JP2015511634A - Systemic immunity or intestinal immunity reinforcement containing neat soy extract as an active ingredient - Google Patents

Abstract

The present invention relates to a systemic immunity and intestinal immunity reinforcing agent comprising a neat soy ethanol-dissolved extract as an active ingredient, and the immunoreinforcing agent comprising the neat soy ethanol-dissolved extract of the present invention ensures safety. By using the prepared food material, there are no toxicity and side effects, and it is possible to provide a low production cost and convenience of use. Further, the immunoreinforcing agent of the present invention, when orally administered with an immunogen, is used for Th1 and Th2 against not only oral infectious pathogenic microorganisms but also parenteral infectious pathogenic microorganisms, viruses, cancer antigens and general antigens. The reaction can be induced to produce effective specific antibodies (blood IgG and small intestine IgA). [Selection] Figure 10

Description

  The present invention was made under the support of the Rural Development Agency of Korea under issue number PJ008325. The research management organization for the issue is the Rural Development Agency, and the name of the research project is “Next Generation BioGreen 21 Project”, The name of the research project is “Optimization of immune enhancer for hepatitis A vaccine (adjuvant)”, the main organization is Korea Food Research Institute, and the research period is 2011.08.01 to 2012.123.31.

  The present invention was made with the support of the Ministry of Knowledge Economy of the Republic of Korea under issue number E0121501. The research management organization for the subject is the Korea Food Research Institute, and the name of the research project is “Korea Food Research Institute Main "Business", the title of research is "Development research of antiallergic foods using T cell regulatory activity of natural products", the main institution is Korea Food Research Institute, and the research period is 2012.1.01-12012.12. 31.

This patent application is filed with the Korean Patent Application No. 10-2012-0031749 filed with the Korean Patent Office on March 28, 2012 and the Korean Patent Application No. 10- filed with the Korean Patent Office on February 21, 2013. Claims priority to 2013-0018895, the disclosure of which is incorporated herein by reference.
The present invention relates to a systemic immunity or intestinal immunity reinforcing agent comprising a neat soy extract as an active ingredient.

  Considering the stability, there is a problem that the immune effect is reduced during the development of the vaccine, which causes a problem in the production of a vaccine that enhances the effect of the immune response. As a solution to such a problem, an immunosuppressant (adjuvant) that enhances an immune response to an antigen by maintaining it in a small amount of an immunogen quickly and powerfully for a long period of time has been developed.

  Immunosuppressants known to date include inorganic salts (eg, aluminum hydroxide, aluminum phosphate and calcium phosphate), immunostimulants; cytokines (eg, IL-2, IL-12, GM-CSF, IFN-α , IFN-γ and IL-18), biomolecules (eg, CD40, CD154, MHC type 1 invariant chain) and LFA ((lymphocyte function-associated antigen) 3), saponins (eg, QS21), MDP (N-acetyl-muramyl-L-alanine-D-isoglutamine) derivatives, CPG-DNA (CpG oligodeoxynucleotides), LPS (Lipopolysaccharides), PL (Monophosphory Lipid A), polyphosphazenes, fat particles (eg Freund, SAF (Syntex Adjuvant Formation) and MF59); liposomes, virosomes, iscoms, Immuning Stimulator G polylactide-co-glycoids microparticles, poloxamers, viruses (e.g., HBcAg, HCcAg and HBsAg), surface active agents and Freund's immunoreinforcing agents or vibriotoxins (For example, LT 63 y LTR72) and includes a variety of bacterial products such as E. coli enterotoxin (enterotoxins), but is not limited thereto.

  Currently, many immunoreinforcing agents such as aluminum salts, calcium salts, CpG-DNA, LPS, and MPL have been developed and studied. However, there are problems in safety by inducing various side effects. That is, the immunoreinforcing agent should not be toxic in terms of safety other than the effect of immune reinforcement, should be excellent in biodegradability, stable, inexpensive and convenient to use. Don't be. Of the currently used immune reinforcing agents, Freund's adjuvant is the most widely used, but cannot be used for the human body, and aluminum salts can be used for the human body. It falls below adjuvant (refer nonpatent literature 1 and nonpatent literature 2).

  The immune function of the intestinal tract centering on the small intestine is called intestinal immunity, and it enhances the immune function of the newborn through colostrum and gravy or enhances the intestinal immunity function by ingesting lactic acid bacteria. Recently, it has been attracting a lot of attention. Intestinal immunity classified into secondary lymphoid tissue system in primary and secondary lymphoid tissue system exists in the mucosal part of the intestinal tract among mucosal lymphoid organs, and protects the body including IgA reaction in intestinal immune system (See Non-Patent Document 3).

  In addition, CT (Cholera Toxin) and LT (E. coli heat-labile enterotoxin) used as oral immunoreinforcing agents are developments of immunoreinforcing agents that are stable and efficient in their own toxicity. Is necessary (see Non-Patent Document 4).

  Throughout this specification, numerous papers and patent documents are referenced and their citations are displayed. The disclosures of the cited articles and patent documents are hereby incorporated by reference in their entirety to more clearly explain the level of the technical field to which the present invention belongs and the contents of the present invention.

Freunds, J.A. et. al. , Soc. Exp. Biol Med. 37,509 (1937) Gupta, R.A. K. et. al. , Vaccine 11, 293-306 (1993) Bienestock, J.A. et. al. , Immunol. , 41, 249-270 (1980) Kim, S .; H. et. al. , J Plant Biotechnol. , 37, 283-291 (2010)

  The inventors of the present invention have made extensive efforts to develop an oral intestinal immunity reinforcing agent that is excellent in safety but has low production cost and convenience in use. As a result, when the neat soy sauce extract is used as an immunoreinforcing agent, it effectively induces Th1 and Th2 immune responses, in particular, effectively induces Th2 immune responses and induces immunogen-specific immune responses. Thus, the present invention was completed by discovering that vaccine efficacy can be greatly improved.

Therefore, an object of the present invention is to provide a systemic immunity or intestinal immunity reinforcing agent.
Another object of the present invention is to provide a vaccine composition for oral administration.
Yet another object of the present invention is to provide a method for inducing a systemic or intestinal immune response.
Other objects and advantages of the invention will become more apparent from the following detailed description of the invention, the claims and the drawings.

According to one aspect of the present invention, the present invention provides a systemic immunity or intestinal immunity reinforcing agent comprising a neat soy extract as an active ingredient, wherein the neat soy extract is an ethanol-dissolved extract extracted with ethanol ( There is provided a systemic immunity or intestinal immunity reinforcing agent characterized by containing an ethanol-soluble component.
According to another aspect of the present invention, the present invention relates to a method for inducing systemic immunity or intestinal immunity reaction comprising the step of administering to a subject a composition containing an ethanol-soluble component from an ethanol extract of neat soy sauce. I will provide a.

