Anti-SARS coronavirus-2 composition and its manufacturing method

Abstract

To provide a novel anti-SARS coronavirus-2 composition that has antiviral activity against SARS coronavirus-2 and a method for producing the same.SOLUTION: An anti-SARS coronavirus-2 composition contains culture extract of Bacillus subtilis var natto. A method for producing the anti-SARS coronavirus-2 composition includes a liquid culture step for culturing the Bacillus subtilis var natto in a liquid medium and a conditioned medium separation step for separating a conditioned medium of the Bacillus subtilis var natto from the culture medium from the liquid culture step; or includes a solid culture step for adding the Bacillus subtilis var natto to beans for solid culture, and a liquid component separation step for adding an extraction solvent to the culture after the solid culture step and then performing solid-liquid separation to separate the liquid component.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an anti-SARS coronavirus-2 composition and a method for producing the same. [Background technology]

[0002] Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (hereafter referred to as SARS-CoV-2). SARS-CoV-2 belongs to the β-coronavirus genus and is thought to be genetically closely related to coronaviruses isolated from Rhinolophus bats.

[0003] Recently, in order to prevent infection with SARS-coronavirus-2, thorough cleaning and disinfection of hands, clothes, etc., which are likely to be contaminated by the virus, has been required, and the effectiveness of ethanol, surfactants, hypochlorous acid water, etc. against SARS-coronavirus-2 is being evaluated.

[0004] For example, Patent Document 1 discloses a SARS-coronavirus-2 inactivating agent containing as active ingredients a linear alkylbenzene sulfonate and at least one polyoxyethylene alkyl ether selected from a polyoxyethylene alkyl ether having 12 carbon atoms in the alkyl group and 7 moles of ethylene oxide added, and a polyoxyethylene alkyl ether having 12 carbon atoms in the alkyl group and 8 moles of ethylene oxide added.

[0005] On the other hand, surfactin, a metabolic product of Bacillus subtilis natto, is a cyclic lipopeptide-type biosurfactant and is known to have an antiviral effect (see, for example, Non-Patent Document 1, etc.). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6795720 [Non-patent literature]

[0007] [Non-Patent Document 1] Vollenbroich D et al., “Mechanism of Inactivation of Enveloped Viruses by the Biosurfactant Surfactin from Bacillus subtilis.”, Biologicals, Vol. 25, pp. 289-297, 1997. Summary of the Invention [Problem to be solved by the invention]

[0008] In addition to existing disinfectants, there is a need for substances that are more effective against SARS-coronavirus-2.

[0009] The present invention has been made in consideration of the above circumstances, and provides a novel anti-SARS coronavirus-2 composition having antiviral activity against SARS coronavirus-2, and a method for producing the same. [Means for solving the problem]

[0010] As a result of extensive research to achieve the above-mentioned objective, the inventors discovered that a natto bacteria culture extract can prevent infection with SARS-coronavirus-2 by fragmenting the spike protein of SARS-coronavirus-2, and thus completed the present invention.

[0011] That is, the present invention includes the following aspects. (1) An anti-SARS coronavirus-2 composition comprising a culture extract of Bacillus subtilis natto. (2) The anti-SARS-coronavirus-2 composition according to (1), wherein the Bacillus subtilis natto culture extract is treated with gamma-polyglutamic acid degrading enzyme. (3) The anti-SARS-coronavirus-2 composition according to (1) or (2), wherein the Bacillus subtilis TTCC903 strain (accession number FERM P-21173) is used as the natto bacteria. (4) A method for producing an anti-SARS-coronavirus-2 composition according to any one of (1) to (3), comprising the steps of: A liquid culture step of culturing the Bacillus subtilis natto in a liquid medium; A culture supernatant separation step of separating the culture supernatant of the Bacillus subtilis natto from the culture liquid after the liquid culture step; or A solid culture step of adding the Bacillus subtilis natto to beans and culturing them in a solid state; A liquid component separation step in which an extraction solvent is added to the culture after the solid culture step, and then solid-liquid separation is performed to separate the liquid component; A manufacturing method comprising: (5) The method according to (4), further comprising, after the liquid component separation step, an enzyme treatment step of adding a gamma-polyglutamic acid degrading enzyme to the liquid component to perform an enzyme treatment. Effect of the Invention

[0012] According to the above-mentioned aspects of the anti-SARS-coronavirus-2 composition and the method for producing the same, it is possible to provide an anti-SARS-coronavirus-2 composition having antiviral activity against SARS-coronavirus-2 and a method for producing the same. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the results of a plaque assay using bovine herpesvirus in Reference Example 1. [Diagram 2] FIG. 1 shows the results of a plaque assay using bovine herpesvirus in Reference Example 2. [Diagram 3] FIG. 1 shows the results of a plaque assay using bovine herpesvirus in Reference Example 3. [Figure 4] FIG. 1 shows the results of a plaque assay using bovine herpesvirus in Reference Example 4. [Diagram 5] FIG. 1 shows the results of a plaque assay using bovine herpesvirus in Reference Example 5. [Figure 6] This figure shows the results of Western blotting using an anti-SARS-CoV-2 spike protein monoclonal antibody as the primary antibody in Example 1 (left), and the results of Western blotting using an anti-SARS-CoV-2 spike protein polyclonal antibody as the primary antibody (right). [Figure 7] This figure shows the Coomassie brilliant blue (CBB) staining image (upper panel) and the results of Western blotting using an anti-SARS-CoV-2 spike protein monoclonal antibody as the primary antibody in Example 2 (lower panel). [Figure 8] FIG. 1 shows the results of a plaque assay using bovine herpesvirus in Reference Example 6. [Figure 9] FIG. 1 shows the results of a plaque assay using SARS-CoV-2 in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] <Anti-SARS coronavirus-2 composition> An anti-SARS-coronavirus-2 composition according to one embodiment of the present invention (hereinafter referred to as "this embodiment") contains a Bacillus subtilis natto culture extract.

