HK1090289B - Antiallergic composition - Google Patents

Description

Antiallergic composition

Technical Field

The present invention relates to an antiallergic composition useful for the prevention and treatment of allergic reactions, particularly to an antiallergic composition containing a lactic acid bacterium having a high antiallergic activity as an active ingredient, a method for obtaining the lactic acid bacterium, and use of the antiallergic composition as foods, beverages, and the like.

Technical Field

Allergy is one of the most prevalent diseases in developed countries. The pathogenesis of allergy is generally divided into 4 types I to IV. Type I allergy is associated with IgE antibodies, and is represented by pollinosis, asthma, urticaria, anaphylactic shock and the like. Type II allergy is related to IgG antibodies and IgM antibodies, and is a cytotoxic reaction due to activation of the complement system, and fetal myeloblastosis and autoimmune hemolytic anemia are among this type. Type III allergy is a tissue injury reaction caused by an antigen-antibody complex, and is represented by Alex reaction, seropathy, and glomerulonephritis. Type IV allergy is delayed allergy (delayed hypersensitivity reaction), is associated with T cells, and is represented by tuberculin reaction and contact dermatitis. Among them, type I, type II and type IV are associated with the occurrence of food allergy. On the other hand, environmental allergies, i.e., pollinosis, atopic dermatitis, bronchial asthma, allergic rhinitis, allergic conjunctivitis, and the like, are mainly type I pathogenesis.

The type I allergy is characterized by the induction of allergen-specific IgE and the release of chemical mediators such as histamine and leukotriene. Immune responses are generated by interactions between various cells, and helper T cells (Th) are one of the cells associated with this. Helper T cells belong to a T cell subset, and function to recognize antigens, produce various cytokines (helper factors), and regulate induction of immune response. Helper T cells are classified into Th1 cells and Th2 cells according to their ability to produce cytokines. Th2 cytokines such as IL-4, IL-5, and IL-13 are essential for inducing class switching of antibodies in B cells, producing IgE antibodies, and the like. Indeed, it is known that Th2 cells are increased in lymphoid cells of allergic patients. The foreign invaded allergen is delivered to T cells from antigen-presenting cells such as dendritic cells and macrophages in a form in which a part of the antigen-presenting cells is bound to mhc ii molecules, and Th2 cells are activated and differentiated. Th2 cytokines released by Th2 cells induce class switching of B cells, producing IgE antibodies that bind to mast cells in tissues and fcrs on the surface of basophils in the blood. When the allergen invades the body for the second time, it is recognized by IgE antibodies bound to the surfaces of mast cells and basophils in the blood, and a bridge is formed between the IgE antibodies. This stimulus acts as a trigger point, causing the mast cells and basophils to release chemical mediators explosively, presenting various allergic symptoms.

Examples of methods for preventing and treating allergy mediated by IgE and chemical mediators include antihistamines which bind to histamine receptors by antagonistic binding to block signal transduction from peripheral nerves, antiallergic agents which reduce symptoms by reducing the activity of chemical mediator-producing cells, steroid agents which reduce inflammation by reducing immunoreactivity, and desensitizing therapy which induces tolerance by periodic injection of allergen. However, any of them has side effects, and the effect is not determined.

In recent years, methods for modulating the production of Th2 cytokine for the inhibition of IgE have received attention. It has been reported that certain bacteria such as tubercle bacillus, hemolytic streptococcus, and lactic acid bacteria enhance Th1 immunity and suppress Th2 immunity, resulting in IgE reduction (clinical immunity, 1999, Vol.32, 454; International Archives of Allergy and Immunology, 1998, Vol.115, 278; Japanese patent laid-open No. Hei 9-2959).

Among them, lactic acid bacteria are useful materials because they are easily used for foods and the like from the viewpoint of safety. However, not all lactic acid bacteria have the effect of enhancing Th1 immunity, and it is necessary to select a useful strain. As an antiallergic lactic acid bacterium, lactobacillus rhamnosus (l.rhamnosus) LGG strain is known, and it has been reported that when ingested by pregnant women, atopic dermatitis of the fetus can be suppressed (Lancet, 2001, 357, page 1076). There are also several patent publications on the use of lactic acid bacteria as antiallergic agents. For example, Japanese patent application laid-open No. 9-2959 discloses the use of lactic acid bacteria as antiallergic agents, wherein the IgE production is 30ng/ml or less when mouse lymphocytes are cultured by adding lactic acid bacteria such as Lactobacillus acidophilus (L.acidophilus), Lactobacillus brevis (L.Brevis), Lactobacillus buchneri (L.Buchnerii), and Lactobacillus casei (L.casei); jp-a 10-309178 discloses the use of bifidobacteria such as bifidobacterium infantis (bifidobacterium. infantis), bifidobacterium breve (bifidobacterium. breve), bifidobacterium longum (bifidobacterium. longum) and bifidobacterium bifidum as antiallergic agents for treating food allergy in particular, and jp-a 2000-95697 discloses the use of lactic acid bacteria such as Enterococcus faecalis (Enterococcus faecium) and Lactobacillus reuteri as type I allergy inhibitors such as allergic bronchial asthma, allergic rhinitis and atopic dermatitis.