  The inventors of the present invention have made extensive efforts to develop an oral intestinal immunity reinforcing agent that is excellent in safety but has low production cost and convenience in use. As a result, when using a neat soy extract containing ethanol-dissolved components extracted with ethanol as an immunopotentiator, it effectively induces Th1 and Th2 immune responses, particularly effectively induces Th2 immune responses. Thus, it was found that an immunogen-specific immune response can be induced to greatly improve the vaccine efficacy.

  Cheonggukjang used in the present invention is a neat soy sauce such as Bacillus (for example, Bacillus amyloliquefaciens, Bacillus methylotrophicas, Bacillus atrophaeus, Bacillus tyrensis, Bacillus subtilis). And Bacillus methylotropicus) or Koclear (eg, Koclear Salsiccia) is inoculated into steamed soybeans and fermented and ripened, and is a traditional Korean fermented food. Neat soy sauce is similar to other traditional fermented foods in South Korea. Compared to the fact that miso ferments steamed soybeans for 40-60 days using salt, Produced through a short fermentation period of only 2-3 days at about 40 ° C. without salt. Cheongyeon sauce is a food that is ingested daily in the Republic of Korea. In developing it as an immunoreinforcement agent, there are no stability problems and side effects due to the intake, and it is convenient to use and inexpensive. In addition, it has an excellent immune enhancing effect and has characteristics that are very suitable for development as an immune reinforcing agent.

The active ingredient used in the present invention is an ethanol-soluble component extracted by treating ethanol with neat soy sauce (for example, neat soy sauce powder). Therefore, the active ingredient used in the present invention is abbreviated as “Neat soy ethanol-dissolved extract”.
The neat soy ethanol-dissolved extract used in the composition of the present invention is a fraction containing an ethanol-dissolved component that is not a precipitate and is a result obtained by treating an ethanol solvent. For example, if an ethanol solvent is processed into neat soy sauce and centrifuged, an ethanol-dissolved component and a precipitate are formed, and the ethanol extract from which the precipitate has been removed is the most desirable neat soy ethanol of the present invention. It is a dissolved extract.
According to a preferred embodiment of the present invention, the ethanol used for producing the neat soy ethanol-dissolved extract is 50% to 99% ethanol. If the concentration of ethanol used is less than 50%, the ethanol-soluble component cannot be sufficiently dissolved in ethanol. If the ethanol concentration exceeds 99%, the price of the ethanol solvent is too high. However, there is a problem in that the extraction efficiency due to the increase in concentration hardly changes. More preferably, the concentration of ethanol used is 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 99%, 60% to 85%, 60% -80%, 60% -75% or 60% -70%.

  The term “extract” as used herein has a meaning commonly used by those skilled in the art as a crude extract as described above, but broadly speaking, the extract is additionally fractionated. Also includes fractionated fractions. That is, the neat soy ethanol-dissolved extract includes not only those obtained using an ethanol solvent, but also those obtained by additionally applying a purification process thereto. For example, fractions obtained by passing the extract through an ultrafiltration membrane having a certain molecular weight cut-off value, various chromatographies (made for separation by size, charge, hydrophobicity or affinity) Fractions obtained through various additional purification methods such as separation by) are also included in the neat soy ethanol-dissolved extract of the present invention.

  According to a preferred embodiment of the present invention, the soy sauce ethanol-dissolved extract used as an active ingredient in the present invention is an extract from which PGA (poly-γ-glutamic acid) has been removed, and more preferably, An extract from which PGA and Levan have been removed.

  The term “removal” used herein with reference to PGA means a state in which PGA is not substantially detected in a neat soy ethanol-dissolved extract. For example, when a neat soy ethanol-dissolved extract is electrophoresed on a gel and the gel is stained with methylene blue, which is a PGA staining dye, the stained band is not observed with the naked eye. The soy sauce ethanol-dissolved extract can be defined as having PGA removed.

  The term “removal” used herein with reference to leban means a state in which levan is not substantially detected in the neat soy extract. For example, if a neat soy ethanol-dissolved extract is oxidized with periodate, then it is reacted with a Schiff reagent, and red or reddish purple color is not observed with the naked eye. A lysed extract can be defined as levan removed.

  The use of neat soy ethanol-soluble fraction as the most desirable active ingredient in the present invention is an approach that is the opposite of existing prior art as far as we know (to our best knowledge). According to the prior art, the immunopotentiating active ingredient of the neat soy extract is known by poly γ-glutamic acid (PGA) and levan, and in order to obtain such an active ingredient, 70% ethanol is neat. A sample such as soy is processed and the precipitate formed therefrom is further purified to obtain PGA or levan. However, in the present invention, an ethanol-dissolved fraction of neat soy sauce is used as an approach method that is completely opposite to this.

  The neat soy sauce used in the present invention can be produced using various microorganisms, and most preferably produced using Bacillus amyloliquefaciens. As demonstrated in the following examples, when producing neat soy sauce using Bacillus amyloliquefaciens, the enzyme activities of α-amylase, β-amylase and protease in the crude enzyme solution of neat soy sauce In addition to being very excellent, spleen cell proliferation activity is also very excellent.

  As used herein, the term 'immunopotentiator' is a pharmacological or immunological substance that alters the effect of a substance such as a drug or a vaccine, Has no direct effect. Mostly contained in the vaccine composition serves to increase the recipient's immune response to the antigen provided with minimal protection of the infected external material. The immunoreinforcing agent is added to the vaccine to stimulate the immune system's response to target the antigen and does not have epidemics. Immunoreinforcing agents act in a variety of ways to present antigens to the immune system.

  As used herein, the term 'immunogen' is a substance that induces a recipient's immune response and has the same meaning as 'antigen'. Examples of viral pathogen antigens utilized in the present invention include Orthomyxviruses such as influenza virus; retroviruses such as RSV (respiratory synchronous virus), SIV (simian immunodefectivity virus) and HIV; EBV (ErpBus) Herpesviruses; CMV (cytomegalovirus) or HSV (herpes simplex viruses); Lentiviruses such as rabies; Romadoviruses such as poliovirus; Picomovirs such as poliovirus; Including antigens from ruses. More specifically, as examples of viral pathogen antigens, examples of HPV antigens include HPV L1, L2, E6 or E7 proteins; examples of HIV antigens include nef, p24, gp120, gp41, tat, rev, pol, env and gp120 T cell and B cell epitopes (Palker et al, J. Immunol., 142: 3612-3619 (1989)). Examples of HBV surface antigens are described in Wu et al, Proc. Natl. Acad. Sci. USA, 86: 4726-4730 (1989). Examples of rotavirus antigens are VP4 (Mackow et al, Proc. Natl. Acad. Sci., USA, 87: 518-522 (1990)) and VP7 (Green et al, J. Virol., 62: 1819-). 1823 (1988); influenza virus antigens include hemagglutinin (HA) and nucleoprotein; HSV antigens include thymidine kinase (Whitley et al, In: New Generation Vaccines, pages 825-854); The avian influenza virus antigen includes hemagglutinin; the swine cholera virus antigen is the envelope; the foot-and-mouth disease virus antigen is envelop; and the Newcastle virus antigen is HN Hemagglutinin-neuraminidase), including the F (Fusion protein).

