JP2009046445A - Virus killing aid and virus killing method - Google Patents

Abstract

【選択図】なしProvided are a virucidal auxiliary agent having a virucidal effect and a virucidal method for use in ozone aeration treatment.
A virucidal auxiliary for use in ozone aeration treatment comprising a compound represented by the following general formula (I). [In the formula (I), x represents an integer of 0 to 2, and R ¹ , R ² , and R ³ each independently represent —H, —OH, or alkylcarbonyl having 1 to 3 carbon atoms. It is a group selected from the group consisting of groups represented by oxy, alkyloxycarbonyl, and alkyl ether, and at least one of R ¹ , R ² , and R ³ is an alkylcarbonyloxy group. ^Further, among ^{R 1, R 2, R 3} , -OH is one or less. ]

[Selection figure] None

Description

  The present invention relates to a virus killing aid and a virus killing method.

Norovirus is taken up by bivalves such as oysters in the sea and is one of the main causes of food poisoning in winter. In particular, livestock oysters in sterilized seawater and chlorinated fungicides are used to purify raw oysters, where norovirus is a problem. However, aseptic breeding is less effective in cold winter water because the amount of oyster drainage is reduced. In addition, since norovirus is resistant to low-concentration chlorinated fungicides, treatment at a high concentration is required to inactivate by this method. High concentration of chlorine degrades food and has a strong odor, so measures such as work environment are necessary.
On the other hand, ozone has a strong oxidizing power and, when decomposed, becomes oxygen and water, and has an environmentally friendly aspect. Therefore, its use is expanded in various fields such as sterilization washing of food and semiconductors and water purification. ing. As a method of ozone treatment, there are generally a method of supplying ozone gas into water to be treated including a treatment object (ozone aeration) and a method of immersing the treatment object in ozone water in which ozone is dissolved (ozone water immersion treatment). It is. Among these, ozone aeration treatment has advantages such as less ozone usage, less water usage, and even greater amount of organic matter to be treated than ozone water treatment.
Sterilization method (for example, Patent Document 1) using ozone at a high concentration (0.1 to 5% by weight in solution concentration) and foods are alternately treated with ozone water and an organic acid solution and / or an alcohol solution. A sterilization method (for example, Patent Document 2) has been proposed. Further, a sterilizing cleaning composition containing ozone water and a surfactant has been proposed, and it is described that the sterilizing effect is promoted by immersing the treatment object in the sterilizing cleaning composition (for example, Patent Document 3). This mainly supplements the cleaning effect on the oil component with a surfactant and aims at the residual effect of ozone. Moreover, in ozone aeration process, it describes about the disinfection promoter which uses the compound which has a specific property (for example, patent document 4).
Japanese Patent Laid-Open No. 2000-109887 Japanese Patent Laid-Open No. 3-164155 JP-A-6-313194 International Publication No. 2007/040260 Pamphlet

However, in all of the existing technologies, the treatment object is a bacterium, and no effective virucidal method using ozone is found.
The present invention has been made in view of the above circumstances, and provides a virucidal aid and a virucidal method that exerts an excellent virucidal effect when used in ozone aeration treatment.

  The virucidal auxiliary used for the ozone aeration treatment of the present invention is characterized by containing a compound represented by the following general formula (I) (hereinafter referred to as (a) component), and is a water-soluble acidic component (hereinafter referred to as ( It is preferable to include b) component)

[In formula (I), x represents an integer of 0 to 2, and R ¹ , R ² and R ³ are each independently represented by —H, —OH or the following general formulas (1) to (3). It is a group selected from the group consisting of groups, and at least one of R ¹ , R ² and R ³ is a group represented by the following general formula (2). ^Further, among ^{R 1, R 2, R 3} , -OH is one or less. ]

[In Formula (1)-(3), R < ⁴ >, R < ⁵ >, R < ⁶ > is a C1-C3 alkyl group. ]

  The virucidal method of the present invention is characterized in that a virucidal treatment is carried out by ozone aeration treatment using a virucidal aid having the components (a) and (b).

  The ozone aeration treatment in the present invention indicates that ozone gas produced using an ozone generator is introduced into water to be treated containing an object to be treated using an air diffuser. In addition, the ozone aeration treatment in the present invention includes circulating the aqueous solution aerated with the component (a) or the aqueous solution containing the components (a) and (b) to the tank containing the object to be treated with ozone. .

  The virus killing in the present invention includes not only making the virus non-viable but also making it lose infectivity.

  According to the present invention, an excellent virucidal effect can be obtained by using a virucidal auxiliary for ozone aeration treatment.

