Virucidal complex microbial composition against livestock infectious viruses

Abstract

The present invention relates to a Bacillus subtilis strain characterized by having a virucidal effect against livestock infectious disease viruses, a Nyalia circulans strain characterized by promoting the virucidal activity of the Bacillus subtilis strain, and a complex microbial preparation composition containing these.

Description

Technology Field

[0001] The present invention relates to a composite microbial composition characterized by having a virucidal effect against livestock infectious disease viruses. Background Technology

[0003] Currently, Korea designates and manages a total of 65 types of infectious livestock diseases under the Infectious Diseases Prevention Act for the purpose of preventing the occurrence or spread of infectious diseases in livestock, based on factors such as transmissibility, pathogenicity, domestic occurrence, and the extent of damage. The 65 types of infectious livestock diseases are classified into 15 types of Type 1 statutory infectious diseases, such as foot-and-mouth disease, highly pathogenic avian influenza, African swine fever, swine fever, and Newcastle disease; 32 types of Type 2 statutory infectious diseases, such as tuberculosis, brucellosis, bovine spongiform encephalopathy, porcine Aujeszky's disease, and rabies; and 18 types of Type 3 statutory infectious diseases, such as bovine leptospirosis, porcine reproductive and respiratory syndrome, and low pathogenic avian influenza.

[0004] Among these, for the 15 types of livestock infectious diseases classified as Class 1 statutory infectious diseases under the Livestock Infectious Disease Prevention Act, the mayor or county head may order the culling of livestock in accordance with the Enforcement Rules of the Livestock Infectious Disease Prevention Act; in particular, regarding six types including rinderpest, bovine pulmonary disease, foot-and-mouth disease, swine cholera, African swine fever, and highly pathogenic avian influenza, culling may be carried out without delay. According to data submitted by the Ministry of Agriculture, Food and Rural Affairs to the National Assembly Agriculture, Food, Rural Affairs and Fisheries Committee, it was found that from 2019 to August 2024, 10,285 cattle, 556,000 pigs, and 47,517,000 chickens were culled and buried due to African swine fever (ASF), foot-and-mouth disease, highly pathogenic avian influenza (AI), and lumpy skin disease.

[0005] Of the 15 types of Class 1 statutory infectious diseases, 14 are viral diseases, and all livestock culled over the past five years were either infected with viral diseases or at risk of infection. Most culled livestock are disposed of through burial, and it is estimated that there are over 4,000 burial sites for culled animals nationwide. The burial of culled animals causes environmental health problems, such as groundwater and soil contamination, due to the leakage of leachate containing pathogens from the burial sites. Furthermore, as costs for executing culling, securing burial sites, and managing them increase, a situation repeatedly occurs where sufficient budget is not allocated to establishing a crucial preventive disease control system. Additionally, while buried animal carcasses can be excavated for incineration or re-burial after three years have passed and requirements are met, most are incinerated, leading to the problem of causing secondary environmental pollution.

[0006] In other words, conventional disposal methods (incineration and burial) for livestock culled due to infectious diseases not only cause environmental problems such as soil, groundwater, and air pollution, but also present issues such as additional cost expenditures associated with burial and post-management. Prior art literature

[0007] Harirchi S. et al., “Bacillales: From taxonomy to biotechnological and industrial perspectives”. Microorganisms, 2022, 10, 2355. The problem to be solved

[0008] The present invention aims to provide a Bacillus subtilis strain characterized by having a virucidal effect against livestock infectious disease viruses, and a Nyalia circulans strain characterized by promoting the virucidal activity of the said Bacillus subtilis strain.

[0009] In addition, the present invention aims to provide a complex microbial composition comprising the above-mentioned Bacillus subtilis strain or culture medium thereof and the above-mentioned Nyalia circulans strain or culture medium thereof, and a method for preparing the same.

[0010] However, the problems that this invention seeks to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below. means of solving the problem

[0012] The present invention relates to Bacillus subtilis (which exhibits a virucidal effect) Bacillus subtilis Provides the YBK-70 strain (accession number KCTC 15571BP) or a culture medium thereof.

[0013] As one embodiment of the present invention, the YBK-70 strain may include 16S rRNA consisting of the nucleotide sequence of SEQ ID NO. 1 and 23S rRNA consisting of the nucleotide sequence of SEQ ID NO. 2.

[0014] In addition, the present invention relates to Nyalia circulans exhibiting a virucidal effect ( Niallia circulans Provides the YBK-04 strain (accession number KCTC 15427BP) or a culture medium thereof.

[0015] As one embodiment of the present invention, the YBK-04 strain may include 16S rRNA consisting of the nucleotide sequence of sequence number 3.

[0016] In addition, the present invention relates to Bacillus subtilis ( Bacillus subtilis ) YBK-70 (KCTC 15571BP) strain or culture thereof and / or Nyalia circulans ( Niallia circulans ) Provides a microbial preparation composition comprising the YBK-004 (KCTC 15427BP) strain or a culture solution thereof.

[0017] In the present invention, the microbial preparation composition exhibits a virucidal effect against porcine epidemic diarrhea virus (PEDV), avian influenza virus (AIV), and porcine reproductive and respiratory syndrome virus (PRRSV), and thus the microbial preparation composition of the present invention can be used for virucidal purposes.

[0018] Accordingly, the present invention provides a virucidal composition comprising the above-mentioned YBK-70 strain or its culture medium and / or the above-mentioned YBK-04 strain or its culture medium as an active ingredient.

[0019] As one embodiment of the present invention, the composite microbial preparation composition or the antiviral composition may further comprise an Actinomyces spp. strain or a culture solution thereof.

[0020] In one embodiment of the present invention, the virus may be porcine epidemic diarrhea virus (PEDV), avian influenza virus (AIV), and / or porcine reproductive and respiratory syndrome virus (PRRSV).

[0021] As another embodiment of the present invention, the microbial preparation composition or the antiviral composition may contain 100 to 500 parts by weight of the YBK-70 strain based on 100 parts by weight of the YBK-04 strain or its culture medium, preferably 200 to 400 parts by weight, and more preferably 300 parts by weight.

[0022] As another embodiment of the present invention, the microbial preparation composition or the antiviral composition may contain 50 to 500 parts by weight of an actinomycete strain based on 100 parts by weight of the YBK-04 strain or its culture medium, preferably 50 to 300 parts by weight, and more preferably 100 to 200 parts by weight.

[0023] In addition, the present invention provides a method for preparing a complex microbial formulation composition comprising the following steps:

[0024] (1) A strain culture preparation step comprising the steps 1) and 2) below; and

[0025] 1) Bacillus subtilis( Bacillus subtilis ) A step of culturing the YBK-70 strain in a nutrient medium containing glucose to obtain the culture solution; and

[0026] 1) Nyalia circulans( Niallia circulans A step of culturing the YBK-004 strain in a nutrient medium containing glucose to obtain the culture solution;

[0027] (2) A step of preparing a complex microbial culture solution by mixing the above YBK-70 strain culture solution and the YBK-04 strain culture solution.

[0028] As one embodiment of the present invention, the method comprises, in step (1) 3) actinomycetes ( Actinomyces The method may further include the step of culturing spp.) in a nutrient medium containing glucose to obtain the culture solution, and the step of further mixing the actinomycete culture solution with the YBK-70 strain culture solution and the YBK-04 strain culture solution in step (2) to prepare a complex microbial culture solution.

