KR20210098650A - The composition of disinfectant for veterinary usage containing thymol - Google Patents

Abstract

The present invention relates to a composite composition of a disinfectant for animal disease prevention containing thymol to disinfect livestock, roads, and livestock-related vehicles for the purpose of preventing animal diseases that cause great economic damage to the livestock industry, such as avian influenza, foot-and-mouth disease, and African swine fever. More specifically, the composite composition includes 1 to 5% by weight of thymol, 7.5 to 12.5% by weight of acetic acid, 15 to 25% by weight of citric acid, 7.5 to 12.5% by weight of phosphoric acid, 5 to 10% by weight of polyoxyethylene lauryl ether, 2 to 6% by weight of polyoxyethylene octylphenyl ether, 4 to 8% by weight of sodium lauryl sulfate solution, 2.5 to 10% by weight of ammonium chloride, and 11 to 55.5% by weight of purified water. The composite composition of a disinfectant for animal disease prevention containing thymol can effectively prevent avian influenza, foot-and-mouth disease, and African swine fever, which are animal diseases that have recently become a global problem, due to its excellent disinfection effect even under sub-zero conditions.

Description

The composition of disinfectant for veterinary usage containing thymol}

The present invention is a disinfectant complex composition for animal disease prevention containing thymol for the prevention of animal diseases such as avian influenza, foot-and-mouth disease and African swine fever. It relates to a disinfectant complex composition for preventing animal diseases containing thymol.

Recently, African swine fever has occurred in Korea following avian influenza and foot and mouth disease, and the use of disinfectants for animal disease prevention to kill these pathogens is increasing significantly. However, despite the use of these disinfectants, diseases continue to occur, and many livestock farms have widespread distrust of disinfectants for animal disease prevention. Disinfectants for domestic animal disease prevention are prescribed to determine the recommended dilution factor by testing the disinfection effect at 4℃ as a standard condition (disinfectant efficacy test guideline, Ministry of Agriculture, Forestry and Livestock Quarantine Office Notice 2018-16), but most animal diseases are quarantined Disinfectants for sterilization have a problem in that the disinfection effect is insufficient in sub-zero conditions in winter because the disinfection effect is greatly reduced in sub-zero conditions. In particular, since avian influenza and foot-and-mouth disease mainly occur in winter due to the characteristics of the virus, when using disinfectants in winter, the concentration is twice the recommended dilution factor tested at 4℃ (that is, if the recommended dilution factor is 200 times, use 100 times in winter) The government recommends and guides its use. Therefore, there is a need to develop a disinfectant for animal disease prevention that has excellent disinfection effect and does not deteriorate even in sub-zero conditions in winter.

Currently, most of the disinfectants used for the prevention of avian influenza, foot-and-mouth disease, and African swine fever in Korea are tri-salt preparations and quaternary ammonium complex disinfectants. As of December 2019, out of a total of 147 disinfectants approved by the Agriculture, Forestry and Livestock Quarantine Headquarters as disinfectants for avian influenza, 56 items were tri-salts and 58 items were quaternary ammonium complexes. Among the disinfectants for dogs, there are 54 tri-salt preparations and 52 quaternary ammonium complex preparations. Of the total 20 approved disinfectants for African swine fever, 11 tri-salt preparations and 5 quaternary ammonium complex preparations are available in Korea. It can be seen that most of the disinfectants approved and used for animal disease control are tri-salt preparations or quaternary ammonium complex preparations.

The tri-salt preparation is an animal disinfectant containing more than 50% by weight of potassium monopersulfate, and has the advantage of having a broad disinfection effect. However, the tri-salt preparation has a problem in that the disinfection effect is lowered under sub-zero conditions. For this reason, in the case of the tri-salt preparation, when used for avian influenza prevention purposes, a 200-fold dilution should be used in spring, summer, and autumn, and a 100-fold dilution should be used in winter.

The quaternary ammonium compound complex is an animal disinfectant containing more than 10% of benzalconium chloride and didecyl dimethyl ammonium chloride. It can be prepared in liquid form and used after diluting with water. While it has the advantage of being convenient to use, there is a problem in that the disinfection effect is greatly reduced in sub-zero conditions. For this reason, the Ministry of Agriculture, Food and Rural Affairs does not recommend the use of quaternary ammonium complex formulations for the purpose of preventing avian influenza in winter.

Patent Document 1: Republic of Korea Patent Publication No. 10-0772054 Patent Document 2: Republic of Korea Patent Publication No. 10-2015-0030525 Patent Document 3: Republic of Korea Patent Publication No. 10-1687337

Therefore, the present invention was invented to solve the problems of the conventional disinfectants for the prevention of animal diseases as described above, and the tri-salt preparation and quaternary ammonium complex used as disinfectants for the prevention of animal diseases such as avian influenza, foot-and-mouth disease and African swine fever. Convenience of use by improving the problem of reducing the disinfection effect of the preparation in sub-zero conditions and by diluting the existing disinfectant at the same concentration throughout the year to improve the inconvenience of diluting it at different concentrations in spring, autumn and winter In addition to the achievements, there is a purpose devised to provide a disinfectant composition for animal disease prevention containing thymol, which can reduce the amount of disinfectant used during winter quarantine, thereby reducing not only the environmental impact but also the cost of the disinfectant.

