KR20170134843A - Universal Virus Binding Protein and Method for Preparing the same - Google Patents

Abstract

The present invention relates to a method of preparing a novel lectin protein by using a biological method, and has invented a biologically converted novel lectin compound by using a fermentation strain, Bacillus Subtilis Natto. Specifically, the present invention has simplified the production step and the manipulation method of the pre-existing extraction process of lectin compounds using a chemical treatment method, and has verified that fermentation products produced according to an optimization method are novel compositions having different forms originated from ConA by using a mass spectrometer. These novel compositions were found to have a stronger affinity to food poisoning-causing virus than commercial lectin proteins, and were found to be superior in terms of neutralizing capacity against virus compared to commercial lectin proteins. Although the pre-existing jack bean-originated lectin protein is known for causing hepatoxicity due to excessive secretion of inflammatory cytokines, a jack bean-originated novel composition has toxicity decreased by 9% at concentration of 1 g/ml, and by 8% at 10 g/ml, thus demonstrating in vitro that hepatoxicity caused by ConA is attenuated by a universal virus-binding protein. Accordingly, the present invention provides a universal virus-binding protein which has strong affinity to food poisoning-causing virus, and attenuates toxicity to hepatocytes; and a method of producing the same through fermentation and extraction.

Description

{Universal Virus Binding Protein and Method for Preparing the Same}

The present invention relates to a generalized viral capture protein and a method for producing the same.

A case in which a harmful virus such as norovirus is detected in a agricultural sample including fruit vegetables has been reported at home and abroad. If you eat foods that do not contain virus-infected food or drink contaminated ground water with drinking water, the virus will infect the human body and cause illness. Although the hygiene status of the modern urban environment has been improved, the number of cases of norovirus infection, which is the cause of food poisoning, has been increasing recently. Centers for Disease Control and Prevention has been developed for the prevention of norovirus infection Reported a total of 348 outbreaks from 1996 to 2000. In order to provide safe food to consumers and to prevent social loss caused by virus infection, it is necessary to detect virus at high speed from the production site of agricultural and marine products and to develop and manufacture source material capable of capturing viruses in various foods and drinking water Simplification of method Development of technology is urgent.

Lectins are carbohydrate binding proteins that bind specifically to monosaccharides or oligosaccharides and are proteins that neutralize and capture viral infections by binding to glycoproteins on the surface of bacteria and viruses. Recently, attempts have been made to inhibit and neutralize viruses using carbohydrate-binding agents, that is, lectins, which bind to viruses. For example, anti-HIV lectins Cyanovirin-N and banlec, anti-IAV lectin ESA-2, anti-HCV lectin Galanthus nivalis agglutinin (GNA) envelope protein N-linked oligosaccharides, inhibiting infection and transmission, and can also be potential candidates for bactericides [5].

Conanavalin A (ConA), derived from soybean, is a protein belonging to the lectin family, which binds to saccharides such as mannose or glucose at the monosaccharide binding site [6]. ConA in physiological conditions is a tetramer and binds selectively to cell surface glycoproteins, including alpha-mannopyranosyl and alpha-glucopyranosyl residues. These features are widely used in biology and biomedicine and are often used as a binding agent for pathogen-physiological reactions, including Dengue virus (DENV), Hepatitis C Virus (HCV), Herpes A coat protein virus such as Herpes Virus (HSV), Human Immunodeficiency Virus (HIV), Influenza A Virus (IAV), murine RNA tumor virus And has recently been reported to have a strong binding capacity with Norovirus, a non-coat protein virus [7-13].

