WO2008023665A1 - Bacterial strain having anti-allergic activity and immunostimulating activity, and beverage, food, anti-allergic agent and immunostimulating agent comprising the bacterial strain - Google Patents

Abstract

Disclosed is a bacterial strain belonging to Lactobacillus brevis subspecies brevis which can proliferate in a sparkling alcoholic beverage, can produce γ-amino butyric acid (GABA) and has an anti-allergic activity and an immunostimulating activity. Also disclosed is an anti-allergic agent comprising a viscous substance secreted from a bacterial strain belonging to Lactobacillus brevis subspecies brevis. Further disclosed is a beverage or food comprising the anti-allergic agent.

Description

 Specification

 Strain having antiallergic action and immunostimulatory action, and beverage, food, antiallergic agent and immunostimulant containing the strain

 Technical field

 [0001] The present invention relates to a strain having an antiallergic action and an immunostimulatory action, and a beverage, food, antiallergic agent and immunostimulant containing the strain. The present invention also relates to an antiallergic agent composed of a viscous substance produced by the strain, and beverages and foods containing the antiallergic agent.

 Background art

 [0002] For treatment of allergic diseases, pharmacotherapy using antihistamines, antiallergic agents, steroids and the like is common. Recently, however, lactic acid bacteria that act on intestinal immunity are attracting attention because they are effective in preventing and treating allergic diseases from the viewpoint of drug therapy and preventive medicine (Patent Documents 1 to 3). Intestinal immunity is an immune mechanism that eliminates pathogenic microorganisms orally taken in orally, and it is thought that if it can suppress intestinal immunity that reacts excessively, it can contribute to the prevention and treatment of allergic diseases.

 [0003] For example, strains of the Bifidobacterium genus infantis, breve, longum, and bifidum are used to treat food allergies. Some have effects (Patent Document 1), Enterococcus faecalis and Ratatobatinoles' Lactobacill us reuteri strains (Patent Document 2), and Lactobacillus paracasei, Some strains of Lactobacillus plantarum and Streptococcus salivarius (Patent Document 3) have been reported to have effects on bronchial asthma, allergic rhinitis, and atopic dermatitis.

[0004] Furthermore, it is known that allergic diseases correlate with physical stress and mental stress, and those with more stress have worse symptoms (Non-patent Document 1). For this reason, in addition to the action of suppressing the immune response in the body, daily prevention and treatment of allergic diseases It is said that it is necessary to relieve stress.

[0005] On the other hand, in the treatment of cancer and infectious diseases, a treatment policy has been established by combining surgical therapy and radiation therapy, centering on chemotherapy using anticancer agents and antibiotics. However, in recent years, immunotherapy that activates reduced immune functions and eliminates pathogenic viruses and malignant tumors has attracted attention. For example, in daily life, the ability of malignant tumor cells and infected cell bodies to develop everywhere in the body NK cells fight these and protect the power of cancer and infectious diseases. Immunotherapy aims to treat various diseases by keeping an eye on the function of the immune system and enhancing it to keep it normal.

[0006] The search for an immunostimulator is effective for enhancing immune function in the living body and maintaining health, and has been performed so far. Among these, immunostimulants produced by microorganisms, mushrooms or plants are known as immunostimulants that have been confirmed to be effective with low toxicity. For example, microbial cell wall polysaccharides or mushroom polysaccharides such as Agarita sashitake are applicable (Patent Documents 4 and 5). Recently, it has been disclosed that the polysaccharide having a dalcoside bond is particularly effective (Patent Documents 6, 7 and 8).

[0007] Patent Document 1: Japanese Patent Laid-Open No. 10-309178

 Patent Document 2: Japanese Unexamined Patent Publication No. 2000-95697

 Patent Document 3: Japanese Patent Laid-Open No. 2005-139160

 Patent Document 4: Japanese Patent Publication No.49-484

 Patent Document 5: Japanese Patent Publication No. 56-46481

 Patent Document 6: Japanese Patent Laid-Open No. 7-184595

 Patent Document 7: Japanese Patent Laid-Open No. 10-194977

 Patent Document 8: Japanese Unexamined Patent Publication No. 2005-220065

 Non-patent document 1: Satoshi Kanno, “For stress and allergic diseases, preparatory school students”, Otolaryngology, 2002, 45, ρ · 204-210

 Disclosure of the invention

 Problems to be solved by the invention

[0008] However, chemically synthesized drugs such as antihistamines, antiallergic agents and steroids, as well as immunomodulators and immunostimulants are directly applied to molecules involved in immune responses in blood. There is a problem that side effects are large because it acts in contact and suppresses the immune reaction necessary to maintain homeostasis. In addition, the activity of lactic acid bacteria reported so far is not practically effective for preventing chronic diseases such as allergic diseases or preventing immune diseases by exerting immunostimulatory action. there were. Furthermore, Lactobacillus brevis subtype brevis (Lactobacil lus brevis) has never been reported for lactic acid bacteria that produce γ-aminobutyric acid (GABA), which has antiallergic and immunostimulatory properties and antistress. Subspecies brevis) and the ability to secrete rice-occupying substances with anti-allenolytic activity have not been reported so far.

 [0009] Therefore, an object of the present invention is to provide ratatonov typhoid that has a stronger antiallergic action and immunostimulatory action than known lactic acid strains, and also has the ability to produce γ-aminobutyric acid (GABA). (Lactobacillus brevis subspecies brevis) (It is also intended to provide a strain belonging to this group. The object of the present invention is to identify the cells of the strain belonging to this ratato brevis subspecies brevis. It is an object of the present invention to provide beverages and foods to be contained, and antiallergic agents and immunostimulants containing these as active ingredients.

 [0010] Furthermore, an object of the present invention is to provide an antiallergic agent consisting of a viscous substance secreted by a strain belonging to the ratatobacillus brevis subspecies brevis, which has a strong antiallergic action with no side effects. Another object of the present invention is to provide drinks and foods containing this antiallergic agent.

 Means for solving the problem

 [0011] In order to achieve the above-mentioned object, the present invention is a strain that can be grown in Lactobacillus brevis subspecies brevis. And provides a strain that produces 7-aminobutyric acid (GABA) and has an antiallergic action and an immunostimulatory action.

[0012] The present inventors have demonstrated the production promoting effect of Thl-type cyto force-in and the suppression of IgE production on spleen cells of strains belonging to subspecies brevis out of Lactobacillus brevis species of lactic acid bacteria. The present inventors have found that IgA production promoting effect is exerted on mouse Peyer's patch cells. There are no reports of lactic acid strains that have both antiallergic and immunostimulatory effects. The antiallergic and immunostimulatory effects of the strain of the present invention have not been reported so far. It was significantly stronger than the reported strain of lactic acid bacteria. Lactic acid bacteria are bacteria that have been used for fermented foods for a long time, and are much safer for the human body than chemically synthesized antiallergic agents and immunostimulants. In addition, the ability of normal lactic acid bacteria to be unable to grow in a foaming alcoholic beverage, the strain of the present invention is capable of growing in an effervescent alcoholic beverage, and therefore this property is used to remove the strain of the present invention. Other lactic acid strains with no activity can be excluded. Furthermore, since the strain of the present invention produces _ァ aminobutyric acid (GA BA), it has an anti-stress action in addition to the above-mentioned action, and is an allergic disease correlated with physical stress and mental stress. It is expected to have a multifaceted and strong effect on the prevention and treatment of cancer and infectious diseases. As such a strain, SBC8027 (FERM BP-10630) force S of ratatobacillus brevis subspecies brevis is particularly preferable.

 [0013] The antiallergic action is preferably a production promoting action of interferon γ and / or interleukin 12.

