TW202128017A - Disinfection composition and method for removing bacterial spores - Google Patents

Abstract

Provided are: a highly safe and versatile disinfection composition in a single dosage form that is capable of effectively and easily removing or killing bacterial spores in a comparatively short time; and a method for removing bacterial spores using the disinfection composition. The disinfection composition is used to remove bacterial spores. The composition is in a single dosage form and contains a germination promoting component and a disinfection component such as cetylpyridinium chloride, benzalkonium chloride, and [epsilon]-polylysine. The germination promoting component contains an amino acid, a sugar, and an inorganic salt. The method for removing bacterial spores comprises a step of bringing the disinfection composition into contact with an object to be treated.

Description

Sterilization composition and method for sterilization of bacterial spores using the same

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

In food processing facilities such as restaurants, nursing facilities, hospital kitchens, or food factories, as a composition for sterilizing or sterilizing tableware, preparation utensils, and various equipment, various sterilization and sterilization have been used and formulated Sterilization of ingredients and bactericides. As such sterilizing and bactericidal agents, alcohols such as ethanol for disinfection, chlorine preparations such as sodium hypochlorite, peroxide preparations such as hydrogen peroxide water, and cationic surfactants such as benzalkonium chloride are widely used. However, although the above-mentioned antibacterial and bactericidal agents used so far show effects on general bacteria or molds, on the other hand, they cannot be expected to have sufficient effects on bacterial spores of the genus Bacillus or Clostridium.

On the other hand, as methods that can effectively sterilize or sterilize bacterial spores, gas sterilization methods using gases such as ethylene oxide, gamma ray irradiation methods, and high-pressure steam sterilization (autoclave) methods are known. Wait. Also, there is known a so-called batch sterilization method, which is a step of repeating heating to a predetermined temperature, leaving it at room temperature for about one night, and then reheating. However, these methods have problems such as complicated operations, bulky devices, or only capable of handling size objects that can be placed in the device.

In addition, as a method that can effectively sterilize or sterilize bacterial spores, a method of acting on sodium hypochlorite or peroxalic acid, a method of acting on aldehydes such as glutaraldehyde or phthalaldehyde (Phtharal), and the like are known. However, the agents used in these methods have toxicity, skin irritation, or strong odor, so there are various problems such as the need to wear protective gear, and it cannot be said to be a highly versatile method.

As a related method and the medicine used, for example, a method of sterilizing bacterial spores treated with a germicide after treatment with a germination inducing substance such as amino acid or sugar, and a two-dose form of the method used Bactericide (Patent Document 1). In addition, there have been proposed methods for killing spore-forming bacteria having a step of contacting spore-forming bacteria with 2,6-pyridinedicarboxylic acid, a step of contacting spore-forming bacteria with a cationic surfactant, and a step of heating the spore-forming bacteria, and its use The composition (Patent Document 2). Furthermore, a fungicide composition for bacterial spores has been proposed, which contains a specific polyalkylene biguanide compound and an alkali agent, and has a pH of 12.5 or higher (Patent Document 3). [Prior Technical Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Laid-Open No. 2004-2229 [Patent Document 2] International Patent Publication No. 2017/017810 [Patent Document 3] Japanese Patent No. 6409201

(The problem to be solved by the invention)

However, the methods proposed in Patent Documents 1 and 2 are complicated because the operations for sterilization and sterilization are multi-stage, and there are problems that sterilization and sterilization take a long time. In addition, the bactericide composition proposed in Patent Document 3 must use a compound with a specific structure and is a strongly alkaline composition, so it cannot be said to be easy to handle, and it is not a highly versatile agent.

The present invention has been completed in view of the problems of this conventional technology, and its subject is to provide an effective and easy sterilization or sterilization process for bacterial spores in a short period of time, and at the same time, it is excellent in safety and versatility. One-dose sterilization composition. In addition, the subject of the present invention is to provide a method for sterilizing bacterial spores using the aforementioned sterilizing composition. (Technical means to solve the problem)

That is, according to the present invention, the sterilization composition shown below is provided. [1] A sterilizing composition for sterilizing bacterial spores, which is a one-dose composition containing germination promoting components and sterilizing components, the germination promoting components containing amino acids, sugars and inorganic Salt. [2] The sterilization composition of [1], wherein the sterilization component is selected from the group consisting of cetylpyridinium chloride, benzalkonium chloride, benzalkonium chloride, ε-polylysine, 1,4-bis(3,3'-(1-decylpyridinium)methoxy)dibromobutane, sorbic acid, potassium sorbate, protamine, monolaurin and nisin At least one of the group. [3] The sterilization composition of [1] or [2], wherein the amino acid is selected from the group consisting of L-alanine, L-aspartic acid, L-tyrosine, and L-proline , At least one of the group consisting of L-valine, L-serine and casein amino acid. [4] The sterilization composition according to any one of [1] to [3], wherein the sugar is selected from D-glucose, D-fructose, D-mannose, D-galactose, maltose, lactose, At least one of the group consisting of sucrose and these caramelization reactants. [5] The sterilization composition according to any one of [1] to [4], wherein the above-mentioned inorganic salt is selected from the group consisting of ammonium chloride, ammonium sulfate, ammonium nitrate, potassium chloride, sodium chloride, potassium nitrate and At least one of the group consisting of sodium nitrate. [6] The sterilization composition according to any one of [1] to [5], which is a liquid composition further containing a liquid medium containing water. [7] The sterilization composition according to [6], wherein the content of the sterilization component is 0.1 to 1,000 mg/L based on the entire sterilization composition. [8] The sterilization composition according to [6] or [7], wherein, based on the entire sterilization composition, the content of the amino acid is 0.0001 to 1% by mass; based on the entire sterilization composition, The content of the above-mentioned sugar is 0.001-10% by mass; the content of the above-mentioned inorganic salt is 0.001-10% by mass based on the entire sterilization composition.

In addition, according to the present invention, there is provided a method for sterilizing bacterial spores as shown below. [9] A method for sterilization of bacterial spores, which has a step of contacting the sterilization composition of any one of the above [6] to [8] with the object to be treated. (Compared to the effect of the previous technology)

According to the present invention, it is possible to provide a one-dose sterilization composition that can effectively and easily sterilize bacterial spores in a short period of time, and is excellent in safety and versatility. Furthermore, according to the present invention, a method for sterilizing bacterial spores using the aforementioned sterilizing composition can be provided.