Because viruses and bacteria have the following differences, the methods and effects of virucidal and sterilization are different. Viruses are a kind of microorganisms that enter cells such as animals and plants, not cells, and use the metabolic function of host cells to create new virus particles and propagate. Therefore, viruses cannot grow alone. Therefore, it is possible to obtain an effect equivalent to that of a generally called virucidal effect by preventing infection of host cells. Other microorganisms, including protozoa, fungi, and bacteria (including mycoplasma, rickettsia, and chlamydia), synthesize and grow their own nucleic acids (both DNA and RNA), proteins, carbohydrates, and lipids, most of which are divided into two parts. A single cell that proliferates.
In general, virucidal and sterilization is a state where it is no longer viable and cannot grow. Bacteria are composed of the following components: That is, the cell wall is composed of lipopolysaccharide, lipoprotein, and peptide glucan, and the cytoplasmic membrane is composed of phospholipid, membrane protein (such as an enzyme), and polysaccharide. Cell components include proteins (enzymes, ribosomal proteins), RNA, DNA and the like. The following four cases can be cited as non-viable cases.
(1) Denaturation of enzymes (bacterial metabolism and synthesis become impossible)
(2) Cell membrane damage (Ca ²⁺ , Mg ²⁺ , RNA, etc. leak from the cell due to changes in cell membrane permeability)
(3) Degradation of RNA and ribosomes (4) Damage to chromosome or its constituent DNA On the other hand, viruses are composed only of DNA or RNA and the envelope protein that encloses it, adsorbing to the host cell, and subsequent DNA or RNA Proliferation is inhibited when entry into the host cell is inhibited. The following two factors can be cited as virus killing factors.
(1) Denaturation or destruction of coat protein (2) DNA and RNA damage

  Because of the difference between bacteria and viruses as described above, even a composition having a bactericidal ability does not necessarily have a virucidal ability. Recently, by adding a specific compound to the liquid to be treated in ozone aeration treatment, it was found that the virucidal effect is specifically improved, and the present invention has been achieved.

(Virus killing aid)
The virus-killing aid of the present invention contains the compound represented by the formula (I) as the component (a).

<(A) component>
In said formula (I), x shows the integer of 0-2, Preferably it is 1.
R ¹ , R ² , and R ³ are each independently a group selected from the group consisting of —H, —OH, and groups represented by the following general formulas (1) to (3), and R ¹ , R ² and at least one of R ³ is a group represented by the general formula (2). ^Further, among ^{R 1, R 2, R 3} , -OH is one or less. When one or two of R ¹ , R ² and R ³ are groups other than the group represented by the formula (2), —H is preferable as the group.

In said formula (1)-(3), R < ⁴ >, R < ⁵ >, R < ⁶ > is a C1-C3 alkyl group, and a methyl group, an ethyl group, n-propyl group, and an isopropyl group are mentioned. Of these, a methyl group and an ethyl group are preferable, and a methyl group is particularly preferable.

Examples of the compound represented by the formula (I) include diacetin, triacetin, propylene glycol diacetate, propyl acetate, 1-ethoxy-2-propanol acetate, and butanediol diacetate. Diacetin has structural isomers, and examples thereof include glycerin-1,3-diacetate and glycerin-1,2-diacetate.
Any one of these compounds may be included in the virus-killing aid of the present invention, and two or more thereof may be included.

  The component (a) of the present invention has a dynamic surface tension of 0.5 m% aqueous solution at 25 ° C. at 100 milliseconds (msec) of 67 mN / m or less and 30 seconds (sec) as follows. It is preferable that the compound has a dynamic surface tension of 55 to 63 mN / m.

Here, the “dynamic surface tension” means the surface tension when a new interface is formed or in a flow / stirring state where the interface is unstable.
Specifically, the bubble formation process when gas is fed from the straw into the water will be described as an example. When gas is supplied through a straw inserted obliquely into water, a hemispherical interface (an interface between water and gas) is first formed from the tip of the straw. At this time, a force (surface tension) for returning the interface to the original and a buoyancy due to the gas act on the interface. Buoyancy increases as the amount of gas in the interface increases. When the buoyancy is greater than the surface tension, the hemispherical interface separates from the tip of the straw to form bubbles and the bubbles rise to the water surface. When such bubble formation is repeated, bubbles gather on the water surface, and foam is formed.
The bubble interface is in an unstable state, but after the bubble is formed (after the supply of gas is stopped), the interface is stabilized with time. Then, the surface tension gradually decreases with this stabilization, and becomes a certain value (equilibrium value).
In this way, the surface tension from when the bubble interface is formed until the surface tension reaches the equilibrium value (until the interface becomes stable) is called dynamic surface tension. It is a changing value.