The technical means of the present invention for achieving the above object is a disinfectant composition for animal disease prevention containing thymol, characterized in that it is composed of all selected thymol, organic acid, inorganic acid, surfactant, cryoprotectant and purified water.

In addition, the composite composition has a composition ratio of 1 to 5% by weight of thymol, 7.5 to 12.5% by weight of acetic acid, 15 to 25% by weight of citric acid, 7.5 to 12.5% by weight of phosphoric acid, 5 to 10% by weight of polyoxyethylene lauryl ether, 2-6 wt% of polyoxyethylene octylphenyl ether, 4-8 wt% of sodium lauryl sulfate solution, 2.5-10 wt% of ammonium chloride and 11-55.5 wt% of purified water.

As described above, the disinfectant complex composition for animal disease prevention containing thymol according to the present invention improves the problem of lowering the disinfection effect in sub-zero conditions, and in the case of existing disinfectants, it should be diluted to different concentrations in spring-autumn and winter. By diluting it to the same concentration throughout the year by improving the inconvenience of cleaning it, it is convenient to use and can relatively reduce the amount of disinfectant used during winter quarantine. there is

1 is a view showing the chemical structure of thymol (Thymol) according to the present invention.

Hereinafter, an embodiment of the present invention will be described in detail based on the accompanying drawings, in which FIG. 1 is a view showing the chemical structure of thymol according to the present invention.

Accordingly, the composite composition of the present invention is a composite composition composed of a mixture of thymol, organic acid, inorganic acid, surfactant, cryoprotectant and purified water. Under the condition of

Here, the organic acid preferably includes one or two or more selected from acetic acid, citric acid, malic acid, and lactic acid.

In addition, the inorganic acid preferably includes one or two or more selected from phosphoric acid, sulfuric acid, and nitric acid.

In addition, the surfactant preferably includes one or two or more selected from reoxyethylene lauryl ether, polyoxyethylene octylphenyl ether, and sodium lauryl sulfate solution.

In addition, it is preferable that the cryoprotectant comprises one or two or more selected from ammonium chloride, calcium chloride, and sodium chloride.

That is, the composite composition has a composition ratio of 1 to 5% by weight of thymol, 7.5 to 12.5% by weight of acetic acid, 15 to 25% by weight of citric acid, 7.5 to 12.5% by weight of phosphoric acid, 5 to 10% by weight of polyoxyethylene lauryl ether, 2 to 6% by weight of polyoxyethylene octylphenyl ether, 4 to 8% by weight of sodium lauryl sulfate solution, 2.5 to 10% by weight of ammonium chloride and 11 to 55.5% by weight of purified water.

As such, when describing each component included in the composite composition in detail, the thymol will preferably be 1 to 5% by weight based on the total composition. At this time, when the thymol is less than 1% by weight, the disinfection effect is insufficient, and when it is 5% by weight or more, there is a problem in manufacturing because it causes difficulty in dissolving.

In the composition of the present invention, acetic acid is preferably used in an amount of 7.5 to 12.5% by weight of the total composition. In the present invention, when the amount of acetic acid is 7.5% by weight or less, the disinfection effect is insufficient. There is a problem in manufacturing by affecting the composition ratio of

Citric acid in the composition of the present invention is preferably used in an amount of 15 to 25% by weight of the total composition. In the present invention, when the amount of citric acid is 15% by weight or less, the disinfection effect is insufficient, and when it is 25% by weight or more, other compositional components There is a problem in manufacturing by affecting the composition ratio of

The phosphoric acid used in the present invention is preferably used in an amount of 7.5 to 12.5% by weight of the total composition, and in the present invention, when the amount of phosphoric acid is 7.5% by weight or less, the disinfection effect is insufficient, and when the amount of phosphoric acid is 12.5% by weight or more There is a problem in manufacturing by affecting the composition ratio of other constituent components.

The polyoxyethylene lauryl ether used in the present invention is preferably used in an amount of 5 to 10% by weight of the total composition, and in the present invention, when the amount of polyoxyethylene lauryl ether is 5% by weight or less, a precipitate is formed There is a problem in the stability of the formulation, and when the amount of polyoxyethylene lauryl ether is 10% by weight or more, there is a problem in manufacturing by affecting the composition ratio of other components.

The polyoxyethylene octylphenyl ether used in the present invention is preferably 2 to 6% by weight of the total composition, and in the present invention, when the amount of polyoxyethyleneoctylphenylether is 2% by weight or less, a precipitate is formed There is a problem in the stability of the formulation, and when the amount of polyoxyethylene octylphenyl ether is 6% by weight or more, there is a problem in manufacturing by affecting the composition ratio of other components.

Sodium lauryl sulfate used in the present invention is preferably used in an amount of 4 to 8% by weight of the total composition, and in the present invention, when the amount of sodium lauryl sulfate is 4% by weight or less, the disinfection effect is insufficient and the amount of sodium lauryl sulfate When it is more than 8% by weight, it takes a long time to dissolve, which is not preferable.

Ammonium chloride used in the present invention is preferably used in an amount of 2.5 to 10% by weight of the total composition, and in the present invention, when the amount of ammonium chloride is 2.5% by weight or less, the disinfection effect is insufficient and the amount of ammonium chloride is 10% by weight % or more, there is a problem in manufacturing by affecting the composition ratio of other components.