The lectin protein extraction process involves protein elution from solubilized lysate, protein precipitation with various concentrations of ammonium sulphate, lyophilization followed by centrifugation, and finally plant-derived lectin production [17]. Such a chemical method has a disadvantage in that the step is complicated, and it is costly and time consuming. In order to extract lectins capable of capturing viruses, it is necessary to simplify the complex extraction process.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

 Boyd M. R. et al. "Discovery of cyanovirin-N, a novel human immunodeficiency virus-activating protein that binds to viral surface envelope glycoprotein gp120: potential applications to microbicide development" Antimicrob. Agents Chemother. 41, 15211530. 1997.  Swanson MD et al. "A lectin isolated from bananas is a potent inhibitor of HIV replication" J Biol Chem. 19: 285 (12): 8646-55. 2010  Sato Y. et al. "Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin" Mar Drugs. 29; 13 (6): 3454-65. 2015  Lauret Izquierdo et al. "Hepatitis C Virus Resistance to Carbohydrate-Binding Agents" PLOS ONE | DOI: 10.1371 / journal.pone.0149064. 2016.  Balzarini J. "Carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses?" Antivir Chem Chemother. 18 (1): 1-11. 2007.  Remy Loris et al. "Legume lectin structure" Biochimica et Biophysica Acta 1383: 936 1998.  Pereira et al. "Binding of Dengue Virus Particles and Dengue Proteins onto Solid Surfaces." ACS applied materials & interfaces 2.9 (2010): 2602-2610.  Lei et al. "Lectin of Concanavalin as an anti-hepatoma therapeutic agent." Journal of biomedical science 16.1 (2009): 1-12.  Izquierdo et al. "Hepatitis C Virus Resistance to Carbohydrate-Binding Agents." PloS one 11.2 (2016): e0149064.  Ito et al. "Inactivation of herpes simplex virus by concanavalin A." Journal of virology 13.6 (1974): 1312-1318.  Botos et al. "Proteins that bind high-mannose sugars of the HIV envelope." Progress in biophysics and molecular biology 88.2 (2005): 233-282.  Klein et al. "Location of ferritin-labeled concanavalin binding to influenza virus and tumor cell surfaces." Journal of virology 10.4 (1972): 844-854.  Calafat et al. "Binding of Concanavalin to the envelope of two murine RNA tumor viruses." Journal of General Virology 14.1 (1972): 103-106.  Isolation of soybean agglutinin (SBA) from soy meal. Journal of Chemical Education, 1982, 59.11: 977

The present inventors have sought to improve the complex and costly drawbacks of the conventional lectin extraction method. As a result, a method of extracting viral capture protein by simplifying the step of producing a koji fermented product by using a fermentation strain was proposed, and the present invention was completed by confirming the antiviral activity of the viral capture protein extracted by the above method.

Accordingly, an object of the present invention is to provide a method for producing a virus-captured protein.

Another object of the present invention is to provide a viral capture protein.

It is another object of the present invention to provide an antiviral composition.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for producing a viral capture protein comprising the steps of:

(a) inoculating a fermentation strain into a culture medium containing soybean meal or soybean extract;

(b) culturing the resulting product of step (a) to produce a soybean fermented product; And

(c) separating the virus-capturing protein from the soybean fermentation product.

The present inventors have sought to improve the complex and costly drawbacks of the conventional lectin extraction method. As a result, a method of extracting viral capture protein by simplifying the step of producing a soybean fermented product using a fermentation strain was proposed, and the antiviral activity of the viral capture protein extracted by the above method was confirmed.

The present invention is designed to easily produce a new protein because its amino acid sequence is different from that of a native lectin protein bound to a virus through a specific fermentation process, And the fermentation process designed to simplify the lectin protein purification method is intended to provide a commercial advantage of lectin production.

The term " lectin " as used herein is a carbohydrate-binding protein, and concanavalin A is mannose or glucose-binding lectin, which is one of the main lectins. In the present specification, 'konkanavalin A' has the same meaning as 'lectin'.

The virus-trapping protein of the present invention has general utility as a virus-binding protein.

The viral capture protein of the present invention is a virus-binding protein and has universal utility for non-enveloped viruses and enveloped viruses.

According to an embodiment of the present invention, the non-coat protein virus is a norovirus or hepatitis A virus.

