JPWO2008023665A1 - Strain having antiallergic action and immunostimulatory action, and beverage, food, antiallergic agent and immunostimulant containing the strain - Google Patents

Abstract

The present invention relates to a strain belonging to Lactobacillus brevis subspecies brevis, which can grow in an effervescent alcoholic beverage, produces γ-aminobutyric acid (GABA), and has an antiallergic action and an immunostimulatory action. provide. The present invention also provides an antiallergic agent comprising a viscous substance secreted by a strain belonging to Lactobacillus brevis subspecies brevis, and beverages and foods containing the antiallergic agent.

Description

  The present invention relates to a strain having an antiallergic effect and an immunostimulatory effect, and beverages, foods, antiallergic agents and immunostimulants 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.

  Treatment of allergic diseases is generally pharmacotherapy using antihistamines, antiallergic agents, steroids and the like. 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 if it is possible to suppress intestinal immunity that reacts excessively, it is thought that it can contribute to the prevention and treatment of allergic diseases.

  For example, strains of the genus Bifidobacterium, infantitis, breve, longum and bifidum are effective in treating food allergies (Patent document 1), Enterococcus faecalis and Lactobacillus reuteri strains (patent document 2), and Lactobacillus paracacti and Lactobacillus p Streptococcus salivarius (Streptococcus sali varius) (patent document 3) has been reported to have an effect on bronchial asthma, allergic rhinitis, atopic dermatitis and the like.

  Furthermore, allergic diseases are known to correlate with physical stress and mental stress, and it is known that a person with more stress has worse symptoms (Non-Patent Document 1). For this reason, it is said that in order to prevent and treat allergic diseases, in addition to the action of suppressing the immune response in the body, it is necessary to eliminate daily stress.

  On the other hand, in the treatment of cancer and infectious diseases, a treatment policy has been established by combining surgical therapy and radiotherapy, mainly chemotherapy using anticancer drugs 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, malignant tumor cells and infected cell bodies are generated everywhere in the living body, but NK cells fight these and protect themselves from cancer and infectious diseases. Immunotherapy aims to treat various diseases by keeping an eye on the function of the immune system and keeping it normal.

  The search for an immunostimulant is effective for improving the immune function in the living body and maintaining health, and has been performed so far. Among these, immunostimulatory substances produced by microorganisms, mushrooms, plants, etc. are known as immunostimulants with low toxicity and confirmed effectiveness. For example, cell wall polysaccharides of microorganisms, or mushroom polysaccharides such as Agaricus and shiitake mushrooms (Patent Documents 4 and 5). Recently, polysaccharides having a glucoside bond have been disclosed as being particularly effective (Patent Documents 6, 7 and 8).

JP-A-10-309178 JP 2000-95697 A JP 2005-139160 A Japanese Patent Publication No.49-484 Japanese Patent Publication No. 56-46481 JP 7-184595 A JP-A-10-194777 JP 2005-220065 PR Satoshi Kanno, “Stress and Allergic Diseases: Targeting Preparatory School Students”, Oto-nose Outlook, 2002, 45, pp. 204-210

  However, chemically synthesized drugs such as antihistamines, antiallergic agents and steroids, as well as immunomodulators and immunostimulants act directly on molecules involved in the immune response in the blood, and immunity necessary for maintaining the homeostasis of the living body. Since the reaction is also suppressed, there is a problem that side effects are large. In addition, the activity of lactic acid bacteria reported so far is not practically effective for preventing chronic diseases such as allergic diseases or for preventing infectious diseases by exerting immunostimulatory action. there were. Furthermore, there has been no report on a strain of lactic acid bacteria that has antiallergic action and immunostimulation and that produces γ-aminobutyric acid (GABA) having antistress action, and Lactobacillus brevis subspecies brevis (Lactobacillus brevis subspecies). Brevis) has never been reported to secrete a viscous substance having an antiallergic action.

  Accordingly, an object of the present invention is to provide a Lactobacillus brevis subspecies brevis having a stronger antiallergic action and immunostimulatory action than previously known lactic acid strains and also having the ability to produce γ-aminobutyric acid (GABA) ( It is to provide a strain belonging to Lactobacillus brevis subspecies brevis). Another object of the present invention is to provide beverages and foods containing bacterial cells belonging to the strain belonging to Lactobacillus brevis subspecies brevis, and antiallergic agents and immunostimulants containing these as active ingredients.

  Furthermore, an object of the present invention is to provide an antiallergic agent comprising a viscous substance secreted by a strain belonging to Lactobacillus brevis subspecies brevis, having no side effects and having a strong antiallergic action. Moreover, the objective of this invention is providing the drink and foodstuff containing this antiallergic agent.

  In order to achieve the above object, the present invention relates to a strain belonging to Lactobacillus brevis subspecies brevis, which can grow in a sparkling alcoholic beverage and contains γ-aminobutyric acid (GABA). Provided is a strain having an antiallergic action and an immunostimulatory action.

  The present inventors have demonstrated that a strain belonging to subspecies brevis among lactic acid bacteria of Lactobacillus brevis species exerts a Th1-type cytokine production promoting action and an IgE production inhibitory action on mouse spleen cells. On the other hand, it has been found that it exerts an IgA production promoting effect. There has been no report of a lactic acid strain having both an antiallergic action and an immunostimulatory action so far, and the antiallergic action and immunostimulatory action of the strain of the present invention are remarkably stronger than those of lactic acid bacteria reported so far. It was a thing. Lactic acid bacteria are bacteria that have been used for fermented foods for a long time, and are much safer to the human body than chemically synthesized antiallergic agents and immunostimulants. In addition, normal lactic acid bacteria cannot grow in effervescent alcoholic beverages, but the strains of the present invention can grow in effervescent alcoholic beverages, and therefore are active from the strains of the present invention using this property. Other lactic acid strains that do not have Furthermore, since the strain of the present invention produces γ-aminobutyric acid (GABA), it has an anti-stress action in addition to the above-described action, and is an allergic disease correlated with physical stress and mental stress, A multifaceted and strong effect can be expected for the prevention and treatment of cancer and infectious diseases. As such a strain, SBC8027 (FERM BP-10630) of Lactobacillus brevis subspecies brevis is particularly preferable.