In the formation of such bubbles, the smaller the amount of gas supplied when the buoyancy becomes greater than the surface tension, the smaller the bubble size and the smaller the buoyancy. That is, the surface tension at this time also becomes small. The subsequent dynamic surface tension gradually decreases from the value of the surface tension at this point, but the manner of change varies depending on the components of the liquid.
In addition, the smaller the equilibrium value, the higher the stability of bubbles and foams, and the more difficult it is to break. Conversely, the larger the equilibrium value, the lower the stability of bubbles and foams, and the more likely they are to break.
Therefore, the component (a) has excellent effects as described later by controlling such characteristics for bubbles generated in the water to be treated by ozone aeration.

The component (a) has a 100 msec dynamic surface tension of preferably 67 mN / m or less, more preferably 64 mN / m or less, and particularly preferably 62 mN / m or less. Although there is no restriction | limiting in particular as a lower limit, 55 mN / m or more is preferable.
Here, the 100 msec dynamic surface tension is the dynamic surface tension after 100 msec from the time when gas supply is started at 0. That is, in the example in which gas is fed from the straw, the dynamic surface tension after 100 msec from the start of gas supply into the straw is shown. When the dynamic surface tension of 100 msec is 67 mN / m or less, the buoyancy becomes larger than the surface tension when the amount of gas supply is small, and the hemispherical interface separates bubbles from the straw tip. That is, fine bubbles are formed.
And by refinement | miniaturization of a bubble, ozone and a process target object and contact efficiency improve, As a result, ozone treatment efficiency improves.

Furthermore, as for (a) component, the 30-sec dynamic surface tension has the preferable range of 55-63 mN / m, and it is more preferable that it is the range of 56-62 mN / m.
Here, the 30 sec dynamic surface tension is the dynamic surface tension after 30 sec from the time when gas supply is started at 0. In general, it takes several tens of hours for the dynamic surface tension to reach an equilibrium value, and the measurement takes time. The 30-second dynamic surface tension employed in the present invention is not necessarily the same as the equilibrium value, but considering the processing time when performing the aeration process, the 30-second dynamic surface tension is sufficient to evaluate the stability of bubbles and foam. It is useful as an index to
When the 30-second dynamic surface tension is in the range of 55 to 63 mN / m, the formed bubbles have appropriate stability. However, if the 30-second dynamic surface tension is less than 55 mN / m, the stability of the bubbles becomes too high, and when the aeration process is performed, the water surface becomes foamed, overflowing, etc., making the process itself difficult. On the other hand, when the 30-second dynamic surface tension exceeds 63 mN / m, the ozone treatment efficiency deteriorates. This is presumed to be due to the low stability of the bubbles and the bubbles breaking before contacting the object to be processed.

  100 msec dynamic surface tension and 30 sec dynamic surface tension are prepared, for example, by dissolving the compound in water to prepare a 0.5 mass% aqueous solution (25 ° C.). It can be measured using a theta t60 (trade name) or the like.

The content of the component (a) in the virus-killing aid of the present invention is not particularly limited, but if it is 0.01% by mass or more, the virus-killing effect is well expressed. Therefore, it is preferable that it is 0.01 mass% or more.
Moreover, the virus killing effect by ozone aeration process cannot be improved as the density | concentration in the to-be-processed water of (a) component is less than 30 ppm. Therefore, the component (a) is contained in the liquid to be treated at 30 ppm or more, preferably 50 ppm or more, more preferably 100 ppm or more.

As described above, the component (a) is such that fine ozone bubbles are formed when the dynamic surface tension of 100 msec is 67 mN / m or less. Contact efficiency is improved. In addition, since the 30-second dynamic surface tension is in the range of 55 to 63 mN / m, it is stable until the formed bubbles come into contact with the object to be processed and is moderately stable to break in a relatively short time. It will have a sex. By these synergistic effects, it is presumed that the contact efficiency between ozone and the object to be treated is improved, ozone oxidation is promoted, and foaming of the water surface is suppressed.
Since the dynamic surface tension satisfies the above condition, fine ozone bubbles having appropriate stability are formed when the aqueous solution containing the ozone oxidation aid is subjected to ozone aeration treatment. Can be promoted. And the reduction and efficiency improvement of the ozone usage-amount in ozone treatment can be achieved.