According to another embodiment of the present invention, the envelope protein virus is selected from the group consisting of dengue virus, hepatitis C virus, herpes virus, human immunodeficiency virus, influenza A virus, murine RNA tumor virus, viral hemorrhagic septicemia virus (VHSV) (VSV), Ebola virus, Zicca virus, MERS Corona virus and Severe acute respiratory syndrome (SARS).

The method of the present invention will be described step by step.

Step (a): Inoculation with the strain of bacteria

First, a fermentation strain is inoculated into a culture medium containing soybean powder or soybean extract.

The bean pulverizer includes beans produced by various processes. For example, beans may be pulverized in various states such as pulverized state, homogenized state, and mash state after processing such as vacuum distillation, freeze drying, or spray drying.

According to one embodiment of the present invention, the bean pulverizer is pulverized or homogenized after drying.

According to another embodiment of the present invention, the bean pulverized product is pulverized after lyophilization.

The soybean extract used in the method for producing the virus-trapping protein of the present invention may be various extracting solvents when soybean is obtained by treating the extracting solvent. Preferably, a polar solvent or a non-polar solvent can be used. Suitable polar solvents are (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, 1-pentanol, Or ethylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO) and (v) dimethyl sulfoxide (DMSO). Suitable nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- But are not limited to, pentane, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2- chloropropane, toluene, 1- chloropropane, chlorobenzene, benzene, diethyl ether, diethylsulfide, Methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF.

More preferably, the extraction solvent used in the present invention is (a) water, (b) anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.) (E) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) hexane and (j) diethyl ether. . Most preferably, the extract of the present invention is obtained by treating soybean with water, ethanol or a combination thereof.

As used herein, the term " extract " means that it is used in the art as a crude extract as described above, but broadly includes fractions obtained by further fractionating the extract. That is, not only the soybean extract obtained by using the above-mentioned extraction solvent, but also the soybean extract obtained by further applying the purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, and a separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) The fraction obtained by the purification method is also included in the soybean extract of the present invention.

The soybean extract used in the present invention may be prepared in powder form by an additional process such as vacuum distillation and freeze drying or spray drying.

According to one embodiment of the present invention, the soybean is Carnavalia ensiformis , Glycine max or Canavalia lineata .

According to an embodiment of the present invention, the culture medium comprises 1 to 20% by mass of a bean pulverizer or a soybean extract.

According to another embodiment of the present invention, the culture medium comprises 1 to 17 mass%, 1 to 14 mass%, 1 to 11 mass%, 1 to 8 mass%, or 2 to 8 mass% of soybean pulverizer or soybean curd extract do.

A variety of solutions can be used to prepare the culture medium containing the soybean pulverizer or the soybean extract. The solution may be diluted in a bacterial culture medium (for example, LB broth miller medium, TSB medium, Nutrient broth medium, BHI medium, etc.), buffer (e.g., Tris buffer, HEPES buffer, etc.] and distilled water.

According to one embodiment of the present invention, the culture medium is a bacterial culture medium.

According to another embodiment of the present invention, the culture medium is an LB broth miller culture medium.

A fermentation strain is inoculated in a culture medium containing the suspension of the soybean pulverizer or the soybean extract.

According to one embodiment, the zymogen that Lactobacillus bacteria (Lactobacillus), A flow Pocono stock (Leuconostoc) genus Bacillus (Bacillus), An by Sela (Weissella) in fermentation one selected from the group consisting of yeast or more Lt; / RTI >

According to another embodiment of the present invention, the fermenting bacteria may be selected from the group consisting of Lactobacillus brevis (KACC 14481), Lactobacillus buchneri (ATCC 4005), Leuconostoc mesenteroides (KCTC 3505), Bacillus subtilis Bacillus subtilis , and Bacillus subtilis < RTI ID = 0.0 > Natto . ≪ / RTI >

Step (b): Preparation of soybean fermentation product

Next, the resultant product of step (a) is cultured to produce a soybean fermented product.