[0015] The anti-SARS-coronavirus-2 composition of this embodiment has antiviral activity against SARS-coronavirus-2, as shown in the Examples described below.

[0016] The Bacillus subtilis natto culture extract contains various components that are metabolic products of the Bacillus subtilis natto, and as shown in the Examples described below, it is presumed that the components contained in the Bacillus subtilis natto culture extract (particularly proteases, more specifically serine proteases) cleave the spike protein of SARS-coronavirus-2, causing SARS-coronavirus-2 to lose its ability to infect cells. However, since the Bacillus subtilis natto culture extract contains a wide variety of components and approximately 80 types of proteases, it is difficult to identify which of these have antiviral activity.

[0017] [Bacillus natto culture extract] The term "natto bacteria culture extract" refers to a substance extracted from the natto bacteria culture after culturing the natto bacteria, and includes various metabolic products of natto bacteria.

[0018] The Bacillus subtilis natto culture extract can be prepared from a culture medium obtained after liquid culture of Bacillus subtilis natto or from a culture product obtained after solid culture of Bacillus subtilis natto.

[0019] A specific example of a method for producing a Bacillus subtilis natto culture extract in the case of liquid culture includes, for example, a method including a liquid culture step and a culture supernatant separation step. In the liquid culture step, the natto bacteria are cultured in a liquid medium. In the culture supernatant separation step, the culture supernatant of the Bacillus subtilis natto is separated from the culture liquid after the liquid culture step.

[0020] In the case of solid culture, for example, it includes a solid culture step and a liquid component separation step. In the solid culture step, Bacillus subtilis natto is added to beans and cultured on a solid state. In the liquid component separation step, an extraction solvent is added to the culture product after the solid culture step, and then solid-liquid separation is carried out to separate the liquid component.

[0021] The method for producing the Bacillus subtilis natto culture extract may be either liquid culture or solid culture as described above. However, solid culture is preferred because it allows the production of a substance having antiviral activity in the Bacillus subtilis natto culture extract at a higher concentration.

[0022] The natto bacteria used in each culture are not particularly limited as long as they belong to the genus Bacillus subtilis natto. However, because they produce a relatively large amount of a substance having antiviral activity, the Bacillus subtilis TTCC903 strain (accession number FERM P-21173), the Bacillus subtilis TTCC940 strain (accession number FERM P-21324), the Bacillus subtilis TTCC904-2 strain (accession number NITE P-01705), or the Bacillus subtilis TTCC2221 strain (accession number NITE P-02380) is preferred, and the Bacillus subtilis TTCC903 strain (accession number FERM P-21173) is more preferred.

[0023] Next, each step in the liquid culture will be described.

[0024] [Liquid culture process] In the liquid culture step, the natto bacteria are cultured in a liquid medium.

[0025] The liquid medium can be appropriately selected and used depending on the type of natto bacteria. Specifically, for example, a medium containing at least a carbon source, a nitrogen source, and inorganic salts and having a pH of usually 6.0 to 9.0, preferably 6.5 to 8.5, can be used. More specific examples of liquid media include tryptic soy broth, LB medium, nutrient broth, and modified media thereof.

[0026] The culture conditions for Bacillus subtilis can be used under the culture conditions commonly used by those skilled in the art. The culture temperature is usually 20°C to 45°C, preferably 35°C to 42°C. The culture method may be shaking culture, stirring culture, or stationary culture, or may be batch culture, semi-batch culture, or continuous culture. Other culture conditions are not particularly limited as long as they allow the proliferation of Bacillus subtilis natto.

[0027] [Culture supernatant separation process] In the culture supernatant separation step, the culture supernatant of the Bacillus subtilis natto is separated from the culture liquid after the liquid culture step.

[0028] Methods for separating the culture supernatant include known methods such as filtration and centrifugation.

[0029] Next, each step in solid culture will be described.

[0030] [Solid culture process] In the solid culture process, Bacillus subtilis natto is added to beans and cultured on a solid state. In the solid culture process, the beans are fermented by Bacillus subtilis natto to obtain natto, and therefore the process can also be called a fermentation process.

[0031] The beans used are preferably in a steamed state. The steamed beans used in the solid culture process may be whole beans or ground beans. The term "whole beans" used here means beans that have not been subjected to a step of cracking the beans.

[0032] The steamed beans can be prepared using a known method. The method for producing the steamed beans specifically includes a soaking step and a steaming step. In the soaking step, when the beans are whole beans, the soaking time can be, for example, 10 hours to 22 hours, and preferably 14 hours to 18 hours. When the beans are ground, the soaking time can be, for example, 2 hours to 5 hours, and preferably 3 hours to 4 hours. In addition, the soaking temperature can be, for example, 15°C to 30°C, and preferably 18°C ​​to 22°C, for both whole beans and ground beans. In the steaming step, the steaming temperature can be, for example, about 100°C to 140°C, and the steaming time can be, for example, 5 minutes to 90 minutes. In addition, the pressure in the steaming step can be 0.02 MPa to 0.28 MPa.

[0033] The beans used in the solid culture are not limited to soybeans (beans belonging to the genus Glycine max), but can be any beans containing protein or protein and sugar and having a stringy property due to fermentation by Bacillus subtilis natto. Specific examples of such beans include beans belonging to the genus Phaseolus, such as white golden beans, red kidney beans, quail beans, tiger beans, and white flower beans. When the beans are soybeans, there is no particular limitation on the soybeans, and they may be domestically or overseas. In addition, a wide range of particle sizes are applicable, including large to very small, and are not limited. In addition, the soybeans are not limited to yellow soybeans, and colored beans such as black soybeans and blue soybeans can also be applied.