  Examples of bacterial pathogen antigens utilized in the present invention include Mycobacterium spp. Helicobacter pylori, Salmonella spp. Shigella spp. , E.C. E. coli, Rickettsia spp. , Listeria spp. Legionella pneumoniae, Pseudomonas spp. Vibrio spp. And an antigen from Borellia burgdorferi. Specifically, examples of bacterial pathogen antigens utilized in the present invention include Shigella sonnei form-1 antigen (Formal et al, Infect. Immun., 34: 746-750 (1981)); cholerae O-antigen (Forrest et al, J. Infect. Dis. 159: 145-146 (1989); E. coli FA / I fimbria antigen (Yamamoto et al, Infect. Immun., 50). : 925-928 (1985)) and non-toxic B subunits of heat-sensitive toxins (Klipstein et al, Infect. Immun., 40: 888-893 (1983)); Bordetella pertussis peractin (Roberts et al. Vacc., 10: 43-48 (1992)); B. pertussis adenylate cyclase-hemolysin (Guiso et al, Micro. Path., 11: (. Fairweather et al, Infect.Immun, 58: 1323-1326 (1990); 23431 (1991)) and fragments C of tetanus toxin Clostridium tetani including).

  Examples of parasitic antigens utilized in the present invention include Plasmodium spp. , Trypanosomes spp. Giardia spp. Boophilus spp. Babesia spp. , Entamoeba spp. Eimeria spp. Laishmania spp. , Schistosome spp. Brugia spp. Fascida spp. , Dirofilaria spp. Wuchereria spp. , And Onchocerea spp. Including antigens of origin. More specifically, examples of parasitic antigens utilized in the present invention include Plasmodium bergerii circucumspozoite antigen and P. aeruginosa. Plasmodium spp. such as the falciparum circusporozoite antigen. Circumsporozoite antigen (Sadoff et al, Sci., 240: 336-337 (1988)); Plasmodium spp. , And galactose-specific lectin from Manemoba histolytica (Mann et al, Proc. Natl. Acad. Acad. Scad. Acad. Scad. Acad. Scad). 88: 3248-3252 (1991)); Leishmania spp. Gp63 (Russellet al, J. Immunol., 140: 1274-1278 (1988)); Brugia malayi paramyosin (Li et al, Mol. Biochem. Parasitol., 49: 315-323 (1991)); and Schistosomams Of triose phosphate isomerase (Shoemaker et al, Proc. Natl. Acad. Sci. USA, 89: 1842-1846 (1992)).

  Examples of cancer antigens utilized in the present invention include prostate specific antigen (Gattuso et al, Human Pathol., 26: 123-126 (1995)), TAG-72 and CEA (carcinoembryonic antigen) (Guadagni et al, Int. J. Biol. Markers, 9: 53-60 (1994)), MAGE-1 and tyrosinase (Coulie et al, J. Immunothera., 14: 104-109 (1993)), p53 (International Publication WO 94/02167). Pamphlet), NY-ESO1 (cancer-testis antigen), AFP (α-feto protein) and cancer antigen 125 (CA-125), EPCA (Early Prostate Cancer) er Antigen).

In vivo immune responses can be broadly divided into Th1 immune responses and Th2 immune responses.
As used herein, the term “Th1 immune response” refers to a reaction that induces a cellular immune response, and “Th2 immune response” refers to a response that promotes a humoral immune response.
The immune reaction measurement in the animal immunized with the present invention is performed, for example, through a method of collecting serum from a target animal to which an immunogen has been administered and confirming the titer of anti-immunogenic antibodies (total IgG, IgG1 and IgG2a). Can be made. Methods for confirming antibody titer include, for example, ELISA (Enzyme-linked immunosorbent assay), lateral movement test (Lateral flow test), MIA (Magnetic immunoassay), and immunoprecipitation (Immunoprecipitation). However, the present invention is not limited thereto, and may be performed by various immunoassay methods known to those skilled in the art. Preferably, an ELISA assay method is used.
Desirably, the intestinal immunity enhancing agent of the present invention promotes the production of immunogen-specific IgG in the blood and immunogen-specific IgA in the small intestine when administered orally with the immunogen.

  According to a preferred embodiment of the present invention, an immunogen and a neat soy ethanol-dissolved extract are administered to a mouse, blood is collected from the orbital venous plexus, the serum is separated and the numerical value of IgG is analyzed, and the small intestine is removed. As a result of the analysis of IgA values, the immunogen and the neat soy ethanol-dissolved extract were administered as compared with the case where only the immunogen (Keyhole Limetic Hemocyanin; KLH) was administered alone. Specific IgG increases (FIG. 6) and the production of immunogen specific IgA increases (FIG. 9). The neat soy ethanol-dissolved extract of the present invention has a strong IgA production activity similar to that of CT (cholera toxin), thereby effectively inducing an intestinal immunity reaction against pathogenic bacteria and viruses. Of course, it also shows that blood IgG production and systemic immune responses that did not appear in the case of CT are well induced.

Desirably, the intestinal immunity enhancing agent of the present invention increases the production of intrasplenic IL-12 when administered orally with the immunogen. This effectively induces a cellular immune response with an activated Th1 response.
Desirably, the intestinal immunity enhancing agent of the present invention increases the production of intrasplenic IL-4 and IL-5 when administered orally with the immunogen. This effectively induces a humoral immune response that produces immunogen-specific antibodies in an activated Th2 response.
Desirably, when the intestinal immunity reinforcing agent of the present invention is orally administered together with the immunogen, the relative amount of IL-4 / IFN-γ in the spleen is 5 to 30 times as compared with the single oral administration of the immunogen. Increase, more desirably 6-20 times, most desirably 7-10 times.
According to a preferred embodiment, when the immunogen and neat soy ethanol-dissolved extract are administered, the production of IL-4 that induces a Th2 response is increased as compared to the immunogen alone. (FIG. 4).