On the other hand, enhancement of Th1 immunity by lactic acid bacteria means activation of cellular immunity such as macrophages, killer T cells, NK cells, etc. by production of IL-12, IFN-. gamma.and the like, thereby preventing viral and bacterial infection, cancer, etc. Specifically, IL-12 produced by macrophages can induce differentiation of undifferentiated helper T cells into Th1 cells, and activate mononuclear macrophage NK cells, thereby achieving resistance to foreign enemies and cancers. Therefore, it was suggested that a lactic acid bacterium which induces the production of strong IL-12 can be used as an immunologically active agent (cancer immunology Immunotherapy, vol.49, p.157, 2000; Japanese patent application laid-open No. 7-228536; Japanese patent application laid-open No. 2002-80364).

In addition, since lactic acid bacteria live and reach the Intestinal tract to exert various effects, it is necessary to be resistant to digestive juices such as gastric acid and bile acid (Symposium on endogenous Flora 3, "endogenous Flora and probiotics," Gakkai Shuppan Center, p.41-55, edited by Tomotari Mitsuoka 1998). In addition, it is known that lactic acid bacteria having high intestinal adhesion can sufficiently exert their effects by staying in the intestinal tract. Therefore, when lactic acid bacteria are orally ingested, it is preferable that the lactic acid bacteria not only have high functionality but also reach the intestinal tract alive.

In recent years, it is considered that many people have changed their constitutions due to allergy such as atopic dermatitis and pollinosis, and this has become a social problem. In which patients with severe symptoms are treated with various agents. However, it is the actual situation that most patients are not treated for a complete period of treatment, and are treated by symptomatic treatment including folk therapy on the basis of ensuring daily life.

In view of such a situation, studies on suppression of allergy by food have been actively conducted recently. As a result, antiallergic effects have been found in various food and drink components, such as Japanese basil oil, fish oil, and special tea polyphenols, and they have been used for treatment.

It is known that the components of foods and drinks having an antiallergic effect have effects similar to those of the above-mentioned specific lactic acid bacteria, and yogurt and lactic acid bacteria drinks using the lactic acid bacteria have been developed as foods and drinks having an antiallergic effect. The prevention or treatment with such antiallergic foods and beverages can be ingested under safe and mild conditions, but daily continuous ingestion is necessary, and daily continuous ingestion in an amount that can be changed is necessary to exert the effect. However, a method for screening and obtaining a strain having a high antiallergic activity has not been clarified so far, and a satisfactory strain has not been obtained yet, and thus it has not been satisfactory for practical use. Furthermore, conventionally, lactic acid bacteria have been used, as in yogurt and lactic acid bacteria beverages, with fermentation with lactic acid bacteria as a primary purpose, while placing antiallergic effects at a secondary level; in addition, since yogurt and lactic acid bacteria beverages are required to be transported under freezing conditions, and most of them cannot be preserved for a long period of time and have a small degree of freedom in handling, they are not satisfactory as foods and drinks which can be continuously taken daily and are effective in preventing or treating allergy. Therefore, there is a need for development of an effective antiallergic composition satisfying these conditions, and a food or beverage using the same.

The object of the present invention is to provide an antiallergic composition useful for the prevention and treatment of allergy, particularly an antiallergic composition containing a lactic acid bacterium having a high antiallergic activity as an active ingredient, a method for obtaining the lactic acid bacterium, and foods, beverages and the like having an antiallergic function. The term "having an antiallergic function" as used herein means that the composition can exert a preventive/therapeutic effect on allergy and/or can improve/alleviate allergic symptoms.

The present inventors have searched for active ingredients thereof for the purpose of preventing and treating environmental allergies such as pollinosis, atopic dermatitis, bronchial asthma, allergic rhinitis, and the like. First, intensive studies have been conducted focusing on lactic acid bacteria in view of safety, and as a result, it has been found that lactic acid bacteria having high antiallergic activity can be obtained by screening out lactic acid bacteria capable of inhibiting activation of Th2 and appropriately balancing activation of Th1, thereby completing the present invention.

That is, the method for obtaining a lactic acid bacterium having a high antiallergic activity of the present invention is a method focusing on the balance of Th1/Th 2. The conventional method is to evaluate the antiallergic activity by measuring IgE, and although the relationship between IgE and allergy is not clear, on the other hand, the degree of IgE cannot be specified in all cases, for example, as in the known hereditary allergic patients, there are those with high IgE and those with low IgE. It is known that, in addition to IgE, the onset of allergy is associated with a combination of events such as an increase in eosinophils and the release of chemokines that cause local infiltration of lymphocytes. Therefore, the present invention has focused on the Th1/Th2 balance that comprehensively explains IgE increase, eosinophil increase, and chemokines, and has succeeded in obtaining a lactic acid bacterium having a high antiallergic activity by developing a method for evaluating and isolating an antiallergic strain using the balance as an index.