According to the present invention, the above culture is cultured at a culture temperature of 25-38 캜 for a culture time of 2-15 days to prepare a soybean fermented product.

According to one embodiment of the present invention, the incubation temperature is 25-37 ° C, 25-36 ° C, 25-35 ° C, 26-35 ° C, 27-35 ° C, 28-35 ° C, 29-35 ° C, Lt; / RTI >

As demonstrated in the following examples, the incubation temperature is 33 ° C.

According to one embodiment of the present invention, the incubation time is 2-14 days, 2-13 days, 2-13 days, 2-12 days, 2-11 days, 2-10 days, 2-9 days or 3- 9 days.

Step (c): Isolation of viral capture protein

Next, the virus-capturing protein is separated from the soybean fermentation product.

Methods for isolating viral capture proteins from soybean fermentations of the present invention can be separated by conventional methods including, for example, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Further, it is separable by a variety of methods known in the art including chromatography (e.g., ion exchange, affinity, hydrophobicity and size exclusion), electrophoresis, SDS-PAGE.

According to the present invention, the concanavalin A present in the soybean has a molecular weight of 48 kDa in the form of a dimer.

The viral capture protein in the fermented soybean of the present invention has a molecular weight of 25 kDa or less in the form of the dimer separated into monomers.

According to another aspect of the present invention, the present invention provides a viral capture protein produced by the above method.

Since the viral capture protein of the present invention is produced by the above production method, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

According to still another aspect of the present invention, there is provided an antiviral composition comprising a fermented product of soybean pulverizer or soybean extract as an active ingredient containing an active ingredient.

The fermented product of the soybean pulverizer or soybean extract is a fermented product produced by a fermentation strain.

According to one embodiment, the zymogen that Lactobacillus bacteria (Lactobacillus), A flow Pocono stock (Leuconostoc) genus Bacillus (Bacillus), An by Sela (Weissella) in fermentation one selected from the group consisting of yeast or more Lt; / RTI >

According to another embodiment of the present invention, the fermenting bacteria may be selected from the group consisting of Lactobacillus brevis (KACC 14481), Lactobacillus buchneri (ATCC 4005), Leuconostoc mesenteroides (KCTC 3505), Bacillus subtilis Bacillus subtilis , and Bacillus subtilis < RTI ID = 0.0 > Natto . ≪ / RTI >

The antiviral composition has a high binding force against viruses.

According to one embodiment of the present invention, the virus is Norovirus or Hepatitis A virus.

As demonstrated in the following examples, the fermented product of the soybean pulverizer or soybean extract has a higher binding force than conventionalconcanavalin A.

The fermented product of the soybean curd pulverized product or the soybean curd extract of the present invention has a high binding force against viruses and thus can be applied to various uses using the same. For example, the fermentation product can be used for virus enrichment. The fermentation product may be concentrated in a column for concentrating the virus to induce binding with the virus. In addition, such a binding force with viruses can be effectively applied to virus detection, diagnosis and sensors, and furthermore, to neutralization and elimination of viruses (for example, disinfectants).

The binding between the fermented product and the virus is characterized by being non-antibody.

The antiviral composition of the present invention can be prepared by mixing conventionalconcanavalin A And exhibit weakened liver toxicity.

As demonstrated in the following examples, the fermented product of the soybean pulverizer or soybean extract exhibits lower cytotoxicity to hepatocytes than conventionalconcanavalin A.

According to one embodiment of the present invention, the antiviral composition is a pharmaceutical composition.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of a fermented product of the soybean curd pulp or soybean curd extract of the present invention described above; And (b) a pharmaceutically acceptable carrier. As used herein, the term " pharmaceutically effective amount " means an amount sufficient to achieve efficacy or activity of a fermentation product of the soybean pulverizer or soybean extract described above.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably administered orally.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.0001-100 mg / kg on an adult basis.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to one embodiment of the present invention, the antiviral composition is a food composition.