[0014] Interferon _γ is a cyto force-in secreted by Thl cells, which inhibits B cells from producing IgE, and also kills killer T cells and macrophages that attack viruses, filamentous fungi, tuberculosis bacteria, etc. Has the effect of increasing the cellular immunity of Interleukin 12 is a cytodynamic force secreted by antigen-presenting cells such as macrophages and has the effect of inducing Thl cells by stimulating NK cells and also induces production of interferon γ by Thl cells. To do. Since the strain of the present invention can promote production of interferon γ and / or interleukin 12 and suppress production of IgE, it is possible to suppress type I allergic reaction. Furthermore, since the strain of the present invention can enhance cellular immunity by shifting the Thl / Th2 balance to the Thl side, it can have an immunostimulatory effect in addition to an antiallergic effect. This prevents virus infection and eliminates cancerous cells.

[0015] The antiallergic action is preferably an IgE production inhibitory action.

[0016] IgE is a causative substance that causes allergic diseases. That is, when an allergen enters, IgE is produced in response to it, and IgE binds to mast cells and basophils to establish sensitization. Thereafter, when the same allergen enters, IgE recognizes the allergen, and inflammatory substances such as histamine are released from mast cells and basophils. this The allergic reaction exhibits various symptoms such as bronchoconstriction and urticaria, and it causes allergic diseases such as hay fever, allergic rhinitis, atopic dermatitis, and asthma. Since the strain of the present invention can suppress the production of IgE, it can suppress the allergic reaction and can be used for the prevention and treatment of the above-mentioned diseases.

 [0017] The immunostimulatory action is more preferably a natural killer cell or killer cell activating action, preferably an IgA production promoting action.

 [0018] IgA is one of immunoglobulins mainly contained in secretions such as intestinal mucus, saliva, tears and breast milk, and plays an important role as a local defense mechanism of mucosa. Since the bacterial strain of the present invention can promote the production of IgA, it can enhance the local defense mechanism of mucosa and can be used for the prevention and treatment of lifestyle diseases such as infectious diseases and allergic diseases.

 [0019] The present invention also provides beverages and foods containing the cells of the above strain. That is, use of the bacterial cells of the strain as a beverage raw material and a food raw material is provided.

 [0020] The bacterial cells of the above strains have an antiallergic action and an immunostimulatory action, and are safe for the human body, so that they can be used as a health food material in beverages and foods. Furthermore, since the above strain produces γ-aminobutyric acid (GABA), it has anti-stress, blood pressure lowering and tranquilizing effects and is highly useful as a health food material.

 [0021] The present invention also provides an antiallergic agent containing the above bacterial strain as an active ingredient. That is, the use of the bacterial strain of the above strain for the production of an antiallergic agent is provided.

 [0022] Since the bacterial cells of the above strain have an interleukin 12 and interferon γ production promoting action and an IgE production inhibitory action, an anti-allergic agent containing this bacterial cell as an active ingredient can be produced. It can be used as an antiallergic agent that is safer than chemically synthesized drugs.

 [0023] Further, the present invention provides an immunostimulant containing a bacterial cell of the above strain as an active ingredient. That is, use of the microbial cell of the said strain for manufacture of an immunostimulant is provided.

[0024] Since the bacterial cells of the above strain have an IgA production promoting action, if an immunostimulant containing this bacterial cell as an active ingredient is produced, immunity superior in safety to chemically synthesized pharmaceuticals is produced. It can be used as an activator. [0025] Furthermore, the present invention provides an antiallergic agent comprising a viscous substance secreted by a strain belonging to Lactobacillus brevis subspecies brevis. That is, the use of a viscous substance secreted by the above strain for the production of an antiallergic agent is provided.

 [0026] The present inventors have found that some strains belonging to subspecies brevis among lactic acid bacteria of the ratatobatiras brevis species secrete a viscous substance, and the viscous substance has an antiallergic action. Since lactic acid bacteria are bacteria that have been used for fermented foods for a long time, the above-mentioned anti-allergic agents are far superior in safety to the human body compared to chemically synthesized anti-allergic agents. The above-mentioned viscous substance is obtained by removing the bacterial cells from the culture solution of the strain, adding alcohol, and precipitating. The strain that secretes this viscous substance is SBC8027 (FERM BP — 10630) Power S Good.

 [0027] The antiallergic agent has an action of promoting production of interferon γ and / or interleukin 12! /, Ability to be preferred.

 [0028] Interferon γ is a cyto force-in secreted by Thl cells, which inhibits B cells from producing IgE, and also kills killer T cells and macrophages that attack viruses, filamentous fungi, and tuberculosis bacteria. Has the effect of increasing the cellular immunity of Interleukin 12 is a cytodynamic force secreted by antigen-presenting cells such as macrophages and has the effect of inducing Thl cells by stimulating NK cells and also induces production of interferon γ by Thl cells. To do. Since the above-mentioned antiallergic agent can promote the production of interferon γ and / or interleukin 12, it is possible to effectively suppress allergic reactions.

 [0029] The present invention also provides beverages and foods containing the antiallergic agent. In other words, the use of the viscous material secreted by the above strains as a beverage ingredient and food ingredient is provided.

 [0030] Since the above-mentioned antiallergic agent is safe for the human body, it can be used as a health food material in beverages and foods.

The invention's effect [0031] According to the present invention, it is possible to provide a strain belonging to Ratatobacillus brevis subspecies brevis, which has both an antiallergic action and an immunostimulatory action stronger than those of lactic acid strains known so far. In addition, since the strain of the present invention can be grown in an effervescent alcoholic beverage, other lactic acid strains having no activity of the strain of the present invention can be removed using this property. _Furthermore , _since the strain of the present invention produces _Ί -aminobutyric acid (GABA), it has an anti-stress action in addition to the above-mentioned action, and is correlated with physical and mental stress. Strong and effective for prevention and treatment of rugged diseases, cancer and infectious diseases. Furthermore, according to the present invention, beverages, foods, and antiallergic agents that contain the bacterial cells of the above strain, have excellent safety, and have antiallergic and immunostimulatory effects can be provided.

 [0032] According to the present invention, a viscous substance secreted by a strain belonging to Ratatobacillus brevis subspecies brevis can be used as an antiallergic agent. Further, according to the present invention, beverages and foods containing this antiallergic agent and having excellent safety can be provided.

 Brief Description of Drawings

 [0033] FIG. 1 shows the amount of interferon sputum produced by mouse spleen cells by the addition of a cell suspension belonging to Ratatobacillus brevis subsp. Brevis.

 FIG. 2 shows the amount of interferon γ produced by the spleen cells of OVA-immunized mice by the addition of OVA and bacterial suspension of each strain.

 FIG. 3 shows the amount of interleukin 12 produced by spleen cells of OVA-immunized mice by the addition of OVA and bacterial suspension of each strain.

 FIG. 4 shows the amount of interleukin 4 produced by spleen cells of OVA-immunized mice by the addition of OVA and cell suspensions of each strain.

 [FIG. 5] Interferon γ / interleukin 4 was calculated and graphed as an index of Thl / Th2 balance of spleen cells of OVA immunized mice.

 FIG. 6 shows the effect of cell suspension of each strain on the production of total IgE induced in spleen cells of OVA-immunized mice by the addition of OVA.

 FIG. 7 shows the effect of cell suspension of each strain on the production of OVA-specific IgE induced in spleen cells of OVA-immunized mice by the addition of OVA.

[FIG. 8] Production of total IgE secreted into the peripheral blood of OVA immunized mice administered intraperitoneally with each strain It shows the amount of production.

 FIG. 9 shows the production of OVA-specific IgE secreted into the peripheral blood of OVA-immunized mice administered with each strain intraperitoneally.

 FIG. 10 shows the amount of IgA produced by adding SBC8027 bacterial suspension.

FIG. 11 shows the amounts of interferon _sputum and interleukin 12 produced by mouse spleen cells by the addition of a viscous substance secreted by SBC8027 belonging to the ratatobatiras brevis subspecies brevis.

 BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, preferred embodiments of the present invention will be described in detail.

 [0035] The strain of the present invention belongs to Lactobacillus brevis subspecies brevis, is capable of growing in a sparkling alcoholic beverage, and produces γ-aminobutyric acid (GABA). Its special feature is that it has antiallergic and immunostimulatory effects.