The immunoreinforcing agent of the present invention can be provided as a pharmaceutical composition and a food composition.
When the immunopotentiator of the present invention is provided as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention is usually used at the time of formulation, and is lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginic acid. , Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. Is not to be done.
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 and the like 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 is administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.
Suitable dosages of the pharmaceutical composition of the present invention include formulation method, mode of administration, patient age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate, and response sensitivity. It can be prescribed in various ways due to such factors.
The dosage of the pharmaceutical composition of the present invention is in the range of 0.00001-100 mg / kg on an adult basis.

The pharmaceutical composition of the present invention is formulated in a unit volume form by formulating with a pharmaceutically acceptable carrier and / or excipient by methods that can be easily carried out by those skilled in the art. Or can be manufactured by being placed in a multi-capacity container. In this case, the dosage form may be in the form of a solution, suspension, syrup or emulsion in oil or an aqueous medium, or in the form of an extract, powder, powder, granule, tablet or capsule. Yes, it may further contain a dispersant or stabilizer.
When the immunoreinforcing agent of the present invention is provided as a food composition, the immunoreinforcing agent of the present invention is expressed as “a food composition for enhancing immunity”.

When the composition of the present invention is produced as a food composition, it contains not only a neat soy ethanol-dissolved extract as an active ingredient, but also ingredients normally added during food production, such as proteins, carbohydrates, fats, nutrients, Contains seasonings and flavors. Examples of the aforementioned carbohydrates are monosaccharides such as sucrose, fructose, etc .; disaccharides such as maltose, sucrose, oligosaccharides, etc .; and polysaccharides such as normal sugars such as dextrin, cyclodextrin and xylitol, sorbitol Sugar alcohols such as erythritol. As a flavoring agent, natural flavoring agents [thaumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.
For example, when the food composition of the present invention is produced as a drink, in addition to the purified soy ethanol extract of the present invention, citric acid, liquid fructose, sugar, sucrose, acetic acid, malic acid, fruit juice, tomato An extract, jujube extract, licorice extract and the like can be further included.

  According to another aspect of the present invention, the present invention provides a vaccine for oral administration for inducing systemic immunity and intestinal immunity comprising as an active ingredient an immunogen protein and a neat soy ethanol-dissolved extract as an intestinal immunity reinforcing agent. A composition is provided.

  According to another aspect of the present invention, the present invention administers to a subject an orally administered vaccine composition comprising an ethanol-soluble component from among (a) an immunogenic protein and (b) an ethanol extract of neat soy sauce. A method for inducing systemic immunity or intestinal immunity response comprising steps is provided.

  As used herein, the term “immunogenic protein” is an epitope, peptide or protein that induces the immune response of the recipient, since this is in common with the contents described in the “immunogen” of the present invention described above. The description is omitted to avoid excessive duplication.

  Suitable dosages of the pharmaceutical composition of the present invention include formulation method, mode of administration, patient age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate, and response sensitivity. It can be prescribed in various ways due to such factors. On the other hand, the oral dose of the pharmaceutical composition of the present invention is desirably 0.001 to 10,000 mg / kg (body weight) per day.

The pharmaceutically acceptable carrier contained in the vaccine of the present invention is usually used at the time of formulation, and is lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginic acid, gelatin, silica. Including, but not limited to, calcium acid, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Absent.
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 and the like in addition to the above components.

  The vaccine composition according to the present invention is formulated using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, surfactants and the like that are commonly used in pharmacology. be able to. Desirably, it is more desirable to formulate as a preparation for oral administration. For example, it is desirable to formulate as a solid preparation for oral administration such as tablets, pills, powders, granules, capsules or liquid preparations such as suspensions, liquids for internal use, emulsions and syrups. When formulated as a solid formulation, the vaccine composition according to the present invention may include one or more excipients such as starch, calcium carbonate, sucrose, lactose, Gelatin or the like can be mixed. When formulated as a liquid formulation, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like can be mixed with the vaccine composition according to the present invention in addition to water or liquid paraffin. .

The features and advantages of the present invention are summarized as follows:
(A) The present invention relates to a systemic immunity and intestinal immunity reinforcing agent comprising a neat soy ethanol-dissolved extract as an active ingredient, a vaccine or a method of inducing systemic immunity and intestinal immunity reaction comprising administering the reinforcing agent or vaccine to a subject I will provide a.
(B) The immunoreinforcing agent containing the neat soy ethanol-dissolved extract of the present invention is free from toxicity and side effects by using food materials with safety, and has low production costs and convenience of use. Can be provided.
(C) The immunoreinforcing agent of the present invention, when orally administered with an antigen, exhibits a Th2 reaction against not only oral infectious pathogenic microorganisms, but also parenteral infectious pathogenic microorganisms, viruses, cancer antigens, general antigens, and the like. It can be induced to produce effective specific antibodies (blood IgG and small intestine IgA).

It is a graph which shows IFN-γ production when spleen cells of C3H / He mice administered orally with an immunogen (KLH) and an immunoreinforcing agent (Kyoei Sho or CT) are cultured in the presence of KLH. a, ab, and b represent the statistical significance of each experimental group at the P <0.05 level. Numerical values in which characters are displayed differently such as a and b mean that they are statistically different from each other at a level of P <0.05 (the probability of making an error is 5% or less). It is a graph which shows IL-12 production | generation at the time of culture | cultivating the spleen cell of the C3H / He mouse which orally administered the immunogen (KLH) and the immuno-reinforcement agent (Keiei soy or CT) in presence of KLH. a, b and c represent the statistical significance of each experimental section at the P <0.05 level. It is a graph which shows IL-4 production | generation when the spleen cell of the C3H / He mouse orally administered with the immunogen (KLH) and the immunoreinforcement agent (Keiyo soy or CT) was cultured in presence of KLH. a and b represent the statistical significance of each experimental section at the P <0.05 level. It is a graph which shows IL-5 production | generation at the time of culture | cultivating the spleen cell of the C3H / He mouse which orally administered the immunogen (KLH) and the immuno-reinforcement agent (Keisei soy or CT) in presence of KLH. a, b and c represent the statistical significance of each experimental section at the P <0.05 level. It is the graph which analyzed the total IgG antibody among the sera of the C3H / He mouse which orally administered the immunogen (KLH) and the immunopotentiating agent (Keisei soy or CT). a and b represent the statistical significance of each experimental section at the P <0.05 level. It is the graph which analyzed the antigen specific IgG antibody among the sera of the C3H / He mouse to which the immunogen (KLH) and the immunoreinforcement agent (Keiyo soy or CT) were orally administered. a, ab and b represent the statistical significance of each experimental section at the P <0.05 level. It is the graph which analyzed the total IgA antibody produced from the small intestine of the C3H / He mouse which orally administered the immunogen (KLH) and the immuno-reinforcement agent (Seiyo soy or CT). a and b represent the statistical significance of each experimental section at the P <0.05 level. It is the graph which analyzed the antigen specific IgA antibody produced from the small intestine of the C3H / He mouse which orally administered the immunogen (KLH) and the immunopotentiating agent (Keiei soy or CT). a and b represent the statistical significance of each experimental section at the P <0.05 level. It is the graph which analyzed the T cell proliferation activity at the time of culture | cultivating the spleen cell of the C3H / He mouse which orally administered the immunogen (KLH) and the immuno-reinforcing agent (Keiyo soy or CT) in presence of KLH. a and b represent the statistical significance of each experimental section at the P <0.05 level. It is a graph which shows the MTT analysis result with respect to the spleen cell of a mouse | mouth. (A) Steamed bean water or 70% ethanol extract, (b) Seiki soy water or 70% ethanol extract was used.