<(B) Component: Water-soluble acidic component>
The water-soluble acidic component (b) is a water-soluble compound having a pH of less than 7.0 when dissolved in about 0.5% in pure water (pH 7.0). In addition, even if the solvent is weakly acidic with a pH of 6 or less, the water-soluble acidic component exhibits water solubility. The pH shown here is a pH value measured using a hydrogen electrode or the like at 25 ° C., but the use temperature of the present invention is not limited to this temperature, and the aqueous solution of the present invention can be used at any temperature. , PH is defined as the value shown at 25 ° C. (hereinafter the same).
For example, an organic acid, an inorganic acid, a water-soluble chelating agent having an acidic group, and the like can be given. Specific examples include (1) to (3) below, but the present invention is not limited thereto.
(1) Examples of organic acids include acetic acid, lactic acid, citric acid, adipic acid, malic acid, succinic acid, maleic acid, fumaric acid, gluconic acid, tartaric acid, glutaric acid, succinic acid, and alkaline agents such as sodium hydroxide. Salt partially neutralized with, etc.
(2) Examples of inorganic acids include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid and the like.
(3) A water-soluble chelating agent having a carboxyl group, phosphoric acid, phosphonic acid group or the like which is an acidic group (acid-dissociable functional group).
Examples of such compounds include citric acid, succinic acid, ethylenediaminetetraacetic acid, nitrosotriacetic acid, diethylenetriaminepentaacetic acid, triethylenetetramine-N, N, N ′, N ″, N ′ ″ N ′ ″-hexaacetic acid, etc. Polycarboxylic acid compounds, hexametaphosphoric acid, pyrophosphoric acid, tripolyphosphoric acid, phosphonic acid compounds such as phytic acid, and phosphonic acid compounds such as 1-hydroxyethane-1,1-diphosphonic acid.
As the component (b) of the present invention, one kind from these compounds may be used, or two or more kinds may be mixed and used. Moreover, it can also be used by partially neutralizing with an alkali agent such as sodium hydroxide so that the pH does not drop too much to cause problems.
And preferably, they are acetic acid, citric acid, succinic acid, adipic acid, phosphoric acid, or a mixture thereof.

The content of the component (b) in the virus-killing aid of the present invention is not particularly defined, but if it is 0.01% by mass or more, the virus-killing effect is well expressed. Therefore, it is preferable that it is 0.01 mass% or more.
Moreover, the virus killing effect by ozone aeration process cannot be improved as the density | concentration in the to-be-processed water of (b) component is less than 30 ppm. Therefore, the component (a) is contained in the liquid to be treated at 30 ppm or more, preferably 50 ppm or more, more preferably 100 ppm or more.

  The component (b) does not have the interfacial scientific properties of the component (a) and has little influence on the state of bubbles, but the virucidal effect of ozone when using a virucidal aid mixed with both components is It can improve synergistically and achieve a reduction in the amount of ozone used, a reduction in the virucidal aid, and an increase in efficiency.

<Optional component>
The virus-killing aid of the present invention has various surfactants, perfumes, enzymes, fluorescent agents, as other components, in a range that does not impair the ozone virus-killing effect, for stability of use and product, You may contain a thickener, a dispersing agent, inorganic salt, alcohols, saccharides, etc.
There is no restriction | limiting in particular as surfactant, According to the objective, it can select suitably from conventionally well-known surfactant, For example, following (1)-(4) etc. are mentioned.
(1) alkylbenzene sulfonic acid, alkyl sulfuric acid, alkyl phenyl ether sulfuric acid, polyoxyethylene alkyl ether sulfuric acid, acylamido alkyl sulfuric acid, alkyl phosphoric acid, polyoxyethylene alkyl ether carboxylic acid, paraffin sulfonic acid, α-olefin sulfonic acid, α- Water-soluble salts such as sulfocarboxylic acids and their esters, and anionic surfactants such as soap.
(2) ethoxylated nonions such as polyoxyalkyl ether and polyoxyalkylphenyl ether, polyglycerin fatty acid ester, glycerin fatty acid ester, propylene glycol fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, glucoside ester, sugar ester, Nonionic surfactants such as sugar active agents such as methyl glucoside ester, ethyl glucoside ester and alkylpolyglucoxide, amide active agents such as alkylamine oxide, alkyldiethanolamide and fatty acid N-alkylglucamide, and alkylamine oxide.
(3) Amphoteric surfactants such as aminocarboxylates such as alkylcarboxybetaines, alkylsulfoxybetaines, alkylamidopropylbetaines, and alkylalaninates, imidazoline derivatives, and alkylamine oxides.
(4) Cationic surfactants such as alkyltrimethylammonium salts and dialkyldimethylammonium salts.
The surfactant may be composed of only one type, or may contain a plurality of types.