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

  Interferon γ is a cytokine secreted by Th1 cells, which inhibits B cells from producing IgE and also has cellular immunity such as killer T cells and macrophages that attack viruses, filamentous fungi, and tuberculosis bacteria. Has an increasing effect. In addition, interleukin 12 is a cytokine secreted by antigen-presenting cells such as macrophages, has an action of inducing Th1 cells by stimulating NK cells, and also induces production of interferon γ by Th1 cells. Since the strain of the present invention can promote the production of interferon γ and / or interleukin 12 and suppress the production of IgE, it is possible to suppress the type I allergic reaction. Furthermore, since the strain of the present invention can enhance cellular immunity by shifting the Th1 / Th2 balance to the Th1 side, it can have an immunostimulatory effect in addition to an antiallergic effect. Thereby, the infection by a virus can be prevented and the cancerous cell can be excluded.

  The antiallergic action is preferably an IgE production inhibitory action.

  IgE is a causative substance that causes allergic diseases. That is, when allergen invades, IgE is produced in response to this, 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 allergic reaction exhibits various symptoms such as bronchoconstriction and urticaria, and causes allergic diseases such as hay fever, allergic rhinitis, atopic dermatitis, and asthma depending on the site of onset. Since the strain of the present invention can suppress the production of IgE, it can suppress allergic reactions and can be used for the prevention and treatment of the above diseases.

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

  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 mucosal local biological defense mechanism. Since the strain of the present invention can promote the production of IgA, it can enhance the local defense mechanism of the mucosa, and can be used for the prevention and treatment of lifestyle diseases such as infectious diseases and allergic diseases.

  Moreover, this invention provides the drink and foodstuff containing the microbial cell of the said strain. That is, use of the bacterial cells of the strain as a beverage raw material and a food raw material is provided.

  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 said strain produces (gamma) -aminobutyric acid (GABA), it has an anti-stress effect | action, a blood pressure lowering effect | action, and a tranquilization effect | action, and its utilization value is high as a health food material.

  Moreover, this invention provides the antiallergic agent which contains the microbial cell of the said strain as an active ingredient. That is, use of the microbial cell of the said strain for manufacture of an antiallergic agent is provided.

  Since the bacterial cells of the above strain have an interleukin 12 and interferon γ production promoting action and an IgE production inhibitory action, if an antiallergic agent containing this bacterial cell as an active ingredient is produced, a chemically synthesized pharmaceutical product It can be used as an antiallergic agent with superior safety.

  Moreover, this invention provides the immunostimulant which contains the microbial cell of the said strain as an active ingredient. That is, use of the microbial cell of the said strain for manufacture of an immunostimulant is provided.

  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, it can be used as an immunostimulator that is safer than chemically synthesized pharmaceuticals. it can.

  Furthermore, the present invention provides an antiallergic agent comprising a viscous substance secreted by a strain belonging to Lactobacillus brevis subspecies brevis (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.

  The present inventors have found that some strains belonging to the subspecies brevis among lactic acid bacteria of the Lactobacillus 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 antiallergic agents are far superior in safety to the human body compared to chemically synthesized antiallergic agents. The above viscous substance is obtained by removing the bacterial cells from the culture solution of the strain and adding alcohol to precipitate the strain. As a strain that secretes this viscous substance, SBC8027 (FERM BP- of Lactobacillus brevis subspecies brevis) is used. 10630) is preferred.

  The antiallergic agent preferably has an action of promoting production of interferon γ and / or interleukin 12.

  Interferon γ is a cytokine secreted by Th1 cells, which inhibits B cells from producing IgE and also has cellular immunity such as killer T cells and macrophages that attack viruses, filamentous fungi, and tuberculosis bacteria. Has an increasing effect. In addition, interleukin 12 is a cytokine secreted by antigen-presenting cells such as macrophages, has an action of inducing Th1 cells by stimulating NK cells, and also induces production of interferon γ by Th1 cells. 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.

  Moreover, this invention provides the drink and foodstuff containing said antiallergic agent. That is, the use of the viscous substance secreted by the strain as a beverage ingredient and food ingredient is provided.

  Since said antiallergic agent is safe with respect to a human body, it can be used by making it contain in a drink and food as a health food material.

  According to the present invention, it is possible to provide a strain belonging to Lactobacillus brevis subspecies brevis having both an antiallergic action and an immunostimulatory action stronger than those of lactic acid strains known so far. Moreover, 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), it has an anti-stress action in addition to the above-described action, and is an allergic disease correlated with physical stress and mental stress, A strong effect can be expected for the prevention and treatment of cancer and infectious diseases. Moreover, according to this invention, the drink, foodstuff, and antiallergic agent which contain the microbial cell of the said strain, are excellent in safety | security, and have an antiallergic action and an immunostimulatory action can be provided.

  According to the present invention, a viscous substance secreted by a strain belonging to Lactobacillus brevis subspecies brevis can be used as an antiallergic agent. Moreover, according to this invention, this antiallergic agent is contained and the drink and foodstuff excellent in safety | security can be provided.

It shows the amount of interferon γ produced by mouse spleen cells by adding a cell suspension belonging to Lactobacillus brevis subspecies brevis. It shows the amount of interferon γ produced by the spleen cells of OVA immunized mice by the addition of OVA and the cell suspension of each strain. It shows the amount of interleukin 12 produced by the spleen cells of OVA immunized mice by the addition of OVA and the cell suspension of each strain. It shows the amount of interleukin 4 produced by the spleen cells of OVA immunized mice by the addition of OVA and the cell suspension of each strain. Interferon γ / interleukin 4 is calculated and graphed as an index of Th1 / Th2 balance of spleen cells of OVA immunized mice. It shows the effect of the 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. 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 is shown. The amount of total IgE secreted into the peripheral blood of OVA immunized mice administered intraperitoneally with each strain is shown. It shows the production amount of OVA-specific IgE secreted into the peripheral blood of OVA immunized mice to which each strain was administered intraperitoneally. It shows the amount of IgA produced by the addition of the cell suspension of SBC8027. It shows the amounts of interferon γ and interleukin 12 produced by mouse spleen cells by the addition of a viscous substance secreted by SBC8027 belonging to Lactobacillus brevis subspecies brevis.

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

  The strain of the present invention belongs to Lactobacillus brevis subspecies brevis, is capable of growing in an effervescent alcoholic beverage, produces γ-aminobutyric acid (GABA), and has an antiallergic effect. And having an immunostimulatory effect.

  In Lactobacillus brevis, there are four subspecies, brevis, gravesensis, otakiensis, and coagulans, respectively. It belongs to the subspecies Brevis. Strains belonging to the subspecies brevis can be separated by using the base sequence of 16S ribosomal DNA and the difference in acid production from the sugar as an index, and can be classified as strains not belonging to subspecies grebsensis, subspecies otachysis and subspecies coagulance.