The composition of the present invention can be provided as a food composition. When the antiviral composition of the present invention is prepared as a food composition, it contains not only the fermented product of the soybean flour or soybean extract as an active ingredient, but also the ingredient normally added during the manufacture of the food, for example, a protein, a carbohydrate , Fats, nutrients, flavoring agents, and flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings. For example, when the food composition of the present invention is prepared as a drink, it may contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract, licorice extract And the like.

The composition for preventing, ameliorating or treating a Th2-mediated immune disease comprising the fermented product of the soybean curd pulp or soybean curd extract of the present invention as an active ingredient can be produced as a health functional food. The health functional food may be any type of food such as a health functional food, a nutritional supplement, a nutrient, a pharmafood, a health supplement, a nutraceutical, a designer food, a food additive, etc. Preferably dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums and ice cream, various soups, drinks, tea, drinks, alcoholic beverages and vitamins And the like.

The health functional food of the present invention includes components that are ordinarily added at the time of food production, and includes, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings. In addition to the above-mentioned food, the food of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), dietary components, synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.) , Alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like.

According to one embodiment of the present invention, the antiviral composition is a composition for feed addition.

The composition of the present invention can be prepared with a composition for feed addition.

The antiviral composition comprising the fermented product of the soybean curd or the soybean curd extract of the present invention as an active ingredient containing the active ingredient can be added to the feed to enhance the antiviral activity of the animal.

The feed additive composition of the present invention may contain phosphate or polyphenol such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, or organic acids such as sodium phosphate, potassium phosphate, acid pyrophosphate or polyphosphate (polymerized phosphate), catechin, , Natural antioxidants such as alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid may be further included.

The composition for feed addition of the present invention may contain additives such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes and other microorganism formulations, nutritional supplements, digestion and absorption enhancers, Additives such as prophylactic agents may be included.

The composition for feed addition of the present invention may be in the form of a dry or liquid preparation, and may contain an excipient for feed addition. Examples of excipients for feed addition include, but are not limited to, zeolite, corn or rice bran.

The composition for feeding a livestock feed of the present invention may be administered to an animal alone or in combination with other feed additives in an edible carrier, and usually a single daily intake or a divided daily intake can be used as is well known in the art .

The animal in which the feed additive composition of the present invention can be used includes animals such as edible wels, cows, calves, pigs, pigs, sheep, goats, horses, rabbits, dogs and cats, domestic chickens, , Poultry such as goose, turkey, quail, small bird, and the like.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for producing a universal viral capture protein by a biological method that is simpler than a conventional chemical treatment method.

(b) The present invention provides a fermented product of soybean curd or soybean curd extract or a universal virus-trapping protein and an antiviral composition containing the same as an effective ingredient, with reduced lectin toxicity.

(c) By using a soybean fermented product that is a non-antibody substance that can replace a virus antibody, it can be utilized for virus neutralization reaction. Further, by using the above-mentioned composition that is easy to manufacture and designed, it is possible to have a manufacturing cost by using the composition as a material for a sensor for detecting a virus and a virus for detecting a virus. In addition, the composition having the virus neutralizing ability can be actively utilized as a composition of a virus disinfectant and an antiviral agent.

FIG. 1 shows the result of comparison between a conventional chemical treatment method and a fermentation product preparation method using a fermentation strain of the biological treatment method of the present invention.
Fig. 2 shows the result of comparing fermentation patterns of fermented soybean curd from 0 to 7 days.
Fig. 3 shows the results of comparing the fermentation patterns on the 0th day and the 4th day of the soybean fermented product.
Figure 4 shows the results for mass value information for peptides identified from soybean fermentations.
FIG. 5 shows the results of relative comparisons of the binding strength between soybean curd extract and food poisoning virus.
Figure 6 shows the result of comparative analysis of the liver toxicity jakdukong extract in vitro (in vitro) level.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Optimization of fermentation production using a strain

