JP7101375B2 - Disinfection composition and method for disinfecting bacterial spores using it - Google Patents

Description

The present invention relates to a sterilizing composition used for sterilizing bacterial spores and a method for sterilizing bacterial spores using the same.

Various sterilizing and sterilizing ingredients are used as a composition for sterilizing or sterilizing tableware, cooking utensils, and various equipment in food handling facilities such as restaurants, nursing homes, hospital kitchens, and food factories. A compounded disinfectant / bactericidal agent is used. Examples of such disinfectant / sterilizing agents include alcohol preparations such as ethanol for disinfection; chlorine-based preparations such as sodium hypochlorite; peroxide preparations such as hydrogen peroxide solution; and cationic surfactants such as benzalkonium chloride. Activators; etc. are widely used. However, while the above-mentioned sterilizing and bactericidal agents that have been widely used so far are effective against general bacteria and fungi, they are sufficiently effective against bacterial spores such as Bacillus and Clostridium. It wasn't what I expected.

On the other hand, as a method capable of effectively sterilizing or sterilizing bacterial spores, a gas sterilization method using a gas such as ethylene oxide, a gamma ray irradiation method, and a high-pressure steam sterilization (autoclave) method are known. Further, there is known a so-called intermittent sterilization method in which a step of heating to a predetermined temperature, leaving it at room temperature for about one night, and then reheating is repeated. However, these methods have problems such as complicated operation, large-scale equipment, or processing of only those having a size that fits inside the equipment.

Further, as a method capable of effectively sterilizing or sterilizing bacterial spores, a method of allowing sodium hypochlorite or acetic acid to act, a method of allowing aldehydes such as glutaraldehyde and phthalal to act, and the like are known. However, since the agents used in these methods have toxicity, skin irritation, or strong odor, there are various problems such as the need to wear protective equipment, and it cannot be said that the method is highly versatile. rice field.

As related methods and agents used therein, for example, a method for sterilizing bacterial spores treated with a germination-inducing substance such as an amino acid or sugar and then treated with a bactericidal agent, and a two-agent type bactericidal agent used therein have been proposed ( Patent Document 1). Further, a spore-killing method having a step of bringing the spore-forming bacterium into contact with dipicolinic acid, a step of bringing the spore-forming bacterium into contact with a cationic surfactant, and a step of heating the spore-forming bacterium, and a composition used thereof have been proposed. (Patent Document 2). Further, a fungicide composition for bacterial spores having a pH of 12.5 or higher, which contains a specific polyalkylene biguanide compound and an alkaline agent, has been proposed (Patent Document 3).

Japanese Unexamined Patent Publication No. 2004-2229 International Publication No. 2017/017810 Japanese Patent No. 6409201

However, in the methods proposed in Patent Documents 1 and 2, since the operation for sterilization and sterilization is multi-step, the operation is complicated and it takes a long time to sterilize and sterilize. was there. Further, the disinfectant composition proposed in Patent Document 3 needs to use a compound having a unique structure, and since it is a strongly alkaline composition, it cannot be said that it is easy to handle, and it is not always versatile. It was not a high-priced drug.

The present invention has been made in view of the problems of the prior art, and the subject thereof is that bacterial spores can be effectively and easily sterilized or sterilized in a relatively short time. At the same time, it is an object of the present invention to provide a one-agent type sterilizing composition having excellent safety and versatility. Further, an object of the present invention is to provide a method for sterilizing bacterial spores using the above-mentioned sterilization composition.

That is, according to the present invention, the sterilization composition shown below is provided.
[1] A sterilizing composition used for eradicating bacterial spores of at least one of Bacillus subtilis and Bacillus cereus , which is a germination promoting component and a sterilizing component . A one-part liquid composition containing a liquid medium containing water and water , wherein the germination promoting component contains amino acids, sugars, and an inorganic salt, the inorganic salt is ammonium chloride, and the sterilizing component. Is at least one selected from the group consisting of ε-polylysine, sorbic acid, potassium sorbate, protamine, monolaurin, and nycin, and the content of the amino acid is 0.001 based on the entire sterilization composition. The sugar content is 0.1 to 1% by mass based on the entire disinfectant composition, and the content of the inorganic salt is based on the entire disinfectant composition. As a disinfectant composition, which is 0.01 to 1% by mass .
[2 ] The amino acid is at least one selected from the group consisting of L-alanine, L-asparagine, L-tyrosine, L-proline, L-valine, L-serine, and casamino acid in the above [1 ]. The disinfectant composition according to the above.
[ 3 ] The sugar is at least one selected from the group consisting of D-glucose, D-fructose, D-mannose, D-galactose, maltose, lactose, sucrose, and caramelized reactants thereof [1]. ] Or [2] .
[4 ] The sterilizing composition according to any one of the above [1] to [3], wherein the content of the sterilizing component is 0.1 to 1,000 mg / L based on the entire sterilizing composition. ..

Further, according to the present invention, the following method for sterilizing bacterial spores is provided.
[ 5 ] A method for sterilizing bacterial spores, which comprises a step of bringing the sterilizing composition according to any one of [ 1 ] to [ 4 ] above into contact with an object to be treated.

According to the present invention, it is possible to effectively and easily sterilize or sterilize bacterial spores in a relatively short time, and to provide a one-agent type sterilization composition excellent in safety and versatility. Can be done. Further, according to the present invention, it is possible to provide a method for sterilizing bacterial spores using the above-mentioned sterilization composition.

