acid or a salt thereof and a cationic surfactant, is a powdered dipicoline It can be prepared by mixing an acid or a salt thereof or a cationic surfactant with water or other solvent, preferably by dissolving.

Examples of the solid germination aid composition include dipicolinic acid or a salt thereof and a cationic surfactant or dipicolinic acid or a salt thereof, a cationic surfactant, 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 germination 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 monoethanolamine, triethanolamine, and triisopropanolamine. As the pH adjuster, 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 using the germination aid composition of the present invention in step 1 and step 2 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 From the viewpoint of further improving the germicidal 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 the stability of a solution containing dipicolinic acid and a cationic surfactant, it is preferably 200 mM or less, more preferably 100 mM or less, more preferably 30 mM or less, more preferably 25 mM or less, more preferably 20 mM or less. It is preferred to be used as is. In addition, when the germination aid composition of the present invention is used in step 1 and step 2 of the germination method of the present invention, the inclusion of dipicolinic acid in the solution containing the spore-forming bacteria and the germination aid composition In view of the above viewpoints, the amount is preferably 0.05 to 200 mM, more preferably 0.5 to 100 mM, more preferably 3 to 30 mM, and more preferably 3 to 25 mM.

When the liquid germination aid composition is used in Step 1 and Step 2 of the germination method of the present invention, the content of the cationic surfactant in the solution containing the spore-forming bacteria and the germination aid composition However, the content of the cationic surfactant in the solution preferably containing dipicolinic acid and the cationic surfactant, more preferably in the aqueous solution containing dipicolinic acid and the cationic surfactant, from the viewpoint of further improving the germicidal effect, Preferably it is 0.1 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 1000 mM or less, more preferably Is 500 mM or less, more preferably 300 mM or less, more preferably 100 mM or less. Further, the content of the cationic surfactant when using the liquid germination aid composition is preferably 0.1 to 1000 mM, more preferably 0.5 to 500 mM, more preferably, taking the above viewpoints together. Is 3 to 300 mM, more preferably 4 to 100 mM, more preferably 6 to 100 mM.

When the liquid germination aid composition is used in Step 1 and Step 2 of the germination method of the present invention, the content of the cationic surfactant in the solution containing the spore-forming bacteria and the germination aid composition However, from the viewpoint of further improving the germicidal effect, it is preferably 0.01% by mass or more, more preferably 0.05% by mass or more, and preferably 10% by mass or less, more preferably 5% by mass or less, and still more preferably. Is 1% by mass or less. In addition, the content of the cationic surfactant when using the liquid germination aid composition is preferably 0.01 to 10% by mass, more preferably 0.05 to 5% by mass, considering the above viewpoints. %, More preferably 0.05 to 1% by mass.

The dipicolinic acid or salt thereof of the present invention can be used either synthetically or by fermentation using a method of extraction from microorganisms. As the cationic surfactant of the present invention, either a commercially available one or a synthetic one can be used.

The germination method and germination aid composition of the present invention can be used for sterilization washing of food processing equipment, sterilization washing of clothing, and the like.

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

<Section 1>
A germination method for performing the following Step 1, Step 2 and Step 3:
Step 1: A step of bringing a spore-forming bacterium into contact with dipicolinic acid or a salt thereof;
Step 2: A step of bringing a spore-forming bacterium into contact with a cationic surfactant; and Step 3: A step of heating the spore-forming bacterium to 50 ° C. or more (however, Step 3 is completed after Step 1 and Step 2 are started) It shall be).

<Section 2>
<Claim 1> The germination method according to <Item 1>, which has a period in which Step 1, Step 2, and Step 3 are performed simultaneously.

<Section 3>
The germination method according to <Item 1>, wherein Step 2 and Step 3 are performed after Step 1 is completed.

<Section 4>
The germination method according to <Item 1>, wherein Step 1 and Step 3 are performed after Step 2 ends.

<Section 5>
<Item 1> to <Item 4>, wherein the cationic surfactant is any one of a primary ammonium salt, a secondary ammonium salt, a tertiary ammonium salt, or a quaternary ammonium salt. How to kill.