A method for extracting lectin using a conventional ammonium sulfate (Sinha, Sharmistha, et al., "Unfolding studies on soybean agglutinin and concanavalin a tetramers: a comparative account." Biophysical journal 88.2 (2005): 1300-1310 .; The development of the modified soybean agglutinin (SBA) from soybean meal, Journal of Chemical Education, 1982, 59.11: 977-) takes 10 days, and the extraction step and the manipulation method are complicated and costly have. To solve this problem, we have developed a method for extracting lectin protein by simplifying the extraction step through the fermentation of Canavalia gladiata using the microorganism strain Bacillus subtilis Natto . As shown in Figure 1, 200 ml (1%) of LB broth miller (LBL407.500, Bioshop, Canada) containing tryptone, yeast extract and sodium chloride, (V / v) of the medium was inoculated into the medium, and then the mixture was stirred at 33 ° C for 7 days to effect fermentation . The fermented supernatant was centrifuged at 12,000 rpm for 20 minutes at 4 ° C to collect the supernatant, and the residue was fractionated using a filter paper (Whatman filter faper 4, 25 nm). The filtrate was filtered through a 10 kDa membrane filter (Sartorius 7554 -95, MASTERFLEX, USA), and the lyophilization was carried out by concentrating the fermentation broth 2.4 times. The solid content thus obtained was subjected to SDS-PAGE as a final product of soybean fermentation, and the molecular weight of the protein was measured.

Example 2: Monitoring the production process of soybean fermented product using protein molecular weight pattern

From 1 to 7 days of fermentation of soybean fermented soybeans, 1 mg of each sample was taken, dissolved in 1 ml of distilled water, and subjected to SDS-PAGE analysis on a 15% polyacrylamide gel. As shown in FIG. 2, the main band was confirmed at 48 kDa molecular weight on the 0th day of fermentation, but the band of 48 kDa molecular weight was decreased from the first day of fermentation, and the band of ConA size of 25 kDa or less was increased. More pronounced at molecular weights below 25 kDa.

Example 3: Peptide Coverage Analysis of Soybean Fermented by Mass Spectrometry

As shown in FIG. 3, the protein was separated by size using 15% SDS-PAGE. As a result, about 48 kDa protein was present as the main protein in the fermentation at day 0, and fermentation was performed for 4 days using Bacillus subtilis natto Three 20-25 KDa proteins were produced. Through peptide coverage analysis, ConA was confirmed to have specific homology with soybean fermented products. The F2, F3 and F4 bands of the soybean powder (column 2 in FIG. 3) and the soybean koji fermented product (column 3 in FIG. 3) were cut and then in gel digestion to obtain 25 mM ABC in 50% ACN solution And then digested with trypsin gold (Promega, V5280) at 50 mM ABC, followed by desalting using Oasis SPE (Waters, 186001828BA) and performing peptide analysis using nanoUPLC-Synapt G2 si (Waters, USA) Respectively. The results obtained by LC-MS were analyzed using PLGS (ProteinLynx Global Server (version 3.0, PLGS, Waters)). As a result, the CVJB cone or Valine A Jack bean (UniProtKB: CVJB Con A , and 97.5%, respectively. In the case of F2, F3 and F4 bands, CVJB concha and valine A and Jack bean (UniProtKB: CVJB Con A and gi72333) were 81.4%, 53.2% and 62.86% (Table 1).

Band name Access number Description Score The same peptide Sequence Cover (%) F0 gi72333 CVJB Concarne Valine A - jack bean 2389.3 23 97.5 F2 gi72333 CVJB Concarne Valine A - jack bean 730.1 9 81.4 F3 gi72333 CVJB Concarne Valine A - jack bean 629.7 8 53.2 F4 gi72333 CVJB Concarne Valine A - jack bean 1341.8 8 62.86

Example 4. Determination of Protein Mass Value Identified from Fermented Soybean Soybean

In order to measure the precise molecular weight of the proteins isolated from the fermented soybean koji, intect protein mass values were measured for protein concentrates having a molecular weight of 30 KDa or less as confirmed in the main band on the 0th day of fermentation and on the fermentation on the 4th day of fermentation of soybean fermentation Were measured. Molecular weight measurement was carried out using an Ultra Performance Liquid Chromatography (UPLC, ACQUITY UPLC I-Class / SYNAPT G2-S HDMS, Waters) for the analysis of fermentation. The analysis conditions are as follows.