  Hereinafter, the present invention will be described in more detail through examples. It is obvious to those skilled in the art that these examples are merely for explaining the present invention more specifically, and that the scope of the present invention is not limited by these examples due to the gist of the present invention. .

Examples Example 1 Preparation of natural product sample In order to examine the possibility of using it as a reinforcing agent, after securing 20 foods and natural products, prepare these pulverized products and add 25 ml of 70% ethanol to each 1 g. Extracted by microwave treatment at 90 W for 5 minutes, and after 12 hours, centrifuged at 10 ° C. and 8,000 rpm for 20 minutes to collect the supernatant. Subsequently, the supernatant was evaporated and concentrated, filtered through a membrane, and used for an activity experiment.

Example 2 Method of selecting spleen cell growth active materials from natural products In order to examine the possibility of utilization as a reinforcing agent, materials that are expected to have an immune enhancing effect are selected from over 20 natural products in an ex vivo experimental system. did. The spleen of a BALB / c mouse (6 weeks old, male) was removed, and a material for increasing immune activity was searched using the primary culture system of the removed spleen cells. That is, a total of 22 kinds of ingredients (licorice, yellow, black sesame, enju, bark, green tea, jujube, wakame, dandelion, pine needles, crab, medicinal beans, sulfur, perilla, neat soy sauce, chlorella, egoma, red beans, koroha, 70% ethanol extract of yellow corn and pepper) at a concentration of 10 μg / ml and 100 μg / ml, respectively, in a 96-well plate at 1.0 × 10 ⁶ cells / well per well Cells were seeded. At this time, ConA (Concanavalin A, 1 μg / ml) and LPS (Lipopolysaccharides, 25 μg / ml) or a natural product extract were added to the culture medium, and RPMI-1640 medium (10% FBS, 100 units / ml penicillin, 100 μg). / Ml streptomycin and 2 mM L-glutamine). After 44 hours of culture, 2 mg / ml MTT (3- (4,5-dimethylthiazolyl-2) -2,5-diphenyltetrazole bromide, Sigma) was treated in an amount of 20 μl per well and incubated in a CO ₂ incubator at 37 ° C. for 4 hours. Left to stand. After 4 hours, 0.01 μM HCl containing 10% SDS (Sodium dodecyl sulfate) was added at 100 μl per well, then left overnight in a 37 ° C. CO ₂ incubator, and the absorbance was measured at 570 nm.
As a result of the MTT test, the spleen cell proliferation activity due to the sample treatment was the highest in neat soy sauce, and it was estimated that it could be used as an immune reinforcing agent (Table 1).

Example 3 Example of Selection of Neat Soy Sauce Excellent in Spleen Cell Proliferating Activity A sample of 12 neat soy sauces purchased in the city was extracted by the same method as in Example 1, and BALB / c mice (6 weeks old, The activity of each sample was compared in the same manner as in Example 2 using male spleen cells. As a result of the experiment, No. 10 Kiyoi Soy expressed relatively high activity at concentrations of 10 μg / ml and 100 μg / ml (Table 2).

Example 4 Separation and characteristics of neat soy sauce strains No. 10 neat soy sauce purchased in the city was identified and identified as shown in Table 3, and neat soy sauce was produced using each strain. The production method is as follows: 500 g of carefully selected beans are soaked at room temperature for 12 hours, boiled at 114 ° C. for 50 minutes, cooled to 50 ° C., and then cultured in TBS (tryptic soy broth) (37 ° C., 24 hours). The liquid was inoculated to 1% (v / w) of soybean amount, fermented at 37 ° C. for 48 hours, and the enzyme activities of α-amylase, β-amylase and protease of each strain were measured. For measurement of enzyme activity, 10 times the amount of distilled water was added to 10 g of neat soy sauce, followed by stirring and extraction for 60 minutes. This was centrifuged at 3,000 rpm for 10 minutes, and then the supernatant was taken. What was filtered by 2 was used as a crude enzyme liquid.

Example 4-1. Measurement of enzyme activity of α-amylase In the case of α-amylase, the blue value method (Fuwa, H., J Biochem., 41: 583-603 (1954)) was modified and measured. After adding 5 ml of 0.04M potassium phosphate buffer (pH 5.9) and 1 ml of the crude enzyme solution to 5 ml of 0.5% starch solution, the mixture was reacted at 25 ° C. for 10 minutes. After 1 ml of the reaction solution was put into a test tube containing 10 ml of 0.5% iodine solution, the color was developed, and the absorbance (A ₆₆₀ ) was measured at ₆₆₀ nm. Unit of enzyme activity (Unit) is 1 minute starch at pH5.9,25 ℃ - was defined as the amount of enzyme required to reduce the _{A 660} of the iodine solution 1%.

Example 4-2. Measurement of β-Amylase Enzyme Activity β-Amylase activity was measured by the dinitrosalic acid method (Bernfeld, P., Methods in Enzymology 1: 149-158 (1955)). The crude enzyme solution using 1% soluble starch as a substrate was mixed at a 1: 1 (v: v) ratio, reacted at 20 ° C. for 10 minutes, added with the same amount of DNS reagent, and boiled for 15 minutes. And heated. After the heating reaction, after cooling with cold water, distilled water was added, the absorbance was measured at 540 nm, and maltose was used as a standard substance. The unit of enzyme activity was defined as the amount of enzyme required for production of 1 mg maltose at pH 4.8 and 20 ° C. for 3 minutes.