<Section 6>
The cationic surfactant is a quaternary ammonium salt such as an alkyltrimethylammonium salt, a dialkyldimethylammonium salt, a benzalkonium salt, an alkylpyridinium salt, or an alkylbenzetonium salt, or a primary ammonium salt such as an alkylamine salt. Item 1. The sprouting method according to any one of <1> to <5>.

<Section 7>
The germination method according to any one of <Item 1> to <Item 6>, wherein the cationic surfactant is a quaternary ammonium salt represented by the general formula (1).

<Section 8>
Cationic surfactant is alkyltrimethylammonium salt, alkyltriethylammonium salt, alkyldimethylethylammonium salt, alkylmethyldiethylammonium salt, dialkyldimethylammonium salt, dialkyldiethylammonium salt, dialkylethylmethylammonium salt, benzalkonium salt, alkyl The germination method according to any one of <Item 1> to <Item 7>, which is at least one selected from the group consisting of a pyridinium salt and an alkylbenzetonium salt.

<Section 9>
The spore killing method according to any one of <1> to <8>, wherein in step 1 and step 2, a spore-forming bacterium, dipicolinic acid or a salt thereof, and a cationic surfactant are contacted in a liquid.

<Section 10>
The germination method according to <9>, wherein the content of dipicolinic acid or a salt thereof in the liquid is 0.05 mM or more and 200 mM or less.

<Section 11>
The germination method according to <Item 9>, wherein the content of the cationic surfactant in the liquid is 0.1 mM or more and 1000 mM or less.

<Section 12>
The concentration of dipicolinic acid or a salt thereof when the spore-forming bacterium contacts dipicolinic acid or a salt thereof is 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 Is 6 mM or more, more preferably 8 mM or more, and preferably 200 mM or less, more preferably 100 mM or less, more preferably 50 mM or less, more preferably 20 mM or less, any one of <Item 1> to <Item 11> Sprouting method.

<Section 13>
The concentration of the cationic surfactant when the spore-forming bacterium and the cationic surfactant are in contact is 0.1 mM or more, more preferably 0.5 mM or more, more preferably 3 mM or more, more preferably 4 mM or more, more preferably Any one of <Item 1> to <Item 12>, which is 6 mM or more, more preferably 8 mM or more, and preferably 1000 mM or less, more preferably 500 mM or less, more preferably 300 mM or less, more preferably 100 mM or less. The germination method as described.

<Section 14>
The concentration of the cationic surfactant when the spore-forming bacteria and the cationic surfactant are in contact is 0.01% by mass or more, more preferably 0.05% by mass or more, and preferably 10% by mass or less, more preferably <5> The germination method according to any one of <1> to <13>, wherein 5 is 5% by mass or less, more preferably 1% by mass or less.

<Section 15>
The germination method according to any one of <Item 1> to <Item 14>, wherein Step 1 and Step 2 are performed independently or simultaneously at 15 ° C. or more and less than 50 ° C.

<Section 16>
Step 1 and Step 2 performed independently of Step 3 are each independently or simultaneously, 15 ° C. or higher, more preferably 20 ° C. or higher, and preferably less than 50 ° C., more preferably 49 ° C. or lower. More preferably, the germination method according to any one of <Item 1> to <Item 15>, wherein the method is performed at 45 ° C. or lower, more preferably 40 ° C. or lower, more preferably 30 ° C. or lower.

<Section 17>
The time of Step 1 performed independently of Step 3, that is, the contact time between the spore-forming bacteria and dipicolinic acid or a salt thereof is preferably 1 second or longer, more preferably 5 seconds or longer, more preferably 30 seconds or longer. 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 or more, more preferably 20 minutes or more, More preferably, it is 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, from <Item 1> to <Item 16> Any one of the germination methods.

<Section 18>
The time of Step 2 when performed independently of Step 3, that is, the contact time between the spore-forming bacteria and the cationic surfactant is preferably 1 second or longer, more preferably 5 seconds or longer, more preferably 30 seconds or longer. 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 or more, more preferably 20 minutes or more, More preferably, it is 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, from <Item 1> to <Item 17> Any one of the germination methods.