[0034] As for the culture conditions, when the steamed beans are pulverized, the fermentation temperature can be 37° C. to 47° C., preferably 38° C. to 44° C. The fermentation time can usually be about 5 hours to 15 hours. On the other hand, when the steamed beans are whole beans, the fermentation temperature can be 37° C. or higher and 47° C. or lower, and preferably 38° C. or higher and 44° C. or lower. The fermentation time can usually be 7 hours or higher and 20 hours or lower. In addition, fermentation can be terminated by lowering the temperature to below 10°C. The additives to be added during the culture and other production conditions conform to known methods for producing natto.

[0035] [Liquid component separation process] In the liquid component separation step, an extraction solvent is added to the culture product after the solid culture step, and then solid-liquid separation is carried out to separate the liquid component.

[0036] The extraction solvent is not particularly limited as long as it can dissolve or disperse the active ingredients contained in the culture without adverse effects such as denaturation. Examples of the extraction solvent include physiological saline and physiological saline with a buffer effect (phosphate buffered saline (PBS), Tris buffered saline (TBS), HEPES buffered saline, etc.).

[0037] Examples of the solid-liquid separation method include known methods such as filtration and centrifugation.

[0038] [Other processes] The liquid component obtained in the solid culture has viscosity because it contains γ-polyglutamic acid produced by the Bacillus subtilis natto by metabolising beans. The liquid component may be used as is, but from the viewpoint of reducing the viscosity and improving the ease of handling, it is preferable to further include an enzyme treatment step in which a γ-polyglutamic acid degrading enzyme is added to the liquid component to perform an enzyme treatment. As the γ-polyglutamic acid decomposition enzyme, for example, PghP, a γ-polyglutamic acid decomposition enzyme possessed by the Bacillus subtilis phage, may be used (see Reference 1: Kimura, K et al., “Characterization of Poly-γ-Glutamate Hydrolase Encoded by a Bacteriophage Genome: Possible Role in Phage Infection of Bacillus subtilis Encapsulated with Poly-γ-Glutamate”, Appl. Environ. Microbiol., Vol. 69, No. 5, pp. 2491-2497, 2003). Alternatively, γ-glutamyl transpeptidase (ggt) that degrades γ-polyglutamic acid to exo-type, or γ-polyglutamyl hydrolase (pghA) that degrades γ-polyglutamic acid to endo-type, which are contained in Bacillus subtilis natto itself, may be used (see Reference Document 2: JP 2003-230384 A). These enzymes may be synthesized using a microorganism or commercially available. Specific examples of enzymes synthesized using a microorganism include a column-purified product obtained by transforming the pghP gene derived from Bacillus subtilis natto bacteriophage (ΦNIT1) into Escherichia coli BL21 strain and highly expressing it.

[0039] The culture supernatant obtained in the liquid culture and the liquid component obtained in the solid culture may be further purified. That is, a purification step may be further included after the culture supernatant separation step or after the liquid component separation step. The purification step also includes a concentration operation for concentrating the active ingredient.

[0040] Examples of purification methods that can be used include salting out using ammonium sulfate or the like, organic solvent precipitation using alcohol or the like, membrane separation using an ultrafiltration membrane (filter) or the like, fractionation using an organic solvent such as acetone fractionation, and chromatographic separation using an ion exchanger, a hydrophobic chromatographic carrier, a gel filtration carrier, or the like, either alone or in appropriate combination.

[0041] [form] In the anti-SARS coronavirus-2 composition of this embodiment, the culture supernatant obtained in the above-mentioned liquid culture and the liquid component obtained in the solid culture may be used as is as the Bacillus subtilis natto culture extract, or a solution diluted with a buffer commonly used by those skilled in the art may be used as the Bacillus subtilis natto culture extract, or may be made into a dry powder. The dry powder can be obtained by subjecting the culture supernatant and the liquid component to the above-mentioned purification step as necessary and then drying them by a drying method such as freeze drying, vacuum heat drying, spray drying, etc.

[0042] The anti-SARS-coronavirus-2 composition of this embodiment may be prepared by further preparing a formulation from the dry powder. The formulation may be in the form of a solid, semi-solid, or liquid. In the case of a solid, examples of the form include powder, granules, pills, pellets, tablets, capsules, and the like. In the case of a semi-solid, examples of the form include a gel. In the case of a liquid, examples of the form include a suspension in which a dry powder is diluted or suspended in a buffer solution such as phosphate buffered saline (PBS).

[0043] [Application] The anti-SARS-coronavirus-2 composition of the present embodiment has excellent antiviral activity against SARS-coronavirus-2, and since Bacillus subtilis natto is a microorganism that has been consumed by humans, it can be suitably used, by further purifying it, as a raw material for a pharmaceutical composition for treating or preventing COVID-19, or as a mouthwash for preventing SARS-coronavirus-2. It can also be incorporated into cleaning agents and disinfectants for hands, clothes, etc. In addition, the anti-SARS-coronavirus-2 composition of this embodiment can also be used as a feed supplement for the purpose of preventing SARS-coronavirus-2 infection in mammals other than humans. EXAMPLES

[0044] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0045] The inventors found that the Bacillus subtilis natto culture extract has antiviral activity against not only SARS-coronavirus-2 but also bovine herpesvirus. Therefore, after examining various conditions using bovine herpesvirus, the inventors examined the antiviral effect against SARS-coronavirus-2 under suitable conditions. The specific test contents are described below.