  “Anti-allergic action” refers to the action of suppressing an allergic reaction. Here, allergy refers to a state in which an antibody is produced in a living body by ingestion or contact of a certain substance, and an excessive antigen-antibody reaction occurs due to reuptake or recontact of the same substance, thereby causing pathological symptoms. An allergic reaction refers to a phenomenon in which an immune reaction, which is a defense mechanism of a living body, attacks own cells that should not be excluded or food that has been ingested. The immune reaction involves antigen-presenting cells, T cells and B cells, and IgG and IgA are mainly produced as humoral immunity, whereas the allergic reaction mainly involves Th2 cells of T cells, IgE is made at concentrations 100 to 10,000 times higher than the normal immune response. This large amount of IgE binds to mast cells and promotes the release of inflammatory substances such as histamine and leukotriene in the mast cells.

  Anti-allergic 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 inflammatory substances, or the action of shifting the Th1 / Th2 balance to the Th1 side. More specifically, IgE production inhibitory action, interferon γ and interleukin 12 production promotion action, and the like can be mentioned.

  The antiallergic action of the above strain is preferably an IgE production inhibitory action, an interferon γ production promoting action and / or an interleukin 12 production promoting action, more preferably two or more of these actions.

  “Immunostimulatory action” refers to strengthening the immunity of a living body to provide resistance to diseases. For example, it activates macrophages, neutrophils, NK cells and the like to enhance innate immune function and promote production of immunoglobulins such as IgA. In particular, the IgA production promoting action can enhance mucosal immunity, and thus can be expected to have a strong effect on the prevention and treatment of infectious diseases and lifestyle-related diseases such as allergic diseases.

  “Effervescent alcoholic beverages” include, for example, beer, miscellaneous sake, and low-malt beer. “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. It is preferable that

  Strains belonging to Lactobacillus 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 a cell bank such as ATCC.

  Among the strains belonging to Lactobacillus brevis subspecies brevis, a strain that can grow in a sparkling alcoholic beverage can be selected according to the method described in JP-A-2003-250557. Specifically, using a genomic DNA of a strain belonging to Lactobacillus brevis subspecies Brevis as a template, a predetermined primer set (from the nucleic acid sequences described in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing of JP-A-2003-250557) A primer set comprising oligonucleotides), the amplified DNA gyrase subunit B gene fragment is cleaved with a restriction enzyme, the restriction enzyme cleavage pattern after acrylamide gel electrophoresis is analyzed, and the strain belonging to group IIb Should be selected. Restriction enzyme cleavage patterns are broadly classified into four groups, but strains that can grow in sparkling alcoholic beverages have been found to belong to group IIb.

  Other than the above method, the strain belonging to Lactobacillus brevis subspecies brevis can be transplanted and cultured in an effervescent alcoholic beverage to examine whether it can proliferate. The culture temperature is suitably 30 ° C., but culturing is possible if it is between 15 ° C. and 45 ° C., preferably 20 ° C. to 37 ° C., particularly around 30 ° C.

  Among the strains belonging to Lactobacillus brevis subspecies brevis, a strain producing γ-aminobutyric acid (hereinafter referred to as GABA) can be selected by analyzing the culture supernatant with an amino acid analyzer or the like and examining the GABA content.

  Among the strains belonging to Lactobacillus brevis subspecies brevis, for example, strains having antiallergic activity include, for example, i) production promoting action of interferon γ and interleukin 12 on mouse spleen cells, ii) ovalbumin (hereinafter OVA) immunized mouse It can be selected by testing the inhibitory effect on IgE production induced by spleen cells, iii) the suppressive action of IgE secreted in the peripheral blood of OVA-immunized mice, and the like.

  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 thereto and cultured for a certain period of time, which is secreted from the spleen cells What is necessary is just to measure the production amount of interferon (gamma) and interleukin 12 by ELISA etc., and to investigate the presence or absence of the production promoting effect of interferon (gamma) and interleukin 12.

  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 for a certain period of time. Culture may be performed, and the production amount of IgE secreted from spleen cells may be measured by ELISA or the like to examine the IgE production inhibitory action.

  In iii), a cell suspension prepared by sterilizing the cells of the test strain is administered 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 is determined by ELISA or the like. What is necessary is just to measure and to compare with the production amount of IgE secreted in the peripheral blood of the OVA mouse which is not administering the bacterial cell suspension intraperitoneally.

  In addition, among strains belonging to Lactobacillus brevis subspecies brevis, a strain having an immunostimulatory effect can be selected by, for example, testing an IgA production promoting effect on mouse Peyer's patch cells.

  Specifically, Peyer's patch cells are separated from the intestinal tract of the mouse and cultured, and then a cell suspension prepared by sterilizing the bacterial cells of the test strain is added and cultured for a certain period of time. What is necessary is just to measure the production amount of secreted IgA by ELISA method, and to investigate the presence or absence of the IgA production promoting effect.

  In addition, SBC8027 belonging to Lactobacillus brevis subspecies brevis, which can be grown in a sparkling alcoholic beverage, produces GABA, and has an antiallergic action (date of trust: June 28, 2006; trust number: FERM BP) -10630) has been deposited with the International Depository Agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, Tsukuba, Ibaraki, Japan, 1st 6th (zip code 305-8565)). Is available.

  The beverage, food and antiallergic agent of the present invention are strains belonging to Lactobacillus brevis subsp. Brevis, can grow in effervescent alcoholic beverages, produce GABA, and have antiallergic and immunostimulatory effects The above-mentioned strain is contained.

  The bacterial cells of the above strains can be added to beverages and foods for the purpose of preventing and treating allergic diseases, cancers and infectious diseases. The said drink and food may consist only of microbial cells of said strain, and may contain the additive normally used in the said field | area. 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 starch, etc. Polysaccharides, sugar alcohols such as erythritol, xylitol, and sorbitol, and vitamins such as vitamin C. These additives may be used alone or in combination.

  In addition, for the purpose of prevention and alleviation of symptoms such as allergic diseases, cancer and infectious diseases, as food additives, food for specified health use, special nutritional food, nutritional supplement, health food, functional food and food for the sick It can also mix | blend with foods and drinks.

  The antiallergic agent and immunostimulant of the present invention contain the above bacterial strain as an active ingredient, and if formulated with the addition of carriers, excipients and / or other additives, the anti-allergic agent has excellent safety. Can be used as an allergic agent. 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, vitamin C Vitamins such as acacia gum, calcium phosphate, alginate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose derivatives, tragacanth, gelatin, syrup, methyl hydroxybenzoate, talc, magnesium stearate, water, mineral oil These additives may be single species or plural species.