The antiallergic composition of the present invention comprises, as active ingredients, lactic acid bacteria capable of balancing the production amounts of interleukin 4(IL-4) as an indicator of Th2 activation and interleukin 12(IL-12) as an indicator of Th1 immune activation: when lymphocytes derived from the spleen of a mouse sensitized with ovalbumin are suspended in a culture medium containing ovalbumin and cultured by adding a lactic acid bacterium to be tested, the production amount of interleukin 12 is the same as that when a lactobacillus paracasei (l.paracasei) KW3110 strain is used as the lactic acid bacterium to be tested, or the production amount of interleukin 12 is 60% or more of that when a lactobacillus paracasei KW3110 strain is used as the lactic acid bacterium to be tested, and the production amount of interleukin 4 is less than 50% of that of a control to which no lactic acid bacterium is added. In the present invention, the lactic acid bacteria having the above-mentioned antiallergic activity of the present invention include lactic acid bacteria having an interleukin 12 production amount of 70% or more as compared with KW3110 strain; lactic acid bacteria containing lactic acid bacteria as active ingredients, wherein the production of interleukin 12 is 70% or more and the production of interleukin 4 is less than 40%. In addition, the lactic acid bacteria with antiallergic activity obtained in the invention also include lactic acid bacteria with antiallergic activity equal to or higher than that of KW4110, KW3925 and KW3926 compared with that of KW4110, KW3925 and KW 3926; lactic acid bacteria with an anti-allergic activity equal to or higher than KW 3925; lactic acid bacteria with antiallergic activity equal to or higher than KW3926 are used as active ingredients.

The present invention also includes a method for obtaining a lactic acid bacterium having a high antiallergic activity, which is an active ingredient of the antiallergic composition of the present invention. The present invention also includes the use of the antiallergic composition of the present invention in foods and beverages, and the use thereof as an antiallergic agent for oral administration by formulation. Lactobacillus casei KW3110 strain, which is one of the active ingredients of the antiallergic composition of the present invention, is a lactic acid bacterium having high adhesion to the intestinal tract and is resistant to digestive juice, and exhibits excellent effects particularly when orally administered as an antiallergic agent.

Lactobacillus paracasei (lactobacillus. paracasei) KW3110 used as an active ingredient of the antiallergic composition of the present invention was deposited as FERM BP-08634 in 20 days 2/2004 at the national institute of integrated industrial and technology, which is an international depository, under the budapest treaty on the microbial collection in patent programs.

The use of the antiallergic composition of the present invention in foods and beverages includes, in particular, providing a beverage having an antiallergic function, which is obtained by using a lactic acid bacterium having a high antiallergic activity, as an active ingredient of the antiallergic composition of the present invention, in a tea beverage. The beverage having an antiallergic function of the present invention can be prepared in a form of good taste, long shelf life and continuous intake per day, and the amount of the continuous intake is an amount that can exert an effect. The lactic acid bacteria having high antiallergic activity of the present invention can be sterilized by heat sterilization or the like, and for example, the lactic acid bacteria of the present invention can be added to a sterilized sealed container to prepare a commercial beverage, so that the flavor of the beverage itself can be maintained, the stability of the beverage product can be maintained, and an antiallergic function can be provided. In addition, when a beverage packed in a sealed container is prepared, the beverage can be prepared by adjusting the concentration of lactic acid bacteria used to maintain the antiallergic function and to increase the function of the beverage to maintain its own flavor, and has a good taste and a long shelf life. In the present invention, from the viewpoint of antiallergic action and storage stability of the product, no milk component is included, that is, various types of non-milk beverages such as soft drinks containing fruit juice and non-milk beverages such as tea drinks are particularly preferable.

The antiallergic composition of the present invention can be used in foods and beverages by combining 2 or more lactic acid bacteria having high antiallergic activity obtained according to the present invention. In this case, various combinations or ratios can be prepared depending on the foods, drinks and lactic acid bacteria. In addition, other antiallergic compositions may also be used in combination or combination.

Disclosure of Invention

That is, the present invention provides an antiallergic composition (claim 1) comprising as an active ingredient a lactic acid bacterium having the following effects: when lymphocytes derived from spleen of a mouse sensitized with ovalbumin are suspended in a culture medium containing ovalbumin and cultured with addition of a lactic acid bacterium to be tested, the production amount of interleukin 12 is equal to or more than 60% of that in the case of using a Lactobacillus paracasei KW3110 strain as the lactic acid bacterium to be tested, or the production amount of interleukin 4 is less than 50% of that in a control without addition of a lactic acid bacterium. The antiallergic composition according to claim 1, which comprises as the active ingredient a lactic acid bacterium having the following effects: when lymphocytes derived from the spleen of a mouse sensitized with ovalbumin are suspended in a culture medium containing ovalbumin and cultured by adding a lactic acid bacterium to be tested, the production amount of interleukin 12 is equal to or more than 75% of that obtained when a Lactobacillus paracasei KW3110 strain is used as the lactic acid bacterium to be tested (claim 2). The antiallergic composition according to claim 1 or claim 2, which comprises as the active ingredient a lactic acid bacterium having the following effects: when lymphocytes derived from the spleen of a mouse sensitized with ovalbumin are suspended in a culture medium containing ovalbumin and cultured by adding a lactic acid bacterium to be tested, the amount of interleukin 4 produced is less than 40% of that of a control to which no lactic acid bacterium is added (claim 3). The antiallergic composition according to any one of claims 1 to 3, characterized by comprising as an active ingredient a lactic acid bacterium having the following effects: when lymphocytes derived from the spleen of a mouse sensitized with ovalbumin are suspended in a culture medium containing ovalbumin and cultured with addition of a lactic acid bacterium to be tested, the production amount of interleukin 12 is equivalent to that in the case of using a Lactobacillus paracasei KW3110 strain as the lactic acid bacterium to be tested, or 75% or more of that in the case of using a Lactobacillus paracasei KW3110 strain as the lactic acid bacterium to be tested, and the production amount of interleukin 4 is less than 40% of that in a control to which no lactic acid bacterium is added (technical scheme 4). The antiallergic composition according to any one of claims 1 to 4, characterized by comprising as an active ingredient a lactic acid bacterium having the following effects: when lymphocytes derived from the spleen of a mouse sensitized with ovalbumin are suspended in a culture medium containing ovalbumin and cultured with addition of a lactic acid bacterium to be tested, the production amount of interleukin 12 is equal to or more than 80% of that obtained when a Lactobacillus paracasei KW3110 strain is used as the lactic acid bacterium to be tested, and the production amount of interleukin 4 is less than 30% of that obtained in a control to which no lactic acid bacterium is added (technical scheme 5). An antiallergic composition, characterized in that the lactic acid bacteria with the production amounts of interleukin 12 and interleukin 4 derived from lymphocytes in the spleen of a mouse as described in any one of claims 1 to 5 are lactobacillus paracasei (lactobacillus. paracasei) KW3110 strain, lactobacillus plantarum KW4110 strain, lactobacillus paracasei KW3925 strain, lactobacillus paracasei KW3926 strain, or streptococcus salivarius (streptococcus. salivariaus) KW3210 strain (claim 6). Disclosed is an antiallergic composition which comprises a Lactobacillus paracasei KW3110 strain as an active ingredient and which exhibits high production amounts of interleukin 12 and interleukin 4 in spleen-derived lymphocytes of a mouse in any one of claims 1 to 5, is resistant to digestive juices, and has high adhesion to the intestinal tract (claim 7). The antiallergic composition according to any one of claims 1 to 7, characterized in that the allergy is pollinosis, bronchial asthma, allergic rhinitis, allergic conjunctivitis, or atopic dermatitis (claim 8). The antiallergic composition according to claim 8 is characterized in that the antiallergic composition is a drug (claim 9). The antiallergic composition according to claim 8 is characterized in that the antiallergic composition is a food or drink (claim 10).

