JP2018203726A - Bactericidal agent, bactericidal method, and bactericidal kit - Google Patents

Abstract

To provide a bactericidal agent, a bactericidal method, and a bactericidal kit having excellent bactericidal effect and high safety.SOLUTION: A bactericidal agent contains 60 v/v% or more of ethanol and nisin as active ingredients. A bactericidal method is a method having a contact process for contacting the bactericidal agent with a bactericidal object. A bactericidal kit has 60 v/v% or more of ethanol and nisin. A content of the ethanol may be 70 v/v% or more. The content of the ethanol may be 80 v/v% or more. The bactericidal agent and the bactericidal kit may be used for foods. The bactericidal object may be foods. The foods may be meat.SELECTED DRAWING: None

Description

  The present invention relates to a disinfectant, a disinfecting method, and a disinfecting kit.

  Nisin is widely known as an antimicrobial peptide derived from lactic acid bacteria. Nisin has been used as a food preservative for various foods because it shows a high sterilizing effect against Gram-positive food-contaminating bacteria and food poisoning bacteria. Although the target food is limited in Japan, Nisin A was approved as a food additive in March 2009. Specifically, “meat”, “whipped cream”, “cheese (excluding processed cheese)”, “processed cheese”, “Western confectionery mainly using cereals and starch”, “Western confectionery”, “sauce” , Mayonnaise, dressing ”,“ egg processed product ”, and“ miso ”total 9 items can be used as a food additive of Nisin A.

  On the other hand, ethanol has been widely used as a disinfectant. By combining nisin and low concentration ethanol of about 30 v / v% or less, there is a synergistic effect of sterilization against bacteria such as Listeria monocytogenes which is Gram-positive bacteria or Escherichia coli which is Gram-negative bacteria. Has already been reported (for example, see Non-Patent Documents 1 and 2).

  Patent Document 1 discloses a sterilizing composition containing nisin and thioctic acid or a salt acceptable for medical or food hygiene as a nisin stabilizer. Furthermore, in the examples, an alcohol preparation comprising this sterilizing composition and 25 w / w% alcohol is described.

JP 2010-270014 A

Brewer R et al., “Enhanced inactivation of Listeria monocytogenes by nisin in the presence of ethanol”, Letters in Applied Microbiology, vol.34, p18-21, 2002. Komsan Phongphakdee and Sunee Nitisinprasert, “Combination Inhibition Activity of Nisin and Ethanol on the Growth Inhibition of Pathogenic Gram Negative Bacteria and Their Application as Disinfectant Solution”, Journal of Food Science, vol.80, nr.10, M2241- M2246, 2015.

  In Non-Patent Documents 1 and 2 and Patent Document 1, the effects of adding nisin and ethanol to a single bacterium are examined, and the sterilization target under various conditions The sterilizing effect on the bacterium has not been studied.

  The present invention has been made in view of the above circumstances, and provides a disinfectant, a disinfecting method and a disinfecting kit having an excellent disinfecting effect and high safety.

  As a result of intensive studies to achieve the above object, the present inventors have used either nisin and high-concentration ethanol, so that either gram-positive bacteria or gram-negative bacteria can be used even in the presence of organic matter such as meat. Also found that it can be effectively sterilized.

That is, the present invention includes the following aspects.
The disinfectant according to the first aspect of the present invention contains 60 v / v% or more ethanol and nisin as active ingredients.
The ethanol content may be 70 v / v% or more.
The ethanol content may be 80 v / v% or more.
The nisin content may be 1.25 ppm or more.
The disinfectant according to the above aspect may be used for food.
The food may be meat.

The sterilization method according to the second aspect of the present invention is a method including a contact step of bringing the sterilization agent according to the first aspect into contact with a sterilization target.
In the contacting step, a sterilizing agent for the food may be applied to the sterilization target.
In the contacting step, the sterilization target may be immersed in a sterilizing agent for the food.
The sterilization target may be food.
The food may be meat.

The sterilization kit according to the third aspect of the present invention comprises 60 v / v% or more ethanol and nisin.
The ethanol content may be 70 v / v% or more.
The ethanol content may be 80 v / v% or more.
The sterilization kit according to the above aspect may be used for food.
The food may be meat.

  According to the above aspect, a disinfectant, a disinfecting method and a disinfecting kit having an excellent disinfecting effect and high safety can be provided.