Purified water in the composite composition according to the present invention is used as an excipient so that the total weight% of the disinfectant composite composition for animal disease prevention containing thymol is 100% by weight according to the sum of the weight% of all components used.

The present invention may be understood more specifically by the following examples and experimental examples, which are intended to illustrate the present invention only and not to limit the scope of the present invention.

[Example 1]

To prepare a composite composition from the composition shown in Table 1 below.

ingredient weight/g thymol 25mg acetic acid 400mg polyoxyethylene lauryl ether 75mg Polyoxyethylene octylphenyl ether 40mg Purified water 460mg 1 g total

The process for preparing the composite composition is as follows.

After acetic acid is put in a mixer, thymol is added to dissolve it, and then polyoxyethylene lauryl ether, polyoxyethylene octylphenyl ether and purified water are added in order, mixed uniformly, subdivided and packaged in a certain amount.

[Example 2]

The following is to prepare a composite composition from the composition shown in Table 2.

ingredient weight/g thymol 25mg citric acid 400mg polyoxyethylene lauryl ether 75mg Polyoxyethylene octylphenyl ether 40mg Purified water 460mg 1 g total

The process for preparing the composite composition is as follows.

It is prepared by putting polyoxyethylene lauryl ether and polyoxyethylene octylphenyl ether in a mixer, adding thymol and stirring, then adding purified water and citric acid to dissolve it, homogenize it, and subdivide and package it by a certain amount.

[Example 3]

To prepare a composite composition as shown in Table 3 below.

ingredient weight/g thymol 25mg phosphoric acid 400mg polyoxyethylene lauryl ether 75mg Polyoxyethylene octylphenyl ether 40mg Purified water 460mg 1 g total

The process for preparing the composite composition is as follows.

It is prepared by putting polyoxyethylene lauryl ether and polyoxyethylene octylphenyl ether in a mixer, adding thymol, stirring, and then adding purified water and phosphoric acid in order, homogenizing, subdividing and packaging a certain amount.

[Example 4]

To prepare a composite composition from the composition shown in Table 4 below.

ingredient weight/g thymol 25mg acetic acid 200mg citric acid 200mg polyoxyethylene lauryl ether 75mg Polyoxyethylene octylphenyl ether 40mg Purified water 460mg 1 g total

The process for preparing the composite composition is as follows.

After acetic acid is put in a mixer, thymol is added to dissolve it, and then citric acid, polyoxyethylene lauryl ether, polyoxyethylene octylphenyl ether and purified water are added in this order, mixed uniformly, subdivided and packaged in a certain amount.

[Example 5]

To prepare a composite composition from the composition shown in Table 5 below.

ingredient weight/g thymol 25mg acetic acid 300mg phosphoric acid 100mg polyoxyethylene lauryl ether 75mg polyoxyethylene octylphenyl ether 40mg Purified water 460mg 1 g total

The process for preparing the composite composition is as follows.

After acetic acid is put in a mixer, thymol is added to dissolve it, and then phosphoric acid, polyoxyethylene lauryl ether, polyoxyethylene octylphenyl ether, and purified water are added in order, mixed uniformly, subdivided and packaged in a certain amount.

[Example 6]

To prepare a composite composition from the composition shown in Table 6 below.

ingredient weight/g thymol 25mg citric acid 300mg phosphoric acid 100mg polyoxyethylene lauryl ether 75mg polyoxyethylene octylphenyl ether 40mg Purified water 460mg 1 g total

The process for preparing the composite composition is as follows.

It is prepared by putting polyoxyethylene lauryl ether and polyoxyethylene octylphenyl ether in a mixer, adding thymol, stirring, and then adding purified water, phosphoric acid and citric acid in order, homogenizing, subdividing and packaging a certain amount.

[Example 7]

To prepare a composite composition from the composition shown in Table 7 below.

ingredient weight/g thymol 25mg acetic acid 100mg citric acid 100mg phosphoric acid 200mg polyoxyethylene lauryl ether 75mg polyoxyethylene octylphenyl ether 40mg Purified water 460mg 1 g total

The process for preparing the composite composition is as follows.

After acetic acid is put in a mixer, thymol is added to dissolve it, and then citric acid, phosphoric acid, polyoxyethylene lauryl ether, polyoxyethylene octylphenyl ether and purified water are added in this order, mixed uniformly, subdivided and packaged in a certain amount. .

[Example 8]

To prepare a composite composition from the composition shown in Table 8 below.

ingredient weight/g thymol 25mg acetic acid 133mg citric acid 133mg phosphoric acid 133mg polyoxyethylene lauryl ether 75mg polyoxyethylene octylphenyl ether 40mg Purified water 461mg 1 g total

The process for preparing the composite composition is as follows.

After acetic acid is put in a mixer, thymol is added to dissolve it, and then citric acid, phosphoric acid, polyoxyethylene lauryl ether, polyoxyethylene octylphenyl ether and purified water are added in this order, mixed uniformly, subdivided and packaged in a certain amount. .

[Example 9]

To prepare a composite composition from the composition shown in Table 9 below.

ingredient weight/g thymol 25mg acetic acid 200mg citric acid 100mg phosphoric acid 100mg polyoxyethylene lauryl ether 75mg polyoxyethylene octylphenyl ether 40mg Purified water 460mg 1 g total

The process for preparing the composite composition is as follows.