<Section 19>
The total contact time for contacting the spore-forming bacterium with dipicolinic acid or a salt thereof in step 1 when having a period to be performed simultaneously with step 3 is preferably 5 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 preferably 90 minutes or less, more preferably 80 minutes or less, more preferably 70 minutes or less, more preferably 65 minutes or less, more preferably The germination method according to any one of <Item 1> to <Item 18>, which is 60 minutes or less, more preferably 50 minutes or less, more preferably 40 minutes or less, and more preferably 35 minutes or less.

<Section 20>
The total contact time of the spore-forming bacteria and the cationic surfactant in step 2 when having a period to be performed simultaneously with step 3 is preferably 5 minutes or more, more preferably 10 minutes or more, more preferably 15 minutes or more, more preferably Is 20 minutes or more, more preferably 25 minutes or more, and preferably 90 minutes or less, more preferably 80 minutes or less, more preferably 70 minutes or less, more preferably 65 minutes or less, more preferably 60 minutes or less, The germination method according to any one of <Item 1> to <Item 19>, more preferably 50 minutes or less, more preferably 40 minutes or less, and more preferably 35 minutes or less.

<Section 21>
The germination method according to any one of <Item 1> to <Item 20>, wherein the time for heating the spore-forming bacterium to 50 ° C. or higher in Step 3 is from 3 minutes to 90 minutes.

<Section 22>
The temperature of step 3 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 it is 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. The sprouting method according to any one of <Item 1> to <Item 21>.

<Section 23>
The time for heating in step 3 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, according to <Item 1> to <Item 22> Any one of the germination methods.

<Section 24>
The sprouting method according to any one of <1> to <23>, wherein the step 1 and the step 2 are performed independently or simultaneously at 15 ° C. or higher and lower than 50 ° C. before the start of the step 3.

<Section 25>
The germination method according to any one of <Item 1> to <Item 24>, wherein Step 1 and Step 2 are performed independently or simultaneously for 1 second to 60 minutes before the start of Step 3.

<Section 26>
The germination method according to any one of <Item 1> to <Item 25>, wherein Step 1 and Step 2 are performed independently in total or simultaneously for 30 minutes to 90 minutes.

<Section 27>
A germicidal aid composition comprising dipicolinic acid or a salt thereof and a cationic surfactant.

<Section 28>
Liquid composition containing dipicolinic acid or a salt thereof, preferably 0.05 mM or more, more preferably 0.5 mM or more, more preferably 3 mM or more, and preferably 200 mM or less, more preferably 100 mM or less, more preferably 30 mM or less. <Section 27> The germination aid composition according to <27>.

<Section 29>
A liquid composition containing a cationic surfactant preferably 0.1 mM or more, more preferably 0.5 mM or more, more preferably 3 mM or more, and preferably 1000 mM or less, more preferably 500 mM or less, more preferably 300 mM or less. The sprouting aid composition according to <Item 27> or <Item 28>.

<Section 30>
It contains dipicolinic acid or a salt thereof, a cationic surfactant, 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 The germination aid composition according to any one of <Item 27> to <Item 29>, wherein the composition is 10 ppm or less, more preferably 1 ppm or less, and more preferably substantially 0 ppm.

Hereinafter, the present invention will be described in more detail with reference to examples.

Preparation of fungus Spore-forming fungus: Bacillus subtilis 168 strain was used as a test strain. An aqueous dispersion having a content of spore-forming bacteria of 10 ⁸ CFU / mL was prepared and used in the experiment. Spore-forming bacteria in this aqueous dispersion were used for experiments after confirming that 95% or more of the spores were formed by microscopic observation.

Preparation of Dipicolinic Acid Aqueous Solution Dipicolinic acid (hereinafter also referred to as DPA) is a reagent (manufactured by Wako Pure Chemical Industries, Ltd., production code 165-05342), and a solvent is Tris-HCl buffer (Wako Pure Chemical Industries) adjusted to 50 mM with ion-exchanged water Kogyo Co., Ltd., production code 318-90225), and sodium hydroxide was used as 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. Unless otherwise specified, the experiment was conducted after adjusting the pH to 7 (24 ° C.).