It is the result of the sterilization test in Test Example 1. Specifically, 70 v / v% ethanol solution, 80 v / v% ethanol solution, 1.25 ppm nisin-containing 70 v / v% ethanol solution (disinfectant 6), or 1.25 ppm nisin-containing 80 v / v% ethanol solution It is a graph which shows the decreasing rate of the microbial cell in the meat piece of the beef thigh which sprayed (sanitizer 7). It is the result of the sterilization test in Test Example 1. Specifically, 70 v / v% ethanol solution, 80 v / v% ethanol solution, 1.25 ppm nisin-containing 70 v / v% ethanol solution (disinfectant 6), or 1.25 ppm nisin-containing 80 v / v% ethanol solution It is a graph which shows the reduction | decrease rate of the microbial cell in the pork thigh which injected (sanitizer 7). It is the result of the sterilization test in Test Example 1. Specifically, 70 v / v% ethanol solution, 80 v / v% ethanol solution, 1.25 ppm nisin-containing 70 v / v% ethanol solution (disinfectant 6), or 1.25 ppm nisin-containing 80 v / v% ethanol solution It is a graph which shows the decreasing rate of the microbial cell in the meat piece of the chicken fillet which sprayed (sanitizer 7). It is a result of the taste test in Test Example 1. Specifically, cooked beef thigh meat pieces (control), 1.25 ppm nisin-containing 70 v / v% ethanol solution (sterilizer 6), or 1.25 ppm nisin content It is a graph which shows the result of the taste test of the meat piece of the beef thigh which inject | poured the 80 v / v% ethanol solution (sanitizer 7), and was cooked. It is a result of the taste test in Test Example 1. Specifically, pork thigh meat pieces (control) that have not been sprayed with a sanitizer, or a 1.25 ppm nisin-containing 70 v / v% ethanol solution (sanitizer 6), or 1.25 ppm nisin It is a graph which shows the result of the taste test of the pork thigh meat piece which inject | poured the 80 v / v% ethanol solution (sanitizer 7), and was cooked. It is a result of the taste test in Test Example 1. Specifically, the meat pieces of cooked chicken fillet meat (control), or 1.25 ppm nisin-containing 70 v / v% ethanol solution (sanitizer 6), or 1.25 ppm nisin contained It is a graph which shows the result of the taste test of the meat piece of the chicken breast meat which injected the 80 v / v% ethanol solution (sanitizer 7), and was cooked. It is a result of the sterilization test in Test Example 2. Specifically, it is a graph showing the rate of decrease of bacterial cells in meat pieces of chicken fillet meat immersed in a 1.25 ppm nisin-containing 20-80 v / v% ethanol solution for 5, 15, or 30 seconds at room temperature. It is a result of the sterilization test in Test Example 2. Specifically, it is a graph showing the decrease rate of bacterial cells in a piece of chicken breast meat immersed in a 20 to 80 v / v% ethanol solution at room temperature for 5, 15, or 30 seconds. It is the result of the sterilization test in Test Example 3. Specifically, it is a graph showing the reduction rate of the cells in the meat pieces of chicken fillet that was immersed in a 1.25 ppm nisin-containing 60-80 ethanol solution for 5, 15, or 30 seconds at 4 ° C. It is the result of the sterilization test in Test Example 4. Specifically, it is a graph which shows the decreasing rate of a microbial cell in each frequency | count at the time of using the 1.25 ppm nisin containing 80v / v% ethanol solution 100 times continuously, and immersing the meat piece of chicken breast meat. It is the result of the taste test in Test Example 5. Specifically, it was immersed in a heat-cooked chicken breast meat piece (control) or an 1.25 ppm nisin-containing 80 v / v% ethanol solution (sanitizer 7), which was not immersed in a disinfectant, and cooked. It is a graph which shows the result of the taste test of the meat piece of chicken fillet. 6 is a graph showing changes over time in the residual activity of nisin in a nisin-containing 70 v / v% ethanol solution (Nisin content: 2 mg / 40 mL) stored under various conditions in Test Example 6. FIG. 6 is a graph showing changes over time in the residual activity of nisin in a nisin-containing 80 v / v% ethanol solution (Nisin content: 2 mg / 40 mL) stored under various conditions in Test Example 6. FIG. 6 is a graph showing changes over time in the residual activity of nisin in a nisin-containing 70 v / v% ethanol solution (Nisin content: 2 mg / 20 mL) stored under various conditions in Test Example 6. FIG. 6 is a graph showing changes over time in the residual activity of nisin in a nisin-containing 80 v / v% ethanol solution (Nisin content: 2 mg / 20 mL) stored under various conditions in Test Example 6. FIG. 6 is a graph showing changes over time in the residual activity of nisin in a nisin-containing 70 v / v% ethanol solution (Nisin content: 2 mg / 10 mL) stored under various conditions in Test Example 6. FIG. 6 is a graph showing changes over time in the residual activity of nisin in a nisin-containing 80 v / v% ethanol solution (Nisin content: 2 mg / 10 mL) stored under various conditions in Test Example 6. FIG. 10 is a graph showing a decrease rate of L. lactis NBRC12007 due to injection of each solution in Test Example 7. 10 is a graph showing the number of viable bacteria on the sink surface separated bacteria after spraying of each solution in Test Example 8. It is a graph which shows the number of living bacteria of the sink surface separation microbe A strain after the injection of each solution in Test example 8. It is a graph which shows the viable count of the sink surface separation microbe B strain after the injection of each solution in Test example 8.

≪Disinfectant≫
The disinfectant according to one embodiment of the present invention contains 60 v / v% or more ethanol and nisin as active ingredients.

The disinfectant according to the present embodiment contains nisin and high-concentration ethanol of 60 v / v% or more, so that gram-positive bacteria can be obtained under conditions where food, residual sebum, and organic matter such as clothing with attached proteins exist. And it has an excellent sterilizing effect against both gram-negative bacteria.
In addition, ethanol and nisin contained in the disinfectant of this embodiment have already been approved for food use as food additives. Therefore, the disinfectant of this embodiment has high safety.

  In general, “sterilization” means to increase the cleanliness by reducing the number of objects such as objects and liquids and microorganisms contained in a limited space. It is considered that the disinfectant according to the present embodiment contains high concentration ethanol of 60 v / v% or more, so that the functional protein of the microorganism can be denatured and the cell membrane or cell wall can be damaged. Furthermore, by containing nisin, after high-concentration ethanol evaporates, it passes through the cell wall damaged by the high-concentration ethanol and reaches the cell membrane of the microorganism. Nisin reaches the low molecular component from the pores formed on the cell membrane. It is speculated that the sterilization effect can be exerted by killing the microorganisms by the outflow and the depletion of ATP.

Moreover, the disinfectant of this embodiment has an excellent disinfecting effect against both gram-positive and gram-negative bacteria.
Specific examples of the Gram-positive bacterium include, for example, Exiguobacterium, Staphylococcus (Staphylococcus aureus, Staphylococcus epidermis) and the like. , Microorganisms belonging to the genus Streptococcus (Streptococcus mutans, etc.), Lactobacillus (Lactobacillus plantarum, etc.); methicillin-resistant Staphylococcus aureus (MRSA), vancomycin Antibiotic-resistant bacteria such as resistant Staphylococcus aureus (VRSA), vancomycin-resistant enterococci (VRE), penicillin-resistant pneumococci (PRSP), and the like are included, but are not limited thereto.
Specific examples of the gram-negative bacterium include, for example, Escherichia (eg, Escherichia coli), Enterobacter, Klebsiella, Bacillus. ) Microorganisms belonging to (Bacillus subtilis, etc.); multidrug-resistant Pseudomonas aeruginosa (MDRP), carbapenem-resistant Pseudomonas aeruginosa, carbapenem-resistant Serratia, third-generation cephalosporin-resistant Escherichia coli, third-generation cephalosporin Examples include, but are not limited to, antibiotic-resistant bacteria such as resistant Klebsiella pneumoniae and multidrug-resistant Acinetobacter.

  In addition, there is no special limitation as a disinfection target object which can use the disinfectant of this embodiment. Specific examples of the sterilization target include, for example, parts of the body of animals including humans such as oral cavity, teeth and periodontals, nasal cavity, fingers, face, and feet; food such as meat, processed meat products, vegetables, and fruits ; Clothing; medical equipment, cooking utensils, hygiene equipment, nursing supplies, baby products, toys, food manufacturing machines, medical equipment, tables, desks, chairs, home furnishings, etc .; household and commercial kitchens, food Examples include, but are not limited to, processing factories, food material manufacturing factories, houses, hotels, restaurants and other indoor areas, medical facilities, hospital rooms, floors, work benches and other places.

The meat is classified into seafood and livestock meat.
Specific examples of seafood include fish such as sweetfish, carp, trout, salmon, walleye pollock, hoki, Thailand, tuna, swordfish, sardine, mackerel, horse mackerel, saury, sea bream, prickly pear, and cruciferous; jawless such as lamprey Examples include: shellfish such as abalone, turban shell, barnacle, oyster, scallop; cephalopods such as octopus and squid; crustacean such as shrimp and crab; and echinoderms such as sea urchin and sea cucumber. In addition, the seafood may be provided for raw consumption or may be provided for heating.
Specifically, for example, livestock meat such as cattle, pigs, horses, sheep, goats, etc .; meat of animals such as sharks, deer, bears; meat of poultry such as chickens, turkeys, quails, ducks, ducks; Examples include, but are not limited to, wild bird meat such as duck, pheasant, sparrow, thrush, and pigeon; meat from other animals such as salmon, frog, kangaroo, turtle, sea pig, whale, and salmon. Moreover, livestock meat may be provided for raw consumption or may be provided for heating.