[0046] [Reference example 1] (Comparative test of the antiviral effect of heated or unheated Bacillus subtilis natto culture extract against bovine herpesvirus) 1. Preparation of Bacillus subtilis natto culture extract Bacillus subtilis TTCC903 strain 1 x 10 3The inoculation was performed on 40 g of boiled soybeans so that the number of bacteria was 100 / g, and the mixture was incubated at 42°C for 18 hours to prepare natto. After stirring 10 g of the obtained natto with a glass rod for 5 minutes, 10 mL of phosphate buffered saline (PBS) was added, and the mixture was mixed by inverting 50 times at room temperature (about 25°C). After mixing, the mixture was centrifuged (3500 rpm, 10 minutes) to obtain a supernatant. The obtained supernatant was then filtered through a φ0.45 filter and a φ0.22 filter to obtain a natto bacteria culture extract. In addition, when 10 g of natto was dispersed with a glass rod, 10 μL of γ-polyglutamic acid decomposition enzyme (PghP) (see Reference 1 above) was added to obtain an enzyme-treated (enzyme treatment +) natto bacteria culture extract. Furthermore, the supernatant that was not subjected to enzyme treatment and a part of the supernatant that was subjected to enzyme treatment were heat-treated at 100°C for 10 minutes. By the above procedure, the following four types of Bacillus subtilis natto culture extracts were obtained. Natto Bacillus Culture Extract No.1: Enzyme treatment +, heat treatment + Natto Bacillus Culture Extract No.2: Enzyme treatment +, heat treatment - Natto Bacillus Culture Extract No.3: Enzyme treatment -, Heat treatment + Natto Bacillus Culture Extract No.4: Enzyme treatment, heat treatment

[0047] 2. Comparative study of the antiviral effect of Bacillus subtilis natto culture extract against bovine herpesvirus Madin-Darby bovine kidney (MDBK) cells were seeded on a 6-well plate and pre-cultured to 100% confluence using MEM or EMEM medium (5 w / v% FBS, 1 v / v% penicillin-streptomycin solution, 1 w / v% L-glutamic acid). Next, 90 μL of the natto extract and 10 μL of a solution containing bovine herpesvirus 1 (IBR Ishikawa (bovine / Japan / 1988)) were mixed and incubated at 37°C for 1 hour. Next, the mixed solution of the natto extract and virus was appropriately diluted with MEM without FBS according to the titer of the virus and inoculated into the MDBK cells at 500 μL / well. The MDBK cells were washed once with PBS before inoculation, and incubated at 37°C for 1 hour after inoculation. After incubation, the cell surface was washed once with PBS, and then 0.8 w / v % methylcellulose medium was overlaid and cultured at 37° C. for 2 to 3 days.

[0048] As shown in Figure 1, the heat-treated Bacillus natto culture extract showed no antiviral effect, regardless of whether it was treated with an enzyme. On the other hand, the non-heat-treated Bacillus natto culture extract showed an antiviral effect, regardless of whether it was treated with an enzyme.

[0049] This result suggests that among the components contained in the Bacillus subtilis natto culture extract, those that are not decomposed by PghP and have relatively low heat resistance are involved in the antiviral effect.

[0050] [Reference example 2] (Confirmation test of the antiviral effect of surfactin against bovine herpesvirus) Next, in order to demonstrate that the component having antiviral effect contained in the Bacillus subtilis natto culture extract is not a known surfactin whose antiviral effect has been confirmed, the following test was carried out.

[0051] 1. Preparation of Surfactin Aqueous Solution Commercially available surfactin (Surfactin Sodium Salt Cat No. 197-12691 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)) was diluted to prepare surfactin aqueous solutions of different concentrations (0.1 mg / mL, 0.01 mg / mL, and 0.001 mg / mL). Some of the surfactin aqueous solutions were heated at 100°C for 10 minutes. By the above procedure, the following six types of surfactin aqueous solutions were prepared. Surfactin aqueous solution No.1: Heat treatment +, concentration 0.1mg / mL Surfactin aqueous solution No. 2: Heat treatment +, concentration 0.01 mg / mL Surfactin aqueous solution No. 3: Heat treatment +, concentration 0.001 mg / mL Surfactin aqueous solution No. 4: Heat treatment, concentration 0.1 mg / mL Surfactin aqueous solution No. 5: Heat treatment, concentration 0.01 mg / mL Surfactin aqueous solution No. 6: Heat treatment, concentration 0.001 mg / mL

[0052] 2. Comparative test of the antiviral effect of surfactin aqueous solution against bovine herpesvirus Next, 90 μL of each surfactin aqueous solution prepared in "1." and 10 μL of a solution containing bovine herpesvirus 1 (IBR Ishikawa (bovine / Japan / 1988)) were mixed and incubated at 37°C for 1 hour. Next, the antiviral effects of the surfactin aqueous solutions against bovine herpesvirus were compared in the same manner as in "2." of Reference Example 1, except that this mixed solution of surfactin aqueous solution and virus was used. The results are shown in Figure 2.

[0053] As shown in Figure 2, it was confirmed that the antiviral effect was concentration-dependent regardless of the presence or absence of heat treatment. In addition, since the antiviral effect in Reference Example 1 was not observed when heated, it was confirmed that the active ingredient in the Bacillus subtilis natto culture extract in Reference Example 1 was not surfactin.

[0054] [Reference example 3] (Relationship between dilution of Bacillus subtilis natto culture extract and antiviral effect against bovine herpesvirus) Next, the dilution ratio of the Bacillus subtilis natto culture extract effective for exerting the antiviral effect was examined.

[0055] 1. Preparation of Bacillus subtilis natto culture extract dilutions For each of Bacillus subtilis natto culture extracts No. 3 and No. 4 in Reference Example 1, a stock solution (1x) and solutions diluted 5x, 10x, 15x, 20x, 50x, 75x, 100x, and 1000x with PBS were prepared. The protein concentration of the stock solution was 1.81 mg / mL.