It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Examples 1 and 2 and Comparative Example 1. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Examples 1 and 2 and Comparative Example 1. It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Examples 3 and 4 and Comparative Example 2. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Examples 3 and 4, and Comparative Example 2. It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Examples 5 and 6. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell number concentration decrease) of Examples 5 and 6. It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Examples 7 and 8. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell number concentration decrease) of Examples 7 and 8. It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Example 9, Comparative Example 3 and 4. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Example 9, Comparative Example 3 and 4. It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Example 10, Comparative Examples 5 and 6. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Example 10, Comparative Examples 5 and 6. It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Example 11 and Comparative Example 7. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Example 11 and Comparative Example 7. It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Example 12 and Comparative Example 8. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Example 12 and Comparative Example 8. It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Example 13 and Comparative Example 9. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Example 13 and Comparative Example 9. It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Example 14 and Comparative Example 10. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Example 14 and Comparative Example 10. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Example 15 and Comparative Example 11. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Example 16 and Comparative Example 12. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Example 17 and Comparative Example 13. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Example 18 and Comparative Example 14. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Example 19 and Comparative Example 15. It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Examples 20 and 21 and Comparative Example 16. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Examples 20 and 21 and Comparative Example 16. It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Examples 22 and 23, and Comparative Example 17. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Examples 22 and 23, and Comparative Example 17. It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Examples 24 and 25, and Comparative Example 18. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Examples 24 and 25, and Comparative Example 18. It is a graph which shows the result of the sterilization effect confirmation test (turbidity reduction) of Example 26 and Comparative Example 19. It is a graph which shows the result of the sterilization effect confirmation test (the viable cell count concentration decrease) of Example 26 and Comparative Example 19.

<Sterilization composition>
Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the following embodiments. The sterilizing composition according to an embodiment of the present invention is a one-agent type composition containing a germination promoting component and a sterilizing component used for sterilizing bacterial spores. The germination promoting component contains amino acids, sugars, and inorganic salts. In addition, "sterilization" in the present specification acts on bacteria (spore-forming bacteria) including bacterial cells (spores) in the state of spores, and significantly reduces or substantially reduces the total number of bacteria. It means to sterilize (kill). Hereinafter, the details of the sterilization composition of the present embodiment will be described.

The germination promoting component is a component that acts on bacterial cells in the state of spores (bacterial spores) to promote germination and lead to vegetative cells. The sterilizing component is a component that acts on at least one of bacterial spores, germinating cells, and vegetative cells to significantly reduce the viable cell count or substantially sterilize the cells. The sterilizing composition of the present embodiment is a so-called one-agent type composition in which both the germination promoting component and the sterilizing component are contained in one formulation. Therefore, the germination and removal of bacterial spores are carried out once by performing an operation for contacting the object to be treated in which bacterial spores are present, such as spraying, spraying, or applying the sterilizing composition of the present embodiment. Bacteria can be advanced and eradicated effectively and easily in a relatively short time. Further, since the disinfectant composition of the present embodiment can substantially constitute the active ingredient only by the food additive or by the food additive and the disinfectant component for medical use, it may come into contact with the skin or be in the mouth. There is almost no problem even if it is set, and it is excellent in safety.

(Bacterial spore)
The bacterial spores to be sterilized in the sterilization composition of the present embodiment show a certain resistance to the environment such as heat and dryness. Such bacterial spores are specific bacteria that form bacterial spores under oligotrophic conditions, commonly referred to as "spore-forming bacteria". Bacterial spores become vegetative cells by germination. Spore-forming bacteria capable of forming bacterial spores are usually present in medical facilities and the like as well as facilities that handle food such as kitchens of restaurants and food factories. Examples of the spore-forming bacterium that forms a bacterial spore to be eradicated by the eradication composition of the present embodiment include Bacillus subtilis, Bacillus cereus, Bacillus megaterium and the like. Bacteria of the genus Bacillus; Bacteria of the genus Clostridium such as Clostridium deficile; Bacteria of the genus Amphibacillus; Bacteria of the genus Sporosarcina; Bacteria of the genus Sporosarcina; Bacteria of the genus (Aeribacillus); bacteria of the genus Alicyclobacillus; bacteria of the genus Sporolactobacillus; and the like.

(Germination promoting ingredient)
Germination promoting components include amino acids, sugars, and inorganic salts. It is considered that by incorporating a germination promoting component containing these components, bacterial spores can be germinated and the coexisting sterilizing component can effectively act. In addition, when any of these three components is not contained, the germination rate is lowered, so that it takes time to sterilize, or the germination is poor and bacterial spores are likely to remain, so that the sterilizing effect is lowered.

As the amino acid, 20 kinds of L-amino acids constituting the protein can be used. Among them, L-alanine (Ala), L-asparagin (Asn), L-tyrosine (Tyr), L-proline (Pro), L-valine (Val), L-serine (Ser), and cazaminoic acid are used. Is preferable. These amino acids can be used alone or in combination of two or more.

As the sugar, a sugar generally blended in a synthetic medium for culturing spore-forming bacteria can be used. Among them, sugars include D-glucose (dextrose), D-fructose (fructose), D-mannose, D-galactose, maltose, lactose (lactose), sucrose (sucrose), and caramelized reactants thereof. It is preferable to use. These sugars can be used alone or in combination of two or more.