  Further, the antiallergic agent of the present invention is composed of a viscous substance secreted by a strain belonging to Lactobacillus brevis subspecies brevis (Lactobacillus brevis subspecies brevis).

  The strain viscous material is obtained by culturing a strain belonging to Lactobacillus brevis subspecies brevis to obtain a culture solution, removing the bacterial cells from the culture solution, and adding a alcohol to precipitate the viscous material, and And a recovery step for removing the liquid and recovering the precipitated viscous material.

  In Lactobacillus brevis, there are four subspecies, brevis, gravesensis, otakiensis, and coagulans, respectively. It belongs to the subspecies Brevis. Strains belonging to subspecies brevis can be separated by using the base sequence of 16S ribosomal DNA and the difference in acid production from sugar as indicators, and are classified as strains that do not belong to subspecies grebsensis, subspecies otakiensis and subspecies coagulance.

  Strains belonging to Lactobacillus 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 a cell bank such as ATCC.

  In the culture step, a strain belonging to Lactobacillus brevis subspecies brevis is, 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% ammonium citrate, 0.01% magnesium sulfate, 0.005% manganese sulfate, 0.2% dipotassium phosphate) Incubation or shaking culture may be performed. The liquid medium can be substituted with fruit juice (apple, grapefruit, grape, etc.).

  In the precipitation step, for example, after filtering the culture solution or centrifuging at 7000 rpm for 10 minutes to precipitate the cells, the alcohol concentration is 35% by volume or more after removing the cells. Alcohol may be added to. The alcohol is preferably a C1-4 lower alcohol, more preferably ethanol or isopropanol. Moreover, 50 volume% is preferable and, as for alcohol concentration, 60 volume% is more preferable.

  In the recovery step, for example, the culture solution to which alcohol has been added in the precipitation step is centrifuged at 7000 rpm for 10 minutes to precipitate a viscous substance, and the supernatant may be removed.

  As an action of the antiallergic agent comprising this viscous substance, it acts on antigen-presenting cells, T cells or mast cells to suppress the production of IgE and release of the above inflammatory substances, and the Th1 / Th2 balance is adjusted to Th1. And, more specifically, an interferon γ production promoting action and an interleukin 12 production promoting action.

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

  In i), spleen cells are isolated and cultured from the spleen of a mouse, sterilized viscous substance is added thereto and cultured for a certain period of time, and the production amounts of interferon γ and interleukin 12 secreted from the spleen cells are determined by ELISA or the like. What is necessary is just to measure and check the presence or absence of the production promotion effect | action of interferon gamma and interleukin 12.

  In ii), spleen cells of OVA immunized mice 2 weeks after the booster are isolated and cultured, and then sterilized viscous substance and OVA are added thereto and cultured for a certain period of time to produce IgE secreted from the spleen cells. The amount may be measured by ELISA or the like to examine the IgE production inhibitory action.

  In iii), a sterilized viscous substance is administered into the abdominal cavity of OVA mice and reared for a certain period of time. The amount of IgE secreted into the peripheral blood is measured by ELISA or the like, and the bacterial cell suspension is administered intraperitoneally. What is necessary is just to compare with the production amount of IgE secreted in the peripheral blood of non-OVA mice.

  SBC8027 (trust date: June 28, 2006; trust number: FERM BP-10630), which secretes a viscous substance having an antiallergic action and belongs to Lactobacillus brevis subspecies brevis, is an international depositary authority. It is deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (1st, 1st East, Tsukuba City, Ibaraki Prefecture, Japan, 1st 6th (zip code 305-8666)).

  The above-mentioned antiallergic agent can be used by freeze-drying the viscous substance recovered in the recovery step, but for example, it may be formulated by adding a carrier, an excipient 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, sugar alcohols such as erythritol, xylitol and sorbitol, vitamin C Vitamins such as acacia gum, calcium phosphate, alginate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose derivatives, tragacanth, gelatin, syrup, methyl hydroxybenzoate, talc, magnesium stearate, water, mineral oil These additives may be single species or plural species.

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

  For example, if the viscous substance recovered in the above recovery step is freeze-dried and added to the target beverage and food, beverages and foods having an antiallergic action can be produced. The beverages and foods may contain only this viscous substance or may contain additives usually used in the art. 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 starch, etc. Polysaccharides, sugar alcohols such as erythritol, xylitol, and sorbitol, and vitamins such as vitamin C. These additives may be used alone or in combination.

  In addition, for the purpose of preventing allergic diseases and alleviating symptoms, food additives such as foods for specified health use, special nutritional foods, nutritional supplements, health foods, functional foods and sick foods You can also

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples.

Example 1
1) About each strain used in the experiment 13 strains (SBC8027 and strains a to l) belonging to Lactobacillus brevis subspecies brevis were isolated by the inventor and used as lyophilized cells until used in the experiment. Stored at ° C. Also, strain X belonging to Lactobacillus rhamnosus was isolated from commercially available yogurt and stored at 4 ° C. as lyophilized cells until used in experiments.

2) Determination of growth ability in beer The growth ability in beer was selected for 13 strains belonging to the aforementioned Lactobacillus brevis subspecies brevis and strain X belonging to Lactobacillus rhamnosus. The selection method was performed according to the method described in JP-A-2003-250557. That is, PCR was performed with a predetermined primer set (primer set consisting of oligonucleotides consisting of the nucleic acid sequences described in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing of JP-A-2003-250557) using the genomic DNA of each lactic acid strain as a template. Then, the amplified DNA gyrase subunit B gene fragment was cleaved with a restriction enzyme, and the restriction enzyme cleavage pattern after acrylamide gel electrophoresis was analyzed for determination. 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 group IIb.

  As a result, it was determined that all 13 strains belonging to Lactobacillus brevis subspecies brevis examined were strains that grew in beer, but strain X belonging to Lactobacillus rhamnosus was a strain that grew in beer. Was not judged. In addition, although the said strain was actually inoculated in beer and the propriety of proliferation was confirmed, it was the same as the determination result of the said method.

3) Evaluation of antiallergic action
i) Preparation of Cell Suspension In order to evaluate the antiallergic action of each of the above strains, a cell suspension of each strain was prepared. First, each strain was subjected to anaerobic conditions (gas composition of N ₂ : CO ₂ : H ₂ = 90: 5: 5), 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% ammonium citrate, 0.01% magnesium sulfate, 0.005% manganese sulfate, 0.2% dipotassium phosphate) After stationary culture for 3 days, the culture was centrifuged at 1,500 rpm for 10 minutes to recover the bacterial cells of each strain. The obtained microbial cells were washed with PBS, freeze-dried, and suspended in PBS to 1 mg / mL. The bacterial cell suspension thus obtained was autoclaved (121 ° C., 15 minutes) and used in the following experiments.

ii) Stimulation of interferon γ production by a strain belonging to Lactobacillus brevis subspecies brevis on mouse spleen cells (in vitro)
13 strains belonging to Lactobacillus brevis subsp. Brevis were added to mouse spleen cells and cultured for a certain period of time, and the amount of interferon γ secreted from the spleen cells was examined to promote the production of interferon γ by each strain. The effect was evaluated.