After acetic acid is put in a mixer, thymol is added to dissolve it, and then citric acid, phosphoric acid, polyoxyethylene lauryl ether, polyoxyethylene octylphenyl ether and purified water are added in this order, mixed uniformly, subdivided and packaged in a certain amount. .

[Example 10]

To prepare a composite composition from the composition shown in Table 10 below.

ingredient weight/g thymol 25mg acetic acid 100mg citric acid 200mg phosphoric acid 100mg polyoxyethylene lauryl ether 75mg polyoxyethylene octylphenyl ether 40mg Purified water 460mg 1 g total

The process for preparing the composite composition is as follows.

After acetic acid is put in a mixer, thymol is added to dissolve it, and then citric acid, phosphoric acid, polyoxyethylene lauryl ether, polyoxyethylene octylphenyl ether and purified water are added in this order, mixed uniformly, subdivided and packaged in a certain amount. .

[Example 11]

To prepare a composite composition from the composition shown in Table 11 below.

ingredient weight/g thymol 25mg acetic acid 240mg citric acid 80mg phosphoric acid 80mg polyoxyethylene lauryl ether 75mg polyoxyethylene octylphenyl ether 40mg Purified water 460mg 1 g total

The process for preparing the composite composition is as follows.

After acetic acid is put in a mixer, thymol is added to dissolve it, and then citric acid, phosphoric acid, polyoxyethylene lauryl ether, polyoxyethylene octylphenyl ether and purified water are added in this order, mixed uniformly, subdivided and packaged in a certain amount. .

[Example 12]

To prepare a composite composition from the composition shown in Table 12 below.

ingredient weight/g thymol 25mg acetic acid 160mg citric acid 160mg phosphoric acid 80mg polyoxyethylene lauryl ether 75mg polyoxyethylene octylphenyl ether 40mg Purified water 460mg 1 g total

The process for preparing the composite composition is as follows.

After acetic acid is put in a mixer, thymol is added to dissolve it, and then citric acid, phosphoric acid, polyoxyethylene lauryl ether, polyoxyethylene octylphenyl ether and purified water are added in this order, mixed uniformly, subdivided and packaged in a certain amount. .

[Example 13]

To prepare a composite composition from the composition shown in Table 13 below.

ingredient weight/g thymol 25mg acetic acid 80mg citric acid 240mg phosphoric acid 80mg polyoxyethylene lauryl ether 75mg polyoxyethylene octylphenyl ether 40mg Purified water 460mg 1 g total

The process for preparing the composite composition is as follows.

After acetic acid is put in a mixer, thymol is added to dissolve it, and then citric acid, phosphoric acid, polyoxyethylene lauryl ether, polyoxyethylene octylphenyl ether and purified water are added in this order, mixed uniformly, subdivided and packaged in a certain amount. .

[Example 14]

To prepare a composite composition from the composition shown in Table 14 below.

ingredient weight/g thymol 25mg acetic acid 100mg citric acid 200mg phosphoric acid 100mg polyoxyethylene lauryl ether 75mg polyoxyethylene octylphenyl ether 40mg Ammonium Chloride 50mg Purified water 410mg 1 g total

The process for preparing the composite composition is as follows.

Put acetic acid in the mixer, add thymol to dissolve it, and then add citric acid, phosphoric acid, polyoxyethylene lauryl ether, polyoxyethylene octylphenyl ether, ammonium chloride and purified water in order, mix evenly, divide and pack will be manufacturing

[Example 15]

To prepare a composite composition from the composition shown in Table 15 below.

ingredient weight/g thymol 25mg acetic acid 100mg citric acid 200mg phosphoric acid 100mg polyoxyethylene lauryl ether 75mg polyoxyethylene octylphenyl ether 40mg calcium chloride 50mg Purified water 410mg 1 g total

The process for preparing the composite composition is as follows.

Put acetic acid in a mixer, add thymol to dissolve it, and then add citric acid, phosphoric acid, polyoxyethylene lauryl ether, polyoxyethylene octylphenyl ether, calcium chloride and purified water in this order, mix uniformly, and divide and pack a certain amount. will do

[Example 16]

To prepare a composite composition from the composition shown in Table 16 below.

ingredient weight/g thymol 25mg acetic acid 100mg citric acid 200mg phosphoric acid 100mg polyoxyethylene lauryl ether 75mg polyoxyethylene octylphenyl ether 40mg sodium chloride 50mg Purified water 410mg 1 g total

The process for preparing the composite composition is as follows.