[0029] As another embodiment of the present invention, in step (1), the culture temperature of each strain may be 30°C to 50°C, and the culture time may be 12 hours to 36 hours.

[0030] As another embodiment of the present invention, in step (2), the mixing may be performed by mixing the YBK-70 strain culture and the YBK-04 strain culture in a ratio of 3:1 (v / v).

[0031] As another embodiment of the present invention, in step (2), the mixing may be performed by mixing the YBK-70 strain culture, the YBK-04 strain culture, and the actinomycete culture in a ratio of 3:1:0.5 to 2 (v / v).

[0032] As another embodiment of the present invention, the total number of microorganisms in the complex microbial preparation composition is 10 6 It may be greater than colony-forming units (CFU) / mL. Effects of the invention

[0034] The invention enables the expression of superior virucidal activity compared to individual strains by mixing novel microbial strains with confirmed virucidal activity in optimal proportions, and in particular, the co-administration of actinomycetes contributes to maximizing the effect. Furthermore, the microbial composition of the present invention in vitro yes in vivo As the efficacy of antiviral activity has been proven even in the environment, the composition of the present invention can be utilized as an environmentally friendly and practical alternative for livestock virus prevention. Brief explanation of the drawing

[0036] FIG. 1 is an image of a Vero cell used to culture Porcine Epidemic Diarrhea Virus (PEDV) to confirm the viricidal effect of a complex microbial dilution prepared according to embodiments of the present invention on Porcine Epidemic Diarrhea Virus (PEDV), in order: an image of a negative control Vero cell with no treatment (Fig. 1a), an image of a pathogen control Vero cell treated only with PEDV solution (Fig. 1b), and an image of a Vero cell treated with a reaction solution in which PEDV solution and the complex microbial dilution were reacted (Fig. 1c). FIG. 2 is an experimental image confirming the virucidal effect of a complex microbial diluent prepared according to embodiments of the present invention against low pathogenic avian influenza virus (AIV), and includes an image of a pathogen control group (Fig. 2a) showing the results of a chicken hemagglutination reaction in the urinary fluid of a developing egg treated only with AIV solution, and an image of a treatment group (Fig. 2b) showing the results of a chicken hemagglutination reaction in the urinary fluid of a developing egg treated with a reaction solution in which AIV solution and a complex microbial diluent were reacted. Figure 3 is an image of MARC-145 cells used to culture Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) to confirm the viricidal effect of a complex microbial diluent prepared according to embodiments of the present invention, in order: an image of a negative control MARC-145 cell with nothing treated (Fig. 3a), an image of a pathogen control MARC-145 cell treated only with PRRSV solution (Fig. 3b), and an image of a MARC-145 cell treated with a reaction solution in which PRRSV solution and the complex microbial diluent were reacted (Fig. 3c). FIG. 4 is a PCR (Polymerase chain reaction) band image confirming the viricidal effect against PEDV by treating the small intestines of three PEDV-infected pigs with a carrier containing the composite microbial composition prepared according to embodiments of the present invention (Lane 1, 2, 3: bands for the small intestine of PEDV-infected pigs (positive), Lane 4, 5, 6: bands after treating the small intestine of PEDV-infected pigs with the composite microbial composition carrier (negative)). FIG. 5 is a PCR band image confirming the virucidal effect against AIV by treating the trachea and lungs of three AIV-infected chickens with a carrier containing the composite microbial composition prepared according to embodiments of the present invention (Lane 1, 2, 3: bands for the trachea and lungs of AIV-infected chickens (positive), Lane 4, 5, 6: bands after treating the trachea and lungs of AIV-infected chickens with the composite microbial composition carrier (negative)). FIG. 6 is a PCR band image confirming the antiviral effect against PRRSV by treating the lungs of three pigs infected with PRRSV with a carrier containing a composite microbial composition prepared according to embodiments of the present invention. Specific details for implementing the invention

[0037] Hereinafter, embodiments and examples of the present invention are described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments and examples described herein. Furthermore, in order to clearly explain the present invention in the drawings, parts unrelated to the explanation have been omitted, and similar parts throughout the specification have been given similar reference numerals.

[0038] Throughout this specification, when a part is described as being "connected" to another part, this includes not only cases where they are "directly connected," but also cases where they are "electrically connected" with other elements interposed between them.

[0039] Throughout this specification, when a component is described as being located "on" another component, this includes not only cases where a component is in contact with another component, but also cases where another component exists between the two components.

[0040] Throughout this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0041] Terms of degree used in this specification, such as “about,” “substantially,” etc., are used to mean at or near the stated value when inherent manufacturing and material tolerances are presented in the stated meaning, and are used to prevent unscrupulous infringers from unfairly exploiting the disclosure in which precise or absolute values ​​are mentioned to aid in understanding the invention.

[0042] The terms “step of” or “step of” as used throughout this specification do not mean “step for”.

[0043] Throughout this specification, the term “combination(s) of these” included in the Markush-type expression means one or more mixtures or combinations selected from the group consisting of the components described in the Markush-type expression, and means including one or more selected from the group consisting of said components.

[0044] Throughout this specification, the description of "A and / or B" means "A or B, or A and B".

[0046] Embodiments of the present invention have been described in detail below, but the present invention may not be limited thereto.

[0048] The first aspect of this invention is Bacillus subtilis deposited under accession number KCTC 15571BP ( Bacillus subtilis ) Provides the YBK-70 strain.

[0049] Bacillus subtilis Bacillus subtilisThe YBK-70 strain was deposited with the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Biotechnology and Bioengineering on August 29, 2023, and was assigned accession number KCTC 15571BP.

[0050] Bacillus subtilis Bacillus subtilis , B. subtilis Bacillus subtilis has long been used as a probiotic for treating intestinal diseases in humans and animals, and has been studied for use as an animal feed fortifier and antifungal in recent decades, but its viricidal effect against livestock infectious disease viruses has not yet been experimentally known.

[0051] In one embodiment of the present invention, the YBK-70 strain may have a virucidal effect against livestock infectious disease viruses, but may not be limited thereto.

[0052] A second aspect of this invention is Nyalia circulans deposited under accession number KCTC 15427BP ( Niallia circulans ) Provides the YBK-04 strain.

[0053] Nyalia Circulans Niallia circulans The YBK-04 strain was deposited with the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Biotechnology and Bioengineering on August 27, 2023, and was assigned accession number KCTC 15427BP.

[0054] Nyalia Circulans Niallia circulans , N. circulans ) is a microorganism commonly found in soil that is used in the industrial production of proteinases.

[0055] In one embodiment of the present invention, the YBK-04 strain may promote the antiviral activity of the YBK-70 strain, but is not limited thereto.

[0056] A third aspect of the present invention provides a complex microbial composition comprising a Bacillus subtilis YBK-70 (KCTC 15571BP) strain or a culture solution thereof and a Nyalia circulans YBK-004 (KCTC 15427BP) strain or a culture solution thereof, which has a virucidal effect against livestock infectious disease viruses.

[0057] Detailed descriptions of parts that overlap with the first and second aspects of the present invention have been omitted, but the descriptions of the first and second aspects of the present invention may be applied in the same way even if such descriptions are omitted in the third aspect of the present invention.