(Preparation of mouse spleen cells)
First, the spleen was aseptically removed from a 6-week-old BALB / c mouse (female) and immersed in an RPMI1640 medium containing 10% FBS. Thereafter, the spleen was transferred to a petri dish and crushed with a pestle, and the suspension of mouse spleen cells was passed through a nylon filter net (Nippon Riken Instruments Co., Ltd.) having an opening of 70 μm and a wire diameter of 39 μm. The suspension of mouse spleen cells that passed through the nylon filter net was centrifuged at 1,500 rpm for 10 minutes, and the supernatant was discarded, followed by erythrocyte hemolysis reagent (0.16M ammonium chloride, Tris-HCl, pH 7.2). ) Was added to the precipitated mouse spleen cells and allowed to stand at room temperature for 5 minutes. Thereafter, fresh 10% FBS-containing RPMI1640 medium was added to wash the mouse spleen cells, centrifuged at 1,500 rpm for 10 minutes, and after discarding the supernatant, the number of cells was 5 × 10 ⁶ cells / mL. RPMI 1640 medium containing 10% FBS was added. The mouse spleen cells thus obtained were used for the following experiments.

(Measurement of interferon γ by ELISA)
Mouse spleen cells were seeded in a 96-well plate so that the cell density was 2.5 × 10 ⁶ cells / mL, and cultured in 10% FBS-containing RPMI1640 medium under conditions of 37 ° C. and 5% CO ₂ . A cell suspension of each strain (final concentration: 10 μg / mL) was added to each well in which mouse spleen cells were cultured, and the amount of interferon γ secreted into the culture supernatant after 72 hours was determined by ELISA. Quantified with. Lipopolysaccharide (hereinafter referred to as LPS) (SIGMA, final concentration: 10 μg / mL) having an interferon γ production promoting action on mouse spleen cells was used as a positive control, and PBS was used as a negative control.

  The ELISA for quantifying interferon γ was performed as follows. First, 50 μL of a primary antibody (Rabbit anti-mouse / rat interferon-γ; BIO SOURCE) prepared to 1.25 μg / mL was added to each well of a 96-well plate (Maxisorp immunoplate; NUNC) at 4 ° C. The solid phase was allowed to stand overnight. Then, this 96-well plate was washed 3 times with Wash Buffer and blocked with 1% bovine serum albumin (BSA; Sigma). Subsequently, 50 μL of each culture supernatant or a preparation of interferon γ whose concentration was previously known was added to each well, reacted with the anti-interferon γ antibody as the primary antibody for 90 minutes, and washed 3 times with Wash Buffer. Subsequently, 50 μL of a secondary antibody (Anti-mouse / rat interferon-γ biotin conjugate; BIO SOURCE) prepared to 0.5 μg / mL was added to each well and allowed to react at room temperature for 90 minutes. Thereafter, each well was washed 5 times with Wash Buffer, streptavidin-HRP (BIO SOURCE) was added to react, washed 5 times with Wash Buffer, and then washed with TMB (tetramethylbenzidine) substrate solution (3, 3 ′, 5 , 5′-Tetramethylbenzidine (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 γ produced by mouse spleen cells was quantified from the standard curve.

  FIG. 1 shows the amount of interferon γ produced by mouse spleen cells by the addition of 13 cell suspensions belonging to Lactobacillus brevis subspecies brevis. As a result, SBC8027 of Lactobacillus brevis subspecies brevis induces the production of interferon γ for mouse spleen cells, and the production amount thereof belongs to LPS and other strains belonging to Lactobacillus brevis subspecies brevis (strains a to l). Was significantly higher than the amount induced by. SBC8027 (accession number: FERM BP-10630; date of accession: June 28, 2006), which has an interferon γ production-inducing action, is an international depositary authority, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Japan) Deposited at 1st, 1st East, Tsukuba City, Ibaraki Prefecture, Central 6 (postal code 305-8666).

iii) SBC8027 promotes Th1 cytokine production, inhibits Th2 cytokine production, and inhibits IgE production on spleen cells of OVA immunized mice (in vitro)
SBC8027, which showed interferon γ production promoting action on mouse spleen cells, promoted Th1 cytokine (interferon γ and interleukin 12) production promotion action, Th2 cytokine (interleukin 4) production inhibitory action and IgE on spleen cells of OVA immunized mice. Production inhibitory action was evaluated. At that time, the effects of strains b and c belonging to Lactobacillus brevis subspecies brevis and strain X belonging to Lactobacillus rhamnosus were also investigated and compared with the effects of SBC8027.

(Preparation of OVA immunized mice)
OVA immunized mice were prepared by intraperitoneally administering OVA to 6-week-old BALB / c mice (female) to artificially induce allergies. Specifically, first, 100 μg of OVA (Ovalbumin, Eggwhite, Purified; Worthington Biochemical Corporation) and 10 mg of aluminum hydroxide were dissolved in 1 mL of PBS to prepare an OVA antigen solution. The first immunization was performed after administration. One week later, an OVA antigen solution was prepared in the same manner as described above, and 200 μL of the solution was again administered into the abdominal cavity of the mouse for booster immunization. The allergic mice were boosted about 1 week later, and this mouse was used as an OVA immunized mouse in the following experiment.

(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.

(Measurement by ELISA of interferon γ, interleukin 12 and interleukin 4)
In order to measure interferon γ, interleukin 12 and interleukin 4, spleen cells of 2.5 × 10 ⁵ OVA immunized mice were seeded in a 96-well plate (cell density was 2.5 × 10 ⁶ cells / cell). mL), 37 ° C., 5% CO ₂ and cultured in 10% FBS-containing RPMI1640 medium. OVA (final concentration: 100 μg / mL) and cell suspension of each strain (final concentration: 1 μg / mL) were added to each well in which spleen cells of OVA-immunized mice were cultured, and cultured after 72 hours. The amount of each cytokine secreted into the supernatant was quantified by ELISA. At that time, a control in which PBS was added instead of the cell suspension was provided.