 [0036] There are four subspecies mosquitoes in Lattobacillus brevis, which are brevis, gravesensis, otakiensis, and coagulans, respectively. The strain belongs to the subspecies Brevis. Strains belonging to subspecies brevis can be separated by using the base sequence and sugar strength of 16S ribosomal DNA, differences in acid production, etc. as indicators, and can be classified as strains not belonging to subspecies grebsensis, subspecies otachysis and subspecies coagulance.

[0037] "Anti-allergic action" refers to an action of suppressing an allergic reaction. Here, allergy refers to a state in which antibodies are produced in the body by ingestion or contact of certain substances, and excessive antigen-antibody reaction occurs due to reingestion or recontact of the same substance, resulting in pathological symptoms. Say. An allergic reaction is a phenomenon in which the immune response, which is the defense mechanism of the living body, should be eliminated by itself! /, A phenomenon that attacks its own cells or ingested food! The immune response involves antigen-presenting cells, T cells, and B cells, and humoral immunity mainly produces IgG and IgA, but allergic reactions mainly involve Th2 cells of T cells. However, IgE is made at a concentration 100 to 10,000 times higher than the normal immune response. This large amount of IgE It promotes the release of inflammatory substances such as histamine and leukotrien in mast cells.

[0038] Antiallergic effects include, for example, the action of acting on antigen-presenting cells, T cells or mast cells to suppress the production of IgE and the release of the above inflammatory substances, and the Thl / Th2 balance on the Thl side. More specifically, IgE production suppression action, interferon _γ and interleukin 12 production promotion action, and the like can be mentioned.

 [0039] Antiallergic effects of the above strains include IgE production inhibitory action, interferon

 It is more preferable to have two or more of these actions, which are preferably promoted by γ production and / or promoted by interleukin 12.

 [0040] "Immunostimulatory action" refers to strengthening the immunity of a living body to make it resistant to diseases. For example, macrophages, neutrophils, sputum cells and the like are activated to enhance the innate immune function and promote the production of immunoglobulins such as IgA. In particular, IgA production promoting action can enhance mucosal immunity, and thus can be expected to have a strong effect on the prevention and treatment of lifestyle diseases such as infectious diseases and allergic diseases.

 [0041] "Effervescent alcoholic beverage" includes, for example, beer, miscellaneous sake, and happoshu. “Can grow in sparkling alcoholic beverages” means that lactic acid bacteria do not die in sparkling alcoholic beverages, and the number of cells increases due to cell division. The alcohol concentration of sparkling alcoholic beverages is 5 % Or more is preferable.

 [0042] Strains belonging to the ratatobacillus brevis subspecies brevis can be easily isolated from the natural world and can be identified by examining the base sequence of 16S ribosomal DNA. It can also be purchased from cell banks such as ATCC.

[0043] Among the strains belonging to Ratatobacillus brevis subspecies brevis, strains capable of growing in effervescent alcoholic beverages can be selected according to the method described in JP-A-2003-250557. Specifically, a genomic DNA of a strain belonging to Lactobacillus brevis subspecies brevis is used as a saddle type and a predetermined primer set (Nucleic acids described in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing of JP-A-2003-250557) PCR with a primer set consisting of oligonucleotides consisting of sequences! /, The amplified DNA gyrase subunit B gene fragment was cleaved with restriction enzymes, and the restriction enzyme cleaved pattern after acrylamide gel electrophoresis And select strains belonging to the group lib. Restriction enzyme cleavage patterns are classified into four groups, and the strains that can grow in sparkling alcoholic beverages have been found to belong to the group lib.

[0044] In addition to the method described above, the strain can also be selected by investigating whether or not the strain belonging to Lactobacillus brevis subspecies brevis is transplanted and cultured in an effervescent alcoholic beverage. The culture temperature can be cultivated as long as it is between 15 ° C to 45 ° C, preferably 20 ° C to 37 ° C, particularly around 30 ° C.

[0045] Among the strains belonging to Ratatobacillus' brevis subspecies brevis, strains producing _ァ aminobutyric acid (hereinafter referred to as GABA) are analyzed by analyzing the culture supernatant with an amino acid analyzer or the like and examining the GABA content. Can be selected.

 [0046] Among the strains belonging to Ratatobacillus' brevis subspecies brevis, strains having an anti-allergic action are, for example, i) production promoting action of interferon γ and interleukin 12 on mouse spleen cells, It can be selected by testing the inhibitory effect of IgE production induced by spleen cells of immunized mice, OVA) the suppressive action of IgE secreted into the peripheral blood of OVA immunized mice, and the like.

[0047] In i), spleen cells are isolated from the spleen of a mouse and cultured, and then a cell suspension prepared by sterilizing the cells of the test strain is added to the spleen cell and cultured for a certain period of time. The production amounts of interferon _γ and interleukin 12 secreted from the blood can be measured by ELISA or the like, and the presence or absence of an interferon γ and interleukin 12 production promoting action can be examined.

 [0048] In ii), spleen cells of an OVA-immunized mouse 2 weeks after the booster were isolated and cultured, and a cell suspension prepared by sterilizing the cells of the test strain and OVA were added thereto. Culture for a certain period of time, measure the production of IgE secreted from spleen cells by ELISA, etc., and examine the IgE production inhibitory action.

 [0049] In iii), a cell suspension prepared by sterilizing the bacterial strain of the test strain was intraperitoneally injected into the abdominal cavity of an OVA mouse, reared for a certain period of time, and the amount of IgE secreted into the peripheral blood was determined. Measure by ELISA etc. and administer the cell suspension intraperitoneally! /, Compare with the production of IgE secreted in the peripheral blood of OVA mice.

[0050] Among the strains belonging to Ratatobacillus' brevis subspecies brevis, it has an immunostimulatory effect. The strain to be selected can be selected, for example, by testing the IgA production promoting effect on mouse Peyer's patch cells.

[0051] Specifically, Peyer's patch cells were isolated from the intestinal tract of the mouse and cultured, and a cell suspension prepared by sterilizing the cells of the test strain was added thereto and cultured for a certain period of time. It is sufficient to measure the production amount of IgA secreted from the plate cells by ELISA and examine the presence or absence of an IgA production promoting action.

 [0052] SBC8027 belonging to Ratatobacillus brevis subspecies brevis, which can be grown in a sparkling alcoholic beverage, produces GABA, and has antiallergic action (date of trust: June 28, 2006; trust number) FERM BP—10630) is an international depositary authority, the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Tsukuba Rokuto 1-chome, 1-chome, 1-Chuo 6 (zip code 305-8566)) Deposited and available

[0053] The beverage, food and antiallergic agent of the present invention are strains belonging to Ratatobacillus brevis subspecies brevis, can be grown in a sparkling alcoholic beverage, produce GABA, and have antiallergic action and immunostimulation. It is characterized by containing the above-mentioned strain having an action.

 [0054] The bacterial cells of the above strains can be added to beverages and foods for the purpose of preventing and treating allergic diseases, cancer, infectious diseases and the like. The beverages and foods may consist only of the bacterial cells of the strains described above, and may contain additives usually used in the field. Examples of the additives include apple fiber, soybean fiber, meat extract, black vinegar extract, gelatin, corn starch, honey, animal and vegetable oils and fats, monosaccharides such as glucose, disaccharides such as sucrose, fructose and mannitol, and dextrose. And polysaccharides such as starch, sugar alcohols such as erythritol, xylitol and sorbitol, and vitamins such as vitamin C. These additives may be used alone or in combination.

 [0055] Further, for the purpose of prevention of allergic diseases, cancer and infectious diseases, and alleviation of symptoms, food additives include foods for specified health use, special nutritional foods, dietary supplements, health foods, functional foods and diseases. It can also mix | blend with food and drinks, such as food for consumers.