In addition, the present invention includes a method for obtaining a lactic acid bacterium having a high antiallergic activity, characterized by isolating a lactic acid bacterium having the following effects: when lymphocytes derived from the spleen of a mouse sensitized with ovalbumin are suspended in a culture medium containing ovalbumin and cultured with addition of a lactic acid bacterium to be tested, the production amount of interleukin 12 is equal to or more than 60% of that obtained when a Lactobacillus paracasei KW3110 strain is used as the lactic acid bacterium to be tested, and the production amount of interleukin 4 is less than 50% of that obtained in a control to which no lactic acid bacterium is added (technical scheme 11). A method for producing a lactic acid bacterium having a high antiallergic activity, characterized by culturing a lactic acid bacterium isolated by the method according to claim 11 in a medium to proliferate the lactic acid bacterium (claim 12). The method for producing a lactic acid bacterium having a high antiallergic activity according to claim 12 is characterized in that the antiallergic activity of a lactic acid bacterium cultured and proliferated in a medium is confirmed (claim 13). The method for producing a lactic acid bacterium having a high antiallergic activity according to claim 13, wherein the antiallergic activity of the proliferated lactic acid bacterium is confirmed by suspending lymphocytes derived from the spleen of a mouse sensitized with ovalbumin in a culture medium containing ovalbumin, and measuring the production amounts of interleukin 12 and interleukin 4 when the test lactic acid bacterium is added and cultured (claim 14). A method for producing a drug or a food or beverage packed in a sealed container, characterized in that a lactic acid bacterium-containing drug or a food or beverage produced by the method for producing a lactic acid bacterium having a high antiallergic activity according to any one of claims 12 to 14 is filled in a container and sealed (claim 15). A method for preventing and/or treating an allergic disease, which is characterized by using the antiallergic composition according to any one of claims 1 to 10 for the treatment of prevention and/or treatment of an allergic disease (claim 16). The method for preventing and/or treating an allergic disease according to claim 16, wherein the allergic disease is pollinosis, bronchial asthma, allergic rhinitis, allergic conjunctivitis, or atopic dermatitis (claim 17). Use of an antiallergic composition according to any one of claims 1 to 10 for the prevention and/or treatment of an allergic disease (claim 18). The use of the antiallergic composition according to claim 18, wherein the prevention and/or treatment of the allergic disease is prevention and/or treatment of pollinosis, bronchial asthma, allergic rhinitis, allergic conjunctivitis, or atopic dermatitis (claim 19).