As the inorganic salt, an inorganic salt generally blended in a synthetic medium for culturing spore-forming bacteria can be used. Among them, as the inorganic salt, it is preferable to use at least one selected from the group consisting of ammonium chloride, ammonium sulfate, ammonium nitrate, potassium chloride, sodium chloride, potassium nitrate, and sodium nitrate.

(Bactericidal component)
As the sterilizing component, a general sterilizing component that can act on ordinary bacteria to sterilize or sterilize can be used. Among them, the sterilizing components include cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride, ε-polylysine, and 1,4-bis (3,3'-(1-decylpyridinium) methyloxy. ) It is preferable to use at least one selected from the group consisting of butane dibromide, sorbic acid, potassium sorbate, protamine, monolaurin, and nisin. Further, considering the influence on the human body, safety and the like, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride, ε-polylysine, sorbic acid, and potassium sorbate are more preferable.

(Liquid medium)
The sterilization composition can be used in various forms such as liquid, paste, gel, milky and solid using a liquid carrier, a gel-like carrier, an emulsion, a solid carrier and the like. Above all, the sterilization composition of the present embodiment is preferably a liquid composition further containing a liquid medium. By using the liquid composition, a wide range of objects to be treated can be sterilized in a short time. The concept of "liquid" in the present specification includes properties similar to liquid such as "paste-like, gel-like, and milky". The liquid composition can be easily sprayed, sprayed or applied by filling, for example, a bottle with a measuring cap, a trigger type spray container, a squeeze type spray container, a pump spray container, a brushed container, or the like. Can be done.

As the liquid medium, it is preferable to use water or a mixed solvent of water and a water-soluble organic solvent. By using a liquid medium containing water, handleability can be improved and corrosion of the object to be treated can be suppressed. As the water-soluble organic solvent, for example, lower alcohols such as ethanol, isopropanol and butanol; polyhydric alcohols such as ethylene glycol, propylene glycol, glycerin and butylene glycol can be used.

When the sterilization composition is a liquid composition containing a liquid medium, the content of amino acids in the sterilization composition may be 0.0001 to 1% by mass based on the entire sterilization composition. It is preferably 0.001 to 0.1% by mass, and more preferably 0.001 to 0.1% by mass. By setting the amino acid content within the above range, bacterial spores can be germinated more efficiently, and sterilization efficiency can be further improved.

When the sterilization composition is a liquid composition containing a liquid medium, the sugar content in the sterilization composition may be 0.001 to 10% by mass based on the entire sterilization composition. It is preferably 0.1 to 1% by mass, and more preferably 0.1 to 1% by mass. By setting the sugar content within the above range, bacterial spores can be germinated more efficiently, and sterilization efficiency can be further improved.

When the sterilization composition is a liquid composition containing a liquid medium, the content of the inorganic salt in the sterilization composition shall be 0.001 to 10% by mass based on the entire sterilization composition. Is preferable, and 0.01 to 1% by mass is more preferable. By setting the content of the inorganic salt within the above range, bacterial spores can be germinated more efficiently, and the sterilization efficiency can be further improved.

When the sterilization composition is a liquid composition containing a liquid medium, the content of the sterilization component in the sterilization composition is 0.1 to 1,000 mg / L based on the entire sterilization composition. It is preferably 1 to 500 mg / L, and more preferably 1 to 500 mg / L. By setting the content of the sterilizing component within the above range, the sterilizing efficiency can be further improved. If the content of the sterilizing component is too small, the sterilizing efficiency may be slightly insufficient. On the other hand, if the content of the sterilizing component is too large, the sterilizing efficiency tends to reach a plateau.

(Other ingredients)
The sterilizing composition of the present embodiment may contain components (other components) other than the above-mentioned components, if necessary, as long as the effects of the present invention are not impaired. Examples of other components include surfactants, pH adjusters, antifoaming agents, antioxidants, emulsifiers and the like.

As the pH adjuster, a general acid or base can be used. Examples of the acid include inorganic acids such as hydrochloric acid and sulfuric acid; organic acids such as lactic acid, citric acid, and salts thereof; and the like. Examples of the base include inorganic bases such as sodium hydroxide and potassium hydroxide; organic bases such as monoethanolamine, triethanolamine and triisopropanolamine; and the like. Among them, as the pH adjuster, it is preferable to use an inorganic acid such as hydrochloric acid or sulfuric acid; or an inorganic base such as sodium hydroxide or potassium hydroxide.

The pH of the sterilizing composition of the present embodiment at 25 ° C. is not particularly limited, but is usually in the neutral range, preferably in the range of pH 5 to 9, in consideration of the sterilizing effect and handleability.

<Sterilization method>
Next, a sterilization method according to an embodiment of the present invention will be described. The sterilization method of the present embodiment includes a step of bringing the above-mentioned sterilization composition into contact with the object to be treated. The object to be treated is an object, equipment, facility or the like in which a bacterial spore to be sterilized by the sterilizing composition is present or may be present on the surface thereof. By contacting the above-mentioned sterilization composition with the object to be treated, bacterial spores can be sterilized, the total number of bacteria can be significantly reduced, or the total number of bacteria can be substantially sterilized (killed).

The object to be treated is not particularly limited as long as it is an object, equipment, facility or the like in which bacterial spores can exist. Specifically, goods such as tableware and cooking utensils; facilities that mainly handle food such as kitchens of restaurants, food factories, backyards of supermarkets; various equipment in food processing factories; various goods and equipment in medical sites, etc. Can be mentioned.