[0058] Nyalia circulans according to the present invention does not have direct antiviral activity, but Bacillus ( Bacillus) When present with microorganisms belonging to the genus, the production of secondary metabolites such as enzymes is increased through mutual synergy, thereby increasing the physiological activity of microorganisms belonging to the genus Bacillus. Accordingly, the complex microbial composition of the present invention can promote the antiviral activity of the Bacillus subtilis strain by composing Nyalia circulans together with the Bacillus subtilis strain.

[0059] The inventors evaluated the virucidal activity against PEDV and AIV using the YBK-70 strain alone through specific experiments and confirmed a virus reduction effect of approximately 4.0 to 4.2 log. This suggests that a single strain alone can exhibit significant virucidal activity (Example 1). Meanwhile, treatment with the YBK-04 strain alone showed a killing effect against PEDV and AIV, but exhibited a relatively low level of virucidal activity (Example 2).

[0060] In addition, the inventors confirmed that a synergistic effect on antiviral activity occurs when the above YBK-70 strain and YBK-04 strain are mixed and treated in a 3:1 ratio (Example 3).

[0061] Furthermore, the inventors confirmed that virucidal activity is maximized when YBK-70, YBK-04, and actinomycetes are mixed and treated in a ratio of 3:1:0.5~2, and this in vivo It was verified through experiments (Examples 4 and 5).

[0062] Accordingly, in one embodiment of the present invention, the composite microbial preparation composition is an actinomycete ( Actinomyces It may additionally include spp.) strains or cultures thereof, but is not limited thereto.

[0063] Actinomycetes are microorganisms that are predominantly distributed in natural environments such as soil, and they possess the ability to decompose various organic substances, including cellulose and lignin. More than 75% of known antibiotics have been isolated from the secondary metabolites of actinomycetes, and about 64% of various physiologically active substances, such as anticancer agents, immunomodulators, and useful enzymes, have been derived from actinomycetes. In addition, experimental results have been reported in which the proliferation of Influenza A virus pdm09 (H1N1) was inhibited when the antiviral effect against Influenza A virus pdm09 (H1N1) was confirmed using culture metabolites of actinomycetes isolated from the ocean.

[0064] In one embodiment of the present invention, the livestock infectious virus may include, but is not limited to, one or more selected from the group consisting of porcine epidemic diarrhea virus (PEDV), avian influenza virus (AIV), and porcine reproductive and respiratory syndrome virus (PRRSV).

[0065] The fourth aspect of the present invention is Bacillus subtilis ( Bacillus subtilis) YBK-70 strain and Nyalia circulans( Niallia circulans A method for preparing a composite microbial composition is provided, comprising: culturing a YBK-004 strain to obtain a Bacillus subtilis culture solution and a Nyalia circulans culture solution; and mixing the Bacillus subtilis culture solution and the Nyalia circulans culture solution to obtain a composite microbial composition.

[0066] In one embodiment of the present invention, the method for preparing the composite microbial composition is an actinomycete ( Actinomyces The method may include, but is not limited to, culturing spp.) to obtain an actinomycete culture solution; and mixing the actinomycete culture solution into the complex microbial composition.

[0067] In one embodiment of the present invention, the culturing step may be performed at about 30°C to about 50°C for about 12 hours to about 36 hours, but is not limited thereto. Most preferably, it is performed at about 40°C for about 24 hours.

[0068] In one embodiment of the present invention, the total number of microorganisms in the composite microbial composition is 10 6 It may be greater than or equal to colony forming units (CFU) / mL, but is not limited thereto. If the amount of microorganisms is less than the total number of microorganisms mentioned above, it may not exhibit a virucidal effect against infectious livestock viruses.

[0069] Detailed descriptions of parts that overlap with the first to third aspects of the present invention have been omitted, but the descriptions of the first to third aspects of the present invention may be applied in the same way even if such descriptions are omitted in the fourth aspect of the present invention.

[0070] The microbial preparation composition of the present invention can be used as a disinfectant, etc., for controlling livestock infectious diseases caused by viruses and preventing the outbreak of livestock infectious diseases caused by viruses, and the microbial preparation can be appropriately formulated to suit its intended use, and specifically can be manufactured in the form of a dry powder or a liquid.

[0071] The present invention will be explained in more detail below using examples, but the following examples are merely illustrative to aid in understanding the present invention, and the content of the present invention is not limited to the following examples.

[0073] [Experimental Methods and Materials]

[0074] 1. Identification of Bacillus subtilis YBK-70 strain and Niallia circulans YBK-04 strain

[0075] Bacillus subtilis Bacillus subtilis The 16S rRNA gene sequences and 23S rRNA gene sequences of the YBK-70 strain from NCBI (National Center for Biotechnology Information) Blast Bacillus subtilis As a result of comparing the gene base sequences and homology of, the most closely related Bacillus subtilis The 16S rRNA gene sequence and 23S rRNA gene sequence matched 99.67% with the strain. Difference from closely related strains of the same species was recognized. B. subtilis It was named strain YBK-70 and deposited with the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Bioscience and Biotechnology on August 29, 2023, and was assigned accession number KCTC 15571BP.

[0076] Nyalia Circulans Niallia circulans ) The 16S rRNA gene sequence of the YBK-04 strain from NCBI Blast Niallia circulans As a result of comparing the gene base sequences and homology of, the most closely related Niallia circulansThe 16S rRNA sequence matched 98.7% with that of the strain. Difference from closely related strains of the same species was recognized. N. circulans It was named YBK-04 and deposited at the National Institute of Bioscience and Biotechnology's Center for Biological Resources on August 27, 2023, and was assigned accession number KCTC 15427BP.

[0077] B. subtilis strain YBK-70 and N. circulans The 16S rRNA and / or 23S rRNA sequences of the YBK-04 strain are shown in Table 1 below.