＜Bacterial elimination composition＞ The following describes embodiments of the present invention, but the present invention is not limited to the following embodiments. The sterilization composition of an embodiment of the present invention is a one-dose composition for sterilizing bacterial spores, containing germination promoting ingredients and sterilizing ingredients. Moreover, the germination promotion component contains amino acid, sugar, and inorganic salt. In addition, the term "sterilization" in this specification means to act on bacteria (spore-forming bacteria) containing bacteria (bacterial spores) in a spore state to significantly reduce the overall bacterial count, or to substantially sterilize (kill them) ). The sterilization composition of this embodiment will be described in detail below.

The germination promotion component acts on the spore state of the bacteria (bacterial spores) to promote germination and guide the vegetative bacteria. In addition, the sterilizing component is a component that acts on at least any one of bacterial spores, germ cells in the process of germination, and vegetative cells to significantly reduce the number of bacteria or substantially sterilize. The sterilization composition of the present embodiment is a so-called one-dose composition containing both a germination promoting component and a sterilization component in one formulation. Therefore, the germination and sterilization of bacterial spores can be carried out by spraying, spreading, or coating the sterilization composition of this embodiment on the treated object with bacterial spores once in contact with each other. It can be sterilized effectively and easily in a short time. Furthermore, the sterilization composition of the present embodiment essentially consists of only food additives or food additives and medical sterilization ingredients constituting the effective ingredients, so even if it touches the skin or is eaten, there are few problems and it is superior in safety. .

(Bacterial spores) Bacterial spores belonging to the sterilization target of the sterilization composition of the present embodiment show certain resistance to environments such as heat or dryness. Such bacterial spores are generally called "spore forming bacteria", which are specific bacteria that form bacterial spores under the condition of lack of nutrients. Bacterial spores become vegetative cells through germination. Spore forming bacteria that can form bacterial spores usually exist in medical facilities in addition to food processing facilities such as the kitchens of restaurants or food factories. Examples of spore-forming bacteria that form the bacterial spores of the sterilization target of the sterilization composition of the present embodiment include the following bacteria: Bacillus subtilis, Bacillus cereus, and Bacillus megaterium Bacteria of the genus Bacillus; Bacteria of the genus Clostridium such as Clostridium difficile; Bacteria of the genus Amphibacillus; Bacteria of the genus Sporosarcina Bacteria of the genus Geobacillus; Bacteria of the genus Aeribacillus; Bacteria of the genus Alicyclobacillus; Bacteria of the genus Sporolactobacillus.

(Germination promoting ingredient) The germination-promoting ingredients include amino acids, sugars and inorganic salts. By containing the germination promoting ingredients containing these ingredients, the bacterial spores can germinate and become a state where the coexisting sterilizing ingredients effectively act. In addition, in the case of not containing any of these three components, the germination is time-consuming due to the reduced germination speed, or germination is poor, and bacterial spores are likely to remain, and the germicidal effect is reduced.

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

As the sugar, sugars generally formulated in a synthetic medium for culturing spore-forming bacteria can be used. Among them, as sugars, it is preferable to use D-glucose (glucose), D-fructose (fructose), D-mannose, D-galactose, maltose, lactose, sucrose, and these caramelization reactants. These sugars can be used singly or in combination of two or more kinds.

As the inorganic salt, an inorganic salt generally formulated 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.

(Sterilization ingredients) As the sterilization component, a general sterilization component that can be used for sterilization or sterilization by acting on bacteria can be used. Among them, as the sterilizing component, it is preferable to use a group selected from the group consisting of cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzalkonium chloride, ε-polylysine, and 1,4 -Bis(3,3'-(1-decylpyridinium)methoxy)dibromobutane, sorbic acid, potassium sorbate, protamine, glyceryl monolaurate and nisin At least one. If further consideration of the impact on the human body or safety, etc., more preferred are cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzalkonium chloride, ε-polylysine, Sorbic acid and potassium sorbate.

(Liquid media) The sterilization composition uses liquid carriers, gel carriers, emulsions, solid carriers, etc., and is used in various forms such as liquid, paste, gel, milk, and solid shapes. Among them, the sterilization composition of the present embodiment is preferably a sterilization composition further containing a liquid medium. By making it into a sterilizing composition, a wide range of processed objects can be sterilized in a short time. In addition, the concept of "liquid" in this specification includes properties based on liquid such as "paste, gel, and milk". The liquid composition can be easily spread, sprayed or coated by filling it in a metered bottle with a cap, a trigger type spray container, a squeeze spray container, a pump spray container, a container with bristles, etc. .

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, the operability can be improved and the corrosion of the processed object 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, butylene glycol, and the like can be used.

When the sterilization composition is a liquid composition containing a liquid medium, the content of the amino acid in the sterilization composition is based on the entire sterilization composition, preferably 0.0001 to 1% by mass, more preferably 0.001 to 0.1% by mass. By setting the content of the amino acid within the above-mentioned range, bacterial spores can germinate 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 sugar in the sterilization composition is based on the entire sterilization composition, preferably 0.001-10% by mass, more preferably 0.1-1% by mass %. By setting the sugar content within the above range, bacterial spores can germinate 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 inorganic salt in the sterilization composition is based on the entire sterilization composition, preferably 0.001-10% by mass, more preferably 0.01-1 quality%. By setting the content of the inorganic salt within the above-mentioned range, bacterial spores can germinate 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 based on the entire sterilization composition, preferably 0.1 to 1,000 mg/L, more preferably 1 ~500mg/L. By setting the content of the sterilization component within the above range, the sterilization efficiency can be further improved. If the content of the sterilization component is too small, the sterilization efficiency may be slightly insufficient. On the other hand, if the content of the sterilization component is too large, the sterilization efficiency tends to reach the upper limit.

(Other ingredients) The antimicrobial composition of the present embodiment may contain components (other components) other than the above-mentioned components as necessary within a range that does not impair the effects of the present invention. Examples of other components include surfactants, pH adjusters, defoamers, antioxidants, and emulsifiers.

As the pH adjuster, general acids or bases can be used. Examples of the acid include inorganic acids such as hydrochloric acid and sulfuric acid, and organic acids such as lactic acid, citric acid, and salts thereof. Examples of the base include inorganic bases such as sodium hydroxide and potassium hydroxide, and organic bases such as monoethanolamine, triethanolamine, and triisopropanolamine. Among them, as the pH adjuster, inorganic acids such as hydrochloric acid and sulfuric acid, and inorganic bases such as sodium hydroxide and potassium hydroxide are preferably used.