Example 4-3. Measurement of enzyme activity of protease The protease activity was measured by partially modifying Anson's method (J. General Physiology 22: 79-89 (1938)). In 1 ml of 0.1M potassium phosphate buffer (pH 6.8), 1.5 ml of 0.6% casein and 0.5 ml of the crude enzyme solution were added and reacted at 37 ° C. for 30 minutes. After the reaction, 3 mL of 0.4M TCA was added and allowed to stand at room temperature for 10 minutes to stop the enzyme reaction. The reaction solution was centrifuged (10,000 rpm, 10 minutes), 0.4 ml Na ₂ CO ₃ 5 ml and 1 N phenol 1 ml were added to 1 ml of the supernatant, and the mixture was colored at 40 ° C. for 30 minutes, at 660 nm. Absorbance was measured. As a standard substance, tyrosine was used, and the unit of enzyme activity was defined as the amount of enzyme required to release 1 μg of tyrosine for 1 minute.
As a result of the enzyme activity measurement experiment, α-amylase, β-amylase, and protease have the highest enzyme activities among the crude enzyme solutions of neat soy sauce produced using Bacillus amyloliquefaciens strain. It appeared high (Table 4).

Example 5 FIG. Comparison of Spleen Cell Proliferation Activity of Neat Soy Sauce Manufactured with Isolated Strains Extracted 7 kinds of Neat Soy Sauces Manufactured with Each Strain Identified from Seiyu Soy Sauce in the Same Method as Example 1 to Prepare Samples Spleen cell proliferation activity of the extract samples was compared in the same manner as in Example 2.
As a result of the experiment, the cell proliferation activity by the sample treatment showed the highest spleen cell proliferation activity of the neat soy extract produced with Bacillus amyloliquefaciens, which was highly likely to be used as an immunoreinforcement agent (Table 5).

Example 6 Animal Experiment Immune Assistance Effect of Neat Soy Sauce by Animal Experiment In order to confirm the immunopotentiating effect of neat soy ethanol dissolved extract (70% ethanol extract) produced by Bacillus amyloliquefaciens, C3H / He (6 The mice were orally immunized with a neat soy ethanol-dissolved extract as a reinforcing agent together with the immunogen. C3H / He mice were divided into 4 groups of 5 mice, and antigens and reinforcing agents were orally administered to each group. As the immunogen, KLH (Keyhole Limeto Hemocyanin) was used, and as the reinforcing agent, neat soy ethanol-dissolved extract or CT (Cholera Toxin) was used. Therefore, group 1 is physiological saline, group 2 is KLH (50 μg / H) and physiological saline, group 3 is KLH (50 μg / H) and neat soy ethanol-dissolved extract (2.8 mg). / H), Group 4 was orally administered KLH (50 μg / H) and CT (10 μg / H) (μg / Head) twice a week for 8 times for 4 weeks, After daily oral administration for 5 days, blood was collected from the orbital venous plexus, and the serum was separated and stored at -70 ° C until analysis. Finally, IgG antibodies in the collected blood serum were analyzed by ELISA (Enzyme-linked immunosorbent assay). In addition, in order to analyze total IgA and immunogen-specific IgA in the small intestine, after removing the mouse small intestine, the upper 10 cm was homogenized with a homogenizer for 1 minute, and then at 10,000 at 4 ° C. Centrifuged at xg for 10 minutes. After centrifugation, the supernatant was collected and analyzed for IgA by ELISA. In order to confirm changes in Th1 and Th2 cytokines, spleen cells were cultured with an immunogen (KLH), and then the supernatant was collected and analyzed for cytokines by ELISA.

Cytokine analysis For analysis of Th1 and Th2 cytokines, KLH was orally administered together with a neat soy ethanol-dissolved extract, and then the spleens of mice were removed and spleen cells that had been collected and treated as a single cell were cultured. Cell culture is performed by culturing 1 ml at a concentration of 5 × 10 ⁶ cells / ml per well in a 24-well plate. At this time, the culture is divided into a medium not added with an immunogen and a medium added with an immunogen. By observing, it was analyzed whether an immune response was induced. The immunogen was 100 μg / ml per well for KLH. For cytokine analysis, cells were cultured for 48 hours (Th1 cytokine) and 72 hours (Th2 cytokine), and the culture medium from which the cells had been removed was collected and stored at −70 ° C. until analysis. Cytokine analysis was performed according to the kit protocol using a cytokine analysis kit manufactured by BD Biosciences (USA).
Briefly, after putting an anti-cytokine antibody in a 96-well plate and allowing it to stand at 4 ° C. overnight, it was washed 3 times with PBST (Phosphate Buffered Saline-Tween 20 added), and 10% FBS (Fetal Bovine Serum) Blocking was performed with added PBS (Phosphate Buffered Saline) for 1 hour. After washing 3 times with PBST, a cell culture solution was added and reacted at room temperature for 2 hours. After 2 hours, the plate was washed 5 times with PBST, treated with anti-cytokine to which biotin was bound and HRP (horse radish peroxidase) to which streptavidin was bound, and reacted for 1 hour. After the wells were washed 7 times with PBST, the substrate was treated by a method such as ELISA described above, and the absorbance was measured.

As a result, IFN-γ and IL-12, which are Th1 cytokines, were analyzed. As a result, IFN-γ showed almost no numerical difference between IFN-γ in the spleen cell culture medium of mice orally administered with an immunoreinforcing agent together with KLH. However, the numerical value of IL-12 increased in the treatment group in which both the neat soy ethanol-dissolved extract and KLH were orally administered (FIGS. 1 and 2). In the case of Th2 cytokine, IL-4 and IL-5 values were higher in the spleen cells of mice treated with neat soy ethanol-dissolved extract and KLH compared to mice orally administered KLH (FIG. 3). And FIG. 4). This is because the production of IFN-γ that induces the Th1 response is not greatly increased, but the production of IL-12 that induces the Th1 response is increased, and the production of IL-4 and IL-5 that induce the Th2 response is increased. Means that
Moreover, as a representative Th2 and Th1 cytokine, focusing on IL-4 and IFN-γ, respectively, and calculating the relative production ratio of IL-4 / IFN-γ, neat soy ethanol-dissolved extract or When CT was administered as an oral immunoreinforcing agent, it appeared 8.2 times higher than the control group. This is because, in the present invention, humoral immunity in which the production of specific antibodies increases as a result of the active Th2 reaction is very actively promoted, and in particular, the production of IgA antibodies in the small intestine by IL-5 is effective. You can see that it was guided. This can actually be confirmed from the results described later (FIG. 8).