The present invention also provides a food or drink having an antiallergic function, characterized in that a lactic acid bacterium having an antiallergic activity according to any one of claims 1 to 7 is added as an active ingredient (claim 20). The food or drink having an antiallergic function according to claim 20, wherein the food or drink to be taken daily may contain 5X 10⁹More than one lactic acid bacteria having anti-allergic activity (scheme 21). The food or drink having an antiallergic function according to claim 20 or 21, characterized in that the food or drink does not contain an ingredient that causes allergy (claim 22). The food or drink having an antiallergic function according to claim 22, wherein the food or drink is a tablet, granule, powder, capsule, or beverage that does not contain an allergen component (claim 23). The food or drink having an antiallergic function according to any of claims 20 to 21, characterized by using a sterilized lactic acid bacterium (claim 24). A beverage having an antiallergic function, characterized in that a lactic acid bacterium having an antiallergic activity according to any one of claims 1 to 7 is added as an active ingredient (claim 25). The beverage having an antiallergic function in claim 22, characterized in that the beverage is a beverage containing no milk componentThe beverage in the container is sealed (claim 26). The beverage having an antiallergic function according to claim 25 or 26 is a fruit juice refreshing beverage or a tea beverage packed in a sealed container (claim 27). The beverage with antiallergic function in any one of claims 25 to 27, characterized in that the lactic acid bacteria is added to the beverage in a sealed container at 10 per 100g of the beverage⁹～10¹¹Range of lactic acid bacteria (claim 28). The beverage having an antiallergic function according to any one of claims 20 to 28, characterized in that the added lactic acid bacteria are lactobacillus paracasei KW3110 or a mutant strain thereof (claim 29). The beverage having an antiallergic function according to any one of claims 20 to 29, characterized in that the food or the beverage is prepared as a health food, a functional food, a food for specific health care, or a food for patients (claim 30). A method for producing a food or drink packed in a sealed container and having an antiallergic function, characterized by comprising adding a lactic acid bacterium having a high antiallergic activity according to any one of claims 1 to 7 or a sterilized lactic acid bacterium thereof to a sterilized food or drink in a substantially sterile state, filling the resulting mixture in a container, and sealing the container (claim 31). A method for measuring antiallergic activity of lactic acid bacteria, characterized in that spleen-derived lymphocytes of a mouse sensitized with ovalbumin are suspended in a culture medium containing ovalbumin, a test lactic acid bacteria is added thereto and cultured, and the amount of interleukin 12 and/or interleukin 4 produced during the culture is measured (claim 32). The method for measuring antiallergic activity of a lactic acid bacterium according to claim 32, wherein the amount of interleukin 12 and/or interleukin 4 produced by adding a lactic acid bacterium to be tested for culture is compared with the case of using Lactobacillus paracasei KW3110 strain to evaluate (claim 33). An antiallergic agent characterized by being prepared by adding a carrier, an excipient and/or other auxiliary agents to the lactic acid bacterium having a high antiallergic activity described in any one of claims 1 to 7 (claim 34). A drug prepared from the lactic acid bacterium having a high antiallergic activity described in any one of claims 1 to 7 (claim 35). A food or drink having an antiallergic function, characterized by using the following means 1Any one of the lactic acid bacteria having high antiallergic activity described in any one of claims 7 is used in place of a part or all of a beverage or food prepared with or containing lactic acid bacteria (claim 36). A method for producing a beverage or food having an antiallergic function, characterized in that a part or all of the lactic acid bacteria having a high antiallergic activity described in any of claims 1 to 7 are used in place of the lactic acid bacteria of the beverage or food prepared with or containing the lactic acid bacteria (claim 37).

The present invention provides an antiallergic composition comprising as the active ingredient a lactic acid bacterium having a high antiallergic activity, which is capable of specifically balancing the production amounts of interleukin 4(IL-4) as an indicator of Th2 activation and interleukin 12(IL-12) as an indicator of Th1 immune activation.

(effective component of lactic acid bacteria of the present invention)

The effective component of the invention, namely the lactic acid bacteria with high antiallergic activity, is the lactic acid bacteria with the following effects: when lymphocytes derived from the spleen of a mouse sensitized with ovalbumin (hereinafter, abbreviated as "OVA") are suspended in a culture medium containing OVA and cultured with addition of a test lactic acid bacterium, the yield of interleukin 12 of the test lactic acid bacterium is equal to or more than 60% of that of the test lactic acid bacterium containing a strain of lactobacillus paracasei KW3110, and the amount of interleukin 4 produced is less than 50% of that of a control containing no lactic acid bacterium. More preferably, the lactic acid bacterium of the present invention is characterized in that when the lymphocytes derived from spleen of mice sensitized with OVA are suspended in a culture medium containing OVA and the test lactic acid bacterium is added thereto for culturing, the production amount of interleukin 12 is equivalent to or 75% or more of that of the test lactic acid bacterium of lactobacillus paracasei KW3110 strain. Further, as a preferable effective ingredient of the present invention, the lactic acid bacterium is characterized in that when lymphocytes derived from spleen of a mouse sensitized with OVA are suspended in a culture medium containing OVA and a test lactic acid bacterium is added thereto and cultured, the amount of interleukin 4 produced is less than 40% of that of a control to which no lactic acid bacterium is added.

The most preferred embodiment of the lactic acid bacteria having high antiallergic activity of the present invention is: when lymphocytes derived from the spleen of a mouse sensitized with ovalbumin (hereinafter, abbreviated as "OVA") are suspended in a culture medium containing OVA and cultured with addition of a test lactic acid bacterium, the yield of interleukin 12 of the test lactic acid bacterium is equal to or more than 80% of that of the test lactic acid bacterium strain Lactobacillus paracasei KW3110, and the amount of interleukin 4 produced is less than 30% of that of a control containing no lactic acid bacterium.