  Examples of the processed meat products include, but are not limited to, processed food products of livestock meat such as hamburger, meatballs, meat loaf, ham, sausage; processed food products of seafood such as kamaboko, tsumire, chikuwa, hampen, fish sausage, etc. Not.

Specifically, examples of the vegetables include Brassicaceae (radish, cabbage, Chinese cabbage, wasabi), Asteraceae (lettuce, chrysanthemum), Celeryceae (carrot, Kintoki carrot, celery, parsley), Eggplant family (tomato, capsicum) , Paprika, potato, eggplant), Aozidae (spinach), liliaceae (onion, scallop, garlic, leek, leek), cucurbitaceae (cilia), and the like, but are not limited thereto. Further, the vegetables may be provided for raw consumption or may be provided for processing.
Specific examples of the fruit include, but are not limited to, strawberry, cherry, grape, sudachi, capos and the like. Moreover, the fruit may be provided for raw consumption or may be provided for processing.

Among these, sanitizing objects include parts of the body of animals including humans, food, clothing with residual sebum and protein, cooking utensils, food manufacturing machines, household and commercial kitchens, food processing plants, and It is preferable that the conditions are such that organic substances such as a food material manufacturing factory exist. As shown in the Examples described later, ethanol alone has low sterilization efficiency, whereas the sterilization agent of this embodiment achieves high sterilization efficiency. In addition, the sterilization target is more preferably food, more preferably meat, and particularly preferably raw meat. By using the disinfectant of this embodiment, meat can be provided for raw consumption.
As the meat, beef, pork or chicken-derived meat is preferable, beef or chicken-derived meat is more preferable, and chicken-derived meat is more preferable.
According to the sterilization method of the present embodiment, when the sterilization target is food (especially meat), the food (particularly meat) while exhibiting an excellent sterilization effect, as shown in Examples described later. ) Can maintain good taste and fragrance.

<Content of ethanol and nisin>
In addition, in the disinfectant of this embodiment, “contains as an active ingredient” means that ethanol and nisin are each contained as a component having a disinfecting effect.

Specifically, the lower limit of the ethanol content in the disinfectant of the present embodiment is preferably 60 v / v% or more, more preferably 70 v / v% or more, and 80 v / v% or more. More preferably.
On the other hand, the upper limit value of the ethanol content in the disinfectant of the present embodiment is not specifically limited, and may be 99 v / v% or less, for example 95 v / v% or less.
When the content of ethanol in the disinfectant of the present embodiment is in the above range, as shown in the examples described later, gram-negative bacteria and gram are obtained due to a synergistic effect with nisin in the presence of organic matter and the like. Excellent sterilization effect can be exerted against any positive bacteria.

The lower limit of the content of nisin in the disinfectant of the present embodiment is not particularly limited as long as it is an amount exhibiting a disinfecting effect. Specifically, the lower limit of the content of nisin in the disinfectant of the present embodiment is preferably 0.5 ppm or more, and more preferably 1.0 ppm or more.
On the other hand, the upper limit of the content of nisin in the disinfectant of the present embodiment is not specifically limited, and is, for example, 2.0 ppm or less, for example 1.8 ppm or less, from the viewpoint of manufacturing cost. Also good. In addition, when the production cost is not particularly limited, the disinfectant of this embodiment is more than 2.0 ppm and may contain nisin.
When the content of nisin in the disinfectant of the present embodiment is in the above range, as shown in the examples described later, gram-negative bacteria and gram are obtained due to a synergistic effect with ethanol in the presence of organic matter and the like. Excellent sterilization effect can be exerted against any positive bacteria.
Details of the components of the disinfectant of this embodiment will be described below.

<Ethanol>
As ethanol contained in the disinfectant of this embodiment, for example, industrial alcohol, absolute ethanol having a purity of 99.5% or more specified in the Japanese Pharmacopoeia, hydrous ethanol containing 4 to 5% water, purity Examples include, but are not limited to, 70-90% ethanol for disinfection. These may be used individually by 1 type and may use 2 or more types together.
The ethanol exemplified above may be used so that the content of ethanol contained in the disinfectant falls within the above range. Moreover, what is necessary is just to select the purity of ethanol suitably according to the kind of target object which uses a disinfection agent. In particular, when the sterilization target is food, it is preferable to use absolute ethanol having a purity of 99.5% or more.

<Nishin>
Examples of nisin contained in the disinfectant of this embodiment include, but are not limited to, nisin A, nisin Z, nisin Q, and the like. The disinfectant of this embodiment may contain one kind of these nisins or may contain two or more kinds.
Among these, nisin A is preferable as the nisin contained in the disinfectant of the present embodiment.

  Nisin A is produced by the lactic acid bacterium Lactococcus lactis and has already been approved and used as a food additive in more than 50 countries including Europe and America. Nisin Z is a protein similar to nisin A. Of the 34 amino acid residues constituting nisin A, the 27th amino acid residue from the N-terminal is a histidine residue in nisin A, but only asparagine residue in nisin Z.

Nisin contained in the disinfectant of the present embodiment can be obtained by culturing and purifying lactic acid bacteria using a known method. Specifically, for example, first, lactic acid bacteria are cultured using MRS medium (Oxoid). Next, the culture supernatant is treated with Amberlite (Amberlite XAD-16, manufactured by Sigma) to adsorb nisin. The amberlite is then washed with distilled water and 40 v / v% ethanol. The nisin is then eluted with 70 v / v% isopropanol containing 0.1 w / v% trifluoroacetic acid. Subsequently, the purified nisin product can be obtained by purifying the elution fraction using a cation exchange column (SP-Sepharose FF, manufactured by GE Healthcare Biosciences). Moreover, the nisin after a cation exchange column can be refine | purified using a reverse phase chromatography as needed, and the refinement | purification degree of nisin can be raised further (reference literature 1: Zendo T et al., " Identification of the Lantibiotic Nisin Q, a New Natural Nisin Variant Produced by Lactococcus lactis 61-14 Isolated from a River in Japan ”, Biosci. Biotechnol. Biochem., Vol. 67, No. 7, p1616-1619, 2003).
A commercially available nisin product may also be used, and specific examples thereof include, but are not limited to, nisin A reagent (containing 2.5% nisin, manufactured by Sigma).
The disinfectant of this embodiment may contain nisin produced using a known method and a commercially available product of nisin, respectively, or may contain two or more kinds.

<Other components>
It is preferable that the disinfectant of this embodiment does not substantially contain nisin stabilizers such as thioctic acid and its analogs.
In the disinfectant of the present embodiment, “substantially free” means that it does not contain any nisin stabilizer, or contains only a trace amount that does not exert the stabilizing effect of nisin. When a small amount of nisin stabilizer is contained, the content thereof may be, for example, less than 1 part by mass with respect to 1 part by mass of nisin.