  Quantification of interferon γ by ELISA was performed in the same procedure as described above. For interleukin-12 quantification, 1 μg / mL Purified anti-mouse IL-12 (p40 / p70) (BD Pharmingen) was used as the primary antibody, and 1 μg / mL Biotin anti-mouse IL-12 was used as the secondary antibody. Using (p40 / p70) (BD Pharmingen), ELISA was performed in the same procedure as the quantification of interferon γ. For the quantification of interleukin-4, 1 μg / mL Monoclonal Anti-mouse IL-4 Antibody (R & D Systems Inc.) was used for the primary antibody, and 1 μg / mL Biotinylated Anti-mouse IL-4AidDyDB (Systems Inc.) and ELISA was performed in the same procedure as the quantification of interferon γ.

  FIG. 2 shows the amount of interferon γ produced by spleen cells of OVA-immunized mice by the addition of OVA and the cell suspension of each strain, FIG. 3 shows the amount of interleukin 12, and FIG. 4 shows the amount of interleukin 4. Is shown. FIG. 5 is a graph obtained by calculating interferon γ / interleukin 4 as an index of Th1 / Th2 balance. As a result, in the control to which OVA and PBS were added, production of interferon γ and interleukin 12 as Th1 type cytokines was hardly observed, and production of interleukin 4 as a Th2 type cytokine was remarkably induced. This suggested that the spleen cells of OVA-immunized mice had a Th1 / Th2 balance shifted to the Th2 side, and allergic reaction was enhanced.

  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 c was added, production of interferon γ and interleukin 12 as Th1-type cytokines was produced. However, it was promoted compared to the strain X belonging to the control and Lactobacillus rhamnosus, and the production of interleukin 4 which is a Th2-type cytokine was remarkably suppressed. Moreover, the action of SBC8027 was more remarkable than that of strain b or strain c, and greatly shifted the Th1 / Th2 balance of spleen cells of OVA immunized mice to the Th1 side. From this result, it was suggested that SBC8027 has a strong antiallergic action.

(Measurement of total IgE and OVA-specific IgE by ELISA)
In order to measure total IgE, spleen cells of 2.5 × 10 ⁵ OVA-immunized mice were seeded in a 96-well plate (cell density was 2.5 × 10 ⁶ cells / mL), 37 ° C., 5% The cells were cultured in RPMI 1640 medium containing 10% FBS under CO ₂ conditions. Thereafter, OVA (final concentration: 100 μg / mL) and cell suspension of each strain (final concentration: 1 μg / mL) were added to each well in which spleen cells of OVA immunized mice were cultured, and 14 days had elapsed. Later, the amount of total IgE secreted into the culture supernatant was quantified by ELISA. For control, PBS was added instead of the bacterial cell suspension, and the amount was similarly determined by ELISA.

On the other hand, in order to measure OVA-specific IgE, a 48-well plate was seeded with 2.5 × 10 ⁶ spleen cells (cell density was 2.5 × 10 ⁶ cells / mL), 37 ° C., 5% The cells were cultured in RPMI 1640 medium containing 10% FBS under CO ₂ conditions. OVA (final concentration: 100 μg / mL) and cell suspension of each strain (final concentration: 1 μg / mL) were added to the spleen cells and cultured for 3 days. The spleen cells were washed three times with a new medium and OVA was performed. The bacterial suspension of each strain was added again to 1 μg / mL and cultured for 11 days, and IgE secreted in the culture supernatant was added to the OVA-specific IgE. As quantified by ELISA. For control, PBS was added instead of the bacterial cell suspension, and the amount was similarly determined by ELISA.

  ELISA for quantifying total IgE and OVA-specific IgE was performed as follows. First, 50 μL of anti-mouse IgE antibody (Mouse IgE ELISA Quantitation Kit; BETHYL Laboratories Inc.) prepared to 10 μg / mL was added to each well of a 96-well plate (Maxisorp immunoplate; NUNC) overnight at 4 ° C. It was solidified by placing. Then, this 96-well plate was washed 3 times with Wash Buffer and blocked with 1% bovine serum albumin (BSA; Sigma). Subsequently, each culture supernatant or a preparation of IgE whose concentration was previously known was added to each well, reacted with anti-mouse IgE antibody for 90 minutes, washed 3 times with Wash Buffer, and then biotinylated kit (Cygnus Technologies, Inc.). 50) of biotinylated OVA prepared in.) Was added to each well and allowed to react at room temperature for 90 minutes. Thereafter, each well was washed 5 times with Wash Buffer, streptavidin-HRP (BIO SOURCE) was added to react, washed 5 times with Wash Buffer, and then washed with TMB (tetramethylbenzidine) substrate solution (3, 3 ′, 5 , 5′-Tetramethylbenzidine (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 IgE preparation, and the amount of IgE produced by spleen cells of OVA mice was quantified from this 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.

  FIG. 6 shows the effect of the 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.

  On the other hand, although the strain b and the strain c and the strain X belonging to Lactobacillus rhamnosus suppressed the total amount of IgE induced by OVA and the production of OVA-specific IgE, they had a weaker effect than SBC8027.

  From the above results, SBC8027 belonging to Lactobacillus brevis subspecies brevis has an action of promoting production of interferon γ and interleukin 12 and an action of suppressing IgE production, and has a stronger anti-allergy compared to previously known strains of lactic acid bacteria. It has been found that it works.

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

  OVA immunized mice were prepared by initial immunization and booster immunization with OVA (Ovalbumin, Eggwhite, Purified; Worthington Biochemical Corporation) as described above, and SBC8027 or bacterial strain suspension of PBS or PBS (control) Was administered intraperitoneally 200 μL once every two days from one week before the first immunization to one week after the booster immunization. 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).

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

  FIG. 8 shows the production amount of total IgE secreted into the peripheral blood of OVA immunized mice to which each strain was administered intraperitoneally. FIG. 9 shows the production amount of OVA-specific IgE secreted into the peripheral blood of OVA immunized mice similarly administered intraperitoneally with each strain. * In the graph indicates that the control is statistically significant at p <0.05.

  As a result, the addition of SBC8027 bacterial 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 effect was statistically significant compared to the control administered with PBS.

  On the other hand, although strain X belonging to Lactobacillus rhamnosus suppresses the production of total IgE and OVA-specific IgE secreted in the peripheral blood of OVA mice, it is weaker than SBC8027 and statistically compared to control. It was not significant.

  From the above results, SBC8027 belonging to Lactobacillus brevis subspecies brevis has an inhibitory effect on IgE production even in vivo, and exhibits a strong antiallergic action compared to known lactic acid bacteria strains. There was found.