The lactic acid bacteria having high antiallergic activity used as the effective ingredient in the present invention can be obtained by isolating lactic acid bacteria having the following effects: when lymphocytes derived from spleen of a mouse sensitized with an allergen are suspended in a medium containing the allergen and cultured by adding a test lactic acid bacterium, the Th 1-induced cytokine production of the test lactic acid bacterium is equal to or more than 60% of that of the test lactic acid bacterium containing Lactobacillus paracasei KW3110 strain, and the amount of interleukin 4 produced is less than 50% of that of a control containing no lactic acid bacterium. That is, it can be obtained by isolating lactic acid bacteria having the following effects: when well-known lactic acid bacteria strains or newly isolated lactic acid bacteria strains are used, lymphocytes derived from spleen of mice sensitized with ovalbumin are suspended in a culture medium containing OVA, and a lactic acid bacterium to be tested is added for culture, the yield of interleukin 12 of the lactic acid bacterium to be tested is basically equal to or more than 60% of that of the lactic acid bacterium to be tested using a Lactobacillus paracasei KW3110 strain, and the production amount of interleukin 4 is less than 50% of that of a control without the addition of lactic acid bacteria. As described above, the lactic acid bacteria used as the active ingredient of the present invention can be easily screened by those skilled in the art to obtain: can be obtained from various known or newly isolated lactic acid bacteria according to the index of the present invention by performing an experimental method for evaluating the antiallergic activity in vitro. Details are as described in example 1.

Lactic acid bacteria having high antiallergic activity to be used as the active ingredient of the present invention, for example, Lactobacillus paracasei KW3110 strain and Lactobacillus casei L14 strain, are commercially available from the milk industry Association of Japan. According to the above-mentioned technical society of milk industry, L14 strain, Lactobacillus casei, is a strain that is known as Lactobacillus paracasei (L.paracasei) in the present invention, because the present inventors analyzed it by RFLP (restriction fragment length polymorphism) and AFLP (amplified fragment length polymorphism) using RiboPrinter manufactured by QUALICON, Inc. Lactobacillus paracasei (lactobacillus. paracasei) KW3110, which is used as an active ingredient of the antiallergic composition in the present invention, is also purchased from the japan dairy technology association as described above, and is deposited as FERM BP-08634 at the organism depositary, national institute of advanced industrial and technology, which is an international depository, under budapest treaty on the microbial deposit in patent programs.

In addition, lactobacillus plantarum KW4110 strain used as an active ingredient of the antiallergic composition of the present invention is available as lactobacillus plantarum JCM1149 strain, lactobacillus paracasei KW3926 strain is available as lactobacillus paracasei JCM8132 strain from JCM (RIKEN), and lactobacillus paracasei KW3925 strain is available as lactobacillus paracasei NRIC1917 strain from NRIC (tokyo agricultural university).

When lactic acid bacteria used as the active ingredient of the present invention are screened, lactic acid bacteria having conventionally known probiotic functions such as intestinal regulation, cholesterol lowering, blood pressure lowering and the like for most lactic acid bacteria can be screened in addition to the antiallergic effect. In addition, when the effective component lactic acid bacteria of the present invention is orally administered, since lactic acid bacteria stay in the intestinal tract to sufficiently exert their effects, lactic acid bacteria having higher effectiveness can be obtained by selecting lactic acid bacteria obtained according to the above criteria, and then performing an in vitro adhesion test on the cell line Caco-2 derived from the human intestinal tract to screen out cells having higher adhesion ability. The lactobacillus paracasei strain KW3110, which is one of the active ingredients of the present invention, is a lactic acid bacterium having resistance to digestive juices and high adhesion to intestinal tracts, and can particularly exhibit the above-mentioned excellent effects when orally administered as an antiallergic agent.

The effective component lactic acid bacteria of the present invention can be used as the effective component of the composition of the present invention by appropriately culturing and proliferating lactic acid bacteria in a culture medium for lactic acid bacteria culture known to those skilled in the art, such as m.r.s. (de Man, Rogosa, sharp) medium, and then subjecting the cultured lactic acid bacteria to live bacteria or sterilization treatment, and optionally freeze-drying or spray-drying to obtain powder. In addition, for process control, if necessary, the antiallergic activity of the obtained lactic acid bacteria cells can be measured. The method for measuring the antiallergic activity is a method in which spleen-derived lymphocytes of an allergen-sensitized mouse are suspended in an allergen-containing medium, and when a test lactic acid bacterium is added and cultured, the amount of produced Th 1-induced cytokines and/or Th 2-induced cytokines is measured.

In the present invention, the lactic acid bacteria having high antiallergic activity obtained by the method of the present invention may be mutated to prepare and use mutant strains having high antiallergic activity. As a means for mutating the strain, a mutant strain having a preferable property, in which an antiallergic activity is increased or other properties are mutated, can be obtained and used by applying a known mutation means such as UV, for example, a method for mutating KW3110 strain of lactic acid bacterium having a high antiallergic activity by a mutation means, and the method for evaluating the antiallergic activity of lactic acid bacterium described in this specification. Further, mutation application can be performed on KW4110 strain, KW3210 strain, KW3925 strain, KW3926 strain, and the like.

(selection of derivative Strain)

In the present invention, strains having different characteristics derived from the lactic acid bacterium strain obtained by the present invention can be selected and used as derivative strains. The method for selecting the strain is described by taking KW3110 as an example and comprises the following steps: the KW3110 strain was cultured in an MRS medium-like medium, and after the culture was incubated at a predetermined temperature and a predetermined number of cells and the culture solution was diluted with fresh MRS medium, the cell suspension was suspended in PBS (prepared by dissolving a tablet manufactured by Dainippoin pharmaceutical) adjusted to pH3.0 with hydrochloric acid at a predetermined concentration in an amount of 10% and cultured at 37 ℃ for 3 hours. The suspension of acid-treated cells was diluted with PBS and transferred to MRS plate medium to form colonies. The formed colonies were selected using color as an index, and 1 strain was selected from among the colonies selected according to color, and cultured in MRS medium for 48 hours. The antiallergic activity of the culture was measured by the method for measuring antiallergic activity of the present invention (the method of example 2), and the antiallergic activity of each colony was determined. By this method, a strain having high antiallergic activity can be selected from among test strains. In the examples of the present invention, the isolated strain was named strain No.90, which was a derivative of KW3110(lactobacillus. paracasei) strain, and deposited as FERM BP-08635 at 20/2 in 2004 at the national institute of integrated and industrial technology, franchised organism depository, which is an international depository, according to budapest treaty on microbial depository in patent programs.