Surfactants The following surfactants were used.
Lauryltrimethylammonium chloride (Coatamine 24P, manufactured by Kao Corporation)
Alkylbenzyldimethylammonium chloride (Sanisol C Kao Co., Ltd.)
Sodium lauryl sulfate (Emar 0, manufactured by Kao Corporation)
Polyoxyethylene (3) sodium lauryl ether sulfate (Emar 20C, manufactured by Kao Corporation)
Polyoxyethylene (2) sodium lauryl ether sulfate (Emar E-27C, manufactured by Kao Corporation)
Sodium dodecylbenzenesulfonate (Neobex G-15, manufactured by Kao Corporation)
Polyoxyethylene lauryl ether (Emulgen 108, manufactured by Kao Corporation)
Polyoxyethylene (23) lauryl ether (Emulgen 123P, manufactured by Kao Corporation)
Polyoxyethylene lauryl ether (Emulgen 130K, manufactured by Kao Corporation)
Polyethylene glycol monolaurate (Emanon 1112, manufactured by Kao Corporation)
Polyoxyethylene hydrogenated castor oil (60E.O.) (Emanon CH-60, manufactured by Kao Corporation)
Lauryl glucoside (Mydoll 12, manufactured by Kao Corporation)
Dodecyltrimethylammonium chloride (manufactured by Tokyo Chemical Industry, hereinafter C12 trimethylammonium chloride)
Tetradecyltrimethylammonium chloride (manufactured by Tokyo Chemical Industry, hereinafter C14 trimethylammonium chloride)
Hexadecyltrimethylammonium chloride (manufactured by Tokyo Chemical Industry, C16 trimethylammonium chloride)
Octadecyltrimethylammonium chloride (manufactured by Tokyo Chemical Industry, hereinafter C18 trimethylammonium chloride)
Dioctadimethylammonium chloride (manufactured by Tokyo Chemical Industry, C8 dimethylammonium chloride)
Didecyldimethylammonium chloride (manufactured by Tokyo Chemical Industry, hereinafter C10 dimethylammonium chloride)
Didodecyldimethylammonium chloride (manufactured by Tokyo Chemical Industry, C12 dimethylammonium chloride)
Ditetradecyldimethylammonium chloride (manufactured by Tokyo Chemical Industry, C14 dimethylammonium chloride)
Dihexadecyldimethylammonium chloride (manufactured by Tokyo Chemical Industry, hereinafter referred to as C16 dimethylammonium chloride)
Dioctadecyldimethylammonium chloride (manufactured by Tokyo Chemical Industry, hereinafter C18 dimethylammonium chloride)

Example 1-1
Bactericidal test Using 10 mM dipicolinic acid aqueous solution (hereinafter also referred to as DPA solution), 0.10% by mass lauryltrimethylammonium chloride and spore-forming bacteria described in Table 1, a spore killing test was performed according to the following procedure, The germicidal effect was evaluated. The evaluation results are shown in Table 1. (In the table, “%” means “mass%”. The same shall apply hereinafter.)

-procedure-

(1) Step 1 and Step 2: Mix 100 μL of an aqueous dispersion of spore-forming bacteria (hereinafter referred to as spore solution; the same in the following examples) with 800 μL of DPA aqueous solution and 100 μL of an aqueous solution of lauryltrimethylammonium chloride. And used as a test solution. The final concentration of DPA was 10 mM, and the final concentration of lauryltrimethylammonium chloride was 0.1% by mass. Here, the final concentration means the concentration in the mixed aqueous solution (in a state of 1 mL in total) (the same applies in the following examples).
(2) Step 1 and Step 2: The above test solution (1) (pH 7.0) was allowed to stand at 24 ° C. for 30 minutes.
(3) Step 3: After the above (2), the test solution was directly heated at 80 ° C. for 30 minutes.
An aluminum block thermostatic chamber MD-01N-110 manufactured by Major Science was used for the heating step.
(4) The test solution heat-treated in (3) is serially diluted with an LP aqueous solution (LP diluted solution (Digo, manufactured by Nippon Pharmaceutical Co., Ltd.)), and 100 μL of each diluted solution is smeared on an LB agar medium (manufactured by BD). And cultured at 37 ° 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.