[0078] division Sequence number Nucleotide sequence (5' → 3') B. subtilis YBK-70_16S rRNA 1 CGCAGGGCGTACCAGCCTATACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTAGGAGCCAGCCGTCGAAAGAGAAAACGTAG B. subtilis YBK-70_23s rRNA 2 TAGGTTGGGTTGGTATCAATATCTGATTCATAGGATATGAGAAGGCAGACCCGGGGAACTGAAACATCTAAGTACCCGGAGGAAGAGAAAGCAAATGCGATTCCCTGAGTAGCGGCGAGCGAAACGGGATTAGCCCAAACCAAGAGGCTTGCCTCTTGGGGTTGTAGGACACTCTGTACGGAGTTACAAAGGAACGAGGTAGATGAAGAGGTCTGGAAAGGCCCGCCATAGGAGGTAACAGCCCTGTAGTCAAAACTTCGTTCTCTCCTGAGTGGATCCTGAGTACGGCGGAACACGTGAAATTCCGTCGGAATCCGGGAGGACCATCTCCCAAGGCTAAATACTCCCTAGTGACCGATAGTGAACCAGTACCGTGAGGGAAAGGTGAAAAGCACCCCGGAAGGGGAGTGAAAGAGATCCTGAAACCGTGTGCCTACAAGTAGTCAGAGCCCGTTAACGGGTGATGGCGTGCCTTTTGTAGAATGAACCGGCGAGTTACGATCCCGTGCAAGGTTAAGCAGAAGATGCGGAGCCGCAGCGAAAGCGAGTCTGAATAGGGCGCATGAGTACGTGGTCGTAGACCCGAAACCAGGTGATCTACCCATGTCCAGGGTGAAGTTCAGGTAACACTGAATGGAGGCCCGAACCCACGCACGTTGAAAAGTGCGGGGATGAGGTGTGGGTAGGGGTGAAATGCCAATCGAACCTGGAGATAGCTGGTTCTCTCCGAAATAGCTTTAGGGCTAGCCTCAAGGTAAGAGTCTTGGAGGTAGAGCACTGATTGGACTAGGGGCCCCTACCGGGTTACCGAATTCAGTCAAACTCCGAATGCCAATGACTTATCCTTGGGAGTCAGACTGCGAGTGATAAGATCCGTAGTCGAAAGGGAAACAGCCCAGACCGCCAGCTAAGGTCCCAAAGTATACGTTAAGTGGAAAAGGATGTGGAGTTGCTTAGACAACCAGGATGTTGGCTTAGAAGCAGCCACCATTTAAAGAGTGCGTAATAGCTCACTGGTCGAGTGACTCTGCGCCGAAAATGTACCGGGGCTAAACGTATCACCGAAGCTGCGGACTGTTCTTCGAACAGTGGTAGGAGAGCGTTCTAAGGGCTGTGAAGCCAGACCGGAAGGACTGGTGGAGCGCTTAGAAGTGAGAATGCCGGTATGAGTAGCGAAAGAGGGGTGAGAATCCCCTCCACCGAATGCCTAAGGTTTCCTGAGGAAGGCTCGTCCGCTCAGGGTTAGTCGGGACCTAAGCCGAGGCCGAAAGGCGTAGGCGATGGACAACAGGTTGATATTCCTGTACCACCTCCTCACCATTTGAGCAATGGGGGGACGCAGGAGGATAGGGTAAGCGCGGTATTGGATATCCGCGTCCAAGCAGTTAGGCTGGGAAATAGGCAAATCCGTTTCCCATAAGGCTGAGCTGTGATGGCGAGCGAAATATAGTAGCGAAGTTCCTGATTCCACACTGCCAAGAAAAGCCTCTAGCGAGGTGAGAGGTGCCCGTACCGCAAACCGACACAGGTAGGCGAGGAGAGAATCCTAAGGTGATCGAGAGAACTCTCGTTAAGGAACTCGGCAAAATGACCCCGTAACTTCGGGAGAAGGGGTGCTCTGTTAGGGTGCAAGCCCGAGAGAGCCGCAGTGAATAGGCCCAGGCGACTGTTTAGCAAAAACACAGGTCTCTGCGAAGCCGTAAGGCGAAGTATAGGGGCTGACGCCTGCCCGGTGCTGGAAGGTTAAGAGGAGCGCTTAGCGTAAGCGAAGGTGCGAATTGAAGCCCCAGTAAACGGCGGCCGTAACTATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCCGCACGAAAGGCGCAACGATCTGGGCACTGTCTCAACGAGAGACTCGGTGAAATTATAGTACCTGTGAAGATGCAGGTTACCCGCGACAGGACGGAAAGACCCCGTGGAGCTTTACTGCAGCCTGATATTGAATGTTGGTACAGCTTGTACAGGATAGGTAGGAGCCTTGGAAACCGGAGCGCCAGCTTCGGTGGAGGCATCGGTGGGATACTACCCTGGCTGTATTGACCTTCTAACCCGCCGCCCTTATCGGGCGGGGAGACAGTGTCAGGTGGGCAGTTTGACTGGGGCGGTCGCCTCCTAAAAGGTAACGGAGGCGCCCAAAGGTTCCCTCAGAATGGTTGGAAATCATTCGCAGAGTGTAAAGGCACAAGGGAGCTTGACTGCGAGACCTACAAGTCGAGCAGGGACGAAAGTCGGGCTTAGTGATCCGGTGGTTCCGCATGGAAGGGCCATCGCTCAACGGATAAAAGCTACCCCGGGGATAACAGGCTTATCTCCCCCAAGAGTCCACATCGACGGGGAGGTTTGGCACCTCGATGTCGGCTCATCGCATCCTGGGGCTGTAGTCGGTCCCAAGGGTTGGGCTGTTCGCCCATTAAAGCGGTACGCGAGCTGGGTTCAGAACGTCGTGAGACAGTTCGGTCCCTATCCGTCGCGGGCGCAGGAAATTTGAGAGGAGCTGTCCTTAGTACGAGAGGACCGGGATGGACGCACCGCTGGTGTACCAGTTGTTCTGCCAAGGGCATCGCTGGGTAGCTATTTCGAACCAGGGCCG N.circulans YBK-04_16s rRNA 3 TGGGCAACCTGCCTGTAAGACTGGCCTAACTTCGGGAAACCGGAGCTAATACCGGATAATCCTTTTCCTCTCATGAGGAAAAGCTGAAAGACGGTTTACGCTGTCACTTACAGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGCGAAGAACAAGTACAAGAGTAACTGCTTGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAAACGTAGGTGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCCCTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGGACTTGAGTGCAGAAGAGAAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTTTGGTCTGTAACTGACGCTGAGGCGAGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGATACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGACCTTACCAGGTCTTGACATCCTCTGACACTCCTAGAGATAGGACGTTCCCTTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAATCCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACTTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCAGCAAAACCGCGAGGTCGAGCAAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTTCCGGGCCTTGTACACACCGCC

[0080] 2. Preparation of Test Substances

[0081] 2-1. Preparation of Strain Culture Solution

[0082] Bacillus subtilis Bacillus subtilis ) YBK-70 (KCTC 15571BP) strain, Nyalia circulans( Niallia circulans ) YBK-004 (KCTC 15427BP) strain, actinomycete( Actinomyces Each strain (spp.) was inoculated into a glucose-containing nutrient medium. Each strain was cultured at 40°C for 24 hours to prepare a culture solution. To distinguish it from the complex microbial culture solution described below, a culture solution containing a single strain is referred to as a single microbial culture solution. The total number of bacteria in the single microbial culture solution is 10 6 It was prepared to have a colony-forming unit (CFU) / mL or higher.

[0084] 2-2. Preparation of Complex Microbial Culture Solution

[0085] A complex microbial culture solution was prepared by mixing two or more selected from the group consisting of YBK-70 strain culture solution, YBK-04 strain culture solution, and actinomycete culture solution at room temperature.

[0086] The complex microbial culture solution was prepared with the following combination.

[0087] - Mix YBK-70 strain culture and YBK-04 strain culture in a 3:1 (v / v) ratio

[0088] - Mix YBK-70 strain culture solution, YBK-04 strain culture solution, and actinomycete culture solution in a ratio of 3:1:0.5

[0089] - Mix YBK-70 strain culture, YBK-04 strain culture, and actinomycete culture in a ratio of 3:1:1

[0090] - Mix YBK-70 strain culture, YBK-04 strain culture, and actinomycete culture in a ratio of 3:1:2

[0091] The total number of bacteria is 10 6 A complex microbial culture solution was prepared with a colony-forming unit (CFU) / mL or higher.