The pH of the sterilization composition of the present embodiment at 25°C is not particularly limited, but considering the sterilization effect and operability, it is usually a neutral region, and preferably a pH in the range of 5-9.

＜Method of sterilization＞ Next, a sterilization method according to an embodiment of the present invention will be explained. The sterilization method of this embodiment has a step of bringing the aforementioned sterilization composition into contact with the object to be treated. The to-be-processed object is an object, equipment, facility, etc., where the bacterial spores of the sterilization target of the sterilization composition are attached to the surface, etc., or are likely to exist. By bringing the above-mentioned sterilization composition into contact with the object to be treated, the bacterial spores can be sterilized, the total bacterial count can be significantly reduced, or the sterilization (killing) can be substantially performed.

There are no particular limitations on the processed object as long as it is an object, equipment, facility, etc., where bacterial spores may exist. Specific examples include items such as tableware and conditioning equipment; facilities that mainly handle food such as kitchens in restaurants, food factories, and supermarkets; various equipment in food processing plants; various items or equipment in medical sites, etc.

When the sterilization composition is in a liquid form, for example, the sterilization composition can be brought into contact with the surface of the object to be treated with an amount of moisture to the whole. As a method of bringing the liquid sterilization composition into contact with the object to be treated, any methods such as spraying, spreading, and coating can be selected. In addition, if the object to be processed is a piping of an equipment or the like, it is also possible to pass a liquid sterilization composition flow through the inside (flow path) of the piping or the like. The time for the sterilization composition to contact the object to be treated is, for example, 0.5 to 10 hours, preferably 1 to 8 hours, more preferably 2 to 6 hours. By bringing the sterilizing composition into contact with the processed object within the above-mentioned time range, a sufficient sterilizing effect can be obtained. In addition, if necessary, heating may be performed in accordance with the state in which the sterilization composition is brought into contact with the object to be processed. The germination of bacterial spores can be promoted by moderate heating, which can further improve the sterilization efficiency. The temperature at the time of heating according to the state in which the sterilization composition is brought into contact with the object to be treated can be, for example, 20 to 40°C, and preferably 25 to 37°C. [Example]

Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited to these examples. In addition, the "parts" and "%" in the examples and comparative examples are referred to as quality standards unless otherwise specified. In addition, sterilized reagents and instruments were used in the test.

<Preparation of spore suspension> (Spore suspension (1)) The spores of Bacillus subtilis ATCC6633 strain were freeze-dried and stored at -80°C to obtain spore powder. After the spore powder was added to 10 mL of ion-exchanged water and suspended, it was heat-treated at 70°C for 20 minutes. After centrifugation at 7,000 rpm and 4°C for 5 minutes and collecting the bacteria, they were resuspended in ion exchange water to _{adjust the OD 650} to 3.00 to obtain a spore suspension (1).

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

(Spore suspension (2)) The spores of Bacillus cereus NBRC15305 strain were freeze-dried and stored at -80°C to obtain spore powder. After the spore powder was added to 10 mL of ion-exchanged water and suspended, it was heat-treated at 70°C for 20 minutes. After centrifugation at 7,000 rpm and 4°C for 5 minutes and collecting the bacteria, they were resuspended in ion exchange water to _{adjust the OD 650} to 3.00 to obtain a spore suspension (2).

＜Preparation of germination promotion liquid＞ (Sprouting promotion liquid (1)) After adding 0.5% L-alanine aqueous solution, 10% D-glucose aqueous solution, and 5.35% ammonium chloride aqueous solution to ion-exchanged water, it is filtered and sterilized, and it is prepared to contain L-alanine, D-glucose and chlorine at a predetermined concentration. The sprouting promotion liquid of ammonium chloride pH 7.0 (1).

(Sprouting promotion liquid (1-1)) After adding 0.5% L-alanine aqueous solution, 10% D-glucose aqueous solution, 5.35% ammonium chloride aqueous solution, and 1mol/L potassium chloride aqueous solution to ion-exchanged water, it is filtered and sterilized, and the preparation contains L- A pH 7.0 germination promoting liquid of alanine, D-glucose, ammonium chloride and potassium chloride (1-1).

(Sprouting promotion liquid (2)) After adding a 10% D-glucose aqueous solution to ion-exchanged water, it is filtered and sterilized to prepare a germination promoting solution (2) containing D-glucose at a predetermined concentration of pH 7.0.

(Germination promotion liquid (3)) After adding 0.5% L-alanine aqueous solution and 10% D-glucose aqueous solution to the ion-exchanged water, it is filtered and sterilized to prepare germination promotion liquid (3 ).

(Sprouting promotion liquid (4)) After adding 0.5% L-alanine aqueous solution to ion-exchanged water, it is filtered and sterilized to prepare a germination promoting solution (4) containing L-alanine at a predetermined concentration and pH 7.0.

(Sprouting promotion liquid (5)) After adding 0.5% L-alanine aqueous solution and 5.35% ammonium chloride aqueous solution to the ion-exchanged water, filter and sterilize to prepare germination promotion liquid (5 ).

<Preparation of sterilization component liquid> Prepare cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), 1,4-bis(3,3'-(1-decylpyridinium)methoxy) as sterilizing ingredients Dibromobutane (trade name "Hygenia (registered trademark)", manufactured by Tama Chemical Industry Co., Ltd.), ε-polylysine, glyceryl monolaurate, and nisin. These sterilization components were dissolved in ion-exchanged water, respectively, to prepare a sterilization component liquid of the type shown below. ‧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 ‧50mg/mL Hygenia aqueous solution ‧100mg/mL Hygenia aqueous solution ‧10mg/mL ε-polylysine aqueous solution ‧50mg/mL ε-polylysine aqueous solution ‧100mg/mL ε-polylysine aqueous solution ‧100mg/mL glyceryl monolaurate aqueous solution ‧50mg/mL Nisin aqueous solution ‧100mg/mL sorbic acid aqueous solution ‧100mg/mL protamine aqueous solution

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

In addition, the suspension was taken out over time, and gradually diluted with the SCDLP liquid medium to obtain a diluted solution. After spreading the obtained dilution on the SCDLP agar medium, it was cultured at 37° C. for 48 hours, and the number of colonies produced was counted to calculate the birth count concentration (CFU/mL). The results are shown in Figure 2. In addition, the cell count concentration (CFU/mL) at the initial stage and 6 hours after the start of shaking is shown in Table 1.