Put acetic acid in a mixer, add thymol to dissolve it, and then add citric acid, phosphoric acid, polyoxyethylene lauryl ether, polyoxyethylene octylphenyl ether, sodium chloride and purified water in this order, mix uniformly, divide and pack a certain amount will do

[Comparative Example 1]

The following Table 17 shows the composition ratio of the commercially available tri-salt disinfectant (Science Easysolic Acid, Quasi-Drug License No. 115-192 for veterinary use).

ingredient weight/g trigonitis 600mg citric acid 200mg sodium chloride 15mg sodium metaphosphate 120mg sulfamic acid 50mg Sodium Olefinsulfonate 15mg 1 g total

[Comparative Example 2]

The following Table 18 shows the composition ratio of the quaternary ammonium complex disinfectant (Science Cold Strong Solution, Quasi-Drug License No. 115-184 for veterinary use) distributed in the market.

ingredient weight/g quaternary ammonium 150mg phosphoric acid 300mg propylene glycol 284mg cinnamon alcohol 50mg Ammonium Chloride 50mg Purified water 166mg 1 g total

Kinds Disinfectant Composite Composition Example 1 Thymol (2.5%) + Acetic Acid (40%) Example 2 Thymol (2.5%) + Citric Acid (40%) Example 3 Thymol (2.5%) + Phosphoric acid (40%) Example 4 Thymol (2.5%) + Acetic Acid (20%) + Citric Acid (20%) Example 5 Thymol (2.5%) + Acetic acid (30%) + Phosphoric acid (10%) Example 6 Thymol (2.5%) + Citric Acid (30%) + Phosphoric Acid (10%) Example 7 Thymol (2.5%) + Acetic acid (10%) + Citric acid (10%) + Phosphoric acid 20%) Example 8 Thymol (2.5%) + acetic acid (13.3%) + citric acid (13.3%) + phosphoric acid (13.3%) Example 9 Thymol (2.5%) + Acetic acid (20%) + Citric acid (10%) + Phosphoric acid (10%) Example 10 Thymol (2.5%) + Acetic acid (10%) + Citric acid (20%) + Phosphoric acid (10%) Example 11 Thymol (2.5%) + Acetic acid (24%) + Citric acid (8%) + Phosphoric acid (8%) Example 12 Thymol (2.5%) + Acetic acid (16%) + Citric acid (16%) + Phosphoric acid (8%) Example 13 Thymol (2.5%) + Acetic acid (8%) + Citric acid (24%) + Phosphoric acid (8%) Example 14 Thymol (2.5%) + Acetic Acid (10%) + Citric Acid (20%) + Phosphoric Acid (10%) + Ammonium Chloride (5%) Example 15 Thymol (2.5%) + Acetic acid (10%) + Citric acid (20%) + Phosphoric acid (10%) + Calcium chloride (5%) Example 16 Thymol (2.5%) + acetic acid (10%) + citric acid (20%) + phosphoric acid (10%) + sodium chloride (5%) Comparative Example 1 Tri-salt (60%) + citric acid (20%) + sodium chloride (1.5%) + sodium metaphosphate (12%) + sulfamic acid (5%) + sodium olefin sulfonate (1.5%) Comparative Example 2 Quaternary ammonium (15%) + phosphoric acid (30%) + propylene glycol (28.4%) + cinnamon alcohol (5%) + ammonium chloride (5%)

<Composite composition of disinfectant for preventing animal diseases containing thymol>

[Experimental Example 1]

1. Experimental method

Examples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 were used to investigate the disinfection effect on the typical bacterial strain, Salmonella typhimurium. The absorbance was compared by culturing 10 each of Salmonella typhimurium in an organic liquid medium according to the notification of the Ministry of Agriculture, Food and Rural Affairs No.

The test group with low absorbance compared to the control group is judged to have inhibited bacterial growth, and when the proliferation of 6 or more of the 10 test groups of each example is inhibited, the example shows the disinfection effect on Salmonella at a dilution factor of 5 times. It was determined that there is

2. Experimental results

In Examples 1, 2, 3, 4, 5, and 6, the number of experimental groups in which bacterial growth was inhibited was 2, 3, 0, 5, 3, and 5, respectively, and the proliferation of less than 6 experimental groups was inhibited. The disinfection effect of the pear dilution on Salmonella was judged as failing.

Examples 7, 8, 9, 10, 11, 12, and 13, the number of experimental groups in which bacterial growth was inhibited was 7, 6, 7, 10, 9, 8, 8, respectively, 6 or more experimental groups were inhibited, The 5-fold dilution was determined to have a disinfecting effect against Salmonella. Of these, Example 10 showed that the proliferation of Salmonella was inhibited in all 10 experimental groups, and thus the efficacy was the most excellent. Examples 7, 8, and 9 may be effective, but the effect is insufficient, Examples 11, 12, and 13 are generally good, but the disinfection effect was low compared to Example 10.

The disinfection effect of the composition of each example is shown in Table 20 below.

composition Experiment
One Experiment
2 Experiment
3 Experiment
4 Experiment
5 Experiment
6 Experiment
7 Experiment
8 Experiment
9 Experiment
10 Dae Jo 0.6124 0.6022 0.6031 0.5996 0.6012 0.6003 0.6399 0.6329 0.6005 0.6066 Example 1 0.3445 0.6015 0.6134 0.6254 0.6348 0.6294 0.6187 0.6361 0.3852 0.6267 Example 2 0.6018 0.6043 0.6182 0.6007 0.6015 0.3397 0.3428 0.6220 0.6130 0.3393 Example 3 0.6216 0.6253 0.6431 0.6036 0.6132 0.6384 0.6294 0.6197 0.6351 0.5993 Example 4 0.6009 0.6034 0.6039 0.6028 0.6021 0.3386 0.3425 0.3409
0.3529 0.3661 Example 5 0.3346 0.6029 0.3409 0.6039 0.6024 0.3419 0.6281 0.6311 0.6082 0.6097 Example 6 0.6401 0.6392 0.6249 0.3421 0.6173 0.3318 0.3319 0.6016 0.3326 0.3341 Example 7 0.3446 0.5986 0.3458 0.3409 0.6035 0.6188 0.3510 0.3528 0.3497 0.3356 Example 8 0.3426 0.5979 0.6287 0.3517 0.3524 0.3436 0.6228 0.3489 0.6311 0.3415 Example 9 0.3529 0.3423 0.3418 0.6072 0.3485 0.6351 0.3420 0.3382 0.3216 0.6116 Example 10 0.3389 0.3432 0.3428 0.3403 0.3392 0.3439 0.3426 0.3498 0.3454 0.3426 Example 11 0.3567 0.3376 0.6003 0.3386 0.3498 0.3383 0.3414 0.3423 0.3512 0.3427 Example 12 0.3542 0.5996 0.3269 0.3406 0.6127 0.3372 0.3218 0.3436 0.3524 0.3319 Example 13 0.3426 0.3298 0.6123 0.3451 0.3399 0.3425 0.6214 0.3516 0.3473 0.3492