When the sterilizing composition is in a liquid state, for example, an amount of the sterilizing composition that wets the surface of the object to be treated may be applied and brought into contact with the sterilizing composition. As a method for bringing the liquid sterilizing composition into contact with the object to be treated, any method such as spraying, spraying, and coating may be selected. Further, if the object to be treated is equipment piping or the like, a liquid sterilizing composition may be circulated inside (flow path) of the piping or the like. The time for contacting the sterilizing composition with the object to be treated is, for example, 0.5 to 10 hours, preferably 1 to 8 hours, and more preferably 2 to 6 hours. By contacting the sterilizing composition with the object to be treated within the above time range, a sufficient sterilizing effect can be obtained. Further, if necessary, the sterilizing composition may be heated in contact with the object to be treated. Appropriate heating promotes germination of bacterial spores and further enhances sterilization efficiency. The temperature when the sterilizing composition is heated in contact with the object to be treated may be, for example, 20 to 40 ° C, preferably 25 to 37 ° C.

Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited to these examples. In addition, "part" and "%" in Examples and Comparative Examples are based on mass unless otherwise specified. In addition, sterilized reagents and instruments were used for the test.

<Preparation of spore suspension>
(Spore suspension (1))
The spores of the Bacillus subtilis ATCC6633 strain were lyophilized and then stored at −80 ° C. to obtain spore powder (spore powder). The spore powder was added to 10 mL of ion-exchanged water, suspended, and then heat-treated at 70 ° C. for 20 minutes. After centrifuging at 7,000 rpm and 4 ° C. for 5 minutes to collect the bacteria, the cells were suspended again in ion-exchanged water to adjust the OD ₆₅₀ to 3.00, and a spore suspension (1) was obtained.

(Spore suspension (1-1))
The spore suspension (1) was diluted 1,000-fold with ion-exchanged water to obtain a spore suspension (1-1) (about 6.0 × 10 ⁴ CFU / mL).

(Spore suspension (2))
The spores of the Bacillus cereus strain NBRC15305 were lyophilized and then stored at −80 ° C. to obtain spore powder (spore powder). The spore powder was added to 10 mL of ion-exchanged water, suspended, and then heat-treated at 70 ° C. for 20 minutes. After centrifuging at 7,000 rpm and 4 ° C. for 5 minutes to collect the bacteria, the cells were suspended again in ion-exchanged water to adjust the OD ₆₅₀ to 3.00, and a spore suspension (2) was obtained.

<Preparation of germination promoting solution>
(Germination promoter (1))
After adding a 0.5% L-alanine aqueous solution, a 10% D-glucose aqueous solution, and a 5.35% ammonium chloride aqueous solution to ion-exchanged water, the mixture is sterilized by filtration to obtain L-alanine, D-glucose, and ammonium chloride. A germination promoting solution (1) having a pH of 7.0 was prepared, each containing a predetermined concentration.

(Germination promoter (1-1))
After adding a 0.5% L-alanine aqueous solution, a 10% D-glucose aqueous solution, a 5.35% ammonium chloride aqueous solution, and a 1 mol / L potassium chloride aqueous solution to ion-exchanged water, the mixture is sterilized by filtration, and L-alanine, D. -A pH 7.0 germination promoter (1-1) containing glucose, ammonium chloride, and potassium chloride at predetermined concentrations was prepared.

(Germination promoter (2))
After adding a 10% D-glucose aqueous solution to ion-exchanged water, the mixture was sterilized by filtration to prepare a germination promoter (2) having a pH of 7.0 and containing D-glucose at a predetermined concentration.

(Germination promoter (3))
After adding 0.5% L-alanine aqueous solution and 10% D-glucose aqueous solution to ion-exchanged water, they are sterilized by filtration, and germination at pH 7.0 containing L-alanine and D-glucose at predetermined concentrations, respectively. Accelerator solution (3) was prepared.

(Germination promoter (4))
After adding a 0.5% L-alanine aqueous solution to ion-exchanged water, the mixture was sterilized by filtration to prepare a germination promoting solution (4) having a pH of 7.0 containing L-alanine at a predetermined concentration.

(Germination promoter (5))
After adding 0.5% L-alanine aqueous solution and 5.35% ammonium chloride aqueous solution to ion-exchanged water, it is sterilized by filtration, and sprouting at pH 7.0 containing L-alanine and ammonium chloride at predetermined concentrations, respectively. Accelerator solution (5) was prepared.

<Preparation of sterilization component liquid>
Cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), 1,4-bis (3,3'-(1-decylpyridinium) methyloxy) butane dibromide (trade name "Hygenia (trade name") Registered trademark) ”, manufactured by Tama Chemical Industry Co., Ltd.), ε-polylysine, monolaurin, and nisin were prepared. Each of these sterilizing components was dissolved in ion-exchanged water to prepare the following types of sterilizing component solutions.
・ 5mg / mL CPC aqueous solution ・ 10mg / mL CPC aqueous solution ・ 50mg / mL CPC aqueous solution ・ 100mg / mL CPC aqueous solution ・ 5mg / mL BAC aqueous solution ・ 10mg / mL BAC aqueous solution ・ 50mg / mL BAC aqueous solution ・ 100mg / mL BAC aqueous solution ・ 5mg / ML Hygenia aqueous solution ・ 50 mg / mL Hygenia aqueous solution ・ 100 mg / mL Hygenia aqueous solution ・ 10 mg / mL ε-polylysine aqueous solution ・ 50 mg / mL ε-polylysine aqueous solution ・ 100 mg / mL ε-polylysine aqueous solution ・ 100 mg / mL monolaurin aqueous solution ・ 50 mg / mL Nisin aqueous solution ・ 100 mg / mL sorbic acid aqueous solution ・ 100 mg / mL protamine aqueous solution