[0056] 2. Comparative test of antiviral effect of diluted extract of Bacillus subtilis culture against bovine herpesvirus Next, the antiviral effects of the diluted solutions of the Bacillus subtilis natto culture extract against bovine herpesvirus were compared in the same manner as in "2." of Reference Example 1, except that the undiluted or diluted Bacillus subtilis natto extract prepared in "1." was used. The results are shown in Figure 3.

[0057] As shown in Figure 3, an antiviral effect was observed in the 20-fold diluted solution, but the antiviral effect was weakened in the 50-fold diluted solution. This result demonstrated that the Bacillus subtilis natto culture extract exhibited sufficient antiviral effect up to a 20-fold dilution.

[0058] [Reference example 4] (Relationship between dilution and reaction temperature of Bacillus subtilis natto culture extract and its antiviral effect against bovine herpesvirus) Next, in addition to the dilution ratio, the optimal reaction temperature with the virus was also examined.

[0059] 1. Preparation of Bacillus subtilis natto culture extract dilutions For Bacillus subtilis natto culture extract No. 4 of Reference Example 1, an undiluted solution (1x) and solutions diluted 10x and 20x with PBS were prepared.

[0060] 2. Comparative test of antiviral effect of diluted extract of Bacillus subtilis culture against bovine herpesvirus Next, the antiviral effects of the diluted solutions of the Bacillus subtilis natto culture extract against bovine herpesvirus were compared in the same manner as in "2." of Reference Example 1, except that the reaction temperature was changed to 37°C, 10°C, and 4°C, using the undiluted solution or the diluted Bacillus subtilis natto extract prepared in "1.". The results are shown in Figure 4. In Figure 4, "PC" stands for positive control, and is a sample prepared by mixing 90 μL of PBS and 10 μL of a solution containing bovine herpesvirus 1 (IBR Ishikawa (bovine / Japan / 1988)) instead of the diluted solution, incubating at 37°C for 1 hour, and then dropping 300 μL (per well of a 12-well plate) of the diluted solution diluted 10,000-fold.

[0061] As shown in Figure 4, at a reaction temperature of 37°C, the undiluted solution and the 10-fold diluted solution showed sufficient antiviral effects, and the 20-fold diluted solution also showed a slightly weak antiviral effect. On the other hand, at 10°C, only the undiluted solution showed a slight antiviral effect.

[0062] From the above, it became clear that the antiviral effect of the Bacillus subtilis natto culture extract is strongly dependent on temperature, with a reaction temperature of around 37°C being particularly suitable.

[0063] [Reference example 5] (Relationship between the type of natto bacteria and their antiviral effect against bovine herpesvirus) Next, we investigated whether natto bacteria culture extracts extracted from various types of natto had similar antiviral effects.

[0064] 1. Preparation of Bacillus subtilis natto culture extract and its dilution In addition to the natto obtained by inoculating Bacillus subtilis TTCC903 strain, the following five types of natto were used to prepare natto bacteria culture extracts in the same manner as in "1." of Reference Example 1. Domestically produced natto: Whole soybean natto (manufactured by Takano Foods Co., Ltd.) produced using Bacillus subtilis TTCC940 strain (accession number: FERM P-21324) Ground natto: Ground natto produced using Bacillus subtilis TTCC904-2 strain (accession number: NITE P-01705) (manufactured by Takano Foods Co., Ltd.) Ultra-small grain natto: Ultra-small grain natto produced using natto bacteria (manufactured by Takano Foods Co., Ltd.) Fermented collagen natto: Fermented collagen natto produced using Bacillus subtilis TTCC2221 strain (accession number: NITE P-02380) Minced natto: extremely small grain natto produced using natto bacteria (manufactured by Takano Foods Co., Ltd.)

[0065] Next, for each Bacillus subtilis natto culture extract, an undiluted solution (1x) and solutions diluted 10x and 20x with PBS were prepared.

[0066] 2. Comparative test of antiviral effect of diluted extract of Bacillus subtilis culture against bovine herpesvirus Next, the antiviral effects against bovine herpesvirus of the diluted solutions of the Bacillus subtilis natto culture extract prepared from each natto were compared in the same manner as in "2." of Reference Example 1, except that the undiluted or diluted Bacillus subtilis natto extract prepared in "1." was used. The results are shown in Figure 5.

[0067] As shown in Figure 5, in the original solution, the natto bacteria culture extract prepared from any natto showed an antiviral effect. In the 10-fold diluted solution, the natto bacteria culture extract prepared from ground and very small grains showed almost no antiviral effect, but the natto bacteria culture extract prepared from domestically produced natto and minced meat showed a slight antiviral effect, and the natto bacteria culture extract prepared from natto obtained by inoculating the TTCC903 strain and fermented collagen natto showed a sufficient antiviral effect. Furthermore, in the 20-fold diluted solution, the natto bacteria culture extract prepared from natto obtained by inoculating the TTCC903 strain and fermented collagen natto showed a slight antiviral effect.

[0068] From the above, it was revealed that although the strength of the antiviral activity differs depending on the type of natto, the antiviral effect is observed in the natto bacteria culture extract prepared from any type of natto. This is presumably due to the difference in the production amount of the component with antiviral activity contained in the natto bacteria culture extract depending on the type of natto.

[0069] [Example 1] (Comparative study of the antiviral effect of heated or unheated natto culture extract against SARS coronavirus-2) Next, the antiviral effects of heated or unheated Bacillus subtilis natto culture extract against SARS-coronavirus-2 were examined.