The disinfectant of the present embodiment may contain other components other than ethanol and nisin as long as the disinfection effect is not hindered.
Specific examples of other components include surfactants (emulsifiers, dispersants (anti-redeposition agents)), chelating agents, wetting agents, abrasives, bactericides (antibacterial agents), viscosity modifiers, solvents ( For example, water, etc.), lubricants, moisturizers, stabilizers, cleaning enzymes, preservatives, antifungal agents, coloring agents, fragrances (including flavoring agents and fragrances), and the like are not limited thereto. .
Moreover, what is necessary is just to select suitably about the other structural component contained in the disinfectant of this embodiment according to the form of the said disinfectant, and the kind of disinfection target object.
It is preferable that the disinfectant of this embodiment consists only of ethanol, nisin, and water.

<Shape and form>
The disinfectant of this embodiment can be liquid, semi-solid, or solid. Specific examples of the shape of the present embodiment include, but are not limited to, liquids, emulsions (emulsions), gels, various dosage forms impregnated with them, and the like.
When the disinfectant of the present embodiment is in a liquid state, specific examples thereof include, for example, a spray container, a push-type container (for example, a type that comes out of liquid), a refill bottle, and the like. However, it is not limited to these.
When the disinfectant of the present embodiment is semi-solid or solid, specifically, for example, a spray container, a push-type container (for example, a type that comes out of a gel), an oral rinse And rinse agents, dentifrices, dipping agents, wipes, paper towels, wet tissues, and the like, but are not limited thereto.

  The storage conditions for the disinfectant of the present embodiment are not particularly limited, and may be stored at a temperature of -20 ° C or higher and 40 ° C or lower, for example. Since the activity of nisin contained in the disinfectant can be kept high for a long period of time, it may be stored preferably at 10 ° C. or less, more preferably at 5 ° C. or less.

<Evaluation method of sterilization effect>
For example, when the disinfectant of the present embodiment is used for food, the disinfecting effect can be evaluated by using the evaluation method shown in Examples described later.
Specifically, for example, first, food such as meat is prepared and prepared to a certain size. Next, before the contact with the disinfectant, the food is transferred to an LB agar plate medium, cultured at 37 ° C. for 18 hours, and the number of bacteria is measured. This is the number of cells before sterilization. Next, the disinfectant of this embodiment is brought into contact with the food (eg, the disinfectant is sprayed on the food or the food is immersed in the disinfectant). Next, the sterilized food is transferred to an LB agar plate medium, cultured at 37 ° C. for 18 hours, and the number of bacteria is measured.
The reduction rate of the number of bacteria, that is, the sterilization rate is calculated using the following formula (1).
Bacteria elimination rate (%) = [1- (Bacteria count after sterilization / Bacteria count before sterilization)] × 100 (1)

  In the above evaluation method, conditions such as ethanol and nisin concentrations, contact time, and contact temperature can be appropriately changed according to the purpose. In addition, the type of culture medium and the culture conditions can be appropriately changed according to the type of microorganism.

  In the above evaluation method, the sterilization rate of the microorganism is preferably 70% or more, and it can be determined that the sterilizing agent has a sterilizing effect. The required sterilization rate varies depending on the field of use, application, or cotyledon method.

≪Sterilization method≫
A sterilization method according to an embodiment of the present invention is a method including a contact step of bringing the sterilizing agent into contact with a sterilization target.

According to the sterilization method of this embodiment, the sterilization target can be sterilized effectively and safely.
Next, the contact step of the sterilization method of this embodiment will be described in detail below.

[Contact process]
In the contact step, the sterilizing agent is brought into contact with the sterilization target.
In the sterilization method of this embodiment, the components and composition of the sterilizing agent used are the same as those described in the above-mentioned sterilizing agent.
As a contact method, it can select suitably according to the shape of a disinfectant and the kind of disinfection target object. Specifically, for example, a method of applying a sterilizing agent to a sterilization target, a method of immersing the sterilization target in a sterilization agent, and adding a sterilization agent to the sterilization target and mixing Methods and the like.
In addition, a method of applying a sterilizing agent to the sterilization target includes a method of spraying a liquid or semi-solid sterilization agent on the sterilization target, a liquid or semi-solid state on the sterilization target, etc. And a method of applying a sterilizing agent such as a solid (for example, a sheet) to a sterilization target.

The contact time can be appropriately selected according to the purpose and the type of the sterilization target.
When the sterilization target is food (especially meat), it is preferable that the contact time is short in order to keep the taste and aroma of the food good. The contact time at this time may be, for example, 5 seconds to 90 seconds, and may be, for example, 15 seconds to 60 seconds.

The contact temperature can be appropriately selected according to the purpose and the type of the sterilization target.
When the sterilization target is food (especially meat), the contact temperature is preferably the same as or lower than room temperature in order to keep the taste and aroma of the food good. The contact temperature at this time may be, for example, 4 ° C. or more and 30 ° C. or less, for example, 4 ° C. or more and 25 ° C. or less.

In the sterilization method of this embodiment, the contact amount of nisin contained in the sterilizing agent with respect to the sterilization target can be appropriately adjusted according to the degree of contamination of the sterilization target.
For example, when the sterilization target is food (especially meat), the contact amount of nisin with respect to the surface area of the food may be about 1 μg / cm ² or more. When the contact amount of nisin with respect to the surface area of the food is not less than the above lower limit, the sterilization effect can be sufficiently exerted.
On the other hand, the upper limit of the contact amount of nisin with respect to the surface area of the food may be an amount that does not exceed the use standard of nisin contained in food as a food additive in Japan.
In addition, even when the sterilization target is other than food, the sterilization effect can be sufficiently exhibited when the contact amount of nisin with respect to the surface area of the sterilization target is not less than the above lower limit. On the other hand, the upper limit of the contact amount of nisin with respect to the surface area of the sterilization target is not particularly limited, and may be an amount that provides an appropriate manufacturing cost.

In the sterilization method of this embodiment, if necessary, after the contact step, a wiping step of wiping off an excess sterilizing agent, a drying step of drying the sterilization target, and the like may be provided.
Examples of the wiping method include, but are not limited to, a method of wiping the disinfectant manually or automatically using a paper towel, a sterilized nonwoven fabric, or the like.
Examples of the drying method include, but are not limited to, natural drying, air drying, fluidized bed drying, low temperature drying, freeze drying, and pressure drying.

In the sterilization method of the present embodiment, when the sterilization target is food (especially meat), a cleaning step of cleaning the food may be provided before the contact step.
Examples of the cleaning method include, but are not limited to, a method of cleaning food using a cleaning agent containing water, a surfactant, and the like. After washing, the food may be used as it is in the contact step. Or you may use for a contact process, after wiping off excess moisture manually or automatically using a paper towel etc.