[0093] 2-3. Preparation of microbial dilution solution Hard water was prepared by adding 0.305 g / L of CaCl2 and 0.139 g / L of MgCl2·6H2O to distilled water. An organic dilution solution was prepared by additionally adding 5 parts by weight of fetal bovine serum to 100 parts by weight of the hard water.

[0094] A microbial dilution solution was prepared by mixing the above organic dilution solution with a single microbial culture solution or a complex microbial culture solution in a 1:1 weight ratio and used in the experiment.

[0096] 3. Preparation of the virus

[0097] 3-1. Porcine Epidemic Diarrhea Virus (PEDV)

[0098] Porcine epidemic diarrhea virus (PEDV, strain 11.29) obtained from the Animal and Plant Quarantine Agency was used in the experiment. PEDV was diluted with maintenance medium (DEME + 10% tryptose phosphate broth + 2.5 μg / mL trypsin), and 2 mL of the diluted solution was inoculated into Vero cell lines and incubated at 37°C at 5% CO2 for 1.5 hours. After incubation, the culture medium was removed, 20 mL of maintenance medium was added, and re-incubation was performed. During incubation, when the cytopathic effect (CPE, a phenomenon in which living viruses proliferate and denature cells) reached approximately 80-90%, the monolayer cells attached to the plate were collected. The collected cell suspension was centrifuged at 750×g for 15 minutes to remove cell debris, and the supernatant was collected to test for the content of the test virus, and 10 7.0 TCID 50 It was disclosed in the test that it was greater than / mL.

[0100] 3-2. Low pathogenic avian influenza virus (AIV)

[0101] Low pathogenic avian influenza virus (AIV) (MS96 / 96(H9N2) strain) obtained from the Animal and Plant Quarantine Agency was used in the experiment. AIV was inoculated into the anortic cavity of 10-day-old developing eggs, and at the point where the proliferation of viable viruses was maximum during subculture (virus titer 10 7.0 EID 50 AIV virus solution collected from (more than / mL) was used in the test.

[0103] 3-3. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

[0104] Porcine reproductive and respiratory syndrome virus (PRRSV) (LMY strain), obtained from the Animal and Plant Quarantine Agency, was used in the experiment. PRRSV was diluted with maintenance medium (MEM + 3% fetal calf serum + 1.78 mM sodium bicarbonate + antibiotics (penicillin 100 U / mL + streptomycin 100 mg / mL)), and 2 mL of the diluted PRRSV solution was inoculated into MARC-145 cell lines and cultured for 3 hours at 37°C and 5% CO2. After culture, the culture medium was removed, 20 mL of maintenance medium was added, and the cells were re-cultured. During culture, when the cytopathic effect (CPE, a phenomenon in which live viruses proliferate and denature cells) reached approximately 80-90%, monolayer cells attached to the plate were collected. After centrifuging the collected cell suspension at 750×g for 15 minutes to remove cell debris, the supernatant was taken and the content of the test virus was tested, and 10 7.0 TCID 50 It was disclosed in the test that it was greater than / mL.

[0106] 4. Experiment to confirm antiviral effect

[0107] In accordance with the Animal and Plant Quarantine Agency Notice No. 2023-48, 'Guidelines for Disinfectant Efficacy Testing,' the virus in the treatment group was 10 per ml compared to the pathogen control 4 TCID 50 (or, EID 50 It was determined that there was a virucidal effect when elimination or inactivation of ) (common logarithm 4) or more was confirmed.

[0108] TCID 50 was calculated using Equation 1 below, and EID 50 It was calculated using Equation 2 below.

[0109] [Equation 1]

[0110]

[0111] [Equation 2]

[0112]

[0113] In the above Equations 1 and 2, L1 is the log interval of the lowest dilution among the experimental groups, L is the log value of each dilution, and S is the sum (%) of the mortality rates of each dilution.

[0114] All experimental results were presented as average values ​​after three repeated experiments.

[0116] 4-1. Experiment to Confirm Viricidal Effect Against PEDV

[0117] PEDV liquid (10 7.0 TCID 50 2.5 mL of the PEDV mixture, prepared by mixing 1.0 mL of the organic diluent with 19.0 mL of the organic diluent, was placed into a test tube containing an equal amount of the test substance and mixed, then reacted for 30 minutes at 40°C while shaking at 10-minute intervals.

[0118] At this time, a pathogen control sample containing only PEDV solution was prepared by performing the same process except for mixing with the test substance.

[0119] After the reaction was completed for 30 minutes, in order to neutralize the efficacy of the test substance, an equal amount of neutralizing solution at 37°C (cell culture medium containing 10% non-activated fetal bovine serum, 5 mL) was immediately added and mixed to neutralize and prepare a neutralized reaction solution.

[0120] Subsequently, the neutralized reaction solution was diluted tenfold (10⁻¹ to 10⁻⁶) using Vero cell culture medium, and 50 μL of the neutralized reaction solution was inoculated into 5 wells per dilution factor on a 96-well tissue culture plate in which a monolayer of Vero cells had been formed. After inoculation, the plates were cultured for 5 days at 37°C and 5% CO₂, and the presence of the virus was finally determined by observing the formation of CPE under a microscope daily. Wells showing CPE in the 96-well microplate were considered positive, and their number was counted. The virus content was calculated using the Kaerber method based on the number of positive wells.

[0122] 4-2. Experiment to Confirm Viricidal Effect Against Low Pathogenic AIV

[0123] After preparing an AIV mixture by mixing 1.0 mL of AIV virus solution with 19.0 mL of organic diluent, 2.5 mL of the mixture was added to a test tube containing an equal amount of test substance at 1-minute intervals and mixed, and then reacted for 30 minutes at 40°C while shaking at 10-minute intervals.

[0124] At this time, the same process was performed for all subjects except for mixing with the test substance, and a pathogen control containing only AIV was included in the treatment.

[0125] After the reaction was completed for 30 minutes, in order to neutralize the efficacy of the test substance, an equal amount of neutralizing solution at 37°C (cell culture medium containing 10% non-activated fetal bovine serum, 5 mL) was immediately added and mixed to neutralize and prepare a neutralized reaction solution.

[0126] Subsequently, 100 μL of each diluted solution of the above-mentioned neutralized reaction solution was inoculated into the anaerobic cavity of 5 developing eggs (10 days old) per dilution factor. After inoculation, the eggs were incubated at 37°C for 5 days, and egg inspection was performed daily. Developing eggs that died within 24 hours of inoculation were considered to have died accidentally and were excluded from the test results. After 5 days of incubation, fluid from the anaerobic cavity of the developing eggs was collected, and the presence or absence of virus was determined by the plate hemagglutination reaction using an equal amount of chicken blood cells (1%). Here, the virus content was calculated using the Kaerber method.

[0128] 4-3. Experiment to Confirm Viralic Effect Against PRRSV

[0129] PPPSV solution (10 7.0 TCID 50 2.5 mL of the PRRSV mixture, prepared by mixing 1.0 mL of the mixture with 19.0 mL of the organic diluent, was placed into a test tube containing an equal amount of the complex microbial diluent of Example 4 and mixed, and then reacted for 30 minutes at 40°C while shaking at 10-minute intervals.