Analysis of total IgG Anti-mouse IgG (SIGMA, Korea) was placed in a 96-well plate at a concentration of 2 μg / ml and allowed to stand at 4 ° C. overnight, and then 100 μl of serum diluted 1 / 10,000 with PBST per well. After the addition, the mixture was reacted at room temperature for 1 hour. Then, an anti-mouse IgG-HRP conjugate (conjugate, SIGMA A-2304) was diluted 1 / 30,000 with PBST, and 100 μl per well was added to the plate, followed by reaction at room temperature for 1 hour. Finally, after treatment with TMB (3,3 ′, 5,5′-tetramethylbenzidine) and 3% H ₂ O ₂ in the light-shielded state, the reaction was performed for 30 minutes, and the reaction was stopped with 2MH ₂ SO ₄ . Absorbance was measured at 450 nm.
As a result, a high number of total IgG antibodies appeared significantly in the experimental group in which the neat soy ethanol-dissolved extract was orally administered as an immunopotentiator, but when CT was treated as an immunopotentiator, the control group And was not significant when compared (FIG. 5).

Analysis of immunogen-specific IgG The immunogen was placed in a 96-well plate at a concentration of 2 μg / ml, reacted at 4 ° C. overnight, and then 100 μl of each serum diluted 1 / 3,000 with PBST was added per well. Thereafter, the reaction was performed at room temperature for 1 hour. After the reaction, anti-mouse IgG-HRP conjugate (SIGMA, USA) was diluted 1 / 10,000 with PBST, added 100 μl per well, and then reacted at room temperature for 1 hour. Finally, TMB and 3% H ₂ O ₂ were added in a light-shielded state, reacted for 30 minutes, and 2MH ₂ SO ₄ was added to stop the reaction, and then the absorbance was measured at 450 nm.
When the neat soy ethanol-dissolved extract was orally administered as an immunopotentiator, the value of the immunogen-specific IgG antibody appeared significantly higher than that of the control group, but in the case of the CT treatment group, There was no significance when compared to the control group (FIG. 6). This shows a tendency almost similar to the results of the previous total IgG antibody analysis, so that when neat soy extract is orally administered with an immunogen as an intestinal immunity enhancer, IgG antibodies are effectively produced in the blood. This indicates that the neat soy ethanol-dissolved extract has an immunopotentiating activity superior to that of CT. In addition, this activity is not caused by oral infection, but pathogenic bacteria that are infected by blood, and even cancer-related antigens, when orally administered together with a neat soy ethanol-dissolved extract, IgG antibodies specific in the blood This means that production can be induced and its availability is expected.

Analysis of total IgA Anti-mouse IgA (BD biosciences, USA) was added to a 96-well plate at a concentration of 2 μg / ml and reacted overnight at 4 ° C. A small intestine sample (the supernatant after homogenized small intestine was centrifuged) was diluted 1/1000 with PBST containing 10% FBS, added at 100 μl per well, and reacted at room temperature for 1 hour. After the reaction, anti-mouse IgA-biotin conjugate (BD biosciences, USA) was diluted in PBST at a concentration of 0.5 μg / ml, and 100 μl per well was added, followed by reaction at room temperature for 1 hour. After the reaction, avidin-peroxidase (avidin-peroxidase, SIGMA, USA) was diluted in PBST at a concentration of 200 ng / ml, added at 100 μl per well, and reacted at room temperature for 1 hour. TMB and 3% H ₂ O ₂ were treated in the light-shielded state, reacted at room temperature for 30 minutes, and 2 μH ₂ SO ₄ was added at 50 μl per well to stop the reaction. The results were analyzed by measuring the absorbance at 450 nm.
As a result, the total IgA values of mice administered orally with KLH and neat soy ethanol-dissolved extract were not different from the experimental group administered orally with KLH alone, but were orally administered with CT as an immunoreinforcement agent. The treatment group was weak compared to the control group, but showed a significant increase (FIG. 7).

Analysis of immunogen-specific IgA Immunogen (KLH) killed in a 96-well plate was added at a concentration of 2 μg / ml and reacted at 4 ° C. overnight. After centrifugation of the homogenized small intestine, the small intestine sample, which was the supernatant, was diluted 1/1000 in PBST containing 10% FBS, added 100 μl per well, and reacted at room temperature for 1 hour. After the reaction, anti-mouse IgA-biotin conjugate (BD biosciences, USA) was diluted in PBST at a concentration of 0.5 μg / ml, and 100 μl per well was added, followed by reaction at room temperature for 1 hour. Avidin-peroxidase (SIGMA, USA) was diluted in PBST at a concentration of 200 ng / ml, added at 100 μl per well, and then reacted at room temperature for 1 hour. TMB and 3% H ₂ O ₂ were treated in the light-shielded state, reacted at room temperature for 30 minutes, and 2 μH ₂ SO ₄ was added at 50 μl per well to stop the reaction. The results were analyzed by measuring the absorbance at 450 nm.
As a result, the value of the immunogen-specific IgA antibody in the treatment group that was orally administered together with KLH and the neat soy ethanol dissolved extract was significantly higher than the control group, and CT was an immunoreinforcing agent. The group treated as indicated a similar tendency (FIG. 8). Therefore, neat soy ethanol-dissolved extract has a strong IgA production activity similar to that of CT, and thus can effectively induce an immune response corresponding to pathogenic bacteria and viruses that enter the intestinal tract. I understand.

Experiment of cell proliferation activity For the experiment of cell proliferation activity, the mouse spleen was removed, collected for each treatment group, made into single cells, and the spleen cells were cultured, and then MTT analysis was performed. A 96-well plate was seeded at 5 × 10 ⁶ cells / ml per well. At this time, RPMI-1640 medium (10% FBS, 100 units / ml penicillin, 100 μg / ml streptomycin and 2 mM L-glutamine) was added with OVA, ConA or a natural product extract and cultured. After 44 hours of culturing, 20 μl of 2 mg / ml MTT was treated per well, and left still in a CO ₂ incubator at 37 ° C. for 4 hours. After adding 100 μl of 0.01M HCl containing 10% SDS per well, the reaction was carried out overnight in a CO ₂ incubator at 37 ° C., and the absorbance was measured at 570 nm.
As a result, as shown in FIG. 9, the spleen cell proliferation of the treatment group using the neat soy ethanol-dissolved extract as an immune enhancer was significantly highest. This is because the neat soy ethanol-dissolved extract improves the antigen-presenting ability of antigen-presenting cells in vivo, and as a result, T cells specific for the antigen are proliferated, thereby enhancing the immune response. Means that