(Ozone aeration treatment method)
The virucidal method of the present invention is an ozone treatment method for treating a treatment object by ozone oxidation, and in the presence of the component (a) and the component (b), ozone is added to untreated water containing the treatment object. It has the process to aerate.
The method of ozone aeration treatment is not particularly limited, and conventionally used methods such as a diffuser plate, a diffused cylinder, and a diffuser can be used. The ozone aeration treatment can be performed, for example, by storing water to be treated containing a virus-killing aid and a treatment target in a container and supplying a gas containing at least ozone (aeration gas) into the water to be treated. . Moreover, you may add a virusicidal adjuvant in to-be-processed water, supplying aeration gas to to-be-processed water. In this step, it is also possible to use a stirring device or the like in order to stir the water to be treated when aeration is performed.
The processing container contains the water to be treated, and the aeration container (processing container) has a strong oxidizing power of ozone. Therefore, the material in contact with the water to be treated is made of glass, Teflon (registered trademark) (polytetrafluoroethylene). ), Titanium, and aluminum or stainless steel treated with ozone (a strong oxide film formed by high-concentration ozone) is preferable.
When using a material such as nitrile rubber, silicon or urethane having low resistance to ozone, it is necessary to pay sufficient attention to deterioration of the processing container.

As the aeration gas used for the ozone aeration treatment, the generated ozone may be used as it is, or may be supplied after being diluted with a dilution gas. The generation method of ozone is not particularly limited, and includes a method of irradiating oxygen with high energy light such as an electron beam, radiation, and ultraviolet rays, a chemical method, an electrolytic method, a discharge method, and the like. Industrially, the silent discharge method is often used because of generation cost and generation amount. For the generation of ozone. Commercially available ozone generators can be used. For example, BO-90 (trade name) manufactured by Bethel Co., Ltd. is commercially available as a low concentration ozone generator, and Navi Engineering Co., Ltd. HO-100 (product) as a high concentration ozone generator. Name) etc. are commercially available. Since ozone is self-degrading, it is desirable to use it immediately after preparation.
As a dilution gas used for the dilution of ozone, a gas inert to ozone or poor in reactivity is preferable, and examples thereof include helium, argon, carbon dioxide, oxygen, air, and nitrogen.

  In the ozone aeration treatment, when the ozone concentration of the aeration gas used for aeration is less than 0.1 vol · ppm, ozone is consumed by reaction with other compounds, the amount of ozone decreases, and the effect is not exhibited. Moreover, when it exceeds 10,000 vol.ppm, a problem arises in the work environment, and facilities such as an exhaust ozone decomposing apparatus are necessary for safety measures. If it is about 500 vol.ppm, it can respond with a simple processing apparatus. Accordingly, the ozone concentration of the aeration gas used for the ozone aeration treatment is preferably 0.1 to 10,000 vol · ppm, and more preferably 1 to 500 vol · ppm.

  Although the pH in the ozone aeration process of this invention is not specifically limited, Usually, it is preferable that it is pH 8.5 or less. If ozone has high alkalinity, self-decomposition is promoted and efficiency deteriorates, and component (a) decomposes under strong acid or strong alkali. Therefore, the virucidal effect is preferably in the vicinity of weakly acidic to weakly alkaline.

  The more preferable pH of the water to be treated is in the range of 2.0 to 6.0, more preferably in the range of pH 3.0 to 5.5, and particularly preferably in the range of pH 3.5 to 5.0. is there. When the pH exceeds 6, the effect of using the component (b) is reduced. When the pH is less than 2, for example, sterilization cleaning of food causes deterioration of the quality of the food itself to be cleaned, which is not preferable.

The treatment time (aeration time) when performing the ozone aeration treatment is not particularly limited, and may be set in consideration of the treatment purpose, the concentration of the target in the water to be treated, the temperature, the treatment volume, and the like.
Further, the treatment temperature at the time of ozone aeration treatment, that is, the temperature of the liquid to be treated is not particularly limited, and can be determined in consideration of the properties of the virucidal object.

<Water>
The water used in the present invention is not particularly limited, and any of pure water, ion exchange water, tap water, well water, river lake water, seawater, and the like can be used.

  There is no restriction | limiting in particular as the target of virus killing, What is necessary is just what is generally ozone-treated. Examples of the processing object include foods, packaging containers, cooking utensils, floors, fingers, piping, and semiconductor devices. Among them, the virus killing method of the present invention is particularly suitable for virus killing of foods, specifically fish and shellfish such as oyster, squid, clam, shrimp, horse mackerel and shirasu; vegetables such as lettuce, cabbage and leeks; strawberries Suitable for killing viruses such as apples. The virucidal method of the present invention is also suitable for water purification.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, an Example does not limit this invention.