<Sterilization effect confirmation test>
(Examples 1 and 2, Comparative Example 1)
Put 18 mL of germination promoter (1) and 20 μL of CPC aqueous solution (10 mg / mL, 100 mg / mL) in a 100 mL Erlenmeyer flask, and then add 2 mL of spore suspension (1) to prepare a suspension for evaluation. Obtained (Examples 1 and 2). The flask was shaken at 37 ° C. and 125 rpm, and the turbidity of the suspension (OD ₆₅₀ ) was measured over time to confirm the germination state of the spores. The measurement results are shown in FIG.

In addition, the suspension was taken out over time and diluted stepwise with SCDLP liquid medium to obtain a diluted solution. After applying the obtained diluted solution to SCDLP agar medium, the cells were cultured at 37 ° C. for 48 hours, the number of grown colonies was measured, and the viable cell count concentration (CFU / mL) was calculated. The results are shown in FIG. Table 1 shows the viable cell count concentration (CFU / mL) at the initial stage and 6 hours after the start of shaking.

Further, the turbidity of the suspension (OD ₆₅₀ ) was measured over time in the same manner as in Examples 1 and 2 above, except that the CPC aqueous solution was not used, and the germination state of the spores was confirmed. The viable cell count concentration (CFU / mL) was calculated (Comparative Example 1). The results are shown in FIGS. 1 and 2. Table 1 shows the viable cell count concentration (CFU / mL) at the initial stage and 6 hours after the start of shaking.

As shown in FIG. 1, in all cases (Examples 1 and 2 and Comparative Example 1), the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 2 and 1, in Examples 1 and 2, the viable cell count concentration decreased sharply with the germination of the spores, and it can be seen that the sterilization proceeded efficiently.

(Examples 3 and 4, Comparative Example 2)
The turbidity of the suspension (OD ₆₅₀ ) was changed over time in the same manner as in Examples 1 and 2 described above, except that the BAC aqueous solution (10 mg / mL, 100 mg / mL) was used instead of the CPC aqueous solution. Along with the measurement, the viable cell count concentration (CFU / mL) was calculated (Examples 3 and 4). In addition, the turbidity of the suspension (OD ₆₅₀ ) was measured over time in the same manner as in Examples 3 and 4 described above, except that the BAC aqueous solution was not used, and the germination state of the spores was confirmed. The viable cell count concentration (CFU / mL) was calculated (Comparative Example 2). The results are shown in FIGS. 3 and 4. Table 2 shows the viable cell count concentration (CFU / mL) at the initial stage and 6 hours after the start of shaking.

As shown in FIG. 3, in all cases (Examples 3 and 4, Comparative Example 2), the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 4 and 2, in Examples 3 and 4, the viable cell count concentration decreased sharply with the germination of the spores, and it can be seen that the eradication proceeded efficiently.

(Examples 5 and 6)
The turbidity of the suspension (OD ₆₅₀ ) was changed over time in the same manner as in Examples 1 and 2 described above, except that the Hygenia aqueous solution (5 mg / mL, 100 mg / mL) was used instead of the CPC aqueous solution. Along with the measurement, the viable cell count concentration (CFU / mL) was calculated (Examples 5 and 6). The results are shown in FIGS. 5 and 6. Table 3 shows the viable cell count concentration (CFU / mL) at the initial stage and 6 hours after the start of shaking.

As shown in FIG. 5, in all cases (Examples 5 and 6), the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 6 and 3, the concentration of viable cells decreased sharply with the germination of spores, and it can be seen that the eradication proceeded efficiently.

(Examples 7 and 8)
The turbidity of the suspension (OD ₆₅₀ ) was changed over time in the same manner as in Examples 1 and 2 described above, except that the ε-polylysine aqueous solution (10 mg / mL, 100 mg / mL) was used instead of the CPC aqueous solution. The germination state of the spores was confirmed, and the viable cell count concentration (CFU / mL) was calculated (Examples 7 and 8). The results are shown in FIGS. 7 and 8. Table 4 shows the viable cell count concentration (CFU / mL) at the initial stage and 6 hours after the start of shaking.

As shown in FIG. 7, in each case (Examples 7 and 8), the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 8 and 4, the concentration of viable cells decreased sharply with the germination of spores, and it can be seen that the eradication proceeded efficiently.

(Example 9, Comparative Examples 3 and 4)
Using a 100 mg / mL CPC aqueous solution, the turbidity of the suspension (OD ₆₅₀ ) was measured over time and the viable cell count concentration (CFU / mL) was calculated in the same manner as in Examples 1 and 2 described above. (Example 9). Further, the turbidity of the suspension (in the same manner as in Example 9 above, except that the germination promoting liquid (2) and the germination promoting liquid (3) were used instead of the germination promoting liquid (1), respectively. OD ₆₅₀ ) was measured over time to confirm the germination state of spores, and the viable cell count concentration (CFU / mL) was calculated (Comparative Examples 3 and 4). The results are shown in FIGS. 9 and 10. Table 5 shows the viable cell count concentration (CFU / mL) at the initial stage and 6 hours after the start of shaking.