≪Sterilization kit≫
A sterilization kit according to an embodiment of the present invention includes 60 v / v% or more ethanol and nisin.

The sterilization kit of this embodiment includes nisin and high-concentration ethanol of 60 v / v% or more, and can be easily sterilized by mixing so that nisin has an arbitrary concentration as necessary. Can be prepared. Moreover, the obtained disinfectant has an excellent disinfecting effect against both gram-positive and gram-negative bacteria.
In addition, ethanol and nisin contained in the sterilization kit of this embodiment have already been approved for food use as food additives. Therefore, the sterilization kit of this embodiment has high safety.

Ethanol and nisin contained in the sterilization kit of the present embodiment are the same as those described in the above-mentioned sterilization agent.
In addition, by mixing ethanol and nisin contained in the sterilization kit of the present embodiment so as to have the content described in the above sterilization agent, a sterilization agent having excellent sterilization properties can be prepared. it can.

  Moreover, the sterilization kit of the present embodiment may further include other components in addition to ethanol and nisin. Examples of other components include the same components as those exemplified as the other components in the above-mentioned disinfectant. Among these, water is preferable as the other component. Moreover, the other component may be provided in the state mixed with ethanol.

Further, the sterilization kit of the present embodiment may further include a lidded container, a spraying container, a push-type container, a brush, or the like.
A disinfectant may be prepared and used appropriately in the container.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to a following example.

[Production Example 1] Production of disinfectant Nisin A reagent (containing 2.5% nisin, manufactured by Sigma) 2 mg each ethanol at 20, 30, 40, 50, 60, 70, 80, or 90 v / v% Dissolved in 40 mL, four types of 1.25 ppm nisin-containing ethanol solutions were produced.
In the following, 1.25 ppm of nisin-containing 20 v / v% ethanol solution was used as the disinfectant 1, 1.25 ppm of nisin-containing 30 v / v% ethanol solution as the disinfectant 2, and 1.25 ppm of nisin-containing 40 v / v. % Ethanol solution was disinfectant 3, 1.25 ppm nisin-containing 50 v / v% ethanol solution was disinfectant 4, 1.25 ppm nisin-containing 60 v / v% ethanol solution was disinfectant 5, and 1.25 ppm nisin. Disinfectant 6 containing 70 v / v% ethanol solution, disinfectant 7 with 1.25 ppm nisin containing 80 v / v% ethanol solution, and disinfectant 8 with 1.25 ppm nisin containing 90 v / v% ethanol solution May be called.

[Test Example 1] Bactericidal test of sterilizing agent for meat and taste test of meat sprayed with sterilizing agent (1) Preparation of meat First, beef thigh, pork thigh and chicken fillet are each 3cm x 3cm square Cut into. Next, the cut meat pieces were dipped once in tap water, and the moisture was wiped off with a paper towel. Next, each piece of meat was transferred to a 1.5 w / v% LB agar plate medium and cultured at 37 ° C. for 18 hours to obtain the number of bacteria before sterilization.

(2) Disinfectant spraying step Next, disinfectants 6 and 7 obtained in Production Example 1 and, as controls, 70 v / v% ethanol solution and 80 v / v% ethanol solution, respectively, were filled in spray bottles. Then, it was sprayed once on the surface of each piece of meat (spraying condition: sprayed from an angle of 45 degrees licked 30 cm above). Subsequently, the meat pieces after spraying were transferred to 1.5 w / v% LB agar plate medium. Next, each LB agar plate medium was inverted, cultured at 37 ° C. for 18 hours, and the number of bacteria was measured. The measurement was performed at least three times. The measurement results of the number of bacteria in each meat are shown in FIG. 1A (beef), FIG. 1B (pork), and FIG. 1C (chicken fillet).

From FIG. 1A, FIG. 1B, and FIG. 1C, the decrease rate of the number of bacteria was low in the meat pieces injected with the 70 v / v% ethanol solution and the 80 v / v% ethanol solution. In addition, the number of bacteria was rather increased in the beef sprayed with 70 v / v% ethanol solution, pork sprayed with 70 v / v% ethanol solution, and chicken fillet meat sprayed with 70 v / v% ethanol solution and 80 v / v% ethanol solution. It was increasing. Therefore, the 70 v / v% ethanol solution and the 80 v / v% ethanol solution have a low sterilizing effect or no sterilizing effect.
On the other hand, in the meat pieces sprayed with the disinfectants 6 and 7, the rate of decrease in the number of bacteria was high. In particular, in beef and chicken fillet, the reduction rate of the number of bacteria was 70% or more in any of the disinfectants 6 and 7. Therefore, it was confirmed that the disinfectants 6 and 7 have a high disinfecting effect.

(3) Taste test Next, each meat piece sprayed with the disinfectant in (2) was left to stand at 4 ° C. for 5 hours, and then cooked. Then, the taste test was done about the meat piece after cooking.
As a control, a piece of meat that had not been sprayed with a disinfectant was used. The umami, texture, aroma (ethanol odor) and bitterness were evaluated by a five-step evaluation. In addition, it means that each evaluation item is strongly presented as the numerical value is large, while each evaluation item is weakly presented as the numerical value is low.
FIG. 2A (beef), FIG. 2B (pork), and FIG. 2C (chicken fillet) show the average values of the results of evaluating each piece of meat, with 8 men and women aged 22-25 as panelists.

As shown in FIG. 2A, compared to the control sterilized non-injected beef thigh meat pieces, the beef thigh meat pieces injected with the sterilizing agents 6 and 7 felt a slightly strong aroma (ethanol odor). On the other hand, no effect on umami was observed.
Also, from FIG. 2B and FIG. 2C, compared to the non-injected pork thigh meat and chicken fillet meat pieces, the pork thigh meat and chicken fillet meat pieces injected with the disinfectant 6 and 7 have aroma (ethanol). Odor) and bitterness were felt slightly strong. On the other hand, as with the meat pieces of beef, no effect on umami was observed.

[Test Example 2] Chicken fillet meat sanitizer immersion test 1
(1) Preparation of chicken fillet First, chicken fillet was cut into approximately 2 cm × 3 cm squares. Subsequently, the moisture of the cut chicken breast meat pieces was wiped off with a paper towel. Next, the meat pieces were transferred to a 1.5 w / v% LB agar plate medium and cultured at 37 ° C. for 18 hours to obtain the number of bacteria before sterilization.

(2) Disinfectant soaking step Next, as a disinfectant 1-7 or as a control, the meat pieces were placed in a container containing 80 mL each of 20, 30, 40, 50, 60, 70 or 80 v / v% ethanol solution. After putting in and stirring well, it was immersed at room temperature for 5, 15, or 30 seconds. The meat pieces were then wiped with a paper towel. Each piece of meat was then transferred to a 1.5 w / v% LB agar plate. Next, each LB agar plate medium was inverted, cultured at 37 ° C. for 18 hours, and the number of bacteria was measured. The measurement was performed at least three times. The measurement results of the number of bacteria under each condition are shown in FIG. 3A (ethanol + nisin) and FIG. 3B (ethanol only).