<Measurement of dynamic surface tension>
(A) In selecting a compound to be used as the component, the 100 msec dynamic surface tension and the 30 sec dynamic surface tension of a 0.5 mass% aqueous solution at 25 ° C. were used for the compound represented by the formula (I) shown in Table 1. It measured by the following method. The results are shown in Table 1.
The dynamic surface tension is measured by dissolving the compounds shown in Table 1 in water to prepare 0.5% by mass aqueous solutions (25 ° C.), and the 100 msec dynamic surface tension and the 30 sec dynamic surface tension of each aqueous solution are Hidehiro. The measurement was performed using a Theta t60 manufactured by Seiki Co., Ltd. As the water used, ultrapure water having a resistivity of 18 MΩ or more purified using GSR-210 manufactured by ADVANTEC was used.

<Preparation of liquid to be treated>
Example 1
A 100 mL glass test tube was charged with 800 μL of feline calicivirus solution (F-9 strain). Triacetin (special grade, manufactured by Kanto Chemical Co., Inc.) and sterilized water were added to the test tube, and the liquid to be treated having a triacetin concentration of 100 ppm was adjusted to 15 mL to obtain a liquid to be treated.
Next, ozone was generated using an ozone gas generator (Ozonizer MODEL YGR-5 manufactured by REI-SEA), and a small pump was used to prepare 60 vol · ppm and 70 mL / min. The prepared ozone gas was passed through an air diffuser (Kinoshita type glass filter 501G No. 4, Kinoshita Rika Kogyo Co., Ltd.), and ozone aeration treatment was performed on the liquid to be treated.
The infectivity measurement (TCID50 method) described later was performed on the liquid to be treated before the start of aeration and the liquid to be treated after aeration for 5 minutes. Moreover, the virus amount was measured about the to-be-processed liquid before the start of aeration, and the to-be-processed liquid after 10 minutes of aeration. The results are shown in Table 2.

(Example 2)
In addition to triacetin, ozone aeration treatment was performed in the same manner as in Example 1 except that succinic acid (special grade, manufactured by Kanto Chemical Co., Inc.) was added to a concentration of 100 ppm. For the liquid to be treated before the start of aeration and the liquid to be treated after aeration for 5 minutes, infectivity was measured as described later. Moreover, the virus amount was measured about the to-be-processed liquid before the start of aeration, and the to-be-processed liquid after 10 minutes of aeration. The results are shown in Table 2.

(Example 3)
The ozone aeration treatment was performed in the same manner as in Example 1 except that the triacetin concentration was adjusted to 50 ppm. For the liquid to be treated before the start of aeration and the liquid to be treated after 5 minutes of aeration, infectivity described later was measured, and the results are shown in Table 2.

Example 4
Ozone aeration treatment was performed in the same manner as in Example 1 except that n-propyl acetate (special grade, manufactured by Kanto Chemical Co., Inc.) concentration was adjusted to 100 ppm instead of triacetin. For the liquid to be treated before the start of aeration and the liquid to be treated after 5 minutes of aeration, infectivity described later was measured, and the results are shown in Table 2.

(Example 5)
Ozone aeration treatment was performed in the same manner as in Example 1 except that the concentration of 1,2-propylene glycol diacetate (manufactured by Tokyo Chemical Industry Co., Ltd.) was adjusted to 100 ppm instead of triacetin. For the liquid to be treated before the start of aeration and the liquid to be treated after 5 minutes of aeration, infectivity described later was measured, and the results are shown in Table 2.

(Comparative Example 1)
Ozone aeration treatment was performed in the same manner as in Example 1 except that triacetin was not added. For the liquid to be treated before the start of aeration and the liquid to be treated after aeration for 5 minutes, infectivity was measured as described later. Moreover, the virus amount was measured about the to-be-processed liquid before the start of aeration, and the to-be-processed liquid after 10 minutes of aeration. The results are shown in Table 2.

(Comparative Example 2)
Ozone aeration treatment was performed in the same manner as in Example 1 except that the monoacetin (special grade, manufactured by Kanto Chemical Co., Inc.) concentration was adjusted to 100 ppm instead of triacetin. For the liquid to be treated before the start of aeration and the liquid to be treated after aeration for 5 minutes, infectivity was measured as described later.