[0070] 1. Preparation of Bacillus subtilis natto culture extract The following four types of Bacillus subtilis natto culture extracts were obtained by the same method as in "1." of Reference Example 1. Natto Bacillus Culture Extract No.1: Enzyme treatment +, heat treatment + Natto Bacillus Culture Extract No.2: Enzyme treatment +, heat treatment - Natto Bacillus Culture Extract No.3: Enzyme treatment -, Heat treatment + Natto Bacillus Culture Extract No.4: Enzyme treatment, heat treatment

[0071] 2. Test to confirm the ability of Bacillus subtilis culture extract to decompose the SARS-coronavirus-2 spike protein 2 μL (0.25 mg / mL) of a solution containing SARS coronavirus-2 spike protein (SARS-CoV-2 (2019-nCoV) Spike RBD-mFc Recombiant Protein (HPLC-verified), Sino Biological, Cat No. 40592-V05H) was mixed with 2 μL of each of the natto bacteria culture extracts prepared in "1." above and incubated at 37 ° C for 1 hour. Next, 3 μL of sample buffer (SB) (Sample Buffer Solution, FUJIFILM, 196-16142) was added to each mixture, and the mixture was heated at 95 ° C for 10 minutes to prepare samples for Western blotting. Next, Western blotting was performed by a known method using human anti-SARS coronavirus-2 spike protein monoclonal antibody (SARS-CoV-2 Spike Antibody (D005), Novus Biologicals, Cat No. NBP2-90989) and rabbit anti-SARS coronavirus-2 spike protein polyclonal antibody (SARS-CoV-2 (COVID-19) Spike Antibody, GeneTex, Cat No. GTX135356) as primary antibodies, and goat anti-human IgG antibody (Proteintech, Cat No. SA00001-17) and peroxidase-labeled anti-rabbit IgG antibody (Promega, W401B) as secondary antibodies. The results of Western blotting using a monoclonal antibody as the primary antibody are shown on the left side of Figure 6, and the results of Western blotting using a polyclonal antibody as the primary antibody are shown on the right side of Figure 6. In the left panel of FIG. 6, "M" is a marker, lanes 1 to 4 are the samples below, and "PC" stands for positive control, which is a sample mixed with an equal amount of PBS instead of the Bacillus subtilis natto culture extract. 1: Sample containing Bacillus subtilis culture extract No. 2 2: Sample containing Bacillus subtilis culture extract No. 4 3: Sample containing Bacillus subtilis culture extract No. 1 4: Sample containing Bacillus subtilis culture extract No. 3

[0072] In addition, in the diagram on the right side of FIG. 6, "M" is a marker, and 5-6 are the following samples. 5: Sample containing Bacillus subtilis natto culture extract No. 4 6: Sample containing Bacillus subtilis culture extract No. 3

[0073] As shown in FIG. 6, it was revealed that the SARS-coronavirus-2 spike protein was fragmented into multiple fragments in the samples mixed with Bacillus subtilis Natto Culture Extract No. 2 and Bacillus subtilis Natto Culture Extract No. 4. Based on these results and previous results using bovine herpesvirus, specifically, the fact that antiviral activity is lost upon heating at 100°C for 10 minutes and that the optimal reaction temperature is around 37°C, it is inferred that the SARS-coronavirus-2 spike protein is fragmented by the protease contained in the Bacillus subtilis natto culture extract.

[0074] [Example 2] (Confirmation test of the antiviral effect of Bacillus subtilis natto culture extract using Bacillus subtilis natto with specific proteases knocked out against SARS coronavirus-2) Next, in order to identify which of the proteases contained in the natto culture extract has antiviral activity against SARS-coronavirus-2, we first investigated the cleavage effect of the natto culture extract on the spike protein of SARS-coronavirus-2 using a natto strain in which at least one of the main proteases, alkaline protease (aprE) and neutral protease (nprE), known to be contained in natto, has been knocked out.

[0075] 1. Preparation of Bacillus subtilis natto culture extract The following strains were incubated at 1 × 10 3 The bacteria were inoculated into 40 g of boiled soybeans so that the number of bacteria was 40 cells / g, and the mixture was incubated at 42° C. for 18 hours to prepare natto. Strain No. 1: Parent strain A Strain No. 2: Parent strain B Strain No. 3: aprE knockout (ΔaprE) strain of parent strain A Strain No. 4: nprE knockout (ΔnprE) strain of parent strain A Strain No. 5: aprE and nprE knockout (ΔaprEΔnprE) strain of parent strain A Strain No. 6: aprE knockout (ΔaprE) strain of parent strain B Strain No. 7: aprE and nprE knockout (ΔaprEΔnprE) strain of parent strain B

[0076] After stirring 10 g of the obtained natto with a glass rod for 5 minutes, 10 mL of phosphate buffered saline (PBS) was added and mixed by inverting 50 times at room temperature (about 25°C). After mixing, centrifugation (3500 rpm, 10 minutes) was performed to obtain a supernatant. The obtained supernatant was then filtered with a φ0.45 filter to obtain a natto liquid. Each natto liquid was used as a natto bacteria culture extract.

[0077] 2. Test to confirm the ability of Bacillus subtilis culture extract to decompose the SARS-coronavirus-2 spike protein 2 μL (0.25 mg / mL) of a solution containing SARS coronavirus-2 spike protein (SARS-CoV-2 (2019-nCoV) Spike RBD-mFc Recombiant Protein (HPLC-verified), Sino Biological, Cat No. 40592-V05H) was mixed with 2 μL of each of the natto bacteria culture extracts prepared in "1." above, and incubated at 37°C for 1 hour. In addition, 2 μL (0.25 mg / mL) of the solution containing the SARS coronavirus-2 spike protein, 2 μL each of the Bacillus subtilis Natto culture extract No. 2 or Bacillus subtilis Natto culture extract No. 4 prepared in “1.” of Example 1, and a protease inhibitor (Halt) at a concentration 10 times the recommended concentration were mixed. TMProtease Inhibitor Cocktail, EDTA-free (100x), Thermo Fisher Scientific KK, Tokyo, Japan) (0.4 μL) was mixed and incubated at 37° C. for 1 hour. As a positive control, a sample was prepared by mixing 2 μL (0.25 mg / mL) of the solution containing the SARS coronavirus-2 spike protein, 2 μL of PBS, and 0.4 μL of the protease inhibitor at a concentration 10 times the recommended concentration, or 2 μL (0.25 mg / mL) of the solution containing the SARS coronavirus-2 spike protein and 2 μL of PBS, and similarly incubated at 37° C. for 1 hour.