4) Evaluation of immunostimulatory action SBC8027, which was found to have a strong antiallergic action in in vitro and in vivo experiments, was evaluated for IgA production promoting action on mouse Peyer's patch cells.

(Preparation of mouse Peyer's patch cells)
First, the intestinal tract was aseptically removed from a 6-week-old BALB / c mouse (female), and the Peyer's patch was removed therefrom with scissors and immersed in RPMI 1640 (GIBCO). Repeat the operation of sucking up the Peyer's plate with a pipette, thoroughly wash the Peyer's plate, transfer the Peyer's plate to an empty petri dish, and then add 10 mL of dispase solution (1.5 mL of 15 mg / mL dispase (GIBCO)) 8.5 mL of RPMI 1640 (GIBCO) was added and allowed to react with stirring for 40 to 45 minutes at 37 ° C. The resulting cell suspension was centrifuged at 1,500 rpm for 10 minutes. Peyer's patch cells were washed by repeating the operation of precipitating Peyer's patch cells, discarding the supernatant, and adding RPMI1640 medium containing 10% FBS, and then the number of Peyer's patch cells was 5 × 10 ⁶ cells / mL. Thus, RPMI1640 medium containing 10% FBS was added and suspended, and used in the following experiments.

(IgA ELISA measurement)
Mouse Peyer's plate cells were seeded in a 96-well plate so that the cell density was 2.5 × 10 ⁶ cells / mL, and cultured in 10% FBS-containing RPMI1640 medium at 37 ° C. and 5% CO ₂ . The cell suspension of each strain (final concentration: 100 μg / mL) was added to each well in which Peyer's patch cells were cultured, and the amount of IgA secreted into the culture supernatant after 7 days was determined by ELISA. Quantified. Lipopolysaccharide (hereinafter, LPS) (SIGMA, final concentration: 10 μg / mL) having IgA production promoting action on mouse spleen cells was used as a positive control, and PBS was used as a negative control.

  IgA was measured using an IgA ELISA kit (Mouse IgA ELISA Quantitation Kit; BETHYL Laboratories Inc.) according to the manufacturer's protocol.

  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, comparable to the action of LPS.

5) Measurement of γ-aminobutyric acid (GABA) production ability SBC8027 belonging to Lactobacillus brevis subspecies brevis was tested for the production ability of γ-aminobutyric acid (hereinafter referred to as GABA). First, SBC8027 was inoculated into 100 mL of a liquid medium (3% malt extract (DIFCO), 2% yeast extract (DIFCO), 0.2% sodium glutamate, pH 6.0) and statically cultured for 4 days. . Thereafter, the culture solution of SBC8027 was centrifuged at 1,500 rpm for 10 minutes to recover the culture supernatant, and the amount of GABA contained in the culture supernatant was quantified by HPLC. The HPLC conditions used are as follows.
-HPLC apparatus: Agilent HPLC 1100
Column used: ZORBAX Eclipse AAA (4.6 × 150 mm, 3.5 μm) (Shimadzu GC)
-Column oven: 40 ° C
・ Flow rate: 1.0 mL / min
Fluorescence detector: Ex. 340 nm, Em. 450nm
HPLC reagent: 10 mg / mL Agilent OPA reagent (0.4 M borate buffer, pH 10.2) (Shimadzu GL Corporation)
-Eluent: A liquid: 40 mM NaH2PO4 (pH 7.8)
Solution B: 45 vol% acetonitrile, 45 vol% MeOH, _{10% H} 2 O
Time table (gradient): See Table 1

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

  From the above results, SBC8027 belonging to Lactobacillus brevis subspecies brevis is a strain that can grow in an effervescent alcoholic beverage, produces GABA having an anti-stress action, and has both an antiallergic action and an immunostimulatory action. It turned out to be.

6) Sensory inspection of the fruit juice lactic acid fermentation broth manufactured using SBC8027 The fruit juice lactic acid fermentation broth was manufactured using SBC8027 which has an antiallergic action, and the sensory test was performed about the fragrance and flavor of the obtained fruit juice lactic acid fermentation broth.

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

Each fruit juice was prepared by diluting grapefruit concentrated juice, apple concentrated fruit juice, white grape concentrated fruit juice and lemon concentrated fruit juice with sterilized water to have a predetermined sugar content, and the pH was adjusted by adding NaOH. Lactic acid fermentation was inoculated with 1.5 × 10 ⁹ cells of SBC8027 per 100 mL of each fruit juice, stirred once a day for 72 hours at 30 ° C., and under stationary conditions except during stirring. 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 index for the growth of SBC8027, and the amount of lactic acid was used as an index for the degree of lactic acid fermentation.

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

  As a result, the lactic acid fermentation of SBC8027 progressed in all the fruit juices used in the test, and the fruit juice lactic acid fermentation liquor was produced. 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.

(Example 2)
1) About each strain used for experiment Lactobacillus brevis subspecies brevis (Lactobacillus brevis subspecies brevis) SBC8027 (FERM BP-10630) was isolated and stored at 4 ° C. as lyophilized cells until used in the experiment. .

2) Evaluation of antiallergic action
i) Preparation of Viscous Substance First, SBC8027 belonging to Lactobacillus brevis subspecies brevis was added to 1.6 L of MRS liquid medium (Difco, composition: 1% proteose peptone, 1% beef extract, 0.5% yeast extract. 3 days after inoculating 2% glucose, 0.1% Tween 80, 0.5% ammonium citrate, 0.01% magnesium sulfate, 0.005% manganese sulfate, 0.2% dipotassium phosphate) The culture was stationary. Thereafter, the culture solution is centrifuged at 7000 rpm for 10 minutes to remove the cells as precipitates, ethanol is added so that the alcohol concentration is 60% by volume, and then the solution is centrifuged at 7000 rpm for 10 minutes to make the viscosity. The material was precipitated and the supernatant was removed. The viscous material thus obtained was dissolved by adding 40 mL of distilled water, applied to a gel filtration column (Sephadex G-100), and fractions having 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.

ii) Preparation of viscous substance solution Next, 1 mg of the lyophilized product of the viscous substance was dissolved in 1 mL of sterilized water and treated with artificial gastric juice and artificial intestinal fluid. First, 0.1 mL of artificial gastric juice (10,000 units / mL pepsin, 2% NaCl) was added and reacted at 37 ° C. for 2 hours, followed by 0.1 mL of artificial intestinal fluid (4% pancreatin, 4% trypsin, 1M NaHCO ₃ ) was added and reacted at 37 ° C. for 2 hours. Thereafter, the enzyme was inactivated by heating at 105 ° C. for 15 minutes to obtain a viscous material solution used in the following experiments. By the treatment with the artificial gastric juice and the artificial intestinal fluid, a viscous substance is ingested orally and a state is obtained when acting on the intestinal membrane mucosa.

iii) Promoting production of interferon γ and interleukin 12 of a strain belonging to Lactobacillus brevis subspecies brevis on mouse spleen cells (in vitro)
Viscous substances treated with artificial gastric juice and artificial intestinal fluid were added to mouse spleen cells and cultured for a certain period of time, and the amount of interferon γ secreted from spleen cells was examined to evaluate the production promoting effect of interferon γ and interleukin 12. .