[0056] The antiallergic agent and the immunostimulant of the present invention contain the above bacterial strains as active ingredients. And can be used as an antiallergic agent with excellent safety if it is formulated by adding carriers, excipients and / or other additives. Examples of pharmaceutically acceptable additives include monosaccharides such as glucose, disaccharides such as sucrose, fructose and mannitol, polysaccharides such as dextrose and starch, and sugar alcohols such as erythritol, xylitol and sorbitol. Vitamins such as vitamin C, acacia gum, calcium phosphate

, Alginate, calcium silicate, microcrystalline cellulose, polybulurpyrrolidone, cellulose derivative, tragacanth, gelatin, syrup, methyl hydroxybenzoate, talc, magnesium stearate, water, mineral oil The product may be a single species or a plurality of species.

 [0057] The antiallergic agent of the present invention comprises a viscous substance secreted by a strain belonging to Lactobacillus brevis subspecies brevis.

 [0058] This strain viscous substance is a step of culturing a strain belonging to Ratatobacillus brevis subsp. Brevis and obtaining a culture solution, and a precipitate for removing the bacterial cells from the culture solution and adding alcohol to precipitate the viscous substance. And a recovery step of removing the supernatant and recovering the precipitated viscous material.

 [0059] Ratatobacillus brevis has four subspecies mosquitoes, which are brevis, gravesensis, otakiensis, and coagulans, respectively. The strain belongs to the subspecies Brevis. Strains belonging to subspecies brevis can be separated using the base sequence and sugar strength of 16S ribosomal DNA, differences in acid production, etc. as indicators, and are classified as strains that do not belong to subspecies grebsensis, subspecies otakiensis and subspecies coagulance .

 [0060] Strains belonging to the ratatobacillus' brevis subspecies brevis can be easily isolated from nature and can be identified by examining the base sequence of 16S ribosomal DNA. It can also be purchased from cell banks such as ATCC.

[0061] In the above culturing step, a strain belonging to Ratatobacillus brevis subspecies brevis, for example, MRS liquid medium (manufactured by Difco, composition: 1% proteose peptone, 1% beef extract, 0.5% yeast extract, 2% glucose, 0.1% Tween 80, 0.5% citrate ammoyu , 0.01% magnesium sulfate, 0.005% manganese sulfate, 0.2% dipotassium phosphate), followed by static culture or shaking culture at room temperature to 37 ° C. The liquid medium can be substituted with fruit juice (apple, grapefruit, grape, etc.).

 [0062] In the precipitation step, for example, the culture solution is filtered, or centrifuged at 7000 rpm for 10 minutes to precipitate the bacterial cells. After removing the bacterial cells, the alcohol concentration is 35 volume%. Alcohol should just be added so that it may become the above. The alcohol is more preferably ethanol or isopropanol, which is preferably a lower alcohol of C; The alcohol concentration is preferably 50% by volume, more preferably 60% by volume.

 [0063] In the recovery step, for example, the culture solution to which alcohol has been added in the precipitation step is centrifuged at 700,000 rpm for 10 minutes to precipitate a viscous substance, and the supernatant may be removed.

[0064] The action of this anti-allergic agent consisting of a viscous substance can act on antigen-presenting cells, T cells or mast cells to suppress the production of IgE and release of the above inflammatory substances, or Thl / Th2 This includes shifting the balance to the Thl side, and more specifically, promoting interferon _γ production and interleukin 12 production.

Yes

 [0065] Among the viscous substances secreted by strains belonging to the ratatobacillus brevis subspecies brevis, those having an anti-allergic action include, for example, i) the production promoting action of interferon γ and interleukin 12 on mouse spleen cells, ii) It can be selected by testing the suppression effect of IgE production induced by spleen cells of ovalbumin (OVA) immune mice, iii) the suppression effect of IgE production secreted into the peripheral blood of OVA immunized mice, etc. .

 [0066] In i), spleen cells are isolated and cultured from the spleen of a mouse, sterilized viscous substance is added to the spleen and cultured for a certain period of time, and the production of interferon γ and intermouth 12 secreted from the spleen cells May be measured by ELISA or the like, and the presence or absence of interferon γ and interleukin 12 production promoting action may be examined.

 [0067] In ii), spleen cells of OVA immunized mice 2 weeks after the booster are isolated and cultured, then sterilized viscous substance and OVA are added thereto, cultured for a certain period of time, and secreted from spleen cells Measure IgE production by ELISA etc. and check IgE production suppression effect! /.

[0068] In iii), a sterilized viscous substance is administered into the abdominal cavity of an OVA mouse and reared for a certain period of time. The amount of IgE secreted in the treetop blood is measured by ELISA, etc., and the cell suspension is administered intraperitoneally, and compared with the production of IgE secreted in the peripheral blood of OVA mice. Good! /

 [0069] In addition, SBC8027 (contract date: June 28, 2006; accession number: FERM BP-10630), which secretes an anti-allergic viscous substance and belongs to ratatobacillus' brevis subspecies brevis, is an international deposit It is deposited with the National Institute of Advanced Industrial Science and Technology Patent Biology Depositary Center (1st, 1st, 1st, 1st, Tsukuba, Ibaraki, Japan) (zip code 305-8566).

 [0070] The above-mentioned antiallergic agent can be used by lyophilizing the viscous substance recovered in the recovery step, but may be formulated by adding a carrier, an excipient and / or other additives, for example. . Examples of pharmaceutically acceptable additives include monosaccharides such as glucose, disaccharides such as sucrose, fructose and mannitol, polysaccharides such as dextrose and starch, sugar alcohols such as erythritol, xylitol and sorbitol, Vitamins such as vitamin C, gum acacia, calcium phosphate, alginate, calcium silicate, microcrystalline cellulose, polybulurpyrrolidone, cellulose derivatives, tragacanth, gelatin, syrup, methyl hydroxybenzoate, talc, magnesium stearate, water, mineral oil These additives may be a single species or a plurality of species! /.

 [0071] The beverage and food of the present invention are characterized by containing an antiallergic agent composed of the above viscous substance.

 [0072] For example, if the viscous substance recovered in the above-described recovery step is freeze-dried and added to the target beverage and food, a beverage and food having an antiallergic action can be produced. The beverages and foods described above may contain additives commonly used in the art, in which only this viscous substance may be added. Examples of this additive include apple fiber, soybean fiber, meat extract, black vinegar extract, gelatin, corn starch, honey, animal and vegetable oils and fats, monosaccharides such as glucose, disaccharides such as sucrose, fructose and mannitol, dextrose and Examples include polysaccharides such as starch, sugar alcohols such as erythritol, xylitol and sorbitol, and vitamins such as vitamin C. These additives may be used alone or in combination.

[0073] Furthermore, as a food additive for the purpose of preventing allergic diseases and alleviating symptoms It can also be added to foods and drinks such as health foods, special nutritional foods, dietary supplements, health foods, functional foods and foods for the sick.

 Example

 Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples.

 [0075] (Example 1)

 1) About each strain used in the experiment

 Lactobacillus brevis subspecies brevi s (13 strains belonging to Lactobacillus brevis subspecies brevi s) (SBC8027 and strains a to l) were isolated by the inventor and stored at 4 ° C as freeze-dried cells until they were used in the experiment. . In addition, strain X belonging to Lactobacill us rhamnosus was isolated from commercially available yogurt and stored at 4 ° C as lyophilized cells until used in experiments.

 [0076] 2) Determination of growth ability in beer

 Proliferation ability in beer was selected for 13 strains belonging to the aforementioned Ratatobacillus brevis subsp. Brevis and strain X belonging to Ratatobacillus' Ramnosus. The selection method was performed according to the method described in Japanese Patent Publication No. 2003-250557. That is, a predetermined primer set using the genomic DNA of each lactic acid strain as a saddle type (a primer set comprising an oligonucleotide comprising the nucleic acid sequences described in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing of JP-A-2003-250557) ), And the amplified DNA gyrase subunit B gene fragment was cleaved with a restriction enzyme and analyzed by analyzing the restriction enzyme cleavage pattern after acrylamide gel electrophoresis. In this method, restriction enzyme cleavage patterns can be broadly classified into four groups, and strains that can grow in beer have been found to belong to the group lib.