[0078] Next, 3 μL of sample buffer (SB) (Sample Buffer Solution, FUJIFILM, 196-16142) was added to each mixture using each Bacillus subtilis culture extract prepared in "1." above, and the mixture was heated at 95° C. for 10 minutes to prepare samples for Western blotting. In addition, for each mixed solution using the Bacillus subtilis culture extract or PBS prepared in "1." of Example 1, 3 μL of sample buffer (SB) (Sample Buffer Solution, manufactured by FUJIFILM, 196-16142) was added, and the mixture was heated at 100° C. for 10 minutes to prepare a sample for Western blotting.

[0079] Next, the prepared samples were subjected to SDS-PAGE electrophoresis by a known method, and then stained with Coomassie brilliant blue (CBB). Western blotting was performed using a human anti-SARS coronavirus-2 spike protein monoclonal antibody (SARS-CoV-2 Spike Antibody (D005), Novus Biologicals, Cat No. NBP2-90989) as the primary antibody and a goat anti-Human IgG antibody (Proteintech, Cat No. SA00001-17) as the secondary antibody. The CBB staining image is shown in the upper part of Figure 7, and the results of Western blotting are shown in the lower part. In Figure 7, "M" is a marker, and lanes 1 to 11 are the following samples. 1: Sample containing natto culture extract using strain No. 1 (parent strain A) 2: Sample containing natto culture extract using strain No. 2 (parent strain B) 3: Sample containing natto culture extract using strain No. 3 (ΔaprE strain of parent strain A) 4: Sample containing natto culture extract using strain No. 4 (ΔnprE strain of parent strain A) 5. Sample containing natto culture extract using strain No. 5 (ΔaprEΔnprE strain of parent strain A) 6: Sample containing Bacillus subtilis natto culture extract using strain No. 6 (parent strain B aprE knockout (ΔaprE) strain) 7: Sample containing natto culture extract using strain No. 7 (ΔaprEΔnprE strain of parent strain B) 8: Sample containing Bacillus subtilis natto culture extract No. 2 and protease inhibitor of Example 1 9: Sample containing Bacillus subtilis natto culture extract No. 4 of Example 1 and protease inhibitor 10: Sample containing PBS and protease inhibitors 11: PBS-containing sample

[0080] As shown in Figure 7, the band of the SARS-coronavirus-2 spike protein disappeared in all samples except for the positive control PBS, the protease inhibitor-containing sample, and the PBS-containing sample. This result indicates that the cleavage of the SARS-coronavirus-2 spike protein by the proteases contained in the Bacillus subtilis natto culture extract is likely not due to aprE and nprE, which are the main proteases contained in the Bacillus subtilis natto culture extract.

[0081] [Reference example 6] (Examination of proteases with antiviral activity using bovine herpesvirus) In order to confirm that serine protease is an active ingredient having antiviral activity contained in the Bacillus subtilis natto culture extract, a plaque assay was performed using bovine herpesvirus in the presence of various protease inhibitors.

[0082] 1. Preparation of Bacillus subtilis natto culture extract dilutions The Bacillus subtilis natto culture extract No. 4 of Reference Example 1 was diluted 10-fold with PBS to prepare a solution.

[0083] 2. Comparative test of antiviral effect of diluted extract of Bacillus subtilis culture against bovine herpesvirus Next, 80 μL of a 10-fold diluted solution of the Bacillus subtilis natto culture extract, 1 μL of a solution containing each protease inhibitor (1-fold concentration), 19 μL of PBS, and 10 μL of a solution containing bovine herpesvirus 1 (IBR Ishikawa (bovine / Japan / 1988)) were mixed and incubated for 1 hour at 37° C. Next, the antiviral effects of the diluted solutions of the Bacillus subtilis natto culture extract against bovine herpesvirus were compared in the same manner as in “2.” of Reference Example 1, except that the 10-fold diluted solution of the Bacillus subtilis natto culture extract, the mixed solution of the inhibitor and the virus were used. As positive controls (PC), 80μL of PBS, 20μL of a solution containing a specific protease inhibitor (20x concentration), and 100μL of a mixed solution containing bovine herpesvirus 1 (IBR Ishikawa (bovine / Japan / 1988)) were incubated at 37℃ for 1 hour, and then 300μL of the solution diluted 10,000 times was dropped (per well of a 12-well plate) (sample in the upper row of FIG. 8), and 80μL of PBS, 10x dilution of Bacillus subtilis natto culture extract, and 100μL of a mixed solution containing bovine herpesvirus 1 (IBR Ishikawa (bovine / Japan / 1988)) were incubated at 37℃ for 1 hour, and then 300μL of the solution diluted 10,000 times was dropped (per well of a 12-well plate) (sample on the far right of the lower row of FIG. 8) were also prepared. The results are shown in FIG. 8.