From FIG. 3A and FIG. 3B, in any of the disinfectants 1 to 7 and the 20 to 80 v / v% ethanol solution, the decrease rate of the bacterial cells as the ethanol concentration increases in the meat pieces having an immersion time of 30 seconds. Also rose.
In addition, when the ethanol concentration was 20 to 50 v / v%, there was no difference in the rate of bacterial cell reduction depending on the presence or absence of nisin. Moreover, it was not possible to achieve a cell reduction rate of 100%.
On the other hand, when the ethanol concentration is 60 to 80 v / v%, compared with the low concentration ethanol solution of 20 to 50 v / v%, the high concentration ethanol solution of 60 to 80 v / v% without nisin has There was an upward trend in the decrease rate. However, a nisin-free ethanol solution could not achieve a cell reduction rate of 100% at any ethanol concentration.
In contrast, the nisin-containing 60-80 v / v% ethanol solution had a higher cell reduction rate than the nisin-free 60-80 v / v% ethanol solution. Furthermore, 100% reduction rate of the bacterial cells could be achieved under the condition of immersing in a 70v / v% and 80v / v% ethanol solution containing nisin for 30 seconds at room temperature.

[Test Example 3] Chicken fillet meat sanitizer immersion test 2
(1) Preparation of chicken fillet First, chicken fillet was cut into approximately 2 cm × 3 cm squares. Subsequently, the moisture of the cut chicken breast meat pieces was wiped off with a paper towel. Next, the meat pieces were transferred to a 1.5 w / v% LB agar plate medium and cultured at 37 ° C. for 18 hours to obtain the number of bacteria before sterilization.

(2) Immersion step of disinfectant Next, after putting meat pieces in a container containing 80 mL of disinfectant 5-7 each and stirring well, they were immersed at 4 ° C. for 5, 15 or 30 seconds. The meat pieces were then wiped with a paper towel. Each piece of meat was then transferred to a 1.5 w / v% LB agar plate. Next, each LB agar plate medium was inverted, cultured at 37 ° C. for 18 hours, and the number of bacteria was measured. The measurement was performed at least three times. The measurement results of the number of bacteria under each condition are shown in FIG.

From FIG. 4, under the condition of 4 ° C., it was possible to achieve a bacterial cell reduction rate of slightly less than 100% (99.2%) by immersing in an 80 v / v% ethanol solution containing nisin for 30 seconds.
3A and 4, it was confirmed that in the ethanol solution of each concentration containing nisin, the bacterial cell reduction rate tends to be slightly lower under the condition of 4 ° C. than under the normal temperature condition.

[Test Example 4] Chicken fillet meat disinfectant continuous immersion test (1) Preparation of chicken fillet First, chicken fillet was cut into approximately 2 cm x 3 cm squares. Subsequently, the moisture of the cut chicken breast meat pieces was wiped off with a paper towel. Next, the meat pieces were transferred to a 1.5 w / v% LB agar plate medium and cultured at 37 ° C. for 18 hours to obtain the number of bacteria before sterilization.

(2) Continuous soaking step of disinfectant Next, the meat pieces were put in a container containing 80 mL of disinfectant 7 and stirred well, and then immersed at room temperature for 30 seconds. The meat pieces were then wiped with a paper towel. Each piece of meat was then transferred to a 1.5 w / v% LB agar plate. Then, a new piece of meat was put into a container containing the same disinfectant 7 again, and immersion for 30 seconds at room temperature and transfer to a 1.5 w / v% LB agar plate medium were continuously performed 100 times. Next, each LB agar plate medium was inverted, cultured at 37 ° C. for 18 hours, and the number of bacteria was measured. The measurement was performed at least three times. The results are shown in FIG.

From FIG. 5, a decrease was observed at the 45th, 55th, and 60th times, but until the 70th immersion, a decrease rate of 100% of the bacterial cells was observed. On the other hand, in the immersion after the 75th time, the cell decrease rate of 100% was not observed.
From this, when sterilizing meat pieces by immersing them in a sterilizing agent, the sterilizing agent is not disposable, and even if it is continuously used up to the 40th time, it can always achieve a reduction rate of 100%. It was confirmed.

[Test Example 5] Taste test of chicken fillet immersed in a disinfectant (1) Disinfecting step of disinfectant 320 mL of disinfectant 7 was dispensed into a plastic container. Next, 3 cm × 14 cm chicken breast meat (60 g) was immersed for 30 seconds at room temperature. Next, the moisture of the soaked chicken fillet was wiped off with a paper towel.

(2) Taste test Next, chicken fillet was cut into approximately 2 cm × 3 cm squares. Next, the cut pieces of chicken fillet were cooked in an oven. Then, the taste test was done about the meat piece after cooking.
As a control, a piece of meat not immersed in a disinfectant was used. The umami, bitterness, fragrance (ethanol odor) and texture were evaluated by a five-step evaluation. In addition, it means that each evaluation item is strongly presented as the numerical value is large, while each evaluation item is weakly presented as the numerical value is low.
FIG. 6 shows an average value of the results of evaluating each piece of meat, with 20 men and women of 22 to 25 years old as panelists.

  From FIG. 6, there was no difference in taste and texture between meat pieces immersed in a disinfectant and meat pieces not immersed in a disinfectant. Moreover, the tendency for bitterness to be somewhat reduced was seen for the piece of meat immersed in the disinfectant compared with the piece of meat not immersed in the disinfectant.

[Test Example 6] Preservative confirmation test of disinfectant Next, as a disinfectant, nisin-containing 70 v / v% or 80 v / v% ethanol solution (Nisin content: 2 mg / 40 mL, 2 mg / 20 mL and 2 mg / (10 mL) was stored at room temperature or 4 ° C. with or without light shielding for a maximum of 18 weeks. On the first day of storage and thereafter every week, the residual activity of nisin in each solution was determined by the agar-well diffusion method. In each elapsed day, the activity measurement was performed at least 3 times or more.

Nisin activity was measured by the agar diffusion method according to the following procedure.
First, the indicator bacterium L. delbrueckii subsp. Bulgaricus JCM 1002T was cultured in MRS medium at 37 ° C. for 18 to 24 hours, and diluted to 1/10 with the same medium. Next, 250 μL of this diluted bacterial solution was mixed with 10 mL of MRS soft agar medium (0.75% (w / v)) kept in a constant temperature (50 ° C.) water bath. Thereafter, this mixed solution was solidified on MRS agar medium (1.5% (w / v)) in a multi-layer. Holes (well, diameter 6 mm) were made using a 2 cork borer. In addition, for the dilution of the nisin-containing ethanol solution as a disinfectant, each concentration of ethanol was used for serial dilution at 2 ⁿ times (n: integer). Thereafter, 65 μL of a test (diluted) solution was injected into each well and cultured at 37 ° C. for 18 hours. The antibacterial activity value was defined as the reciprocal value of the highest dilution concentration in which a growth inhibition circle was recognized, and the unit was expressed in Arbitrary Unit (AU) / mL.