Column (A) (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) were used at a column temperature of 60 ° C, ACQUITY BEH300 C18 (1.7 ㎛ × 2.1 × 50 ㎜) The mass spectrometer was analyzed in an ESI power mode. The analysis conditions were capillary voltage 3.0 kV, cone voltage 30 V, source temperature 120 캜, and scan time 0.5 s. ESI prot 1.0, deconvoluted MW (Da) and std value as shown in Table 2 were set.

In the F2, F3 and F4 bands, 81.4%, 53.2% and 62.86% of the CVJB concha, valine A and jack bean (UniProtKB: CVJB Con A and gi72333) F2 is a monomeric ConA protein predicted to be the protein in the F2 region of FIG. 4 (26213.96 Da), which is confirmed in the determination of the amount of the inactive protein mass, although the exact cleavage site can not be confirmed due to the trypsin recognizing the residue , And the mass value of the F3 band was calculated to be 21175.48 Da, which is the protein (21176 Da) of the F3 region of FIG. 4, which is confirmed upon measurement of the mass of the atactic protein. In the case of the F4 band, the mass value was calculated to be 18796.86 Da, which was predicted to be 18796.8 Da in the F4 region of FIG. 4 (FIG. 4 and Table 2).

Charge (+) Peak (m / z) MW (Da) Deconvoluted MW (Da) F2 29 904.9487 26214.2820 26213.9637 + 0.7529 28 937.2229 26214.0188 27 971.8983 26214.0397 26 1009.2819 26215.1229 25 1049.5328 26213.1215 24 1093.2675 26214.2294 23 1140.7080 26213.1019 22 1192.5895 26214.7943 21 1249.2468 26213.0160 20 1311.7194 26214.2292 19 1380.5923 26212.1028 18 1457.2847 26212.9816 17 1543.1046 26215.64332 F3 28 757.2388 21174.4640 21175.4820 +/- 0.9101 27 785.3299 21176.6929 26 815.4147 21174.5783 25 848.0338 21175.6465 24 883.3274 21175.6670 23 921.6443 21174.6362 22 963.4954 21174.7241 21 1009.4067 21176.3739 20 1059.8358 21176.5572 19 1121.5699 21290.6772 18 1177.6122 21178.8766 17 1246.6261 21175.5087 F4 21 896.0964 18796.8576 18796.8607 ± 0.4439 20 940.8870 18797.5812 19 990.3096 18796.7315 18 1045.2678 18796.6774 17 1106.6982 18796.7344 16 1175.8607 18797.6441 15 1254.1100 18796.5309 14 1343.6151 18796.5002 13 1446.8917 18796.4888 12 1567.3868 18796.5463 11 1709.7998 18796.7104 10 1880.6688 18796.6086 9 2089.5081 18796.5014

Example 5. Measurement of binding force between soybean fermented food and food poisoning virus

Blitz (Forte BIO) instrument was used to measure the binding force between food poisoning virus and soybean fermented product. The biochip used in Blitz was fixed on the chip using AR2G (Amine Reactive), and the binding force with soybean fermented product was measured on the fourth day. The reaction conditions were initial baseline 60 seconds; Custom 150 seconds; Loading 210 seconds; Custom 180 seconds; Baseline 60 seconds; Association 180 seconds; And disassociation of 180 seconds.