[0130] At this time, a pathogen control sample containing only PRRSV was prepared by performing the same process except for mixing with the complex microbial dilution.

[0131] After the reaction was completed for 30 minutes, in order to neutralize the efficacy of the complex microbial dilution, an equal amount of neutralizing solution (5 mL of cell culture medium containing 10% non-activated fetal bovine serum) at 37°C was immediately added and mixed to neutralize the reaction solution, thereby preparing a neutralized reaction solution.

[0132] Subsequently, the above neutralized reaction solution is diluted in tenfold increments using MARC-145 cell culture medium (10 -1 ~10 -650 μL of the neutralized reaction solution was inoculated into 5 wells per dilution factor into a 96-well tissue culture plate in which a monolayer of MARC-145 cells had been formed. After inoculation, the plates were cultured at 37°C and 5% CO2 for 5 days, and the presence of the virus was finally determined by observing the formation of CPE under a microscope daily. Wells showing CPE in the 96-well microplate were considered positive, and their number was counted. The virus content was calculated using the Kaerber method based on the number of positive wells.

[0134] [Example]

[0135] Example 1. Bacillus subtilis Antiviral effects of YBK-70

[0136] 1-1. Viral action against PEDV

[0137] Results of 3 repeated tests, Bacillus subtilis The PEDV titer of the YBK-70 treatment group is the control group (10 6.5 TCID 50 It showed a decrease of 4.0–4.2 log compared to / mL, and the median was 4.0 log.

[0138] repeat Treatment area Dilution factor Neutralizing solution dilution factor (number of positive cases / total number of inoculated cases) TCID 50 Log reduction median 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 Treatment area 1 / 2 4 / 5 3 / 5 2 / 5 1 / 5 0 / 5 0 / 5 2.5 4.0 4.0 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 2 Treatment area 1 / 2 4 / 5 3 / 5 2 / 5 0 / 5 0 / 5 0 / 5 2.3 4.2 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 3 Treatment area 1 / 2 4 / 5 3 / 5 2 / 5 1 / 5 0 / 5 0 / 5 2.5 4.0 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5

[0139] * Treatment group: Treated with microbial dilution and virus solution, Pathogen control: Treated with virus solution only. ** Log reduction: Pathogen-controlled TCID 50 TCID in processing section 50 limit value

[0141] 1-2. Viricidal effect against low pathogenic AIV

[0142] Results of 3 repeated tests, Bacillus subtilis The AIV titer of the YBK-70 treatment group is the control group (10 6.5 EID 50It showed a decrease of 4.0–4.2 log compared to / mL, and the median was 4.2 log.

[0143] repeat Treatment area Dilution factor Neutralizing solution dilution factor (number of positive cases / total number of inoculated cases) EID 50 Log reduction median 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 Treatment area 1 / 2 4 / 5 3 / 5 2 / 5 0 / 5 0 / 5 0 / 5 2.3 4.2 4.2 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 2 Treatment area 1 / 2 4 / 5 3 / 5 2 / 5 0 / 5 0 / 5 0 / 5 2.3 4.2 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 3 Treatment area 1 / 2 4 / 5 3 / 5 2 / 5 1 / 5 0 / 5 0 / 5 2.5 4.0 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5

[0144] * Treatment group: Treated with microbial dilution and virus solution, Pathogen control: Treated with virus solution only. ** Log reduction: Pathogen control EID 50 EID in treatment zone 50 limit value

[0146] Example 2. Niallis curculans Antiviral effects of YBK-04

[0147] 2-1. Viral action against PEDV

[0148] Results of 3 repeated tests, Niallia circulans The PEDV titer of the YBK-04 treatment group is the control group (10 6.5 TCID 50 It showed a decrease of 3.4 to 3.6 log compared to / mL, and the median was 3.6 log.

[0149] repeat Treatment area Dilution factor Neutralizing solution dilution factor (number of positive cases / total number of inoculated cases) TCID 50 Log reduction median 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 Treatment area 1 / 2 5 / 5 4 / 5 2 / 5 1 / 5 0 / 5 0 / 5 2.9 3.6 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 2 Treatment area 1 / 2 5 / 5 4 / 5 3 / 5 1 / 5 0 / 5 0 / 5 3.1 3.4 3.6 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 3 Treatment area 1 / 2 5 / 5 4 / 5 2 / 5 1 / 5 0 / 5 0 / 5 2.9 3.6 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5

[0151] 2-2. Viralcidal effects against low pathogenic AIV

[0152] Results of 3 repeated tests, Niallia circulans The AIV titer of the YBK-04 treatment group is the control group (10 6.5 TCID 50 It showed a decrease of 3.4 to 3.6 log compared to / mL, and the median was 3.6 log.

[0153] repeat Treatment area Dilution factor Neutralizing solution dilution factor (number of positive cases / total number of inoculated cases) EID 50 Log reduction median 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 Treatment area 1 / 2 5 / 5 4 / 5 2 / 5 1 / 5 0 / 5 0 / 5 2.9 3.6 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 2 Treatment area 1 / 2 5 / 5 4 / 5 3 / 5 1 / 5 0 / 5 0 / 5 3.1 3.4 3.6 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 3 Treatment area 1 / 2 5 / 5 4 / 5 2 / 5 1 / 5 0 / 5 0 / 5 2.9 3.6 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5

[0155] Example 3. B. subtilis YBK-70 and N. curculans Antiviral effects of YBK-04 combination

[0156] 3-1. B. subtilis YBK-70 and N. curculans Ratio of YBK-04 combination

[0157] Based on the antiviral effects of the YBK-70 and YBK-04 strains confirmed in Examples 1 and 2, each strain culture medium was mixed in a 3:1 (v / v) ratio and used.

[0158] virus Reduction Ratio(YBk-70 / YBK-04) YBK-70 YBK-04 PEDV 10 4.0 (10×10 3 ) 10 3.4 (2.5×10 3 ) 4 AIV 10 4.2 (1.6×10 4 ) 10 3.6 (4.0×10 3 ) 4

[0160] 3-2. Viral action against PEDV

[0161] As a result of three repeated tests, the PEDV titer of the test substance treatment group was the control group (10 6.5 TCID 50 It showed a decrease of 4.8–5.0 log compared to / mL, and the median was 4.8 log.

[0162] repeat Treatment area Dilution factor Neutralizing solution dilution factor (number of positive cases / total number of inoculated cases) EID 50 Log reduction median 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 Treatment area 1 / 2 3 / 5 2 / 5 1 / 5 0 / 5 0 / 5 0 / 5 1.7 4.8 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 2 Treatment area 1 / 2 3 / 5 2 / 5 1 / 5 0 / 5 0 / 5 0 / 5 1.7 4.8 4.8 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 3 Treatment area 1 / 2 4 / 5 1 / 5 0 / 5 0 / 5 0 / 5 0 / 5 1.5 5.0 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5

[0164] 3-3. Viricidal effect against low pathogenic AIV

[0165] As a result of three repeated tests, the AIV titer of the test substance treatment group was the control group (10 6.5 EID 50 It showed a decrease of 5.0–5.2 log compared to / mL, and the median was 5.0 log.