[0084] (Type of protease inhibitor) Aprotinin: serine protease (Cayman Chemical, Michigan USA, CAT No. 14716) AEBSF.HCl: serine protease (AdipoGen Life Sciences, Inc., San Diego, USA, CAT No. AG-CR1-6well3610-M050) Pepstatin A: Aspartic protease (Merck KGaA, Darmstadt, Germany, Cat No. P5318-5MG) Leupeptin: serine and cysteine ​​protease (PEPTIDE INSTITUTE, INC. Osaka, Japan, Cat No. 336-40413) E-64: Cysteine ​​protease (Cayman Chemical, Cat No. 10007963) Bestatin.HCl: Aminopeptidase (Sigma-Aldrich, Tokyo, Japan, Cat No. B8385-1MG) Inhibitor cocktail: a mixture of the above protease inhibitors, manufactured by Thermo Fisher Scientific, product name "Halt Protease Inhibitor Cocktail", Aprotinin concentration in the original solution: 80 μM, AEBSF.HCl concentration: 100 mM, Pepstatin A concentration: 1 mM, Leupeptin concentration: 2 mM, E-64 concentration: 1.5 mM, Bestatin.HCl concentration: 5 mM *All protease inhibitors are EDTA-free. In addition, except for the "Inhibitor cocktail," the powdered products were diluted to match the concentrations of each protease in the inhibitor cocktail stock solution.

[0085] As shown in Figure 8, in the sample containing the serine protease AEBSF.HCl and the inhibitor cocktail, plaques appeared and antiviral activity was lost. This suggests that, among the proteases contained in the Bacillus subtilis natto culture extract, serine proteases are likely to suppress viral cell infection. Incidentally, aprotinin is also a serine protease, but since it was prepared on a different day than the time of use, it did not work well as an inhibitor and was unable to inhibit the protease contained in the Bacillus subtilis natto culture extract, which is presumably why no plaque formation was observed.

[0086] [Example 3] (Comparative study of the antiviral effect of heated or unheated natto culture extract against SARS coronavirus-2) Next, we investigated whether the antiviral activity of Bacillus subtilis natto culture extract was observed in a test system using SARS coronavirus-2.

[0087] 1. Preparation of Bacillus subtilis natto culture extract The following two types of Bacillus subtilis natto culture extracts were obtained by the same method as in "1." of Reference Example 1. Natto Bacillus Culture Extract No.1: Enzyme treatment +, heat treatment + Natto Bacillus Culture Extract No.2: Enzyme treatment +, heat treatment - Natto Bacillus Culture Extract No.3: Enzyme treatment -, Heat treatment + Natto Bacillus Culture Extract No.4: Enzyme treatment, heat treatment

[0088] 2. Comparative study of the antiviral effect of Bacillus subtilis natto culture extract against SARS coronavirus-2 Vero-TMPRSS2 cells were seeded on a 6-well plate and pre-cultured in MEM (High-glucose) medium (2 w / v% FBS, 1 v / v% penicillin-streptomycin solution) to 100% confluence. Then, 90 μL of Bacillus subtilis natto culture extract and SARS coronavirus-2 AS01090-2 (SARS-CoV-2 / Hu / DP / Kng / 19-027, LC528233, 2.0 × 10 6 PFU) was mixed with 10 μL of a solution containing 100 μL of natto extract and the virus was incubated at 37° C. for 1 hour. Next, the mixed solution of the natto extract and the virus was appropriately diluted with FBS-free MEM according to the titer of the virus and inoculated onto Vero-TMPRSS2 cells at 500 μL / well. The Vero-TMPRSS2 cells were washed once with PBS before inoculation, and incubated at 37° C. for 1 hour after inoculation. After the incubation, the cell surface was washed once with PBS, and then 1 w / v% methylcellulose medium was overlaid and cultured at 37° C. for 3 days. The results are shown in FIG. 9. In FIG. 9, T1 to T12 are samples with the compositions shown in Table 1 below.

[0089] [Table 1]

[0090] As shown in FIG. 9, in the samples (T8, T9, T11, and T12) using the Bacillus subtilis natto culture extract that had not been subjected to heat treatment, no plaque formation was observed, and significant antiviral activity was confirmed.

[0091] From the above, it was inferred that the proteases (especially serine proteases) contained in the natto bacteria culture extract cleave and degrade the spike protein of SARS-coronavirus-2, thereby losing the ability of SARS-coronavirus-2 to infect cells. [Industrial Applicability]

[0092] According to the anti-SARS-coronavirus-2 composition and the method for producing the same of the present embodiment, it is possible to provide an anti-SARS-coronavirus-2 composition having antiviral activity against SARS-coronavirus-2 and a method for producing the same. [Accession number]

[0093] "Bacillus subtilis TTCC903 strain" was deposited on February 2, 2007 with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number FERM P-21173.

[0094] "Bacillus subtilis TTCC940 strain" was deposited on July 23, 2007 with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number FERM P-21324.

[0095] "Bacillus subtilis TTCC904-2 strain" was deposited on September 9, 2013 at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number NITE P-01705.

[0096] "Bacillus subtilis TTCC2221 strain" was deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on November 30, 2016 under the accession number NITE P-02380.

Claims

1. Contains natto bacteria culture extract, The anti-SARS coronavirus-2 composition, wherein the natto bacteria is Bacillus subtilis TTCC903 strain (accession number FERM P-21173).

2. The anti-SARS coronavirus-2 composition described in claim 1, which is a dry powder.

3. A method for producing the anti-SARS coronavirus-2 composition according to claim 1 or 2, comprising: A liquid culture step of culturing the Bacillus subtilis natto in a liquid medium; A culture supernatant separation step of separating the culture supernatant of the Bacillus subtilis natto from the culture liquid after the liquid culture step; or A solid culture step of adding the Bacillus subtilis natto to beans and culturing them in a solid state; A liquid component separation step of adding an extraction solvent to the culture after the solid culture step, and then performing solid-liquid separation to separate the liquid component; A manufacturing method comprising:

4. The manufacturing method described in claim 3, wherein the beans are steamed beans.

5. The method according to claim 3, further comprising, after the liquid component separation step, an enzyme treatment step of adding a γ-polyglutamic acid degrading enzyme to the liquid component to perform an enzyme treatment.

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