Furthermore, except that the CPC aqueous solution was not used, the rest was carried out in the same manner as in Examples 1 and 2. The turbidity (OD ₆₅₀ ) of the suspension was measured over time to confirm the germination state of the spores, and at the same time, the birth count concentration (CFU/mL ) (Comparative Example 1). The results are shown in Figures 1 and 2. In addition, the cell count concentration (CFU/mL) at the initial stage and 6 hours after the start of shaking is shown in Table 1.

10⁶ 1,000 實施例2 0.005 0.1 0.268 100 44

10⁶ 2,157 比較例1 0.005 0.1 0.268 — 52

10⁶ 13

10⁶ [Table 1] L-Alanine (%) glucose(%) Ammonium chloride (%) CPC (mg/L) Concentration of bacterial count (CFU/mL) Early 6 hours later Example 1 0.005 0.1 0.268 10 53

10 ⁶ 1,000 Example 2 0.005 0.1 0.268 100 44

10 ⁶ 2,157 Comparative example 1 0.005 0.1 0.268 — 52

10 ⁶ 13

10 ⁶

As shown in Fig. 1, in all cases (Examples 1 and 2, Comparative Example 1), turbidity decreased over time, and spore germination was confirmed. In addition, as shown in Fig. 2 and Table 1, in Examples 1 and 2, the bacterial count concentration drastically decreased as the spores germinated, and it can be seen that sterilization was performed efficiently.

(Examples 3 and 4, Comparative Example 2) Except for replacing the CPC aqueous solution and using the BAC aqueous solution (10 mg/mL, 100 mg/mL), the rest was the same as the above Examples 1 and 2, and the turbidity of the suspension was measured over time (OD ₆₅₀ ), and calculate the concentration of birth bacteria (CFU/mL) at the same time (Examples 3 and 4). In addition, except that the BAC aqueous solution was not used, the same procedures as in Examples 3 and 4 above were carried out. The turbidity (OD ₆₅₀ ) of the suspension was measured over time to confirm the germination state of the spores, and the number of birth bacteria (CFU/mL) was calculated at the same time. (Comparative Example 2). The results are shown in Figures 3 and 4. In addition, the cell count concentration (CFU/mL) at the initial stage and 6 hours after the start of shaking is shown in Table 2.

10⁶ 5,600 實施例4 0.005 0.1 0.268 100 59

10⁶ 6,400 比較例2 0.005 0.1 0.268 — 72.5

10⁶ 10.4

10⁵ [Table 2] L-Alanine (%) glucose(%) Ammonium chloride (%) BAC (mg/L) Concentration of bacterial count (CFU/mL) Early 6 hours later Example 3 0.005 0.1 0.268 10 54.5

10 ⁶ 5,600 Example 4 0.005 0.1 0.268 100 59

10 ⁶ 6,400 Comparative example 2 0.005 0.1 0.268 — 72.5

10 ⁶ 10.4

10 ⁵

As shown in FIG. 3, in any case (Examples 3 and 4, Comparative Example 2), turbidity decreased over time, and spore germination was confirmed. In addition, as shown in Figs. 4 and Table 2, in Examples 3 and 4, as the spores germinated, the bacterial count concentration drastically decreased, and it can be seen that sterilization was performed efficiently.

(Examples 5 and 6) Except for replacing the CPC aqueous solution and using the Hygenia aqueous solution (5mg/mL, 10mg/mL), the rest was the same as the above Examples 1 and 2, and the turbidity of the suspension (OD ₆₅₀ ) was measured over time. , And calculate the concentration of birth bacteria (CFU/mL) at the same time (Examples 5 and 6). The results are shown in Figures 5 and 6. In addition, the cell count concentration (CFU/mL) at the initial stage and 6 hours after the start of shaking is shown in Table 3.

10⁶ 3,083 實施例6 0.005 0.1 0.268 100 77.5

10⁶ 3,883 [table 3] L-Alanine (%) glucose(%) Ammonium chloride (%) Hygenia (mg/L) Concentration of bacterial count (CFU/mL) Early 6 hours later Example 5 0.005 0.1 0.268 5 83.5

10 ⁶ 3,083 Example 6 0.005 0.1 0.268 100 77.5

10 ⁶ 3,883

As shown in FIG. 5, in either case (Examples 5 and 6), turbidity decreased over time, and spore germination was confirmed. In addition, as shown in Fig. 6 and Table 3, the concentration of the number of bacteria decreased rapidly as the spores germinated, and it can be seen that the bacteria were efficiently sterilized.

(Examples 7 and 8) Except that instead of the CPC aqueous solution, the ε-polylysine aqueous solution (10 mg/mL, 100 mg/mL) was used, and the rest was the same as the above Examples 1 and 2, and the turbidity of the suspension was measured over time. The degree (OD ₆₅₀ ) was used to confirm the germination state of the spores, and at the same time calculate the birth count concentration (CFU/mL) (Examples 7 and 8). The results are shown in Figures 7 and 8. In addition, the cell count concentration (CFU/mL) at the initial stage and 6 hours after the start of shaking is shown in Table 4.

10⁶ 8,633 實施例8 0.005 0.1 0.268 100 67

10⁶ 1,950 [Table 4] L-Alanine (%) glucose(%) Ammonium chloride (%) ε-Polylysine (mg/L) Concentration of bacterial count (CFU/mL) Early 6 hours later Example 7 0.005 0.1 0.268 10 75

10 ⁶ 8,633 Example 8 0.005 0.1 0.268 100 67

10 ⁶ 1,950

As shown in Fig. 7, turbidity decreased over time in all cases (Examples 7 and 8), and spore germination was confirmed. In addition, as shown in Fig. 8 and Table 4, as the spores germinated, the concentration of the number of bacteria decreased sharply, and it was found that the sterilization was carried out efficiently.

(Example 9, Comparative Examples 3 and 4) Using 100mg/mL CPC aqueous solution, the same as the above Examples 1 and 2, the turbidity (OD ₆₅₀ ) of the suspension was measured over time, and the birth count concentration (CFU) was calculated at the same time. /mL) (Example 9). In addition, except instead of the germination promoting liquid (1), the germination promoting liquid (2) and the germination promoting liquid (3) were used separately, the rest was performed in the same manner as in Example 9, and the turbidity (OD ₆₅₀ ) of the suspension was measured over time. Confirm the germination state of the spores, and calculate the birth count concentration (CFU/mL) at the same time (Comparative Examples 3 and 4). The results are shown in Figures 9 and 10. In addition, the cell count concentration (CFU/mL) at the initial stage and 6 hours after the start of shaking is shown in Table 5.