[0166] repeat Treatment area Dilution factor Neutralizing solution dilution factor (number of positive cases / total number of inoculated cases) EID 50 Log reduction median 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 Treatment area 1 / 2 3 / 5 1 / 5 0 / 5 0 / 5 0 / 5 0 / 5 1.3 5.2 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 2 Treatment area 1 / 2 4 / 5 1 / 5 0 / 5 0 / 5 0 / 5 0 / 5 1.5 5.0 5.0 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 3 Treatment area 1 / 2 4 / 5 1 / 5 0 / 5 0 / 5 0 / 5 0 / 5 1.5 5.0 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5

[0167] From the above results, B. subtilis YBK-70 and N. circulans It was confirmed that the combination of YBK-04 exhibited an enhanced antiviral effect compared to each strain alone.

[0169] Example 4. Viral-killing effect of complex microbial dilution

[0170] 4-1. 3 : 1 : 0.5 ( B. subtilis YBK-70 : N. curculans YBK-04 : Actinomyces sp.) Mixed formulation

[0171] 4-1-1. Viral action against PEDV

[0172] As a result of three repeated tests, the PEDV titer of the complex composition treatment group with a ratio of 3:1:0.5 was higher than that of the control group (10 6.5 TCID50 It showed a decrease of 5.4 to 5.6 log compared to / mL, and the median was 5.4 log.

[0173] repeat Treatment area Dilution factor Neutralizing solution dilution factor (number of positive cases / total number of inoculated cases) TCID 50 Log reduction median 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 Treatment area 1 / 2 2 / 5 1 / 5 0 / 5 0 / 5 0 / 5 0 / 5 1.1 5.4 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 2 Treatment area 1 / 2 2 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0.9 5.6 5.4 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 3 Treatment area 1 / 2 2 / 5 1 / 5 0 / 5 0 / 5 0 / 5 0 / 5 1.1 5.4 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5

[0175] 4-1-2. Viralcidal effects against low pathogenic AIV

[0176] As a result of three repeated tests, the AIV titer of the complex composition treatment group with a ratio of 3:1:0.5 was higher than that of the control group (10 6.5 EID 50 It showed a decrease of 5.4 to 5.6 log compared to / mL, and the median was 5.6 log.

[0177] repeat Treatment area Dilution factor Neutralizing solution dilution factor (number of positive cases / total number of inoculated cases) EID 50 Log reduction median 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 Treatment area 1 / 2 2 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0.9 5.6 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 2 Treatment area 1 / 2 2 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0.9 5.6 5.6 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 3 Treatment area 1 / 2 2 / 5 1 / 5 0 / 5 0 / 5 0 / 5 0 / 5 1.1 5.4 Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5

[0179] 4-2. 3 : 1 : 1 ( B. subtilis YBK-70 : N. curculans YBK-04 : Actinomyces sp.) Mixed formulation

[0180] 4-2-1. Viral action against PEDV

[0181] As a result of three repeated tests, the PEDV titer of the group treated with a 3:1:1 ratio composite composition was the control group (10 6.5 TCID 50 It showed a decrease of more than 6.0 log compared to / mL. In other words, the virus titer of the treatment group was 10 0.5 TCID 50 It appeared at levels below / mL, indicating that it was almost completely eradicated.

[0182] Figure 1 shows a photograph of Vero cells observed under a microscope. The negative control group, which was treated with nothing, was cultured with only Vero cells, resulting in a clear cellular appearance (Figure 1a). In contrast, the pathogen control group, in which only PEDV was cultured in Vero cells, showed CPE as the virus denatured the cells (Figure 1b). Meanwhile, in the treatment group inoculated with Vero cells using a reaction solution prepared by reacting the mixed microbial dilution with PEDV, no denaturation occurred in the cells, appearing similar to the negative control group, confirming that the PEDV was completely eradicated (Figure 1c).

[0183] repeat Treatment area Dilution factor Neutralizing solution dilution factor (number of positive cases / total number of inoculated cases) TCID 50 Log reduction median 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 6.0≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 2 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 6.0≥ 6.0≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 3 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 6.0≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5

[0185] 4-2-2. Viralicidal effect against low pathogenic AIV

[0186] As a result of three repeated tests, the AIV titer of the group treated with a 3:1:1 ratio composite composition was the control group (10 6.5 EID 50 It showed a decrease of more than 6.0 log compared to / mL. In other words, the virus titer of the treatment group was 10 0.5 EID 50 It appeared at levels below / mL, indicating that it was almost completely eradicated.

[0187] Figure 2 is a photograph showing the hemolysis of chicken blood cells after placing the urinary fluid into a 96-well microplate and dropping chicken blood cells onto it. In the case of the pathogen control group, in which only AIV was inoculated into the urinary cavity of developing eggs, all chicken blood cells were hemolyzed (Figure 2a). In contrast, in the case of the treatment group, in which the reaction solution prepared by reacting the complex microbial dilution with AIV was inoculated into the urinary cavity, no hemolysis occurred in the chicken blood cells; instead, the cells settled at the bottom of each well of the 96-well plate and showed an agglutination state, indicating that the AIV was completely eradicated (Figure 2b).

[0188] repeat Treatment area Dilution factor Neutralizing solution dilution factor (number of positive cases / total number of inoculated cases) EID 50 Log reduction median 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 6.0≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 2 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 6.0≥ 6.0≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 3 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 6.0≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5

[0190] 4-2-3. Viral action against PRRSV

[0191] As a result of the 3-times repeated test, the PRRSV titer in the control group was 10 5.9 TCID 50 It was greater than / mL, and the PRRSV titer of the treatment group was 10 0.5 TCID 50 Appeared at / mL or less, the reduction in PRRSV in the treatment group compared to the control group was 10 5.4 It appeared to be more than double.

[0192] repeat Treatment area Dilution factor Neutralizing solution dilution factor (Number of positives / Total number of inoculated) TCID 50 Log reduction 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 5.4≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 4 / 5 3 / 5 5.9 2 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 5.6≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 3 / 5 6.1 3 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 5.4≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 4 / 5 3 / 5 5.9

[0193] Figure 3 shows a micrograph of MARC-145 cells. The negative control group, which was treated with nothing, was cultured with only MARC-145 cells, resulting in a clear cell profile (Figure 3a). In contrast, the pathogen control group, in which only PRRSV was cultured on MARC-145 cells, showed CPE as the virus denatured the cells (Figure 3b). Meanwhile, in the treatment group inoculated with MARC-145 cells using a reaction solution prepared by reacting the mixed microbial dilution with PRRSV, no cell denaturation occurred, exhibiting the same appearance as the negative control group, confirming that the PRRSV was completely eradicated (Figure 3c).

[0195] 4-3. 3 : 1 : 2 ( B. subtilis YBK-70 : N. curculans YBK-04 : Actinomyces sp.) Mixed formulation

[0196] 4-3-1. Viral action against PEDV

[0197] As a result of three repeated tests, the PEDV titer of the group treated with a 3:1:2 ratio composite composition was the control group (10 6.5 TCID 50 It showed a decrease of more than 6.0 log compared to / mL. In other words, the virus titer of the treatment group was 10 0.5 TCID 50 It appeared at levels below / mL, indicating that it was almost completely eradicated.