 [0077] As a result, all the 13 strains belonging to Ratatobacillus brevis subspecies brevis examined were determined to be strains that grew in beer S, strains belonging to Ratatobacillus rhamnosus

X was not determined to be a strain that grew in beer. In addition, the above-mentioned strain was actually inoculated into beer and the ability to confirm the growth was the same as the determination result of the above method.

[0078] 3) Evaluation of antiallergic action i) Preparation of cell suspension

 In order to evaluate the antiallergic action of each of the above strains, cell suspensions of each strain were prepared. First, each strain is subjected to anaerobic conditions (N: CO: H = 90: 5: 5 gas composition), MRS

 2 2 2

 Liquid medium (Difco, composition: 1% proteose peptone, 1% beef extract, 0.5% fermented mother extract, 2% glucose, 0.1% Tween 80, 0.5% ammonium oxalate, 0.01 (Magnesium sulfate, 0.005% manganese sulfate, 0.2% dipotassium phosphate) for 3 days, and the culture is centrifuged at 1,500 rpm for 10 minutes. Recovered. The obtained bacterial cells were washed with PBS, freeze-dried, and suspended in PBS so as to be 1 mg / mL. The bacterial cell suspension thus obtained was autoclaved (121 ° C, 15 minutes) and used in the following experiments.

[0079] ii) Stimulation of interferon _γ production by strains belonging to Ratatobacillus brevis subsp. Brevis on mouse spleen cells (in vitro)

 By adding a cell suspension of 13 strains belonging to Ratatobacillus brevis subspecies brevis to mouse spleen cells and culturing for a certain period of time, the amount of interferon γ secreted from the spleen cells was determined to determine the interferon γ of each strain. The production promoting effect was evaluated.

 [0080] (Preparation of mouse spleen cells)

First, the spleen was aseptically removed from a 6-week-old BALB mouse (female) and immersed in an RP MI1640 medium containing 10% FBS. Thereafter, the spleen was transferred to a petri dish and crushed with a pestle, and the mouse spleen cell suspension was passed through a nylon filter net (Nippon Riken Kikai Co., Ltd.) having an opening of 70 Hm and a wire diameter of 39 Hm. The mouse spleen cell suspension that passed through this nylon filter net was centrifuged at 1,500 rpm for 10 minutes, and the supernatant was discarded, followed by erythrocyte lysis reagent (0.16M ammonium chloride, Tris'HC1, pH7. 2) was added to the precipitated mouse spleen cells and allowed to stand at room temperature for 5 minutes. Then, the addition of new 10% FBS-containing RPMI1640 medium were washed mouse spleen cells, 1, 500 and centrifuged 10 minutes at a rotation, as the number of cells after the supernatant was discarded becomes 5 X 10 ⁶ cells / mL RPMI1640 medium containing 10% FBS was added. The mouse spleen cells thus obtained were used for the following experiments.

 [0081] (Measurement of interferon γ by ELISA)

Place mouse spleen cells in a 96-well plate so that the cell density is 2.5 x 10 ⁶ cells / mL. The cells were seeded and cultured in RPMI 1640 medium containing 10% FBS under conditions of 37 ° C and 5% CO. Ma

 2

After culturing the mouse spleen cells, add the cell suspension of each strain (final concentration: 10 g / mU to each well) and the amount of interferon γ secreted into the culture supernatant after 72 hours The positive control was negative for lipopolysaccharide (LPS) (SIGMA, final concentration: 10 g / mL), which has an interferon _γ production promoting effect on mouse spleen cells. PBS was used as a control.

 [0082] ELISA for quantifying interferon γ was performed as follows. First,

1. Add 50 L of the primary antibody (Rabbit anti-mouse / rat inteneron-y; BIO SOURCE) prepared to 25 g / mL to each well of a 96 well plate (Maxisorp immunoplate; NUNC), 4 ° The solid phase was established by leaving it to stand in C. The 96-well plate was then washed 3 times with Wash Buffer and blocked with 1% ushi serum albumin (BSA; Sigma). Subsequently, each culture supernatant or a preparation of interferon _gamma whose concentration is known in advance is added to each well in an amount of 50 HL, and reacted with the primary anti-interferon gamma antibody for 90 minutes. After washing three times, add 50 L of secondary antibody, Anti-mouse / rat interferon-γ biotin conjugate (BIO SOURCE), prepared to 0.5 gZ mL to each well, and react at room temperature for 90 minutes I let you. Then, each well is washed 5 times with Wash Buffer, added with streptavidin HRP (BIO SOURCE), reacted, washed 5 times with Wash Buffer, and then TMB (tetramethylbenzidine) poor greed 3, 5, 5 — Tetramethylbenzidin e (TMB) Liquid Substrate System (SIGMA) was added and reacted. After sufficient color development, 50 μL of 2N sulfuric acid was added to each well to stop the reaction, and the absorbance at 450 nm was measured. A standard curve was prepared from the absorbance of the interferon γ preparation, and the amount of interferon Ί produced by mouse spleen cells was quantified from this standard curve.

[0083] FIG. 1 shows the amount of interferon γ produced by mouse spleen cells by the addition of a cell suspension of 13 strains belonging to Ratatobacillus brevis subsp. Brevis. As a result, Ratatobacillus brevis subspecies brevis SBC8027 induces the production of interferon _{γ in} mouse spleen cells, and the production amount is LPS and other strains belonging to Ratata brevis subspecies brevis (strain). a ~ l) significantly higher compared to the amount induced by V, that was. SBC8027 (accession number: F ERM BP—10630; date of accession: June 28, 2006), which has the effect of inducing interferon γ production, is the International Depository Agency, National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary. Deposited at 1st, 1st, 1st, Tsukuba, Ibaraki, Japan (6th postal code: 305-8566).

 [0084] iii) SBC8027 promotes Thl site force-in production, suppresses Th2 site force-in production and suppresses IgE production (in vitro) on spleen cells of OVA immunized mice

 SBC8027, which showed interferon γ production promoting action on mouse spleen cells, promoted Thl site force-in (interferon γ and interleukin 12) production and Th2 site force-in (interleukin 12) on spleen cells of OVA immunized mice. 4) Production suppression The production and IgE production suppression effects were evaluated. At that time, the actions of strains b and c belonging to Ratatobathiras' brevis subspecies brevis and strain X belonging to Ratatobathiras rhamnosus were also investigated and compared with the effects of SBC8027.

 [0085] (Creation of OVA immunized mice)

 OVA immunized mice were prepared by artificially inducing allergies by intraperitoneally administering OVA to 6-week-old BALB / c mice (female). Specifically, first, lOO g OVA Ovalbumin ^ Eggwhite ^ Puriiied (Worthington Biochemical Corporation) and 10 mg aluminum hydroxide were dissolved in lmL PBS to prepare an OVA antigen solution. First immunization was performed by intraperitoneal administration. One week later, an OVA antigen solution was prepared in the same manner as described above, and 200 L of the solution was administered into the abdominal cavity of the mouse again for additional immunization. The mice that had been boosted were allergic about one week later, and this mouse was used as an OVA immunized mouse in the following experiments.

 [0086] (Preparation of spleen cells of OVA immunized mice)

 Spleens were aseptically removed from OVA immunized mice 2 weeks after the booster immunization, and spleen cells were prepared therefrom by the same procedure as described above.

 [0087] (Measurement of interferon γ, interleukin 12 and interleukin 4 by ELISA)

To measure interferon γ, interleukin 12 and interleukin 4, 2.5 × 10 ⁵ spleen cells of OVA immunized mice were seeded on 96-well plates (cell density 2.5 x 10 ⁶ cells / mL), 37. C, 5% CO, containing 10% FBS RPMI164

 2

 Cultured in 0 medium. Cultivate spleen cells of OVA immunized mice! /, And add OVA (final concentration: 100 g / mL) and cell suspension of each strain (final concentration: 111 g / mL) to each well. Then, after 72 hours, the amount of each site force-in secreted into the culture supernatant was quantified by ELISA. At that time, a control in which PBS was added instead of the cell suspension was provided.