As shown in FIG. 9, in all cases (Example 9, Comparative Examples 3 and 4), the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 10 and 5, in Example 9, the viable cell count concentration decreased sharply with the germination of the spores, and it can be seen that the eradication proceeded efficiently. On the other hand, in Comparative Examples 3 and 4 containing no one of the germination promoting components, the germination rate was slow, germination was not effective, and the viable cell count concentration did not decrease so much. From this, it can be seen that the germination promoting component was insufficient (deficient).

(Example 10, Comparative Examples 5 and 6)
Using a 100 mg / mL CPC aqueous solution, the turbidity of the suspension (OD ₆₅₀ ) was measured over time and the viable cell count concentration (CFU / mL) was calculated in the same manner as in Examples 1 and 2 described above. (Example 10). Further, the turbidity of the suspension (in the same manner as in Example 10 above, except that the germination promoting liquid (4) and the germination promoting liquid (5) were used instead of the germination promoting liquid (1), respectively. OD ₆₅₀ ) was measured over time to confirm the germination state of spores, and the viable cell count concentration (CFU / mL) was calculated (Comparative Examples 5 and 6). The results are shown in FIGS. 11 and 12. Table 6 shows the viable cell count concentration (CFU / mL) at the initial stage and 6 hours after the start of shaking.

As shown in FIG. 11, in all cases (Example 10, Comparative Examples 5 and 6), the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 12 and 6, in Example 10, the viable cell count concentration decreased sharply with the germination of the spores, and it can be seen that the eradication proceeded efficiently. On the other hand, in Comparative Examples 5 and 6 containing no one of the germination promoting components, the germination rate was slow, germination was not effective, and the viable cell count concentration did not decrease so much. From this, it can be seen that the germination promoting component was insufficient (deficient).

(Example 11, Comparative Example 7)
The turbidity (OD ₆₅₀ ) of the suspension was measured over time in the same manner as in Examples 1 and 2 described above, except that the monolaurin aqueous solution (100 mg / mL) was used instead of the CPC aqueous solution. The viable cell count concentration (CFU / mL) was calculated (Example 11). In addition, the turbidity of the suspension (OD ₆₅₀ ) was measured over time to confirm the germination state of the spores and the viable bacteria in the same manner as in Example 11 above, except that the monolaurin aqueous solution was not used. The number concentration (CFU / mL) was calculated (Comparative Example 7). The results are shown in FIGS. 13 and 14. Table 7 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

As shown in FIG. 13, in Example 11, the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 14 and 7, in Example 11, it can be seen that the viable cell count concentration decreased sharply with the germination of the spores, and the sterilization proceeded efficiently.

(Example 12, Comparative Example 8)
The turbidity (OD ₆₅₀ ) of the suspension was measured over time in the same manner as in Examples 1 and 2 described above, except that the aqueous solution of Nisin (50 mg / mL) was used instead of the aqueous solution of CPC. The viable cell count concentration (CFU / mL) was calculated (Example 12). In addition, the turbidity of the suspension (OD ₆₅₀ ) was measured over time to confirm the germination state of the spores and the viable bacteria in the same manner as in Example 12 described above, except that the aqueous solution of Nisin was not used. The number concentration (CFU / mL) was calculated (Comparative Example 8). The results are shown in FIGS. 15 and 16. Table 8 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

As shown in FIG. 15, in Example 12, the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 16 and 8, in Example 12, the viable cell count concentration decreased sharply with the germination of the spores, and it can be seen that the eradication proceeded efficiently.

(Example 13, Comparative Example 9)
The turbidity (OD ₆₅₀ ) of the suspension was measured over time in the same manner as in Examples 1 and 2 described above, except that the aqueous solution of sorbic acid (100 mg / mL) was used instead of the aqueous solution of CPC. , The viable cell count concentration (CFU / mL) was calculated (Example 13). In addition, the turbidity of the suspension (OD ₆₅₀ ) was measured over time in the same manner as in Example 13 described above, except that the aqueous solution of sorbic acid was not used, and the germination state of the spores was confirmed and raw. The bacterial count concentration (CFU / mL) was calculated (Comparative Example 9). The results are shown in FIGS. 17 and 18. Table 9 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

As shown in FIG. 17, in Example 13, the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 18 and 9, in Example 13, the viable cell count concentration decreased sharply with the germination of the spores, and it can be seen that the eradication proceeded efficiently.

(Example 14, Comparative Example 10)
The turbidity (OD ₆₅₀ ) of the suspension was measured over time in the same manner as in Examples 1 and 2 described above, except that the aqueous protamine solution (100 mg / mL) was used instead of the aqueous CPC solution. The viable cell count concentration (CFU / mL) was calculated (Example 14). In addition, the turbidity of the suspension (OD ₆₅₀ ) was measured over time to confirm the germination state of the spores and the viable bacteria in the same manner as in Example 14 described above, except that the protamine aqueous solution was not used. The number concentration (CFU / mL) was calculated (Comparative Example 10). The results are shown in FIGS. 19 and 20. Table 10 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

As shown in FIG. 19, in Example 14, the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 20 and 10, in Example 14, the viable cell count concentration decreased sharply with the germination of the spores, and it can be seen that the eradication proceeded efficiently.