10⁶ 3,800 比較例3 — 0.1 — 100 62

10⁶ 34

10⁶ 比較例4 0.005 0.1 — 100 84

10⁶ 57.5

10⁴ [table 5] L-Alanine (%) glucose(%) Ammonium chloride (%) CPC (mg/L) Concentration of bacterial count (CFU/mL) Early 6 hours later Example 9 0.005 0.1 0.268 100 51

10 ⁶ 3,800 Comparative example 3 — 0.1 — 100 62

10 ⁶ 34

10 ⁶ Comparative example 4 0.005 0.1 — 100 84

10 ⁶ 57.5

10 ⁴

As shown in Fig. 9, in all cases (Example 9, Comparative Examples 3 and 4), turbidity decreased over time, and spore germination was confirmed. In addition, as shown in Figs. 10 and Table 5, in Example 9, the concentration of the number of bacteria decreased sharply as the spores germinated, and it can be seen that the bacteria were efficiently sterilized. In contrast, Comparative Examples 3 and 4, which did not contain one of the germination promoting components, had a slow germination speed, did not germinate effectively, and did not decrease the number of bacteria. Therefore, it can be seen that the germination promoting component is insufficient (insufficient).

(Example 10, Comparative Examples 5 and 6) A 100mg/mL CPC aqueous solution was used, and the rest was the same as the above Examples 1 and 2. The turbidity (OD ₆₅₀ ) of the suspension was measured over time, and the birth count concentration was calculated at the same time ( CFU/mL) (Example 10). In addition, except that the germination promoting liquid (4) and the germination promoting liquid (5) were used instead of the germination promoting liquid (1), the rest was performed in the same manner as in Example 10, and the turbidity (OD ₆₅₀ ) of the suspension was measured over time. Confirm the germination state of the spores, and calculate the birth count concentration (CFU/mL) at the same time (Comparative Examples 5 and 6). The results are shown in Figures 11 and 12. In addition, the cell count concentration (CFU/mL) at the initial stage and 6 hours after the start of shaking is shown in Table 6.

10⁶ 20,950 比較例5 0.005 — — 100 54

10⁶ 12.7

10⁵ 比較例6 0.005 — 0.268 100 18.6

10⁶ 61

10⁴ [Table 6] L-Alanine (%) glucose(%) Ammonium chloride (%) CPC (mg/L) Concentration of bacterial count (CFU/mL) Early 6 hours later Example 10 0.005 0.1 0.268 100 67

10 ⁶ 20,950 Comparative example 5 0.005 — — 100 54

10 ⁶ 12.7

10 ⁵ Comparative example 6 0.005 — 0.268 100 18.6

10 ⁶ 61

10 ⁴

As shown in FIG. 11, in all cases (Example 10, Comparative Examples 5 and 6), turbidity decreased over time, and spore germination was confirmed. In addition, as shown in Figs. 12 and Table 6, in Example 10, the bacterial count concentration drastically decreased as the spores germinated, and it can be seen that sterilization was performed efficiently. In contrast, the comparative examples 5 and 6 which did not contain one of the germination promoting components had a slow germination speed, did not germinate effectively, and did not decrease the number of bacteria. Therefore, it can be seen that the germination promoting component is insufficient (insufficient).

(Example 11, Comparative Example 7) Except that instead of the CPC aqueous solution, the glycerol monolaurate aqueous solution (100 mg/mL) was used, the rest was performed in the same manner as the above Examples 1 and 2, and the turbidity (OD) of the suspension was measured over time. ₆₅₀ ), and at the same time calculate the concentration of birth bacteria (CFU/mL) (Example 11). In addition, except that the aqueous solution of glyceryl monolaurate was not used, the rest was performed in the same manner as in Example 11. The turbidity (OD ₆₅₀ ) of the suspension was measured over time to confirm the germination state of the spores, and at the same time, the birth count concentration (CFU/ mL) (Comparative Example 7). The results are shown in Figures 13 and 14. In addition, the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking is shown in Table 7.

10⁶ 34,450 比較例7 0.005 0.1 0.286 — 71

10⁶ 37.7

10⁶ [Table 7] L-Alanine (%) glucose(%) Ammonium chloride (%) Glyceryl monolaurate (mg/L) Concentration of bacterial count (CFU/mL) Early 3 hours later Example 11 0.005 0.1 0.268 100 63

10 ⁶ 34,450 Comparative example 7 0.005 0.1 0.286 — 71

10 ⁶ 37.7

10 ⁶

As shown in FIG. 13, the turbidity of the Example 11 system decreased over time, and spore germination was confirmed. In addition, as shown in Figs. 14 and Table 7, in Example 11, the number of bacteria in the system of Example 11 drastically decreased as the spores germinated, and it can be seen that sterilization was performed efficiently.

(Example 12, Comparative Example 8) Except that instead of the CPC aqueous solution, the nisin aqueous solution (50 mg/mL) was used, the rest was the same as the above Examples 1 and 2, and the turbidity (OD _{650) of the suspension was measured over time.} ), and at the same time calculate the concentration of birth bacteria (CFU/mL) (Example 12). In addition, except that the nisin aqueous solution was not used, the rest was performed in the same manner as in Example 12. The turbidity (OD ₆₅₀ ) of the suspension was measured over time to confirm the germination state of the spores, and the number of birth bacteria (CFU/mL) ) (Comparative Example 8). The results are shown in Figures 15 and 16. In addition, the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking is shown in Table 8.

10⁶ 68,000 比較例8 0.005 0.1 0.286 — 81

10⁶ 46.4

10⁶ [Table 8] L-Alanine (%) glucose(%) Ammonium chloride (%) Nisin (mg/L) Concentration of bacterial count (CFU/mL) Early 3 hours later Example 12 0.005 0.1 0.268 50 66

10 ⁶ 68,000 Comparative example 8 0.005 0.1 0.286 — 81

10 ⁶ 46.4

10 ⁶

As shown in Fig. 15, in Example 12, the turbidity decreased over time, and spore germination was confirmed. In addition, as shown in Figs. 16 and Table 8, in Example 12, the bacterial count concentration drastically decreased as the spores germinated, and it can be seen that sterilization was performed efficiently.