 [0088] Quantification of interferon γ by ELISA was performed in the same procedure as described above. For quantitation of interleukin-12, 1 μg / mL Purified anti-mouse

Interferon using IL-12 (p40 / p70) (BD Pharmingen) and 1 g / mL Biotin anti-mouse IL-12 (p40 / p70) (BD Pharmingen) as the secondary antibody. ELISA was performed according to the same procedure as for quantification of _γ . For the quantification of interleukin-4, 1g / mL Monoclonal Anti-mouse IL-4 Antibody (R & D Systems Inc.) was used for the primary antibody, and 1g / mL Biotinylated Anti-mouse IL- for the secondary antibody. 4 ELISA was performed using Antibody (R & D Systems Inc.) in the same manner as the quantification of interferon γ.

[0089] Fig. 2 shows the amount of interferon γ produced by the spleen cells of OVA-immunized mice by the addition of OVA and cell suspension of each strain, Fig. 3 shows the amount of interleukin 12, and Fig. 4 shows the amount of interleukin. The amount of 4 is shown. Fig. 5 is a graph showing the calculation of interferon _γ / interleukin 4 as an index of Thl / Th2 balance. As a result, in the control supplemented with OV Α and PBS, the production of interferon γ and interleukin 12 that are Thl-type site force-in was hardly observed, and the production of interleukin 4 that is a Th2-type site force-in was remarkable. Was guided to. This suggests that the spleen cells of OVA immunized mice have a Thl / Th2 balance shifted to the Th2 side and allergic reaction is enhanced.

[0090] On the other hand, in the treatment group to which the cell suspension of OVA and SBC8027 was added and in the treatment group to which the cell suspension of OVA and strain b or strain c was added, the interface was a Thl type site force-in. Γ and interleukin 12 productivity control and the production of interleukin 4 which is a Th2-type site force-in was significantly suppressed compared to strain X belonging to Ratatobacillus rhamnosus. The action of SBC8027 is more than the action of strain b or strain c. It was remarkable and greatly shifted the Thl / Th2 balance of spleen cells of OVA immunized mice to the Thl side. This result suggests that SBC8027 has a strong antiallergic action.

[0091] (Measurement of total IgE and OVA-specific IgE by ELISA)

In order to measure total IgE, 96 spleen plates were seeded with 2.5 x 10 ⁵ OVA immunized mouse spleen cells (cell density 2.5 x 10 ⁶ cells / mL). C, 5% CO,

 2

 The cells were cultured in RPMI1640 medium containing 10% FBS. Then, OVA (final concentration: 100 g / mL) and cell suspension of each strain (final concentration: 1 g / mL) were added to each well in which spleen cells of OVA-immunized mice were cultured. The amount of total IgE secreted into the culture supernatant after the passage of days was quantified by ELISA. As a control, PBS was added instead of the cell suspension, and quantified by ELISA in the same manner.

[0092] On the other hand, in order to measure OVA-specific IgE, 48 wenole plates were seeded with 2.5 x 10 ⁶ spleen cells (cell density was 2.5 x 10 ⁶ cells / mL). 37. C, 5% CO,

 2

 The cells were cultured in RPMI1640 medium containing 10% FBS. OVA (final concentration: 100 g / mL) and bacterial suspension of each strain (final concentration: 1 μg / mL) are added to the spleen cells and cultured for 3 days, and the spleen cells are washed 3 times with new medium. OVA was removed, and the bacterial suspension of each strain was added to the spleen cells after washing again to a concentration of 1 g / mL, and cultured for 11 days. The IgE secreted in the culture supernatant was then removed. Quantified by ELISA as OVA-specific IgE. As a control, PBS was added in place of the cell suspension, and quantified by ELISA in the same manner.

[0093] ELISA for quantifying total IgE and OVA-specific IgE was performed as follows. First, 96 mouse refection of anti-mouse IgE antibody (Mouse IgE ELISA Quantitation Kit; BETHYL Laboratories Inc.) prepared to 10 g / mL. 50 L of each well (Maxisorp immunoplate; NUNC) was added to each well and left at 4 ° C for solid phase. The 96-well plate was then washed 3 times with Wash Buffer and blocked with 1% urine serum albumin (BSA; Sigma). Subsequently, each culture supernatant or IgE preparation with a known concentration is added to each well, reacted with anti-mouse IgE antibody for 90 minutes, washed 3 times with Wash Buffer, and then Biotinylation Kit (Cygnus Technology). , Inc.), add 50 μL of biotinylated OVA to each weinole, The reaction was allowed to proceed for 90 minutes at room temperature. Then, each well was washed 5 times with Wash Buffer, added with streptavidin-HRP (BIO SOURCE), reacted, washed 5 times with Wash Buffer, and then washed with TMB (tetramethylbenzidine) substrate solution (3, 3 ', 5 , 5'-Tetram ethylbenzidine (TMB) Liquid Substrate System (SIGMA) was added for reaction. After sufficient color development, 50 L of 2N sulfuric acid was added to each well to stop the reaction, and the absorbance at 450 nm was measured. A standard curve was prepared from the absorbance of the IgE preparation, and the amount of IgE produced by spleen cells of OVA mice was quantified from the standard curve. The amount of OVA-specific IgE was expressed as a relative value to the absorbance of the control (negative control) because there was no OVA-specific IgE preparation.

 [0094] Fig. 6 shows the effect of the bacterial cell suspension of each strain on the production of total IgE induced in the spleen cells of OVA-immunized mice by the addition of OVA. FIG. 7 shows the effect of the cell suspension of each strain on the production of OVA-specific IgE induced in the spleen cells of OVA-immunized mice by the addition of OVA.

 As a result, the addition of SBC8027 cell suspension suppressed the total IgE amount induced by OVA and the OVA-specific IgE by about 15% and about 90%, respectively.

 [0096] On the other hand, although strain b and strain c and strain X belonging to Ratatobacillus rhamnosus suppressed the total amount of OVA-induced IgE and production of OVA-specific IgE, they had weaker effects than SBC8027. .

[0097] Based on the above results, SBC8027 belonging to Ratatobacillus brevis subspecies brevis has an interferon _γ and interleukin 12 production promoting action and an IgE production inhibitory action, compared with the strains of lactic acid bacteria known so far. It has been found that it exhibits a strong antiallergic action.

 [0098] iv) Inhibitory effect of SBC8027 on IgE production in OVA immunized mice (in vivo)

 SBC8027, which was found to have a strong antiallergic effect in in vitro experiments, was evaluated in vivo for its inhibitory effect on IgE production in OVA immunized mice. At that time, the effect of strain X belonging to Lactobacillus rhamnosus was also investigated and compared with that of SBC8027.

[0099] OVA immunized mice were prepared as described above using OVA (Ovalbumin, Eggwhite, Purified; Wor initial immunity and effort at thington Biochemical Corporation)! ] SBC8027 or strain X suspension or PBS (control) once every two days from one week before the first immunization to one week after the booster immunization It was administered intraperitoneally. Each bacterial cell suspension was prepared by suspending the lyophilized bacterial cells as described above in PBS to 1 mg / mL and autoclaving (121 ° C, 15 minutes).

 [0100] One week after the booster immunization, blood was collected from the tail vein of the mouse, and the amount of total IgE and OVA-specific IgE in the serum separated therefrom was measured by ELISA. Measurement of total IgE in serum and OVA-specific IgE by LISA was performed in the same manner as described above.

 [0101] FIG. 8 shows the production of total IgE secreted into the peripheral blood of OVA-immunized mice given each strain intraperitoneally. FIG. 9 shows the production of OVA-specific IgE secreted into the peripheral blood of an OVA immune mouse to which each strain was similarly administered intraperitoneally. * In the graph indicates statistical significance with respect to control at p <0.05.

 [0102] As a result, the addition of SBC8027 bacterial cell suspension suppressed the total IgE amount secreted into the peripheral blood of OVA mice by about 80%, and the OVA-specific IgE amount by about 60%. This action was statistically significant compared to the control receiving PBS.