Comparative Examples 1-1 and 1-2
In the procedure (1) of Example 1-1, the aqueous solution shown in Table 1 was used instead of the aqueous solution containing the final concentration of 10 mM DPA and the final concentration of 0.1% by mass of the cationic surfactant. Otherwise, the same procedure as in Example 1-1 was performed.

Example 2-1 and comparative examples 2-1 to 2-10:
An aqueous solution obtained by mixing each surfactant aqueous solution shown in Table 2 with the DPA aqueous solution was used. The final concentration of DPA in the aqueous solution was 10 mM, and the final concentration of each surfactant was 0.1% by mass. A germination test was performed under the same conditions as in Example 1-1 to evaluate the germination effect. The evaluation results are shown in Table 2.

Examples 3-1 to 3-10:
An aqueous solution in which each surfactant aqueous solution shown in Table 3 was mixed with the DPA aqueous solution was used. The final concentration of DPA and each surfactant in the aqueous solution was 10 mM. A germination test was performed under the same conditions as in Example 1-1 to evaluate the germination effect. The evaluation results are shown in Table 3.

Comparative Examples 3-1 to 3-3:
The germicidal effect was evaluated under the same conditions as in Examples 3-1, 3-3 and 3-4 except that the temperature in step 3 was RT (25 ° C.).

Examples 4-1 to 4-13:
An aqueous solution in which each surfactant aqueous solution shown in Table 4 was mixed with the DPA aqueous solution was used. The final concentration of DPA in the aqueous solution was 10 mM, and the final concentration of each surfactant was the concentration shown in Table 4. A germination test was performed under the same conditions as in Example 1-1 to evaluate the germination effect. The evaluation results are shown in Table 4.

Examples 5-1 to 5-3:
An aqueous solution in which a C18 trimethylammonium chloride aqueous solution was mixed with a DPA aqueous solution was used. The final concentration of DPA in the aqueous solution was 10 mM, and the final concentration of C18 trimethylammonium chloride was 20 mM. The heating temperature in the procedure (3) was set to the temperature shown in Table 5. A germination test was performed under the same conditions as in Example 1-1 to evaluate the germination effect. The evaluation results are shown in Table 5.

Examples 6-1 to 6-4
An aqueous solution in which a C18 trimethylammonium chloride aqueous solution was mixed with a DPA aqueous solution was used. The final concentration of DPA in the aqueous solution was 10 mM, and the final concentration of C18 trimethylammonium chloride was 1 mM. The pH (24 ° C.) of the test solution in procedures (1) and (2) was set to the value shown in Table 6. A germination test was performed under the same conditions as in Example 1-1 to evaluate the germination effect. The evaluation results are shown in Table 6. Example 6-2 is a buffer system of phthalic acid and sodium hydroxide, Example 6-3 is a buffer system of boric acid and sodium hydroxide, and Example 6-4 is a buffer system of potassium chloride and sodium hydroxide. Using.

Example 7-1
-procedure-
(1) Step 1: 100 μL of spore solution was mixed with 900 μL of DPA aqueous solution to obtain a test solution (pH 7.0). The final concentration of DPA was 10 mM.
(2) The test solution was allowed to stand at 24 ° C. for 30 minutes.
(3) The DPA solution was removed from the spores by the following method. The test solution in Step 1 was centrifuged at 15 krpm for 5 minutes in a centrifuge (Table Top Micro Cooling Centrifuge 3500, manufactured by Kubota Corporation), and the supernatant was removed.
(4) Step 2: The supernatant was removed, and immediately, 1 mL of a C16 trimethylammonium chloride aqueous solution was added to a final concentration of 10 mM, and the mixture was allowed to stand at 24 ° C. for 30 minutes.
(5) Step 3: After the above (4), the test solution was heated at 80 ° C. for 30 minutes.
(6) The test solution of (5) was serially diluted with sterilized water, 100 μL of each diluted solution was smeared on LB agar medium (manufactured by BD), and cultured at 37 ° 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).