<Measurement of infectivity>
(Measuring method)
About the obtained to-be-processed liquid, 0.5 mL is extract | collected from each to-be-processed liquid before an ozone aeration process and after the ozone aeration process for 5 minutes, 2% MEM culture medium (MEM Earle's, manufacturer: GIBCO, 2%) Each was added to 4.5 mL (containing fetal calf serum). 20 μL was collected from the 2% MEM medium to which the liquid to be treated was added, and added to 180 μL of the 2% MEM medium. This process was repeated to prepare a liquid to be treated which was diluted 10 times.
In advance, 10% MEM medium was extracted from a plate (96 well) in which CRFK cells (cat kidney cells) were cultured using 10% MEM medium, and 180 μL of 2% MEM medium was added per well instead. The plate was then placed in a CO ₂ incubator and left at 37 ° C. for 1 hour. Thereafter, the plate was taken out, and 20 μL of the above-mentioned serially diluted solution to be treated was added to CRFK cells in the well of the plate (added to 4 wells for each serial dilution). The plate was placed in a CO ₂ incubator and left at 35 ° C. for 1 to 5 days, and then observed with a microscope. Infection was determined when cell deformation was observed at 50% or more (2 out of 4 wells). The threshold of infection and non-infection was confirmed, and the lowest infected concentration was defined as the infectivity of the virus.

(Evaluation methods)
○: Non-infected (no more than 1 virus-infected cell in 4 wells)
×: Infected (two or more virus-infected cells in 4 wells)
The relative concentration is set so that the virus concentration in the solution to be treated before dilution is 1, and the concentration of the virus solution after serial dilution is 1E-01 (10 ⁻¹ ), 1E-02 (10 ⁻² ), 1E-03 (10 ^{− 3} ) ...

<Measurement of viral load>
(Measuring method)
About the obtained liquid to be treated, the amount of virus before ozone aeration treatment and after ozone aeration treatment for 10 minutes was measured by a real-time PCR method.
RNA was extracted from the ozone-aerated liquid by the method described later, and cDNA was synthesized using the extracted RNA. Furthermore, DNA synthesis was performed using cDNA, and fluorescence intensity was monitored over time by real-time PCR.
Regarding the fluorescence intensity during the amplification of DNA, the relative virus concentration was calculated using a calibration curve prepared using feline calicivirus with a known relative concentration. And the logarithmic reduction coefficient of the virus was calculated | required by following formula (1) using the amount of viruses before ozone aeration processing, and the amount of viruses after 10-minute ozone aeration processing.
Log reduction coefficient of virus = Log (initial virus relative concentration) −Log (virus relative concentration after 10 minutes ozone aeration treatment) Formula (1)

(RNA extraction)
Reagent kit A: RNA extraction was performed by the following procedure using QIAamp Viral RNA Mimi Kit (manufactured by QIAGEN) and ethanol (for molecular biology, manufactured by Wako Pure Chemical Industries, Ltd.). Reagents other than ethanol used in reagent preparation are constituent reagents of reagent kit A.
[Reagent preparation]
(1) Preparation of Carrier RNA-Buffer AVL Buffer AVL 310 μL was added to Carrier RNA 310 μg and completely dissolved, and then Buffer AVL was added to make a total volume of 560 μL.
(2) Preparation of Buffer AW1 25 mL of ethanol was added to 19 mL of AW1.
(3) Preparation of Buffer AW2 30 mL of ethanol was added to 13 mL of AW2.

[Extract]
In Examples 1 and 2 and Comparative Example 1, the liquid to be treated before aeration and the liquid to be treated after 10 minutes of aeration treatment were collected, and a dispersion diluted to 1/10 with 2% MEM medium was used as the sample stock solution. did.
140 μL of the sample stock solution and 560 μL of Carrier RNA-Buffer AVL were placed in a 1.5 mL microtube, mixed for 15 seconds with a vortex mixer, allowed to stand at room temperature for 10 minutes, and then spun down. Ethanol (560 μL) was added to the microtube, mixed with a vortex mixer for 15 seconds, allowed to stand at room temperature for 10 minutes, and then spun down. 630 μL of the mixed solution in the microtube was placed in a QIAamp spin column. The spin column was set in a 2 ml collection tube and centrifuged (8100 rpm, 1 minute). The spin column was set in a new 2 mL collection tube, 630 μL of the mixed solution in the microtube was added, and centrifugation was similarly performed.
Subsequently, 500 μL of Buffer AW1 was added to the QIAamp spin column, and centrifugation (8100 rpm, 1 minute) was performed. Subsequently, 500 μL of Buffer AW2 was added to the QIAamp spin column, and centrifugation (13200 rpm, 3 minutes) was performed.
The QIAamp spin column was transferred to a new 2 mL collection tube and centrifuged (13200 rpm, 1 minute).
The QIAamp spin column was set in a new 1.5 mL collection tube, 60 μL of AWE was added, and the mixture was allowed to stand at room temperature, followed by centrifugation (8100 rpm, 1 minute) to obtain extracted RNA as a filtrate.