 [0103] On the other hand, strain X belonging to Ratatobacillus rhamnosus suppresses the production of total IgE and OVA-specific IgE secreted in the peripheral blood of OVA mice, but has a weaker effect than SBC8027, and It was not statistically significant.

 [0104] Based on the above results, SBC8027, which belongs to Ratatobacillus brevis subspecies brevis, has an inhibitory effect on IgE production even in vivo, and has a stronger anti-allergic effect compared to previously known strains of lactic acid bacteria. It was found that

 [0105] 4) Evaluation of immunostimulatory effect

 SB C8027, which was found to have a strong antiallergic effect in in vitro and in vivo experiments, was evaluated for its IgA production promoting effect on mouse Peyer's patch cells.

 [0106] (Preparation of mouse Peyer's patch cells)

First, the intestinal tract was aseptically removed from a 6-week-old BALB mouse (female), and then the Peyer's patch was removed with scissors and immersed in RPMI 1640 (GIBCO). Repeat the operation of sucking up the Peyer plate with the pipette to thoroughly wash the Peyer plate and remove the Peyer plate. After transferring to an empty petri dish, add 10 mL of dispase solution (1.5 mL of 15 mg / mL dispase (GIBCO) and 8.5 mL of RPMI 1640 (GIBCO), and continue at 37 ° C for 40-45 minutes. The cell suspension thus obtained was centrifuged at 1,500 rpm for 10 minutes to precipitate Peyer's patch cells, and the supernatant was discarded, followed by addition of RPMI1640 medium containing 10% FBS. by repeating the operation to wash the Peyer's patch cells. Thereafter, number suspended 5 X 10 ⁶ cells / mL in comprising as the addition of 10% FBS RPMI 1640 medium containing of Peyer's patch cells were used in the following experiments .

[0107] (IgA ELISA measurement)

Inoculate mouse Peyer's plate cells on a 96-well plate so that the cell density is 2.5 x 10 ⁶ cells / mL, and culture in RPMI1640 medium containing 10% FBS at 37 ° C and 5% CO.

 2

 It was. The cell suspension of each strain (final concentration: 10 011 g / mL) was added to each well in which noel plate cells were cultured, and the amount of IgA secreted into the culture supernatant after 7 days Was quantified by ELISA. Lipopolysaccharide (hereinafter LPS) (SIGMA, final concentration: 10 g / mL) having an IgA production promoting action on mouse spleen cells was used as a positive control, and PBS was used as a negative control.

[0108] IgA ELISA kit (Mouse IgA ELISA Quantitation Kit)

 Quantified using BETHYL Laboratories Inc.) according to the manufacturer's protocol

Yes

 [0109] Fig. 10 shows the amount of IgA produced by the addition of the cell suspension of SBC8027. As a result, SBC8027 induced IgA production on mouse Peyer's patch cells, and the production amount was significantly higher than that of the control, and was comparable to the action of LPS.

 [0110] 5) Measurement of γ-aminobutyric acid (GABA) productivity

SBC8027 belonging to Ratatobacillus brevis subspecies brevis was tested for the ability to produce γ-aminobutyric acid (GABA). First, SBC8027 was inoculated into lOOmL liquid medium (3% malt extract (DIFCO), 2% yeast extract (DIFCO), 0.2% sodium glutamate, pH 6.0) and left to stand for 4 days. did. Thereafter, the culture solution of SBC8027 is centrifuged at 500 rpm for 10 minutes to recover the culture supernatant, and the GABA contained in the culture supernatant is recovered. The amount was quantified by HPLC. The HPLC conditions used are as follows.

 • HPLC system: Agilent HPLC 1100

 'Column used: ZORBAX eclipse ΑΑΑ (4.6 x 150mm, 3.5m) (Shimadzu GE)

 • Column oven: 40 ° C

 • Fluent. OmL min

 • Fluorescence detector: Ex. 340nm, Em. 450nm

 • HPLC reagent: lOmg / mL Agilent OPA reagent (0.4M borate buffer, pHIO

2) (Shimadzu GL Corporation)

 • Eluent: Liquid A: 40 mM NaH2P04 (pH 7.8)

 Liquid B: 45% by volume acetonitrile, 45% by volume ^^ 〇 ^ 1, 10% H 2 O

 2

 • Timetable (gradient): see Table 1

[0111] [Table 1]

As a result, SBC8027 produced 479.3 mol / L of GABA in the culture supernatant.

[0113] Based on the above results, SBC8027 belonging to Ratatobacillus' brevis subspecies brevis can be grown in effervescent alcoholic beverages, producing GABA with anti-stress action, and further has anti-allergic and immunostimulatory effects. It became clear that it was a strain having both.

[0114] 6) Sensory test of lactic acid fermentation broth manufactured using SBC8027

 A fruit juice lactic acid fermentation broth was produced using SBC8027, which has an antiallergic action, and a sensory test was conducted on the aroma and flavor of the obtained fruit juice lactic acid fermentation broth.

[0115] Table 2 shows the fruit type, sugar content, and pH of the fruit juice used for lactic acid fermentation. [0116] [Table 2]

[0117] Grapefruit concentrated juice, apple concentrated fruit juice, white grape concentrated fruit juice and lemon concentrated fruit juice are each diluted with sterilized water to have a predetermined sugar content, and pH is added with NaOH. It was adjusted. Lactic acid fermentation is inoculated with 1.5 × 10 ⁹ cells of SB C8027 per lOOmL of each juice, stirred once a day for 72 hours at 30 ° C, and under static conditions except during stirring. It was. After completion of fermentation, the turbidity of each lactic acid fermentation broth was measured with a spectrophotometer (Tytec Corp.), and the amount of lactic acid was measured with F-kit D- / L-lactic acid (JK International Corp.). The turbidity of each lactic acid fermentation broth was used as an indicator of SBC8027 growth, and the amount of lactic acid was used as an indicator of the degree of lactic acid fermentation.

 [0118] Table 3 shows the turbidity, lactic acid amount, and sensory test results of each lactic acid fermentation broth.

[0119] [Table 3]

As a result, the lactic acid fermentation of SBC8027 progressed with all the juices used in the test, leading to the production of a lactic acid fermentation broth. In particular, in fermentation using apple juice, thickening occurs, a mild fragrance is added, and it has desirable properties as a lactic acid fermented beverage. [0121] (Example 2)

 1) About each strain used in the experiment

 SBC8027 (FERM BP-10630) of Lactobacillus brevis subspecies brevi s was isolated by the inventor and stored at 4 ° C as lyophilized cells until use in experiments.

[0122] 2) Evaluation of antiallergic action

 i) Preparation of viscous substances

 First, SBC8027 belonging to Ratatobathiras brevis subspecies brevis, 1.6L MRS liquid medium (Difco, composition: 1% proteose peptone, 1% beef extract, 0.5% fermented maternal extract, 2% glucose, 0.1% Tween 80, 0.5% ammonium citrate, 0.01% magnesium sulfate, 0.005% manganese sulfate, 0.2% dipotassium phosphate) and left to stand for 3 days. . After that, the culture solution is centrifuged at 7000 rpm for 10 minutes to remove the cells as a sediment, ethanol is added so that the alcohol concentration becomes 60% by volume, and then it is centrifuged at 7000 rpm for 10 minutes. Then, the viscous substance was precipitated and the supernatant was removed. The viscous material thus obtained was dissolved in 40 mL of distilled water, applied to a gel filtration column (Seph adex G-100), and fractions with a molecular weight of 100,000 or more were collected and lyophilized. . As a result, 720 mg of a viscous material was obtained by dry weight.

[0123] ii) Preparation of viscous substance solution

 Next, 1 mg of the lyophilized product of the viscous substance was dissolved in 1 mL of sterile water and treated with artificial gastric juice and artificial intestinal fluid. First, add 0.1 mL artificial gastric fluid (10,000 units / mL pepsin, 2% NaCl), react for 2 hours at 37 ° C, then continue with 0.1 mL artificial intestinal fluid (4% noncreatine, 4% Trypsin, 1M NaHCO 3) was added and reacted at 37 ° C. for 2 hours.