Example 7-2
-procedure-
(1) Step 2: 100 μL of the spore solution was mixed with 900 μL of a C16 trimethylammonium chloride aqueous solution to obtain a test solution (pH 7.0). The final concentration of C16 trimethylammonium chloride was 10 mM.
(2) The test solution was allowed to stand at 24 ° C. for 30 minutes.
(3) The C16 trimethylammonium chloride solution was removed from the spores by the following method. The test solution of (2) was centrifuged using a centrifuge at 15 krpm for 5 minutes, and the supernatant was removed.
(4) Step 1: The supernatant was removed, and immediately, 1 mL of a DPA aqueous solution was added to a final concentration of 10 mM, and the mixture was allowed to stand at 24 ° C. for 30 minutes.
(5) After the above (4), the test solution was heated at 80 ° C. for 30 minutes.
(6) The test solution of (5) was serially diluted with sterilized water, 100 μL of each diluted solution was smeared on LB agar medium (manufactured by BD), and cultured at 37 ° 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).

Example 7-3
-procedure-
(1) Step 1 and Step 2: 100 μL of the spore solution was mixed with 900 μL of an aqueous solution of DPA and C16 trimethylammonium chloride to obtain a test solution (pH 7.0). The final concentration of DPA and C16 trimethylammonium chloride was 10 mM.
(2) Immediately after (1), the test solution was heated at 80 ° C. for 30 minutes.
(3) The test solution of (2) was serially diluted with sterilized water, 100 μL of each diluted solution was smeared on LB agar medium (manufactured by BD), and cultured at 37 ° C. for 16 hours.
(4) After culturing in (3), the number of viable bacteria X (CFU / mL) was determined from the number of colonies that had grown.

Comparative Example 7-1
-procedure-
(1) 100 μL of the spore solution was mixed with 900 μL of the DPA solution to obtain a test solution (pH 7.0). The final concentration of DPA was 10 mM.
(2) Immediately after (1), the test solution was heated at 80 ° C. for 30 minutes.
(3) The DPA solution was removed from the spores by the following method.
The test solution of (2) was centrifuged using a centrifuge at 15 krpm for 5 minutes, and the supernatant was removed.
(4) After removing the supernatant, 1 mL of a C16 trimethylammonium chloride aqueous solution was immediately added to a final concentration of 10 mM, and the mixture was allowed to stand at 24 ° C. for 30 minutes.
(5) The test solution of (4) was serially diluted with sterilized water, 100 μL of each diluted solution was smeared on an LB agar medium (manufactured by BD), and cultured at 37 ° C. for 16 hours.
(6) The viable cell count X (CFU / mL) was determined from the number of colonies that had grown after the culture of (5).

Comparative Example 7-2
-procedure-
(1) Step 3: 100 μL of the spore solution was heated at 80 ° C. for 30 minutes.
(2) Step 2: After (1), after cooling to room temperature, immediately mixed with 900 μL of C16 trimethylammonium chloride aqueous solution and allowed to stand for 30 minutes to obtain a test solution (pH 7.0). The final concentration of C16 trimethylammonium chloride was 10 mM.
(3) The test solution of (2) was serially diluted with sterilized water, 100 μL of each diluted solution was smeared on LB agar medium (manufactured by BD), and cultured at 37 ° C. for 16 hours.
(4) After culturing in (3), the number of viable bacteria X (CFU / mL) was determined from the number of colonies that had grown.

Example 8-1:
A germination test was conducted in the same manner as in Example 3-1.

Comparative Example 8-1:
An isophthalic acid aqueous solution having the same concentration was used instead of the DPA aqueous solution. Other than that was carried out similarly to Example 8-1.