<Disinfection Effect of Example Composition>

[Experimental Example 2]

1. Experimental method

In order to evaluate the disinfection effect in sub-zero conditions, the efficacy tests of the compositions of Examples 10, 14, 15 and 16 were carried out at -5 ° C in accordance with the Notification No.

go. Prepare a water bath for sub-zero conditions

After adding ethanol to 40% distilled water, it was stored in a freezing chamber set at -10°C so that the temperature was -5°C or lower.

me. Preparation of Published Virus for Examination

Avian influenza virus H9N2 was received from the Agriculture, Forestry and Livestock Quarantine Headquarters, and the live virus during subculture was used, but the virus was collected at the time of maximum virus growth and stored in ice water for a short time until just before use.

all. virus dilution

The virus was diluted using 5% organic matter (FBS).

La. Dilution of Disinfectant (Examples 10, 14, 15 and 16 Compositions)

Before the reaction with the virus diluent, the compositions of Examples 10, 14, 15, and 16 were diluted with 5% organic material (FBS), respectively, and stored in a constant temperature water bath at -5°C or less for 20 to 30 minutes.

mind. reaction of disinfectant

1) 1.0 mL of virus solution at 4°C was mixed with 19 mL of virus dilution solution.

2) Disinfectant reaction time was measured for 10 minutes from the time the reaction solution (disinfectant dilution + virus solution) reached -5℃, and shaken every 5 minutes in the middle (two test tubes for each dilution of disinfectant were prepared, one It was used for temperature measurement, and one was used for disinfectant reaction).

3) As the pathogen control group, hard water without disinfectant dilution was used.

4) For the toxicity control group, a disinfectant diluent diluted with hard water was used.

bar. neutralization reaction

When the reaction of the disinfectant was finished, 1 mL of the reaction solution was taken out in order to neutralize the efficacy of the disinfectant, put into 1 mL of a neutralizing medium (PBS containing non-assimilated 10% fetal bovine serum) at 37°C and mixed.

buy. Determination of virus propagation

1) Dilute the neutralizing solution to the stock solution, 10 ^-1 ,10 ^-2 ,10 ^-3 ,10 ^-4 ,10 ^-5 using PBS, and 0.2 mL of the neutralized reaction solution for 5 9-11 day embryonated eggs per dilution factor was inoculated into the allantoic cavity.

2) After inoculation, cultured at 37° C. for 5 days, censoring was carried out daily, and embryonated eggs that died within 24 hours of inoculation were considered accidental and excluded from the test results.

3) All inoculated eggs that died within 5 days from 24 hours after inoculation were stored at 4°C.

4) Allantoic fluid was collected from all embryonated eggs that survived until 5 days after inoculation and from dead inoculated eggs stored at 4℃, and hemagglutination reaction was performed using 1% chicken red blood cells to determine the presence or absence of virus and virus titer. did.

Oh. Virus content calculation

It was calculated by Karber method.

ruler. control test

1) The pathogen control group was tested without disinfectant under hard water conditions, and it was confirmed that the ^{pathogen titer was more than 210 5} EID _{50 /mL in the neutralization reaction step.}

2) In the toxicity control group, it was confirmed that egg toxicity by the disinfectant did not occur.

car. result judgment

The disinfectant test result was treated as pass (effective) when a decrease of ^{10 4} (minimum 4 log ₁₀ ) or more of the pathogen was confirmed compared to the pathogen control group.

2. Test results

Examples 10, 14, 15 and 16 The results of the disinfection effect test on the avian influenza virus (Avian influenza virus H9N2) under sub-zero conditions of the compositions are shown in Table 21 below.

composition test strain Exam conditions pass or not Example 10 AI virus H9N2 Sub-zero conditions (-5℃) fail Example 14 AI virus H9N2 Sub-zero conditions (-5℃) pass Example 15 AI virus H9N2 Sub-zero conditions (-5℃) fail Example 16 AI virus H9N2 Sub-zero conditions (-5℃) fail

<Disinfection effect of compositions of Examples 10, 14, 15 and 16 under sub-zero conditions>

[Experimental Example 3]

1. Experimental method

In order to evaluate whether the disinfection effect decreases compared to room temperature under sub-zero conditions, Example 14, The disinfection effect test of the compositions of Comparative Example 1 and Comparative Example 2 was performed as follows. The test method at room temperature standard condition of 4°C was carried out as follows, and the test method at -5°C, the sub-zero standard condition, was carried out in the same manner as [Experimental Example 2].