 Three

 Thereafter, the enzyme was inactivated by heating at 105 ° C. for 15 minutes to obtain a viscous material solution used in the following experiment. By treatment with the artificial gastric juice and artificial intestinal fluid, a viscous substance is ingested orally and a state is reached when it acts on the intestinal mucosa.

[0124] iii) Promoting the production of interferon _gamma and interleukin 12 by strains belonging to Ratatobacillus brevis subspecies brevis on mouse spleen cells (in vitro)

Viscous substances treated with artificial gastric juice and artificial intestinal fluid are added to mouse spleen cells for a certain period of time By culturing and examining the amount of interferon γ secreted from the spleen cells, the production promoting action of interferon _γ and interleukin 12 was evaluated.

 [0125] (Preparation of mouse spleen cells)

First, the spleen was aseptically removed from a 6-week-old BALB mouse (female) and immersed in an RP MI1640 medium containing 10% FBS. Thereafter, the spleen was transferred to a petri dish and crushed with a pestle, and the mouse spleen cell suspension was passed through a nylon filter net (Nippon Riken Kikai Co., Ltd.) having an opening of 70 Hm and a wire diameter of 39 Hm. The mouse spleen cell suspension that passed through this nylon filter net was centrifuged at 1,500 rpm for 10 minutes, and the supernatant was discarded, followed by erythrocyte lysis reagent (0.16M ammonium chloride, Tris'HC1, pH7. 2) was added to the precipitated mouse spleen cells and allowed to stand at room temperature for 5 minutes. Then, the addition of new 10% FBS-containing RPMI1640 medium were washed mouse spleen cells, 1, 500 and centrifuged 10 minutes at a rotation, as the number of cells after the supernatant was discarded becomes 5 X 10 ⁶ cells / mL RPMI1640 medium containing 10% FBS was added. The mouse spleen cells thus obtained were used for the following experiments.

 [0126] (Measurement of interferon γ and interleukin 12 by ELISA)

Mouse spleen cells were seeded on a 96-well plate so that the cell density was 2.5 × 10 ⁶ cells / mL, and cultured in RPMI1640 medium containing 10% FBS at 37 ° C. and 5% CO. Ma

 2

To each well in which mouse spleen cells are cultured, add a cell suspension of each strain (final concentration: 1 11 g / mU), and measure the amount of interferon γ secreted into the culture supernatant after 72 hours. As a positive control, lipopolysaccharide (hereinafter referred to as LPS) that promotes the production of interferon _gamma and interleukin 12 in mouse spleen cells (SIGMA, final concentration: 10 g / mL) PBS was used as a negative control.

 [0127] ELISA for quantifying interferon γ was performed as follows. First,

1. Add 50 L of the primary antibody (Rabbit anti-mouse / rat inteneron-y; BIO SOURCE) prepared to 25 g mL to each well of a 96 well plate (Maxisorp immunoplate; NUNC) at 4 ° C. The solid phase was established by standing still. The 96-well plate was then washed 3 times with Wash Buffer and blocked with 1% ushi serum albumin (BSA; Sigma). Subsequently, each culture supernatant or the concentration is clear in advance. 50 mg HL of each interferon gamma preparation was added to each well, reacted with the primary anti-interferon gamma antibody for 90 minutes, washed 3 times with Wash Buffer, and then prepared to 0.5 gZ mL. A secondary antibody, Anti-mouse / rat interferon-γ biotin conjugate (BIO SOURCE) was added to each well in an amount of 50 L and allowed to react at room temperature for 90 minutes. Then, each well is washed 5 times with Wash Buffer, added with streptavidin HRP (BIO SOURCE), reacted, washed 5 times with Wash Buffer, and then TMB (tetramethylbenzidine) poor greed 3, 5, 5 — Tetramethylbenzidin e (TMB) Liquid Substrate System (SIGMA) was added and reacted. After sufficient color development, 50 μL of 2N sulfuric acid was added to each well to stop the reaction, and the absorbance at 450 nm was measured. A standard curve was prepared from the absorbance of the interferon γ preparation, and the amount of interferon Ί produced by mouse spleen cells was quantified from this standard curve.

 [0128] For the quantification of interleukin 12, 1 ag / mL Purified anti-mouse IL—12 (p40 / p70) (BD Pharmingen) was used for the primary antibody and 1 μg / mL for the secondary antibody. (7) ELISA was performed using Biotin anti-mouse IL-12 (p40 / p70) (BD Pharmingen) in the same manner as the quantification of interferon γ.

 FIG. 11 shows the amounts of interferon γ and interleukin 12 produced by mouse spleen cells by addition of a viscous substance secreted by SBC8207 belonging to Ratatobacillus brevis subspecies brevis.

 [0130] As a result, the viscous substance secreted by SBC8207 belonging to Ratatobatilas' brevis subspecies brevis induces the production of interferon γ and interleukin 12 in mouse spleen cells, both of which are produced by LPS. It was significantly higher than the amount induced.

 [0131] From the above results, it was found that the viscous substance secreted by the strain belonging to Ratatobatilas' brevis subsp. Brevis has an antiallergic effect.

 Industrial applicability

[0132] Since the strain of the present invention can grow in an effervescent alcoholic beverage, other lactic acid strains having no activity can be excluded from the strain of the present invention using this property. Furthermore, since the strain of the present invention produces γ-aminobutyric acid (GABA), in addition to the above-mentioned action, It also has a stress action, and can be expected to have a strong effect on the prevention and treatment of allergic diseases, cancer and infectious diseases that are correlated with physical and mental stress. In addition, according to the present invention, beverages, foods and antiallergic agents which contain the cells of the above strain, have excellent safety, and have antiallergic and immunostimulatory effects can be provided.

 Further, according to the present invention, a viscous substance secreted by a strain belonging to Ratatobacillus brevis subspecies brevis can be used as an antiallergic agent, and beverages and foods containing this antiallergic agent and having excellent safety can be provided.

Claims

The scope of the claims

 [I] A strain belonging to Lactobacillus brevis subspecies brevi s,

 Can grow in sparkling alcoholic beverages,

 Produces aminoaminobutyric acid (GABA),

 A strain having antiallergic action and immunostimulatory action.

[2] The strain according to claim 1, wherein the antiallergic effect is a production promoting effect of interferon γ and / or interleukin 12.

[3] The strain according to claim 1, wherein the antiallergic action is an IgE production inhibitory action.

[4] The strain according to claim 1, wherein the immunostimulatory action is an IgA production promoting action.

[5] The strain according to claim 1, wherein the immunostimulatory action is a natural killer cell or killer cell activating action.

 [6] The strain according to any one of claims;! To 5, which is SBC8027 (FERM BP-10630) of Lactobacillus brevis subspecies brevis.

 [7] The use of the bacterial cell according to any one of claims 6 to 6 as a beverage ingredient.

[8] Use of the bacterial cell according to any one of claims 1 to 6 as a food material.

[9] Use of the bacterial cell of the strain according to any one of claims 6 to 6 for the production of an antiallergic agent.

 [10] Use of the bacterial cell of the strain according to any one of claims;! To 6 for the production of an immunostimulant.

 [I I] Use of a viscous substance secreted by the strain according to any one of claims 1 to 6 for the production of an antiallergic agent.

 [12] The use according to claim 11, wherein the viscous substance is a viscous substance obtained by removing cells from the culture solution of the strain and adding alcohol to precipitate.

[13] The use according to claim 11 or 12, wherein the antiallergic agent has an action of promoting production of interferon γ and / or interleukin 12.

[14] The strain is Lactobacillus brevis subs 14. Use according to any one of claims 11 to 13 which is SBC8027 (FERM BP-10630) of pecies brevis).

[15] Use of a viscous substance secreted by the strain according to any one of claims 6 to 6 as a beverage ingredient.

 [16] Use of a viscous substance secreted by the strain according to any one of claims 1 to 6 as a food ingredient.