(Reverse transcription reaction) RT reaction: cDNA synthesis from RNA Reagent kit B: A reaction solution was prepared using RiverTra Plus (Toyobo Co., Ltd.) and the extracted RNA. Reagents other than the extracted RNA used in the preparation of the reaction solution are constituent reagents of reagent kit B.
[Preparation of reaction solution]
A PCR tube was charged with 9 μL of RNase-free water, 1 μL of random primer, and 2 μL of RNA extract, incubated for 5 minutes in a 65 ° C. water bath, and then rapidly cooled on ice.
Subsequently, 4 μL of 5XRT buffer, 2 μL of 10 mM-4dNTP, 1 μL of 10 U / μL-RNase inhibitor, and 1 μL of RiverTraACE were added to the PCR tube to prepare a reaction solution.
[CDNA synthesis]
CDNA was synthesized from the prepared reaction solution under the following conditions.
Reaction conditions: annealing 30 ° C. for 10 minutes, reverse transcription 42 ° C. for 60 minutes, denaturation 85 ° C. for 5 minutes Equipment used: RC320 (manufactured by Astec Co., Ltd.)

(DNA synthesis from cDNA)
[Sample solution]
Reagent kit C: A sample solution was prepared using LightCycler Fast Start DNA Master SYBRGreen I (Roche), primer (F), primer (R), and cDNA. Note that the following reagents (1) to (3) used in the preparation of the sample solution are constituent reagents of the reagent kit C.
(1) H ₂ O balance (2) MgCl ₂ 2μL
(3) SYBGreen I 2 μL
(4) Primer (F) 0.4 μL
Sequence: 5'-3 '
Base sequence: ACCCGAGCAAGGAACAATGGGT
(A, T, C, and G in the base sequence represent adenine, thymine, cytosine, and guanine, respectively, and so on.)
(5) Primer (R) 0.4 μL
Sequence: 5'-3 '
Base sequence: GAGCAAAGGGCCTAAGGATTGT
(6) Reverse transcript (cDNA) 2 μL
A sample solution was prepared with reagents, primers and cDNA as described above.
The total amount of (1) to (6) was 20 μL, and (4) and (5) were each adjusted to 0.2 μmol in the sample solution.
[Real-time PCR]
The obtained sample solution was synthesized under the following conditions, and monitored using a detection wavelength of 530 nm.
A calibration curve was prepared by determining the number of cycles of each relative concentration from the fluorescence intensity at a detection wavelength of 530 nm using feline calicivirus having a known relative concentration.
Reaction conditions: 95 ° C., 10 minutes PCR reaction: 45 cycles (95 ° C. 10 seconds, 63 ° C. 10 seconds, 72 ° C. 5 seconds)
Equipment used: LightCycler (Roche)

From the results of Table 1, in each of Measurement Examples 1 to 5, the values of 100 msec dynamic surface tension and 30 sec surface tension were preferable values for the virucidal aid of the present invention. In particular, it can be said that triacetin is more preferable as a compound having the characteristics that the 100 msec dynamic surface tension is relatively low and the 30 sec dynamic surface tension is relatively high.
Examples 1-5 were uninfected against CRFK cells at any dilution factor. On the other hand, from the results of Comparative Example 1, it was not possible to significantly reduce the infectivity of the virus only by ozone aeration treatment without adding a virus-killing aid. Further, Example 1 reduced the amount of virus compared to Comparative Example 1. In Example 2, it was found that the amount of virus can be further reduced by adding the water-soluble acidic component (b) to the component (a).

Claims (3)

A virus killing aid used for ozone aeration treatment, comprising a compound represented by the following general formula (I):

[In formula (I), x represents an integer of 0 to 2, and R ¹ , R ² and R ³ are each independently represented by —H, —OH or the following general formulas (1) to (3). It is a group selected from the group consisting of groups, and at least one of R ¹ , R ² and R ³ is a group represented by the following general formula (2). ^Further, among ^{R 1, R 2, R 3} , -OH is one or less. ]

[In Formula (1)-(3), R < ⁴ >, R < ⁵ >, R < ⁶ > is a C1-C3 alkyl group. ]   The virus-killing assistant according to claim 1, wherein the virus-killing assistant contains a water-soluble acidic component. A virus killing method, wherein the virus killing agent according to claim 1 or 2 is used for a virus killing treatment by ozone aeration treatment.