Further, a nisin-containing 70 v / v% or 80 v / v% ethanol solution (Nisin content: 2 mg / 40 mL) was stored at −15 ° C. under light-shielded conditions for 9 weeks. The remaining activity of nisin in each solution was determined by the agar diffusion method on the first day of storage and thereafter every week. In each elapsed day, the activity measurement was performed at least 3 times or more.
The respective results are shown in FIGS. 7A to 7F.
FIG. 7A is a graph showing changes over time in the residual activity of nisin in a nisin-containing 70 v / v% ethanol solution (Nisin content: 2 mg / 40 mL) stored under various conditions.
FIG. 7B is a graph showing changes over time in the residual activity of nisin in a nisin-containing 80 v / v% ethanol solution (Nisin content: 2 mg / 40 mL) stored under various conditions.
FIG. 7C is a graph showing changes over time in the residual activity of nisin in a nisin-containing 70 v / v% ethanol solution (Nisin content: 2 mg / 20 mL) stored under various conditions.
FIG. 7D is a graph showing changes over time in the residual activity of nisin in a nisin-containing 80 v / v% ethanol solution (Nisin content: 2 mg / 20 mL) stored under various conditions.
FIG. 7E is a graph showing changes over time in the residual activity of nisin in a nisin-containing 70 v / v% ethanol solution (Nisin content: 2 mg / 10 mL) stored under various conditions.
FIG. 7F is a graph showing changes over time in the residual activity of nisin in a nisin-containing 80 v / v% ethanol solution (Nisin content: 2 mg / 10 mL) stored under various conditions.

  From FIG. 7A and FIG. 7B, in the nisin-containing ethanol solution having a nisin content of 2 mg / 40 mL, the nisin-containing ethanol solution (without light shielding) stored at room temperature was 80 v / v in 2 weeks after preparation with 70 v / v% ethanol. In v% ethanol, it was deactivated 4 weeks after preparation. The nisin-containing 70v / v% ethanol solution (with light shielding) stored at 4 ° C is 8 weeks after preparation, and the nisin-containing 80v / v% ethanol solution (with light shielding) stored at 4 ° C is 4 weeks after preparation (18 weeks after preparation). 1/8 activity immediately after the preparation was confirmed), and nisin-containing 70 v / v% and 80 v / v% ethanol solution (with light-shielded) stored at -15 ° C., activity of about 6000 AU / mL from preparation to 6 weeks Was maintained. Further, when the light-shielding condition and the light-shielding-free condition were compared with each other at room temperature storage and 4 ° C. storage, high activity was maintained under the light-shielding condition.

  From FIG. 7C and FIG. 7D, in the nisin-containing ethanol solution having a nisin content of 2 mg / 20 mL, the nisin-containing ethanol solution stored at room temperature (without light shielding) has a significant activity in 2 weeks after preparation with 70 v / v% ethanol. It was inactivated by 4 weeks, and inactivated by 80 v / v% ethanol in 2 weeks. On the other hand, in the nisin-containing ethanol solution stored at 4 ° C., both 70 v / v% ethanol and 80 v / v% ethanol were not inactivated by the fourth week after preparation. In the nisin-containing ethanol solution having a nisin content of 2 mg / 20 mL, both 70 v / v% ethanol and 80 v / v% ethanol tended to lack nisin activity stability.

  From FIG. 7E and FIG. 7F, in the nisin-containing ethanol solution having a nisin content of 2 mg / 10 mL, the activity of nisin-containing 70 v / v% ethanol solution (no light-shielding) stored at room temperature significantly increased in the second week after preparation. However, the nisin-containing 70 v / v% ethanol solution (without light shielding) stored at 4 ° C. maintained the activity immediately after the preparation from the preparation to the 4th week. Moreover, the nisin-containing 80 v / v% ethanol solution (without light shielding) stored at room temperature showed a significant decrease in activity in the second week after preparation and was deactivated in six weeks, but the nisin-containing 80 v / v% stored at 4 ° C. In the ethanol solution (without light shielding), the activity immediately after the preparation was maintained until 4 weeks from the preparation, and 50% of the activity was maintained until the 8th week. In addition, it became clear that the activity of the nisin-containing 80 v / v% ethanol solution (with light-shielding) stored at room temperature significantly decreased in the second week after preparation and gradually decreased thereafter.

  From the above, it was revealed that the light-shielding condition at 4 ° C. is optimal as the storage condition for the nisin-containing ethanol solution.

10 ⁿ times the injection test indicator bacteria solution in PBS for Test Example 7] of disinfectant L. lactis NBRC12007 (nisin producing bacteria) was serially diluted (n an integer) (the number of viable bacteria by smearing plate culture method (Determined) 10 ⁻⁵ dilution (100 μL) was added dropwise to 1.5% (w / v) GM17 agar plate medium and smeared with a conage bar until the bacterial solution was dry. Thereafter, the agar plate medium smeared with the bacterial solution was mixed with a nisin-containing PBS solution (2 mg / 40 mL), 20 v / v% ethanol alone, a nisin-containing 20 v / v% ethanol solution (2 mg / 40 mL), 70 v / v% ethanol alone. 80 v / v% ethanol alone, nisin-containing 70 v / v% ethanol solution (2 mg / 40 mL), or nisin-containing 80 v / v% ethanol solution (2 mg / 40 mL) was sprayed and allowed to stand at room temperature for 1 minute. . Then, the agar plate medium which injected each solution was cultured at 30 degreeC for about 18 hours, each solution was sprayed, and the number of bacteria after culture | cultivation (X2) was measured. In addition, the number of bacteria (X1) before jetting each solution was measured in advance. Accordingly, the ratio {(X1-X2) / X1 × 100} of the number of bacteria (X1-X2) decreased after culturing each solution with respect to the number of bacteria (X1) before spraying each solution is the rate of decrease in the number of bacteria (%) And the result is shown in FIG.

  From FIG. 8, in the nisin-containing PBS solution (2 mg / 40 mL), 20 v / v% ethanol alone, and the nisin-containing 20 v / v% ethanol solution (2 mg / 40 mL), the bacterial count reduction rate was 36.4%, 43.2% and 41%. On the other hand, with 70 v / v% ethanol alone, 80 v / v% ethanol alone, nisin-containing 70 v / v% ethanol solution (2 mg / 40 mL) and nisin-containing 80 v / v% ethanol solution (2 mg / 40 mL) Was 100%.

  From the above, it was confirmed that nisin-containing 70 v / v% ethanol solution (2 mg / 40 mL) and nisin-containing 80 v / v% ethanol solution (2 mg / 40 mL) are useful as disinfectants.