(Preparation of mouse spleen cells)
First, the spleen was aseptically removed from a 6-week-old BALB / c mouse (female) and immersed in an RPMI1640 medium containing 10% FBS. Thereafter, the spleen was transferred to a petri dish and crushed with a pestle, and the suspension of mouse spleen cells was passed through a nylon filter net (Nippon Riken Instruments Co., Ltd.) having an opening of 70 μm and a wire diameter of 39 μm. The suspension of mouse spleen cells that passed through the nylon filter net was centrifuged at 1,500 rpm for 10 minutes, and the supernatant was discarded, followed by erythrocyte hemolysis reagent (0.16M ammonium chloride, Tris-HCl, pH 7.2). ) Was added to the precipitated mouse spleen cells and allowed to stand at room temperature for 5 minutes. Thereafter, fresh 10% FBS-containing RPMI1640 medium was added to wash the mouse spleen cells, centrifuged at 1,500 rpm for 10 minutes, and after discarding the supernatant, the number of cells was 5 × 10 ⁶ cells / mL. RPMI 1640 medium containing 10% FBS was added. The mouse spleen cells thus obtained were used for the following experiments.

(Measurement of interferon γ and interleukin 12 by ELISA)
Mouse spleen cells were seeded in a 96-well plate so that the cell density was 2.5 × 10 ⁶ cells / mL, and cultured in 10% FBS-containing RPMI1640 medium under conditions of 37 ° C. and 5% CO ₂ . A cell suspension of each strain (final concentration: 1 μg / mL) was added to each well in which mouse spleen cells were cultured, and the amount of interferon γ secreted into the culture supernatant after 72 hours was determined by ELISA. Quantified with. For positive control, lipopolysaccharide (hereinafter LPS) (SIGMA, final concentration: 10 μg / mL) that promotes production of interferon γ and interleukin 12 on mouse spleen cells, and for negative control, PBS It was used.

  The ELISA for quantifying interferon γ was performed as follows. First, 50 μL of a primary antibody (Rabbit anti-mouse / rat interferon-γ; BIO SOURCE) prepared to 1.25 μg / mL was added to each well of a 96-well plate (Maxisorp immunoplate; NUNC) at 4 ° C. The solid phase was allowed to stand overnight. Then, this 96-well plate was washed 3 times with Wash Buffer and blocked with 1% bovine serum albumin (BSA; Sigma). Subsequently, 50 μL of each culture supernatant or a preparation of interferon γ whose concentration was previously known was added to each well, reacted with the anti-interferon γ antibody as the primary antibody for 90 minutes, and washed 3 times with Wash Buffer. Subsequently, 50 μL of a secondary antibody (Anti-mouse / rat interferon-γ biotin conjugate; BIO SOURCE) prepared to 0.5 μg / mL was added to each well and allowed to react at room temperature for 90 minutes. Thereafter, each well was washed 5 times with Wash Buffer, streptavidin-HRP (BIO SOURCE) was added to react, washed 5 times with Wash Buffer, and then washed with TMB (tetramethylbenzidine) substrate solution (3, 3 ′, 5 , 5′-Tetramethylbenzidine (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 γ produced by mouse spleen cells was quantified from the standard curve.

  As for the quantification of interleukin 12, 1 μg / mL Purified anti-mouse IL-12 (p40 / p70) (BD Pharmingen) was used as the primary antibody, and 1 μg / mL Biotin anti-mouse IL as the secondary antibody. ELISA was performed in the same procedure as for the determination of interferon γ using -12 (p40 / p70) (BD Pharmingen).

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

  As a result, the viscous substance secreted by SBC8207 belonging to Lactobacillus brevis subspecies brevis induces the production of interferon γ and interleukin 12 in mouse spleen cells, both of which are the amounts induced by LPS. In comparison, it was remarkably high.

  From the above results, it was found that the viscous substance secreted by the strain belonging to Lactobacillus brevis subspecies brevis has an antiallergic action.

  Since the strain of the present invention can be grown in a sparkling 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), it has an anti-stress action in addition to the above-described action, and is an allergic disease correlated with physical stress and mental stress, A strong effect can be expected for the prevention and treatment of cancer and infectious diseases. Moreover, according to this invention, the drink, foodstuff, and antiallergic agent which contain the microbial cell of the said strain, are excellent in safety | security, and have an antiallergic action and an immunostimulatory action can be provided.

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

Claims (16)

A strain belonging to Lactobacillus brevis subspecies brevis,
Can grow in sparkling alcoholic beverages,
producing γ-aminobutyric acid (GABA),
A strain having antiallergic action and immunostimulatory action.   The strain according to claim 1, wherein the antiallergic action is an action of promoting production of interferon γ and / or interleukin 12.   The strain according to claim 1, wherein the antiallergic action is an IgE production inhibitory action.   The strain according to claim 1, wherein the immunostimulatory action is an IgA production promoting action.   The strain according to claim 1, wherein the immunostimulatory action is a natural killer cell or killer cell activating action.   The strain according to any one of claims 1 to 5, which is SBC8027 (FERM BP-10630) of Lactobacillus brevis subspecies brevis (Lactobacillus brevis subspecies brevis).   Use of the bacterial cell according to any one of claims 1 to 6 as a beverage raw material.   Use of the bacterial cell according to any one of claims 1 to 6 as a food material.   Use of the bacterial cell of the strain according to any one of claims 1 to 6 for the production of an antiallergic agent.   Use of the microbial cell of the strain as described in any one of Claims 1-6 for manufacture of an immunostimulant.   Use of the viscous substance which the strain as described in any one of Claims 1-6 secretes for manufacture of an antiallergic agent.   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.   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.   The use according to any one of claims 11 to 13, wherein the strain is SBC8027 (FERM BP-10630) of Lactobacillus brevis subspecies brevis.   Use of the viscous substance secreted by the strain according to any one of claims 1 to 6 as a beverage ingredient.   Use of the viscous substance secreted by the strain according to any one of claims 1 to 6 as a food material.

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