go. Published virus preparation

After receiving avian influenza virus H9N2 from the Agriculture, Forestry and Livestock Quarantine Headquarters, thawing the frozen virus and storing it in ice water for a short time until just before use.

me. virus dilution

The virus was diluted with hard water ( _{containing 0.305 g of CaCl 2 and} 0.139 g (w/v) of MgCl ₂ .6H _{2 O in 1 liter of distilled water).}

all. Dilution of disinfectant (Example 14, Comparative Example 1, Comparative Example 2 composition)

The compositions of Example 10, Comparative Example 1, and Comparative Example 2 were each diluted in hard water and used.

La. reaction of disinfectant

1) 1.0 mL of 4°C virus solution was mixed with 19 mL of hard water.

2) Put 2.5 mL of the prepared virus solution into a test tube containing the same amount of diluent solution at 4°C, mix (5 mL in total), and then react at 4°C for 30 minutes, shaking well every 10 minutes in the middle.

3) As the pathogen control group, hard water without disinfectant dilution was used.

4) Toxicity control group used a disinfectant diluent diluted with hard water.

mind. neutralization reaction

When the reaction of the disinfectant was finished, 1 mL of the reaction solution was taken out to neutralize the efficacy of the disinfectant, and 37 equal volumes of neutralizing medium (PBS containing non-assimilated 10% fetal bovine serum) was added to 1 mL and mixed.

bar. Determination of virus propagation

1) Dilute the neutralizing solution to the stock solution, 10 ^-1 ,10 ^-2 ,10 ^-3 ,10 ^-4 ,10 ^-5 using PBS, and 0.2 mL of the neutralized reaction solution for 5 9-11 day embryonated eggs per dilution factor was inoculated into the allantoic cavity.

2) After inoculation, cultured at 37° C. for 5 days, censoring was carried out daily, and embryonated eggs that died within 24 hours of inoculation were considered accidental and excluded from the test results.

3) All inoculated eggs that died within 5 days from 24 hours after inoculation were stored at 4°C.

4) Allantoic fluid was collected from all embryonated eggs that survived until 5 days after inoculation and dead inoculated eggs stored at 4°C, and hemagglutination reaction was performed using 1% chicken red blood cells to determine the presence or absence of virus and virus titer. .

buy. Virus content calculation

It was calculated by Karber method, etc.

Oh. control test

1) The pathogen control group was tested without disinfectant under hard water conditions, and it was confirmed that the ^{pathogen titer was more than 210 5} EID _{50 /mL in the neutralization reaction step.}

2) In the toxicity control group, it was confirmed that egg toxicity by the disinfectant did not occur.

ruler. result judgment

The disinfectant test result confirms the effective dilution factor compared to the pathogen control group. In other words, the effective dilution factor was the dilution factor in which a decrease of ^{10 4} (minimum 4 log10) or more of the pathogen was confirmed compared to the pathogen control group.

2. Test results

Example 10, Comparative Example 1 and Comparative Example 2 The results of the disinfection effect test on the avian influenza virus (Avian influenza virus H9N2) at room temperature and sub-zero conditions of the compositions are shown in Table 22 below.

composition effective dilution factor note Room temperature condition (4℃) Sub-zero conditions (-5℃) Example 14 1,875 times 1,875 times No reduction in disinfection effect Comparative Example 1 1,250 times 750 times Decreased disinfection effect Comparative Example 2 625 times 500 times Decreased disinfection effect

<Disinfecting effect of the composition of Example 14, Comparative Example 1 and Comparative Example 2 at room temperature and sub-zero conditions>

[Experimental Example 4]

1. Experimental method

Example 14 In order to examine the disinfection effect of the composition on African swine fever virus (ASFV), the disinfection effect evaluation was requested to Wageningen Bioveterinary Research, an ASF virus disinfection efficacy testing institution certified by OIE (International Office of Waterworks).

go. Published Virus: Netherlands ’86 ASFV

me. Test Cells: Porcine Alveolar Macrophage (PAM) cells

all. Cell culture: RPMI 1640 with 5% FBS and 1% antibiotic (penicillin-streptomycin)

La. Virus and Disinfectant Diluent: Hard Water with 5% FBS Organics

※WHO hard water conditions (autoclave sterilization: 121℃, 15 minutes)

CaCl2 0.304g

MgCl2 0.139g

mind. Neutralizer: RPMI + 10% FBS + 1% antibiotics (penicillin, streptomycin) (stored at 37°C)

bar. preparation of virus

The virus titer used in the test should be greater than or equal ^{to 10 7} TCID _{50 /} ml (+/-0.2 log) to determine the extent of ^{a 4 10} log reduction. The virus is diluted in a ratio of 1 mL of virus solution + 19 mL of hard water containing 5% FBS.

buy. Prepare disinfectant dilutions

1) Disinfectant dilution factor: Prepare the disinfectant dilution solution in advance and add virus suspension to make the desired dilution factor. The desired dilution factor is 1:125, 1:250, 1:375 Dilution of the disinfectant is using sterile hard water containing 5% FBS. to carry out

2) On the day of the test, prepare a disinfectant solution diluted with sterile hard water containing 5% organic FBS and store at 4℃ for 30 minutes.