(Example 15, Comparative Example 11)
18 mL of germination promoter (1-1) and 20 μL of CPC aqueous solution (50 mg / mL) are placed in a 100 mL Erlenmeyer flask, and then 2 mL of spore suspension (1-1) is added to prepare a suspension for evaluation. Obtained (Example 15). The flask was shaken at 37 ° C. and 125 rpm. The suspension was removed over time and diluted stepwise with SCDLP liquid medium to give a diluted solution. After applying the obtained diluted solution to SCDLP agar medium, the cells were cultured at 37 ° C. for 48 hours, the number of grown colonies was measured, and the viable cell count concentration (CFU / mL) was calculated. The results are shown in FIG. Table 11 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

Further, the viable cell count concentration (CFU / mL) in the suspension was calculated over time in the same manner as in Example 15 above, except that the CPC aqueous solution was not used (Comparative Example 11). The results are shown in FIG. Table 11 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

As shown in FIGS. 21 and 11, even when the viable cell count concentration is more realistic (about 6.0 × 10 ³ CFU / mL), the viable cell count concentration drops sharply in Example 15. It can be seen that the sterilization proceeded efficiently.

(Example 16, Comparative Example 12)
The viable cell count concentration (CFU / mL) in the suspension was calculated over time in the same manner as in Example 15 above, except that the BAC aqueous solution (50 mg / mL) was used instead of the CPC aqueous solution. (Example 16). In addition, the viable cell count concentration (CFU / mL) in the suspension was calculated over time in the same manner as in Example 16 above, except that the BAC aqueous solution was not used (Comparative Example 12). The results are shown in FIG. Table 12 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

As shown in FIGS. 22 and 12, even when a more realistic viable cell count concentration (about 6.0 × 10 ³ CFU / mL) is used, the viable cell count concentration drops sharply in Example 16. It can be seen that the sterilization proceeded efficiently.

(Example 17, Comparative Example 13)
The viable cell count concentration (CFU / mL) in the suspension was calculated over time in the same manner as in Example 15 above, except that the Hygenia aqueous solution (5 mg / mL) was used instead of the CPC aqueous solution. (Example 17). In addition, the viable cell count concentration (CFU / mL) in the suspension was calculated over time in the same manner as in Example 17 above, except that the Hygenia aqueous solution was not used (Comparative Example 13). The results are shown in FIG. Table 13 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

As shown in FIGS. 23 and 13, even when a more realistic viable cell count concentration (about 6.0 × 10 ³ CFU / mL) is used, the viable cell count concentration drops sharply in Example 17. It can be seen that the sterilization proceeded efficiently.

(Example 18, Comparative Example 14)
The viable cell count concentration (CFU / mL) in the suspension was changed over time in the same manner as in Example 15 above, except that the ε-polylysine aqueous solution (5 mg / mL) was used instead of the CPC aqueous solution. Calculated (Example 18). In addition, the viable cell count concentration (CFU / mL) in the suspension was calculated over time in the same manner as in Example 18 above, except that the ε-polylysine aqueous solution was not used (Comparative Example 14). The results are shown in FIG. Table 14 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

As shown in FIGS. 24 and 14, even when a more realistic viable cell count concentration (about 6.0 × 10 ³ CFU / mL) is used, the viable cell count concentration drops sharply in Example 18. It can be seen that the sterilization proceeded efficiently.

(Example 19, Comparative Example 15)
18 mL of germination promoter (1) and 20 μL of nisin aqueous solution (50 mg / mL) were placed in a 100 mL Erlenmeyer flask, and then 2 mL of spore suspension (1-1) was added to obtain a suspension for evaluation. (Example 19). The flask was shaken at 37 ° C. and 125 rpm. The suspension was removed over time and diluted stepwise with SCDLP liquid medium to give a diluted solution. After applying the obtained diluted solution to SCDLP agar medium, the cells were cultured at 37 ° C. for 48 hours, the number of grown colonies was measured, and the viable cell count concentration (CFU / mL) was calculated. The results are shown in FIG. Table 15 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

Further, the viable cell count concentration (CFU / mL) in the suspension was calculated over time in the same manner as in Example 19 above, except that the aqueous nisin solution was not used (Comparative Example 15). The results are shown in FIG. Table 15 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

As shown in FIGS. 25 and 15, even when a more realistic viable cell count concentration (about 6.0 × 10 ³ CFU / mL) is used, the viable cell count concentration drops sharply in Example 19. It can be seen that the sterilization proceeded efficiently.

(Examples 20 and 21, Comparative Example 16)
Put 18 mL of germination promoter (1) and 20 μL of CPC aqueous solution (5 mg / mL, 50 mg / mL) in a 100 mL Erlenmeyer flask, and then add 2 mL of spore suspension (2) to prepare a suspension for evaluation. Obtained (Examples 20 and 21). The flask was shaken at 37 ° C. and 125 rpm, and the turbidity of the suspension (OD ₆₅₀ ) was measured over time to confirm the germination state of the spores. The measurement result is shown in FIG.