(Example 13, Comparative Example 9) Except for replacing the CPC aqueous solution and using the sorbic acid aqueous solution (100 mg/mL), the rest was performed in the same manner as the above Examples 1 and 2, and the turbidity (OD ₆₅₀ ) of the suspension was measured over time. At the same time, the number of birth bacteria (CFU/mL) was calculated (Example 13). In addition, except that the aqueous solution of sorbic acid was not used, the rest was performed in the same manner as in Example 13. The turbidity (OD ₆₅₀ ) of the suspension was measured over time to confirm the germination state of the spores, and at the same time, the birth count concentration (CFU/mL) was calculated ( Comparative Example 9). The results are shown in Figures 17 and 18. In addition, Table 9 shows the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking.

10⁶ 23

10⁵ 比較例9 0.005 0.1 0.286 — 56

10⁶ 23.7

10⁶ [Table 9] L-Alanine (%) glucose(%) Ammonium chloride (%) Sorbic acid (mg/L) Concentration of bacterial count (CFU/mL) Early 3 hours later Example 13 0.005 0.1 0.268 100 55

10 ⁶ twenty three

10 ⁵ Comparative example 9 0.005 0.1 0.286 — 56

10 ⁶ 23.7

10 ⁶

As shown in FIG. 17, the turbidity of Example 13 decreased over time, and spore germination was confirmed. In addition, as shown in Figs. 18 and Table 9, in Example 13, the bacterial count concentration drastically decreased as the spores germinated, and it can be seen that sterilization was performed efficiently.

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

10⁶ 98,000 比較例10 0.005 0.1 0.286 — 58

10⁶ 25.6

10⁶ [Table 10] L-Alanine (%) glucose(%) Ammonium chloride (%) Protamine (mg/L) Concentration of bacterial count (CFU/mL) Early 3 hours later Example 14 0.005 0.1 0.268 100 43

10 ⁶ 98,000 Comparative example 10 0.005 0.1 0.286 — 58

10 ⁶ 25.6

10 ⁶

As shown in FIG. 19, the turbidity of Example 14 decreased over time, and spore germination was confirmed. In addition, as shown in Figs. 20 and Table 10, in Example 14, the bacterial count concentration drastically decreased as the spores germinated, and it can be seen that sterilization was performed efficiently.

(Example 15, Comparative Example 11) After placing 18 mL of the germination promoting solution (1-1) and 20 μL of the CPC aqueous solution (50 mg/mL) in a 100 mL Erlenmeyer flask, 2 mL of the spore suspension (1-1) was added to obtain an evaluation suspension (Example 15). The flask was shaken at 37°C and 125 rpm. The suspension was taken out over time, and diluted stepwise with SCDLP liquid medium to obtain a diluted solution. After spreading the obtained dilution on the SCDLP agar medium, it was cultured at 37° C. for 48 hours, and the number of colonies produced was counted to calculate the birth count concentration (CFU/mL). The results are shown in Figure 21. In addition, the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking is shown in Table 11.

In addition, except that the CPC aqueous solution was not used, the same procedure was performed as in Example 15 above, and the number of bacteria in the suspension (CFU/mL) was calculated over time (Comparative Example 11). The results are shown in Figure 21. In addition, the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking is shown in Table 11.

[Table 11] L-Alanine (%) glucose(%) Ammonium chloride (%) Potassium chloride (%) CPC (mg/L) Concentration of bacterial count (CFU/mL) Early 3 hours later Example 15 0.005 0.1 0.268 0.373 50 6,700 110 Comparative example 11 0.005 0.1 0.268 0.373 — 3,750 1,970

As shown in Figure 21 and Table 11, even if it is set to a more realistic bacterial count concentration (approximately 6.0×10 ³ CFU/mL), the bacterial count concentration of Example 15 drops sharply. bacteria.

(Example 16, Comparative Example 12) Except for replacing the CPC aqueous solution and using the BAC aqueous solution (50 mg/mL), the same procedure was performed as in Example 15 above, and the number of bacteria in the suspension (CFU/mL) was calculated over time (Example 16). In addition, except that the BAC aqueous solution was not used, the same procedure as in Example 16 was performed, and the number of bacteria in the suspension (CFU/mL) was calculated over time (Comparative Example 12). The results are shown in Figure 22. In addition, the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking is shown in Table 12.

[Table 12] L-Alanine (%) glucose(%) Ammonium chloride (%) Potassium chloride (%) BAC (mg/L) Concentration of bacterial count (CFU/mL) Early 3 hours later Example 16 0.005 0.1 0.268 0.373 50 7,350 110 Comparative example 12 0.005 0.1 0.268 0.373 — 6,800 2,300

As shown in Figure 22 and Table 12, even in the case of a more realistic bacterial count concentration (approximately 6.0×10 ³ CFU/mL), the bacterial count concentration of the Example 16 system drastically decreased, and it can be seen that the removal was carried out efficiently. bacteria.

(Example 17, Comparative Example 13) Except for replacing the CPC aqueous solution and using the Hygenia aqueous solution (5 mg/mL), the rest was performed in the same manner as in Example 15 above, and the bacterial count concentration (CFU/mL) in the suspension was calculated over time (Example 17). In addition, except that the Hygenia aqueous solution was not used, the same procedure was performed as in Example 17 above, and the number of bacteria in the suspension (CFU/mL) was calculated over time (Comparative Example 13). The results are shown in Figure 23. In addition, the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking is shown in Table 13.

[Table 13] L-Alanine (%) glucose(%) Ammonium chloride (%) Potassium chloride (%) Hygenia (mg/L) Concentration of bacterial count (CFU/mL) Early 3 hours later Example 17 0.005 0.1 0.268 0.373 5 5,900 1 Comparative example 13 0.005 0.1 0.268 0.373 — 3,500 2,530

As shown in Figure 23 and Table 13, even if it is set to a more realistic cell count concentration (approximately 6.0×10 ³ CFU/mL), the cell count concentration of Example 17 is drastically reduced, and it can be seen that removal is carried out efficiently. bacteria.

(Example 18, Comparative Example 14) Except for replacing the CPC aqueous solution and using the ε-polylysine aqueous solution (5 mg/mL), the rest was the same as in Example 15 above, and the concentration of the number of bacteria in the suspension (CFU/mL) was calculated over time (Example 18 ). In addition, except that the ε-polylysine aqueous solution was not used, the same procedure as in Example 18 was performed, and the number of bacteria in the suspension (CFU/mL) was calculated over time (Comparative Example 14). The results are shown in Figure 24. In addition, the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking is shown in Table 14.