[0198] repeat Treatment area Dilution factor Neutralizing solution dilution factor (number of positive cases / total number of inoculated cases) TCID 50 Log reduction median 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 6.0≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 2 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 6.0≥ 6.0≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 3 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 6.0≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5

[0200] 4-3-2. Viralicidal effect against low pathogenic AIV

[0201] As a result of three repeated tests, the AIV titer of the complex composition treatment group with a ratio of 3:1:2 was the control group (10 6.5 EID 50 It showed a decrease of more than 6.0 log compared to / mL. In other words, the virus titer of the treatment group was 10 0.5 EID 50 It appeared at levels below / mL, indicating that it was almost completely eradicated.

[0202] repeat Treatment area Dilution factor Neutralizing solution dilution factor (number of positive cases / total number of inoculated cases) EID 50 Log reduction median 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 6.0≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 2 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 6.0≥ 6.0≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5 3 Treatment area 1 / 2 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 ≤0.5 6.0≥ Pathogen control - 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 5 / 5 6.5

[0203] From the above results, Bacillus subtilis YBK-70, Niallia circulans YBK-04, Actinomyces It was confirmed that the antiviral effect of the composite composition containing sp. was further enhanced due to the addition of actinomycetes.

[0205] Example 5. In vivo Confirmation of the antiviral effect of the complex microbial composition

[0206] In previous experiments, it was confirmed that the highest level of viricidal effect against both PEDV and AIV could be obtained when YBK-70, YBK-04, and actinomycete culture solutions were mixed in a weight ratio of 3:1:1 or 3:1:3. Below, a mixture prepared by mixing the above strain culture solutions in a 3:1:1 ratio was selected as a representative example to verify whether it exhibits a viricidal effect in virus-infected animals.

[0208] 5-1. Viral action against Porcine Epidemic Diarrhea Virus (PEDV)

[0209] First, a composite microbial carrier containing a composite microbial composition was prepared. After challenging pigs with PEDV, the small intestines of three pigs confirmed to be infected were collected. A portion of each collected small intestine was placed in a reactor with the composite microbial carrier and treated for 24 hours. The presence of PEDV was confirmed in the pig small intestines treated in the reactor and in the pig small intestines not treated in the reactor using a Porcine epidemic diarrhea PCR kit.

[0210] Lanes 1 to 3 of FIG. 4 show the PCR results for pig small intestines that were not treated with the reactor, and Lanes 4 to 6 show the PCR results for pig small intestines that were treated with the reactor. PEDV was detected only in Lanes 1 to 3 and was not detected in Lanes 4 to 6. That is, all three pigs were infected with PEDV, and it was confirmed that PEDV was completely eradicated during the process of treatment with a complex microbial carrier in the reactor.

[0212] 5-2. Viricidal effect against low pathogenic avian influenza virus (AIV)

[0213] A composite microbial carrier containing a composite microbial composition was prepared. After challenging broiler chickens with low-pathogenic AIV, the tracheas and lungs of three broiler chickens confirmed to be infected were collected. Each collected portion of the trachea and lungs was placed in a reactor with the composite microbial carrier and treated for 24 hours. The presence of AIV was confirmed in the tracheas and lungs treated in the reactor and in the tracheas and lungs not treated in the reactor using an Avian influenza virus PCR kit.

[0214] Lanes 1 to 3 of FIG. 5 are PCR band images of the trachea and lungs of broiler chickens that were not treated with the reactor, and Lanes 4 to 6 are PCR band images of the trachea and lungs that were treated with the reactor. AIV was detected only in Lanes 1 to 3 and was not detected in Lanes 4 to 6. That is, all three broiler chickens were infected with AIV, and it was confirmed that AIV was completely eradicated during the process of treatment with a complex microbial carrier in the reactor.

[0216] 5-3. Viral action against Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

[0217] A composite microbial carrier containing a composite microbial composition was prepared. After challenging pigs with PRRSV, lungs were collected from three pigs confirmed to be infected. The collected portions of lungs were placed in a reactor with the composite microbial carrier and treated for 24 hours. The presence of PRRSV was confirmed in the pig lungs treated in the reactor and in the pig lungs not treated in the reactor using a Porcine reproductive and respiratory syndrome virus PCR kit.

[0218] Lanes 1 to 3 of FIG. 6 show the PCR results for pig lungs that were not treated with the reactor, and Lanes 4 to 6 show the PCR results for pig lungs that were treated with the reactor. PRRSV was detected only in Lanes 1 to 3 and was not detected in Lanes 4 to 6. That is, all three pigs were infected with PRRSV, and it was confirmed that PRRSV was completely eradicated during the process of treatment with a composite microbial carrier in the reactor.

[0220] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical concept or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0221] The scope of the present invention is defined by the claims set forth below rather than by the detailed description above, and all modifications or variations derived from the meaning and scope of the claims and the concept of equivalents thereof should be interpreted as being included within the scope of the present invention.

[0223] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC15571BP Date of Deposit: 2023-08-29 Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC15427BP Date of Deposit: 2023-08-27

Claims

Claim 1 A Bacillus subtilis YBK-70 strain with accession number KCTC 15571BP, ​​wherein the YBK-70 strain has a viricidal effect against porcine epidemic diarrhea virus, avian influenza virus, and / or porcine reproductive and respiratory syndrome virus. Claim 2 In claim 1, the strain is a YBK-70 strain comprising 16S rRNA consisting of the nucleotide sequence of SEQ ID NO.

1. Claim 3 In claim 1, the strain is a YBK-70 strain comprising 23S rRNA consisting of the nucleotide sequence of SEQ ID NO.

2. Claim 4 A virucidal composition comprising the Bacillus subtilis YBK-70 strain of accession number KCTC 15571BP or a culture medium thereof, wherein the virus is a porcine epidemic diarrhea virus (PEDV) and / or an avian influenza virus (AIV). Claim 5 A virucidal composition according to claim 4, wherein the composition further comprises the Niallia circulans YBK-04 strain of accession number KCTC 15427BP or a culture medium thereof, and the virus is PEDV, AIV, and / or Porcine reproductive and respiratory syndrome virus. Claim 6 A virucidal composition according to claim 5, wherein the composition comprises 100 to 300 parts by weight of a YBK-70 strain or its culture solution based on (100 parts by weight) a YBK-04 strain or its culture solution. Claim 7 In paragraph 5, the above composition is an actinomycete ( Actinomyces A virucidal composition comprising additionally containing a strain (spp.) or a culture solution thereof. Claim 8 A virucidal composition according to claim 7, wherein the composition comprises 50 to 200 parts by weight of an actinomycete strain or a culture solution thereof based on (100 parts by weight) a YBK-04 strain or a culture solution thereof. Claim 9 Bacillus subtilis (accession number KCTC 15571BP) Bacillus subtilis A pharmaceutical composition for the prevention or treatment of viral infectious diseases comprising a YBK-70 strain or a culture medium thereof, wherein the virus is a porcine epidemic diarrhea virus (PEDV) and / or an avian influenza virus (AIV). Claim 10 A pharmaceutical composition according to claim 9, wherein the composition further comprises the Niallia circulans YBK-04 strain of accession number KCTC 15427BP or a culture medium thereof, and the virus is PEDV, AIV, and / or Porcine reproductive and respiratory syndrome virus. Claim 11 In item 10, the above composition is an actinomycete ( Actinomyces A pharmaceutical composition comprising additionally containing a strain of spp.) or a culture medium thereof.

Images