In addition, the suspension was taken out over time and diluted stepwise with SCDLP liquid medium to obtain a diluted solution. After applying the obtained diluted solution to SCDLP agar medium, the cells were cultured at 37 ° C. for 48 hours, the number of grown colonies was measured, and the viable cell count concentration (CFU / mL) was calculated. The results are shown in FIG. Table 16 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

Further, the turbidity of the suspension (OD ₆₅₀ ) was measured over time in the same manner as in Examples 20 and 21 except that the CPC aqueous solution was not used, and the germination state of the spores was confirmed. The viable cell count concentration (CFU / mL) was calculated (Comparative Example 16). The results are shown in FIGS. 26 and 27. Table 16 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

As shown in FIG. 26, in all cases (Examples 20 and 21, Comparative Example 16), the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 27 and 16, in Examples 20 and 21, the viable cell count concentration decreased sharply with the germination of the spores, and it can be seen that the eradication proceeded efficiently.

(Examples 22 and 23, Comparative Example 17)
The turbidity of the suspension (OD ₆₅₀ ) was changed over time in the same manner as in Examples 20 and 21 described above, except that the BAC aqueous solution (5 mg / mL, 50 mg / mL) was used instead of the CPC aqueous solution. Along with the measurement, the viable cell count concentration (CFU / mL) was calculated (Examples 22 and 23). In addition, the turbidity (OD ₆₅₀ ) of the suspension was measured over time in the same manner as in Examples 22 and 23 described above, except that the BAC aqueous solution was not used, and the germination state of the spores was confirmed. The viable cell count concentration (CFU / mL) was calculated (Comparative Example 17). The results are shown in FIGS. 28 and 29. Table 17 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

As shown in FIG. 28, in all cases (Examples 22 and 23, Comparative Example 17), the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 29 and 17, in Examples 22 and 23, the viable cell count concentration decreased sharply with the germination of the spores, and it can be seen that the eradication proceeded efficiently.

(Examples 24 and 25, Comparative Example 18)
The turbidity of the suspension (OD ₆₅₀ ) was changed over time in the same manner as in Examples 20 and 21 described above, except that the Hygenia aqueous solution (5 mg / mL, 50 mg / mL) was used instead of the CPC aqueous solution. Along with the measurement, the viable cell count concentration (CFU / mL) was calculated (Examples 24 and 25). In addition, the turbidity of the suspension (OD ₆₅₀ ) was measured over time in the same manner as in Examples 24 and 25 described above, except that the Hygenia aqueous solution was not used, and the germination state of the spores was confirmed. The viable cell count concentration (CFU / mL) was calculated (Comparative Example 18). The results are shown in FIGS. 30 and 31. Table 18 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

As shown in FIG. 30, in each case (Examples 24 and 25, Comparative Example 18), the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 31 and 18, in Examples 24 and 25, the viable cell count concentration decreased sharply with the germination of the spores, and it can be seen that the eradication proceeded efficiently.

(Example 26, Comparative Example 19)
The turbidity of the suspension (OD ₆₅₀ ) is measured over time in the same manner as in Examples 20 and 21 described above, except that the ε-polylysine aqueous solution (50 mg / mL) was used instead of the CPC aqueous solution. At the same time, the viable cell count concentration (CFU / mL) was calculated (Example 26). In addition, the turbidity of the suspension (OD ₆₅₀ ) was measured over time in the same manner as in Example 26 described above, except that the ε-polylysine aqueous solution was not used, and the germination state of the spores was confirmed. The viable cell count concentration (CFU / mL) was calculated (Comparative Example 19). The results are shown in FIGS. 32 and 33. Table 19 shows the viable cell count concentration (CFU / mL) at the initial stage and 3 hours after the start of shaking.

As shown in FIG. 32, in all cases (Example 26, Comparative Example 19), the turbidity decreased with time, and germination of spores could be confirmed. Further, as shown in FIGS. 33 and 19, in Example 26, the viable cell count concentration decreased sharply with the germination of the spores, and it can be seen that the eradication proceeded efficiently.

The sterilizing composition of the present invention is useful as a highly safe composition capable of sterilizing bacterial spores, which was conventionally difficult to sterilize, by a simple operation.

Claims (5)

Bacillus subtilis and Bacillus cereus of at least one of the spore-forming bacteria A sterilizing composition used for sterilizing bacterial spores.
Germination promoting ingredient,Sterilization component, And a liquid medium containing waterOne-dose containingLiquidIt is a composition
The germination promoting component contains amino acids, sugars, and inorganic salts.
The inorganic salt is ammonium chloridethe law of nature,
The sterilizing component is at least one selected from the group consisting of ε-polylysine, sorbic acid, potassium sorbate, protamine, monolaurin, and nisin.
The content of the amino acid is 0.001 to 0.1% by mass based on the entire sterilization composition.
The sugar content is 0.1 to 1% by mass based on the entire sterilization composition.,
The content of the inorganic salt is 0.01 to 1% by mass based on the entire sterilization composition. Sterilization composition. The eradication according to claim 1, wherein the amino acid is at least one selected from the group consisting of L-alanine, L-asparagine, L-tyrosine, L-proline, L-valine, L-serine, and casamino acid. Composition. Claim 1 or 2 wherein the sugar is at least one selected from the group consisting of D-glucose, D-fructose, D-mannose, D-galactose, maltose, lactose, sucrose, and caramelized reactants thereof. The disinfectant composition described. The sterilizing composition according to any one of claims 1 to 3, wherein the content of the sterilizing component is 0.1 to 1,000 mg / L based on the entire sterilizing composition. A method for sterilizing bacterial spores, which comprises a step of bringing the sterilizing composition according to any one of claims 1 to 4 into contact with an object to be treated.

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