[Table 14] L-Alanine (%) glucose(%) Ammonium chloride (%) Potassium chloride (%) ε-Polylysine (mg/L) Concentration of bacterial count (CFU/mL) Early 3 hours later Example 18 0.005 0.1 0.268 0.373 5 3,600 1 Comparative example 14 0.005 0.1 0.268 0.373 — 1,600 1,280

As shown in Fig. 24 and Table 14, even if it is set to a more realistic cell count concentration (approximately 6.0×10 ³ CFU/mL), the cell count concentration of the Example 18 system drops sharply, and it can be seen that the removal is carried out efficiently. bacteria.

(Example 19, Comparative Example 15) After placing 18 mL of the germination promoting solution (1) and 20 μL of the nisin aqueous solution (50 mg/mL) in a 100 mL Erlenmeyer flask, 2 mL of the spore suspension (1-1) was added to obtain an evaluation suspension (Example 19). The flask was shaken at 37°C and 125 rpm. The suspension was taken out over time, and diluted stepwise with SCDLP liquid medium to obtain a diluted solution. After spreading the obtained dilution on the SCDLP agar medium, it was cultured at 37° C. for 48 hours, and the number of colonies produced was counted to calculate the birth count concentration (CFU/mL). The results are shown in Figure 25. In addition, the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking is shown in Table 15.

In addition, except that the nisin aqueous solution was not used, the same procedure as in Example 19 was performed, and the number of bacteria in the suspension (CFU/mL) was calculated over time (Comparative Example 15). The results are shown in Figure 25. In addition, the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking is shown in Table 15.

[Table 15] L-Alanine (%) glucose(%) Ammonium chloride (%) Nisin (mg/L) Concentration of bacterial count (CFU/mL) Early 3 hours later Example 19 0.005 0.1 0.268 50 6,800 1 Comparative example 15 0.005 0.1 0.268 — 6,850 605

As shown in Fig. 25 and Table 15, even if it is set to a more realistic cell count concentration (approximately 6.0×10 ³ CFU/mL), the cell count concentration of Example 19 is drastically reduced, and it can be seen that removal is carried out efficiently. bacteria.

(Examples 20 and 21, Comparative Example 16) After placing 18 mL of the germination promoting liquid (1) and 20 μL of the CPC aqueous solution (5 mg/mL, 50 mg/mL) into a 100 mL Erlenmeyer flask, the spore suspension (2) 2 mL was added. Suspensions for evaluation (Examples 20 and 21). The flask was shaken at 37°C and 125 rpm, and the turbidity (OD ₆₅₀ ) of the suspension was measured over time to confirm the germination state of the spores. The measurement results are shown in Figure 26.

In addition, the suspension was taken out over time, and gradually diluted with the SCDLP liquid medium to obtain a diluted solution. After spreading the obtained dilution on the SCDLP agar medium, it was cultured at 37° C. for 48 hours, and the number of colonies produced was counted, and the concentration of birth bacteria (CFU/mL) was calculated. The results are shown in Figure 27. In addition, the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking is shown in Table 16.

Furthermore, except that the CPC aqueous solution was not used, the rest was carried out in the same manner as in Examples 20 and 21. The turbidity (OD ₆₅₀ ) of the suspension was measured over time to confirm the germination state of the spores, and at the same time, the birth count concentration (CFU/mL ) (Comparative Example 16). The results are shown in Figures 26 and 27. In addition, the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking is shown in Table 16.

10⁵ 1,970 實施例21 0.005 0.1 0.268 50 81

10⁵ 4,715 比較例16 0.005 0.1 0.286 — 87

10⁵ 27.3

10⁵ [Table 16] L-Alanine (%) glucose(%) Ammonium chloride (%) CPC (mg/L) Concentration of bacterial count (CFU/mL) Early 3 hours later Example 20 0.005 0.1 0.268 5 98

10 ⁵ 1,970 Example 21 0.005 0.1 0.268 50 81

10 ⁵ 4,715 Comparative example 16 0.005 0.1 0.286 — 87

10 ⁵ 27.3

10 ⁵

As shown in FIG. 26, in all cases (Examples 20 and 21, and Comparative Example 16), turbidity decreased over time, and spore germination was confirmed. In addition, as shown in Figs. 27 and Table 16, in Examples 20 and 21, the bacterial count concentration drastically decreased as the spores germinated, and it can be seen that sterilization was performed efficiently.

(Examples 22 and 23, Comparative Example 17) Except for replacing the CPC aqueous solution and using the BAC aqueous solution (5mg/mL, 50mg/mL), the rest was the same as the above Examples 20 and 21, and the turbidity of the suspension was measured over time. (OD ₆₅₀ ), and calculate the birth count concentration (CFU/mL) at the same time (Examples 22 and 23). In addition, except that the BAC aqueous solution was not used, the rest was performed in the same manner as in Examples 22 and 23. The turbidity (OD ₆₅₀ ) of the suspension was measured over time to confirm the germination state of the spores, and at the same time, the birth count concentration (CFU/mL) was calculated. (Comparative Example 17). The results are shown in Figures 28 and 29. In addition, the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking is shown in Table 17.

10⁶ 1,500 實施例23 0.005 0.1 0.268 50 71

10⁵ 17,250 比較例17 0.005 0.1 0.286 — 18

10⁶ 23.3

10⁵ [Table 17] L-Alanine (%) glucose(%) Ammonium chloride (%) BAC (mg/L) Concentration of bacterial count (CFU/mL) Early 3 hours later Example 22 0.005 0.1 0.268 5 16

10 ⁶ 1,500 Example 23 0.005 0.1 0.268 50 71

10 ⁵ 17,250 Comparative example 17 0.005 0.1 0.286 — 18

10 ⁶ 23.3

10 ⁵

As shown in FIG. 28, turbidity decreased over time in all cases (Examples 22 and 23, and Comparative Example 17), and spore germination was confirmed. In addition, as shown in Figs. 29 and Table 17, in Examples 22 and 23, the bacterial count concentration drastically decreased as the spores germinated, and it can be seen that sterilization was performed efficiently.

(Examples 24 and 25, Comparative Example 18) Except that instead of the CPC aqueous solution, Hygenia aqueous solution (5mg/mL, 50mg/mL) was used, the rest was the same as the above Examples 20 and 21, and the turbidity of the suspension was measured over time. (OD ₆₅₀ ), and calculate the birth count concentration (CFU/mL) at the same time (Examples 24 and 25). In addition, except that the Hygenia aqueous solution was not used, the same procedures as in Examples 24 and 25 were carried out. The turbidity (OD ₆₅₀ ) of the suspension was measured over time to confirm the germination state of the spores, and at the same time, the birth count concentration (CFU/mL) was calculated. (Comparative Example 18). The results are shown in Figures 30 and 31. In addition, the cell count concentration (CFU/mL) at the initial stage and 3 hours after the start of shaking is shown in Table 18.