(preparation of antibody)

In the present invention, in order to manage the quality control and production process of the lactic acid bacteria product having a high antiallergic activity of the present invention, it is necessary to isolate and detect the lactic acid bacteria having a high antiallergic activity of the present invention, and to identify and examine the content of the lactic acid bacteria. Antibodies can be prepared and used for separating and detecting the lactic acid bacteria. Specific examples of the antibody capable of specifically binding to the lactic acid bacterium having a high antiallergic activity of the present invention include immunospecific antibodies such as monoclonal antibodies and polyclonal antibodies. They can be prepared by a conventional method using the above-mentioned lactic acid bacteria as an antigen, but among them, monoclonal antibodies are more preferable from the viewpoint of specificity. Methods for producing monoclonal antibodies and polyclonal antibodies can be performed according to known methods. For example, reference may be made to Koehler, g. and c.milstein, Nature: 256, 495-. That is, the monoclonal antibody is produced by immunizing a mouse with the lactic acid bacterium of the present invention, fusing spleen cells of the mouse with mouse myeloma cells, and producing the antibody from the resulting hybridoma. When the lactic acid bacterium of the present invention is detected using an antibody that specifically binds to the lactic acid bacterium of the present invention, the detection can be carried out by an immunological detection method using a known antibody. Examples of the immunological measurement method include known immunological measurement methods such as the RIA method, the ELISA method, and the fluorescent antibody method.

(antiallergic action of lactic acid bacteria as an active ingredient of the present invention)

Allergy is the production of IgE antibodies by antigen stimulation, which bind to Fc receptors on the surface of mast cells in tissue and basophils in the blood. Upon the second antigen stimulation (re-invasion of allergen), IgE antibodies bound to the surface of mast cells and basophils in the blood are recognized, and a bridge is formed between the IgE antibodies. This stimulus acts as a trigger point, causing the mast cells and basophils to release chemical mediators explosively, presenting with symptoms of various allergies. Therefore, it is necessary to suppress IgE for the treatment and prevention of allergy, and for this purpose, it is necessary to enhance Th1 immunosuppression Th2 immunity.

The lactic acid bacteria of the present invention can strongly induce the production of interleukin 12(IL-12) as a Th1 immune index and strongly inhibit the production of interleukin 4(IL-4) as a Th2 immune index in an in vitro system using mouse lymphocytes. Therefore, the lactic acid bacteria of the present invention inhibit the production of IgE antibodies by enhancing Th1 immunity and suppressing Th2 immunity, and have therapeutic and prophylactic effects on allergy based on this mechanism of action.

Therefore, the antiallergic composition of the present invention exerts a specific effect on environmental allergies such as pollinosis, atopic dermatitis, bronchial asthma, allergic rhinitis, bronchial asthma, and the like. In addition, the lactic acid bacteria of the present invention have not only the above-mentioned antiallergic effect but also the conventionally known probiotic functions of most lactic acid bacteria, such as an intestinal tract-regulating effect, a cholesterol-lowering effect, and a blood-pressure-lowering effect. It has been confirmed that KW3110 strain, which is one of the effective lactic acid bacteria of the present invention, has excellent intestinal cell adhesion and tolerance to gastric acid and bile acid in addition to strong antiallergic and immunostimulatory activities, and its effect is also expected from the viewpoint of the aforementioned probiotic function.

(administration of the antiallergic composition of the present invention)

The antiallergic composition of the present invention is used as an antiallergic agent by mixing the active ingredient of the present invention with a physiologically acceptable carrier, excipient, binder, or drug release agent, and formulating the mixture into a preparation. The antiallergic agent of the present invention can be administered orally or parenterally. Examples of the preparations for oral administration include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. Examples of parenteral preparations include injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections), drops, external preparations (e.g., nasal preparations, transdermal preparations, and ointments), and suppositories (e.g., rectal suppositories and vaginal suppositories). These preparations can be prepared by adding pharmaceutically acceptable excipients and additives by means generally used in the art. In order to allow the antiallergic composition of the present invention to exert its function in vivo in a timely and effective manner, it is preferable to control the time for starting elution and to add a bitter taste masking agent, and to improve stability against oxygen and humidity, for example, a coating treatment product coated with a coating agent containing yeast cell walls as a main component as described in patent 3349677, and a capsule encapsulated in a soft capsule or a hard capsule according to a conventional method are preferably used in pharmaceutical preparations, health foods, and the like. Examples of pharmaceutically acceptable excipients and additives include carriers, binders, flavors, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, and the like. Examples of the pharmaceutically acceptable carriers include magnesium carbonate, magnesium stearate, talc, granulated sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting-point paraffin, cacao butter, and the like.