Oh. Preparation of Cells Required for Immunoperoxidase Monolayer Assay (IPMA Assay: Immunoperoxidase monolayer assay): After preparing cells by taking primary cells from pig lungs, store them in liquid nitrogen. If necessary, thaw the cells at 1x10 ⁶ /ml and aliquot them into a 96-well plate and incubate.

ruler. test procedure

1) Mix 2.5ml of virus solution and 2.5ml of disinfectant dilution of each dilution.

2) React the mixture prepared in this way at 4℃±1℃ for 30 minutes±10 seconds under standard test conditions, and mix every 10 minutes.

3) Immediately after the reaction, 1 ml of the mixed solution is taken to neutralize the mixed disinfectant, and then added to 1 ml of the neutralizing medium stored at 37°C.

4) Perform 10-fold serial dilution of the mixed solution in 6 steps from 10 ^{-2 to 10 -7} using a neutralizing medium at 37°C.

5) Use the diluted solution immediately or store it at -70℃.

car. Immunoenzyme tomography (IPMA assay, end point titration)

1) After thawing, inoculate 100 μl of each dilution into a 96 well plate (8 times).

2) Add 100 μl of PAM to this.

3) Incubate for 4 days in a 37 °C, 5% CO _{2 humidified incubator.}

4) After 4 days of culture, wash, dry and freeze the plate.

5) Fix the cells, wash again, and then stain with ASF-HIS, Mouse-anti-Swine IgG/HRPO conjugate and AEC (3-Amino-9-ethylcarbazole).

6) Read the plate under a microscope and calculate the titer by the Spearman Karber method.

card. control

1) In order to check whether the titer of the virus used is correctly used, add it to hard water and 5% FBS without disinfectant, mix well, and use it as a pathogen control.

2) Positive controls are included in the test as 1% and 2% NaOH (with hard water and 5% FBS). At least 4 log TCID50/ml should decrease after 30 minutes of reaction in 2% NaOH.

3) In order to confirm the cytotoxicity of the disinfectant, a disinfectant control group is included, and if rare cytotoxicity is observed in the disinfectant when evaluated by the immunoenzyme tomography (IPMA) test, retest.

get on. result judgment

^{As a result of the disinfectant efficacy test, when the virus titer is reduced by at least 4 10} logs by disinfectant treatment at 4 ° C for 30 minutes compared to the pathogen control group, it is judged to be effective, and the dilution factor for which the effect is confirmed is the effective dilution factor. .

2. Test results

Example 14 The disinfecting effect of the composition on African swine fever virus (ASFV) was determined as an effective disinfectant in a 250-fold dilution. The results of the disinfection effect test are shown in Table 23 below.

dilution factor 125 times 250 times 500 times organic matter 5% FCS 5% FCS 5% FCS temperature 4℃ 4℃ 4℃ contact time 30 minutes 30 minutes 30 minutes ASFV titer (log ₁₀ )

2.50

2.50

2.50 5.13 disinfection effect pass pass fail

<Example 14 Disinfecting Effect of Composition on African Swine Fever Virus (ASFV)>

As described above, the disinfectant complex composition for animal disease prevention containing thymol according to the present invention improves the problem of lowering the disinfection effect in sub-zero conditions, so that in the case of existing disinfectants, it should be diluted to different concentrations in spring and autumn and winter. By improving the inconvenience and diluting it to the same concentration throughout the year, in addition to the convenience of use, it is possible to relatively reduce the amount of disinfectant used during winter quarantine, thereby reducing not only the environmental impact but also the cost of the disinfectant. This is a very promising invention.

Claims (6)

A disinfectant composition for animal disease prevention containing thymol, characterized in that it is composed of all selected thymol, organic acid, inorganic acid, surfactant, cryoprotectant and purified water.
The method of claim 1,
The composite composition has a composition ratio of 1 to 5% by weight of thymol, 7.5 to 12.5% by weight of acetic acid, 15 to 25% by weight of citric acid, 7.5 to 12.5% by weight of phosphoric acid, 5 to 10% by weight of polyoxyethylene lauryl ether, polyoxy Ethylene octylphenyl ether 2-6% by weight, sodium lauryl sulfate solution 4-8% by weight, ammonium chloride 2.5-10% by weight, and purified water 11-55.5% by weight of thymol-containing disinfectant for animal disease prevention composite composition.
The method of claim 1,
The organic acid is acetic acid, citric acid, malic acid, a disinfectant composite composition for preventing animal diseases containing thymol containing thymol, characterized in that it comprises one or two or more selected from lactic acid.
The method of claim 1,
Inorganic acid is a disinfectant composition for animal disease prevention containing thymol, characterized in that it comprises one or two or more selected from phosphoric acid, sulfuric acid, and nitric acid.
The method of claim 1,
The surfactant is polyoxyethylene lauryl ether, polyoxyethylene octyl phenyl ether, and a disinfectant composite composition for animal disease prevention containing thymol, characterized in that it comprises one or two or more selected from sodium lauryl sulfate solution.
The method of claim 1,
The cryoprotectant is a disinfectant complex composition for animal disease prevention containing thymol, characterized in that it comprises one or two or more selected from ammonium chloride, calcium chloride, and sodium chloride.

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