10⁶ 1,650 實施例25 0.005 0.1 0.268 50 67

10⁵ 29,350 比較例18 0.005 0.1 0.286 — 84

10⁵ 12.9

10⁵ [Table 18] L-Alanine (%) glucose(%) Ammonium chloride (%) Hygenia (mg/L) Concentration of bacterial count (CFU/mL) Early 3 hours later Example 24 0.005 0.1 0.268 5 13

10 ⁶ 1,650 Example 25 0.005 0.1 0.268 50 67

10 ⁵ 29,350 Comparative Example 18 0.005 0.1 0.286 — 84

10 ⁵ 12.9

10 ⁵

As shown in FIG. 30, in all cases (Examples 24 and 25, and Comparative Example 18), the turbidity decreased over time, and spore germination was confirmed. In addition, as shown in Fig. 31 and Table 18, in Examples 24 and 25, the bacterial count concentration drastically decreased as the spores germinated, and it can be seen that sterilization was performed efficiently.

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

10⁶ 102,000 比較例19 0.005 0.1 0.286 — 18

10⁶ 26.2

10⁵ [Table 19] L-Alanine (%) glucose(%) Ammonium chloride (%) ε-Polylysine (mg/L) Concentration of bacterial count (CFU/mL) Early 3 hours later Example 26 0.005 0.1 0.268 50 20

10 ⁶ 102,000 Comparative Example 19 0.005 0.1 0.286 — 18

10 ⁶ 26.2

10 ⁵

As shown in FIG. 32, turbidity decreased over time in all cases (Example 26, Comparative Example 19), and spore germination was confirmed. In addition, as shown in Fig. 33 and Table 19, in Example 26, the bacterial count concentration drastically decreased as the spores germinated, and it can be seen that the sterilization was carried out efficiently. (Industrial availability)

The sterilizing composition of the present invention can be used as a highly safe composition that can sterilize bacterial spores that are difficult to sterilize in the past by simple operation.

Fig. 1 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Examples 1 and 2 and Comparative Example 1. Fig. 2 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of microorganisms) of Examples 1 and 2 and Comparative Example 1. 3 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Examples 3 and 4 and Comparative Example 2. FIG. Fig. 4 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Examples 3 and 4 and Comparative Example 2. Fig. 5 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Examples 5 and 6. Fig. 6 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Examples 5 and 6. Fig. 7 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Examples 7 and 8. Fig. 8 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Examples 7 and 8. Fig. 9 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Example 9 and Comparative Examples 3 and 4. Fig. 10 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Example 9 and Comparative Examples 3 and 4. Fig. 11 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Example 10 and Comparative Examples 5 and 6. Fig. 12 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Example 10 and Comparative Examples 5 and 6. FIG. 13 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Example 11 and Comparative Example 7. FIG. Fig. 14 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Example 11 and Comparative Example 7. 15 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Example 12 and Comparative Example 8. FIG. Fig. 16 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Example 12 and Comparative Example 8. Fig. 17 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Example 13 and Comparative Example 9. 18 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Example 13 and Comparative Example 9. 19 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Example 14 and Comparative Example 10. Fig. 20 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Example 14 and Comparative Example 10. Fig. 21 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Example 15 and Comparative Example 11. Fig. 22 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Example 16 and Comparative Example 12. Fig. 23 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Example 17 and Comparative Example 13. Fig. 24 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Example 18 and Comparative Example 14. Fig. 25 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Example 19 and Comparative Example 15. FIG. 26 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Examples 20 and 21 and Comparative Example 16. FIG. Fig. 27 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Examples 20 and 21 and Comparative Example 16. Fig. 28 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Examples 22 and 23 and Comparative Example 17. Fig. 29 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Examples 22 and 23 and Comparative Example 17. FIG. 30 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Examples 24 and 25 and Comparative Example 18. FIG. FIG. 31 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Examples 24 and 25 and Comparative Example 18. FIG. Fig. 32 is a graph showing the results of the sterilization effect confirmation test (reduction in turbidity) of Example 26 and Comparative Example 19. FIG. 33 is a graph showing the results of the sterilization effect confirmation test (decrease in the number of bacteria) in Example 26 and Comparative Example 19. FIG.

Claims (9)

A sterilizing composition used to sterilize bacterial spores, It is a one-dose composition containing germination promoting ingredients and sterilizing ingredients, The said germination promotion component contains amino acid, sugar, and inorganic salt. Such as the sterilization composition of claim 1, wherein the aforementioned sterilization component is selected from the group consisting of cetylpyridinium chloride, benzalkonium chloride, benzalkonium chloride, ε-polylysine, 1,4 -Bis(3,3'-(1-decylpyridinium)methoxy)dibromobutane, sorbic acid, potassium sorbate, protamine, glyceryl monolaurate and nisin At least one. The sterilization composition of claim 1 or 2, wherein the above-mentioned amino acid is selected from L-alanine, L-aspartic acid, L-tyrosine, L-proline, and L-valine At least one of the group consisting of acid, L-serine and casein amino acid. The sterilization composition of claim 1 or 2, wherein the sugar is selected from the group consisting of D-glucose, D-fructose, D-mannose, D-galactose, maltose, lactose, sucrose and these caramelized reactants At least one of the constituent groups. The sterilization composition of claim 1 or 2, wherein the inorganic salt is at least one selected from the group consisting of ammonium chloride, ammonium sulfate, ammonium nitrate, potassium chloride, sodium chloride, potassium nitrate, and sodium nitrate. Such as the sterilization composition of claim 1, which is a liquid composition further containing a liquid medium containing water. Such as the sterilization composition of claim 6, wherein, based on the entire sterilization composition, the content of the aforementioned sterilization component is 0.1 to 1,000 mg/L. Such as the sterilization composition of claim 6 or 7, wherein, based on the entire sterilization composition, the content of the above-mentioned amino acid is 0.0001 to 1% by mass; Based on the entire sterilization composition, the content of the above-mentioned sugar is 0.001-10% by mass; The content of the above-mentioned inorganic salt is 0.001-10% by mass based on the entire sterilization composition. A method for sterilization of bacterial spores has a step of contacting the sterilization composition of any one of claims 6 to 8 with the object to be treated.

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