[Test Example 8]
1. Bactericidal test of disinfectant against sink surface isolates Bacteria (hereinafter sometimes referred to as "sink surface isolates") were collected from the sink surface of our laboratory with a cotton swab, and 100 μL of PBS was dispensed 1 The mixture was stirred in a 5 mL Eppendorf tube to obtain an indicator fungus solution. Subsequently, the index bacterial solution was serially diluted 10 ⁿ times (n: integer) with PBS (the number of viable bacteria was determined by a smear plate culture method). Thereafter, a 1/10 dilution (100 μL) was added dropwise to a 1.5% (w / v) LB agar plate medium, and smeared with a conage bar until the bacterial solution was dry. The components contained in the LB agar plate medium include 0.5% (w / v) Bact (registered trademark) Yeast Extract, 1% (w / v) Bact (registered trademark) Tryptone (manufactured by BD Difco) and 1 % (W / v) NaCl. Subsequently, 80 v / v% ethanol alone using a spray bottle and a nisin-containing 80 v / v% ethanol solution (2 mg / 40 mL) stored at 4 ° C. under light-shielded conditions for 8 weeks were applied to each smeared LB agar plate medium. Were sprayed once (about 0.8 mL). In this experiment, a simple injection suitable for practical use was assumed, and the injection was performed at an angle of 45 ° obliquely 3 cm above the LB agar plate medium. Further, 10 ^-1 dropwise diluted bacterial suspension (100 [mu] L), and the blank in this experiment the LB agar plates were smeared until cell solution has dried, 18 hours of culture at 37 ° C. with inverted each agar plate plates, bacteria Number was measured. The viable cell count (CFU) per plate is shown in FIG.

  From FIG. 9, the viable cell count was significantly reduced in the nisin-containing 80 v / v% ethanol solution (2 mg / 40 mL), compared with the viable cell count when the 80 v / v% ethanol solution was injected. As a result, the synergistic antibacterial effect of ethanol and nisin on the bacteria attached to the sink surface and its effectiveness were confirmed.

2. Bacterial species identification of sink surface isolates The bacteria used as blanks in the above "1." were purified to obtain single colonies of two sink surface isolates. Chromosomal DNA was extracted according to a conventional method using the obtained single colonies of the two kinds of sink surface isolates. Next, 16S ribosomal (16Sr) DNA was amplified from the extracted DNA by PCR, and sequence analysis was performed. The sequence analysis of 16Sr DNA (V3-V4 region) of the sink surface isolate was performed using a DNA sequencer, and the homology analysis of the obtained data was performed using H-Inv DB BLAST (http://www.h-invitational.jp). /hinv/blast/blasttop.cgi), and two species of sink surface isolates were identified from the sequence data from which 96% or more homology was obtained. Highly homologous bacterial species (homology 96% or higher) are shown in Table 1 below.

  Table 1 suggests that the sink surface isolate A strain may belong to the genus Enterobacter or Klebsiella, and the sink surface isolate B strain may belong to the genus Exiguobacterium. The Enterobacter genus is a gram-negative non-spore-forming anaerobic gonococcus and is one of the causative bacteria of opportunistic infections and urinary tract infections. The genus Klebsiella is a gram-negative gonococcus attenuated bacterium, causing a fungal change phenomenon and causing an infectious disease. Exiguobacterium spp. Is a Gram-positive bacilli and has a wide growth temperature from −2.5 ° C. to 50 ° C .;

3. Spray test of disinfectant against sink surface isolate A and B strains Except for using sink surface isolate A strain and B strain, perform the disinfectant spray test using the same method as "1." went. In addition, a nisin-containing 80 v / v% ethanol solution (2 mg / 40 mL) was sprayed on each of two LB agar plate media smeared with sink surface separated bacteria A and B, respectively. FIG. 10A shows the results for the sink surface isolate A strain, and FIG. 10B shows the results for the sink surface isolate B strain.

From FIG. 10A, the number of viable bacteria was significantly increased in the nisin-containing 80 v / v% ethanol solution (2 mg / 40 mL) in the sink surface isolate A strain compared to the viable cell count in the injection of the 80 v / v% ethanol solution alone. Diminished. Further, from FIG. 10B, in the sink surface isolate B strain, no viable bacteria were detected in the 80 v / v% ethanol solution alone and the nisin-containing 80 v / v% ethanol solution (2 mg / 40 mL).
In addition, in a nisin-containing 80 v / v% ethanol solution (2 mg / 40 mL, activity value: 15754AU / mL) stored at 4 ° C. for 18 weeks under light-shielded conditions, nisin was isolated from the sink surface isolate A strain by coexistence with ethanol. And a sufficient synergistic antibacterial effect against B strain (not shown).

The disinfectant according to the present embodiment contains nisin and high-concentration ethanol of 60 v / v% or more, and thus has an excellent disinfecting effect against both gram-positive bacteria and gram-negative bacteria.
In addition, ethanol and nisin contained in the disinfectant of this embodiment have already been approved for food use as food additives. Therefore, the disinfectant of this embodiment has high safety.
Moreover, according to the sterilization method of the present embodiment, the sterilization target can be sterilized effectively and safely.
In addition, the sterilization kit of this embodiment includes nisin and high-concentration ethanol of 60 v / v% or more, and can be easily removed by mixing the nisin to an arbitrary concentration as necessary. A fungus can be prepared. Moreover, the disinfectant prepared using the disinfecting kit of this embodiment has an excellent disinfecting effect against both gram-positive and gram-negative bacteria.

Claims (16)

  A disinfectant containing 60 v / v% or more of ethanol and nisin as active ingredients.   The disinfectant according to claim 1, wherein the ethanol content is 70 v / v% or more.   The disinfectant according to claim 1 or 2, wherein the ethanol content is 80 v / v% or more.   The disinfectant according to any one of claims 1 to 3, wherein the nisin content is 1.25 ppm or more.   The disinfectant according to any one of claims 1 to 4, which is used for food.   The disinfectant according to claim 5, wherein the food is meat.   A sterilization method comprising a contact step of bringing the sterilizing agent according to any one of claims 1 to 6 into contact with a sterilization target.   The sterilization method according to claim 7, wherein the sterilizing agent is applied to the sterilization object in the contact step.   The sterilization method according to claim 7, wherein the sterilization object is immersed in the sterilization agent in the contact step.   The sterilization method according to any one of claims 7 to 9, wherein the sterilization target is a food.   The sterilization method according to claim 10, wherein the food is meat. 60% v / v or more ethanol,
A sterilization kit comprising Nisin.   The sterilization kit according to claim 12, wherein the ethanol content is 70 v / v% or more.   The sterilization kit according to claim 12 or 13, wherein the ethanol content is 80 v / v% or more.   The sterilization kit according to any one of claims 12 to 14, which is used for food.   The sterilization kit according to claim 15, wherein the food is meat.

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