Example 9-1
(1) Step 1 and Step 2: 100 μL of the spore solution was mixed with 800 μL of the DPA aqueous solution and 100 μL of the C16 trimethylammonium chloride aqueous solution, respectively, to obtain a test solution (pH 7.0). The final concentration of DPA and C16 trimethylammonium chloride was 10 mM.
(2) Step 3: Immediately after (1) above, the test solution was immediately heated at 80 ° C. for 30 minutes. An aluminum block thermostatic chamber MD-01N-110 manufactured by Major Science was used for the heating step.
(3) The test solution heat-treated in (2) above is serially diluted with an LP aqueous solution (LP diluted solution (Digo, manufactured by Nippon Pharmaceutical Co., Ltd.)), and each diluted solution is added to an LB agar medium (BD) by 100 μL. It was smeared and cultured at 37 ° C. for 16 hours.
(4) After culturing in (3), the number of viable bacteria X (CFU / mL) was determined from the number of colonies that had grown.

Comparative Example 9-1
The same operation as in Example 9-1 was performed, but the heating in Step 3 was not performed and the mixture was allowed to stand at room temperature for 30 minutes.

Comparative Example 9-2
In Example 9-1 (1), the spore solution was contacted with purified water in place of the DPA and cationic surfactant aqueous solution of Step 1 and Step 2. The heating in step 3 was performed in the same manner as in Example 9-1.

Table 9 shows the results of Example 9-1 and Comparative Examples 9-1 and 9-2.

Examples 10-1 to 10-2
An aqueous solution in which a C16 trimethylammonium chloride aqueous solution was mixed with a DPA aqueous solution was used. The final concentration of DPA was the concentration shown in Table 10, and the final concentration of C16 trimethylammonium chloride was 10 mM. A germination test was performed under the same conditions as in Example 1-1 to evaluate the germination effect. Table 10 shows the evaluation results.

The sprouting 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

A germination method comprising the following step 1, step 2 and step 3:
Step 1: A step of bringing a spore-forming bacterium into contact with dipicolinic acid or a salt thereof;
Step 2: A step of bringing a spore-forming bacterium into contact with a cationic surfactant; and Step 3: A step of heating the spore-forming bacterium to 50 ° C. or more (however, Step 3 is completed after Step 1 and Step 2 are started) It shall be).
The germination method of Claim 1 which has a period which performs the said process 1, the process 2, and the process 3 simultaneously.
The germination method according to claim 1 or 2, wherein the cationic surfactant is a quaternary ammonium salt.
The germination method according to claim 3, wherein the cationic surfactant is a quaternary ammonium salt represented by the general formula (1).

Wherein any one of R 11 ~ R 14, hydroxyl group, an ester group, an amide group hydrocarbon group having 3 or more carbon atoms which may have a remaining three of R 11 ~ R 14 Represents a methyl group or an ethyl group, and X − represents an inorganic or organic anionic compound. ]
The germination method according to claim 3, wherein the quaternary ammonium salt is at least one selected from the group consisting of an alkyltrimethylammonium salt, a dialkyldimethylammonium salt and a benzalkonium salt. *
The spore killing method according to any one of claims 1 to 5, wherein, in the step 1 and the step 2, a spore-forming bacterium, dipicolinic acid or a salt thereof, and a cationic surfactant are contacted in a liquid.
The germicidal method according to claim 6, wherein the content of dipicolinic acid or a salt thereof in the liquid is 0.05 mM or more and 200 mM or less.
The germination method according to claim 6 or 7, wherein the content of the cationic surfactant in the liquid is 0.1 mM or more and 1000 mM or less.
The spore killing method according to any one of claims 1 to 8, wherein the time for heating the spore-forming bacteria to 50 ° C or higher in the step 3 is 3 minutes or more and 90 minutes or less.
A germicidal aid composition comprising dipicolinic acid or a salt thereof and a cationic surfactant.
The germicidal aid composition according to claim 10, which is a liquid composition containing dipicolinic acid or a salt thereof in an amount of 0.05 mM to 200 mM.
The germicidal aid composition according to claim 10 or 11, which is a liquid composition containing a cationic surfactant in an amount of 0.1 mM to 1000 mM.