As shown in Fig. 5, Norovirus showed commercial value of Con A and 5.776e ^-6 KD, and fermented by soybean fermentation showed a KD value of 5.894e- ⁷ . That is, when the concentration of ConA and the soybean koji fermented at the same concentration were measured, the binding capacity of soybean koji fermented product was better than that of ConA. In addition, the binding strength of hepatitis A virus (HAV) and Con A was 9.264e ^-5 KD, and the hepatitis A virus and soybean fermentation products were measured to have a KD value of 1.133e ^-6 KD. Therefore, it was confirmed that the fermented soybean koji had better binding force against food poisoning virus than Con A.

Example 6. Evaluation of Hepatotoxicity of Soybean Fermented Soybean

HepG2 cells were plated in 96-well plates at 5 × 10 ⁴ cells / well and stabilized by incubation in a 5% CO ₂ incubator at 37 ° C. for 24 hours. ConA and soybean fermented products were treated with each concentration (1100 μg / ml) and cultured in a 5% CO ₂ incubator at 37 ° C. for 48 hours. Microplate reader was prepared by the method described in CCK-8 (Cell counting Kit-8) The absorbance at 450 nm was measured using a VersaMax ELISA Micro plate reader. The cell survival rate of the ConA and soybean fermentation treatment groups was expressed as a percentage with 100% of the cell survival rate of the control group not treated with ConA and the soybean fermented product .

As shown in FIG. 6, when HepG2 cells were treated with ConA at a concentration of 1-100 μg / ml, it was confirmed that the cell viability was 84% at a concentration of 1 μg / ml and the toxicity was very weak at 10 μg / ml ConA treatment decreased the cell survival rate to 74%. In contrast, the cell viability was 93% at the concentration of 1 ㎍ / ㎖ and 88% at the concentration of 10 ㎍ / ㎖, which is the same concentration as that of ConA, where cell viability decreased to less than 80% It was confirmed at the cellular level that the treatment of water alleviated liver toxicity caused by ConA.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (13)

A method for producing a virus-trapping protein comprising the steps of:
(a) inoculating a fermentation strain into a culture medium containing soybean meal or soybean extract; And
(b) culturing the resulting product of step (a) to produce a soybean fermented product; And
(c) separating the virus-captured protein from the soybean fermentation product.
The method of claim 1, wherein the virus is selected from the group consisting of norovirus, hepatitis A virus, dengue virus, hepatitis C virus, hepatitis E virus, herpes virus, human immunodeficiency virus, influenza A virus, murine Viral hemorrhagic septicemia virus (VSV), vesicular stomatitis virus (VSV), Ebola virus, Zicca virus, MERS Corona virus or Severe acute respiratory syndrome (SARS) ≪ / RTI >
The method according to claim 1, wherein the soybean is Canavalia gladiata , Glycine max or Canavalia lineata .
The method according to claim 1, wherein the culture medium of step (a) comprises 1 to 20% by mass of a bean pulverizer or a soybean extract.
The method of claim 1, wherein one selected from the zymogen Lactobacillus bacteria (Lactobacillus), A flow Pocono stock (Leuconostoc) genus Bacillus (Bacillus), An by Sela (Weissella) in the group consisting of a yeast, which in step (a) Or more of the fermentation broth.
6. The method according to claim 5, wherein the step (a) comprises culturing the strain of Lactobacillus brevis (KACC 14481), Lactobacillus buchneri (ATCC 4005), Leuconostoc mesenteroides (KCTC 3505) , Bacillus subtilis and Bacillus subtilis Natto. ≪ Desc / Clms Page number 19 >
The method according to claim 1, wherein the culturing of step (b) is performed for 1-20 days.
A viral capture protein produced by the method of any one of claims 1 to 7.
An antiviral composition comprising, as an active ingredient, a fermented product of Canavalia gladiata pulverizer or soybean extract.
10. The method of claim 9, wherein the virus is Norovirus or Hepatitis A virus.
10. The composition of claim 9, wherein the antiviral composition is a pharmaceutical composition.
10. The composition of claim 9, wherein the antiviral composition is a food composition.
10. The composition of claim 9, wherein the antiviral composition is a feed additive composition.

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