Example 7 Component confirmation example of neat soy ethanol-dissolved extract having immunopotentiating effect Example 7-1. Manufacture of a neat soy extract A neat soy was produced from Bacillus amyloliquefaciens, which was determined to have the best immune enhancement effect in the above examples. Distilled water or 1,000 ml of 70% ethanol was added to 100 g of powdered neat soy sauce, extracted by microwave treatment at 90 W for 5 minutes, and after 12 hours, 20 minutes under conditions of 10 ° C. and 8,000 rpm. The supernatant was recovered by centrifugation. The collected supernatant was lyophilized and used as a sample.
Since the neat soy ethanol-dissolved extract generally referred to in the present specification is a component dissolved in ethanol among the components of neat soy sauce, components such as polyγ-glutamic acid (PGA) and levan Is the extracted product. According to the prior art, in order to separate PGA or levan, the sample is treated with 70% ethanol and the precipitate formed therefrom is additionally purified to give PGA (Bioscience, Biotechnology, and Biochemistry, Vol 65 516-521, 2001) or Levan (Clinical & Experimental allergy, Vol 36, 94-101).
On the other hand, in the present invention, contrary to the prior art, the neat soy ethanol extract used in the present invention is substantially composed of PGA and levan in order to use the 70% ethanol-soluble fraction of neat soy sauce. It can be said that it was removed.

Example 7-2. Measurement of immunopotentiating effect The immunopotentiating effect of the neat soy extract was measured using MTT analysis in the same manner as in Example 2. For this purpose, the spleen of the mouse was removed and then made into single cells, and the spleen cells were cultured. The cell culture was adjusted to a concentration of 1 × 10 ⁶ cells / ml, and 200 μl of cells were collected in a 96-well plate, and cultured separately in a medium with or without a neat soy extract. The neat soy extract was cultured at a concentration of 100 μg / ml per well. After 72 hours of culture, 20 μl of 2 mg / ml MTT was added to each well and placed in a CO ₂ incubator at 37 ° C. for 4 hours. After 4 hours, 0.01 M HCl containing 10% SDS was added at 100 μl per well, and then left overnight in a CO ₂ incubator at 37 ° C., and the absorbance was measured at 595 nm.
As can be confirmed in FIG. 10, the immunopotentiating activity effect could be observed with 70% ethanol neat soy extract. It can be seen that this is an effect exhibited by a new neat soy extract in a state in which PGA or levan known to have conventional immune enhancing activity has been substantially removed.

Example 7-3. Measurement of molecular weight by gel electrophoresis The molecular weight was measured by using a concentration gradient SDS-page method and preparing a neat soy extract as a sample at 500 μg / ml. 6.5 μl of neat soy extract (500 μg / ml) was mixed with 3.5 μl of 4 × sample buffer and then electrophoresed on a 4-12% concentration gradient SDS-gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue (G-250), then decolorized, and stained with 0.5% methylene blue / 3% acetic acid to compare the molecular weight.
As a result of the experiment, a substance specifically stained with methylene blue was detected only with the water extract, which is considered to be a PGA having a large molecular weight. Therefore, it was found that the immune enhancing substance obtained from 70% ethanol purified soy extract is a substance other than the PGA.

  As described above, specific portions of the present invention have been described in detail. For those skilled in the art, such a specific technique is merely a preferred embodiment, and the scope of the present invention is not limited thereby. Is obvious. Accordingly, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (19)

In the systemic immunity or intestinal immunity reinforcing agent containing the neat soy extract as an active ingredient,
A systemic immunity or intestinal immunity reinforcing agent, wherein the neat soy extract contains an ethanol-soluble component extracted with ethanol.   The reinforcing agent according to claim 1, wherein the ethanol is 50% to 99% ethanol.   The reinforcing agent according to claim 1 or 2, wherein the neat soy extract is an extract from which PGA is removed.   The reinforcing agent according to claim 1 or 2, wherein the neat soy extract is an extract from which PGA and levan are removed.   The reinforcing agent according to claim 1 or 2, wherein the neat soy sauce is produced using Bacillus amyloliquefaciens.   The reinforcing agent according to claim 1 or 2, which promotes the production of immunogen-specific IgG in blood and immunogen-specific IgA in the small intestine when orally administered with the immunogenic protein.   The intestinal immunity reinforcing agent according to claim 1 or 2, which increases the production of IL-12 in the spleen when the reinforcing agent is orally administered together with an immunogenic protein.   The intestinal immunity reinforcing agent according to claim 1 or 2, wherein the reinforcing agent increases the production of IL-4 and IL-5 in the spleen when orally administered with the immunogenic protein.   When the reinforcing agent is orally administered with the immunogenic protein, the relative production ratio of IL-4 / IFN-γ in the spleen is increased by 5 to 30 times compared to the oral administration of the immunogenic protein alone. 8. The intestinal tract immune reinforcing agent according to 8. (A) an immunogenic protein and (b) a neat soy extract as a systemic immunity or intestinal immunity reinforcing agent as an active ingredient, and the neat soy extract contains an ethanol-soluble component extracted with ethanol A vaccine composition for oral administration for inducing systemic immunity or intestinal immunity.   The vaccine composition for oral administration according to claim 10, wherein the ethanol is 50% to 99% ethanol.   The vaccine composition for oral administration according to claim 10 or 11, wherein the neat soy extract as the systemic immunity or intestinal immunity reinforcing agent is an extract from which PGA is removed.   The vaccine composition for oral administration according to claim 10 or 11, wherein the neat soy extract as the systemic immunity or intestinal immunity reinforcing agent is an extract from which PGA and levan are removed.   12. A composition according to claim 10 or 11 wherein the reinforcing agent promotes the production of immunogen specific IgG in the blood and immunogen specific IgA in the small intestine when administered orally with the immunogenic protein.   12. A composition according to claim 10 or 11, wherein the reinforcing agent increases the production of intrasplenic IL-12 when administered orally with an immunogenic protein.   12. A composition according to claim 10 or 11, wherein said reinforcing agent increases production of intrasplenic IL-4 and IL-5 when administered orally with an immunogenic protein.   17. When the reinforcing agent is administered orally with an immunogenic protein, the relative production ratio of IL-4 / IFN-γ in the spleen is increased by a factor of 5 to 30 as compared to single oral administration of the immunogen. A composition according to 1.   A method for inducing systemic immunity or intestinal immunity reaction, comprising a step of administering to a subject a composition containing an ethanol-soluble component from an ethanol extract of neat soy sauce.   Systemic immunity or intestinal immunity response to a specific immunogen protein comprising the step of administering to an object a vaccine composition for oral administration containing an ethanol-soluble component from (a) an immunogenic protein and (b) an ethanol extract of neat soy sauce Guidance method.