The formulation may be prepared as follows. That is, the oral preparation can be prepared by adding, as an active ingredient, for example, an excipient (e.g., lactose, white sugar, starch, mannitol), a disintegrant (e.g., calcium carbonate, calcium carboxymethylcellulose), a binder (e.g., α -starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropyl cellulose) or a lubricant (e.g., talc, magnesium stearate, polyethylene glycol 6000), compression molding, and coating or the like by a known method as needed for the purpose of taste masking, enteric solubility, sustainability, or the like. Examples of the coating agent include ethyl cellulose, hydroxymethyl cellulose, polyethylene glycol, cellulose acetate phthalate, hydroxypropyl phthalate, and eudragit (methacrylic acid acrylate copolymer, manufactured by Rhom Germany Co., Ltd.).

Injections are prepared by dissolving and suspending the active ingredient in an aqueous solvent (e.g., distilled water, physiological saline, Ringer's solution, etc.) or an oily solvent (e.g., olive oil, sesame oil, cottonseed oil, vegetable oil such as corn oil, or propylene alcohol), together with a dispersant (e.g., Tween 80(Atlas Powder, usa), HCO60(Nikko Chemicals), polyethylene glycol, carboxymethylcellulose, sodium alginate, etc.), a preservative (e.g., methyl hydroxybenzoate, propylhydroxybenzoate, benzyl alcohol, chlorobutanol, phenol), and an isotonic agent (e.g., sodium chloride, glycerol, sorbitol, glucose, invert sugar), etc., and emulsifying the resulting solution in an injection. At this time, additives such as dissolution aids (e.g., sodium salicylate, sodium acetate), stabilizers (e.g., human serum albumin), painless agents (e.g., benzalkonium chloride, procaine hydrochloride), and the like are added as desired.

The preparation of the external preparation can be prepared by preparing the effective components into solid, semisolid or liquid composition. For example, the solid composition is prepared by mixing the active ingredients directly or with excipients (e.g., lactose, mannitol, starch, microcrystalline cellulose, white sugar), thickeners (e.g., natural gums, cellulose derivatives, acrylic polymers), and the like, and making into powder. The liquid composition can be prepared in the same manner as in the case of an injection. The semisolid composition may be in the form of an aqueous or oily gel or ointment. Additionally, these compositions can each include pH adjusting agents (e.g., carbonic acid, phosphoric acid, citric acid, hydrochloric acid, sodium hydroxide), preservatives (e.g., propyl paraben, chlorobutanol, benzalkonium chloride), and the like. The suppository can be prepared into oily or aqueous solid, semisolid or liquid composition. The oily base used in the composition includes, for example, a glyceride of a higher fatty acid [ e.g., cacao butter, ウイテプゾル -type (manufactured by Dynamit Nobel Co.) ], a medium fatty acid [ e.g., Miglioes-type (manufactured by Dynamit Nobel Co.) ], or a vegetable oil (e.g., sesame oil, soybean oil, cottonseed oil). The aqueous base agent comprises polyethylene glycol and propylene glycol. In addition, examples of the hydrocolloid base include natural gums, cellulose derivatives, vinyl polymers, and acrylic polymers.

(use of food and drink)

The antiallergic composition of the present invention can be used in combination with a food or drink as a food or drink having an antiallergic function. When the antiallergic composition of the present invention is used in combination with a food or beverage, an effective amount of the active ingredient may be added or combined at the stage of producing a raw material or a product of the food or beverage. The "effective amount of the active ingredient" as used herein means that the content of the active ingredient to be taken in a normal food or drink amount is in the following range.

That is, the determination of the effective amount of the effective ingredient in the food or drink of the present invention is dependent on the subject, the age and weight of the subject, symptoms, administration time, dosage form, administration method, combination of the drugs, and the like. For example, the active ingredient of the present invention is administered orally as a drug at 0.1 to 100mg/kg body weight (preferably 1 to 10mg/kg body weight) per adult, and administered parenterally at 0.01 to 10mg/kg body weight (preferably 0.1 to 1mg/kg body weight) 1 to 3 times a day. The pharmaceutical agents having other action mechanisms to be used in combination with the active ingredient of the present invention are also appropriately selected based on the clinical amount thereof.

When the effective amount of the active ingredient of the present invention in a food or beverage is expressed by the number of lactic acid bacteria, the daily intake is preferably 5 × 10⁹More than one, more preferably 1X 10 per day¹⁰More than one, most preferably 5 × 10¹⁰More than one. Therefore, the number of lactic acid bacteria contained in each food is determined according to the amount of food or drink that is usually taken daily. For example, when ingested as a food (yogurt), 1 adult per dayThe amount of (b) is in the range of 50 to 500g, preferably 60 to 200g, and the active ingredient of the present invention can be incorporated into food in such an amount.

In the present invention, the active ingredient of the antiallergic composition of the present invention may be incorporated into a food as it is or in the form of the above-mentioned preparation. More specifically, the food or drink of the present invention can be used in various forms: the active ingredient of the present invention can be mixed with appropriate raw materials to prepare foods and drinks as it is, or further mixed with various proteins, saccharides, fats, trace elements, vitamins, etc. to prepare a liquid, semi-liquid or solid, or further added to or mixed with general foods and drinks.

In the field of foods and beverages using lactic acid bacteria, they are classified into dairy products, meats, breads, beverages, and vegetables according to their practical schemes. By using the lactic acid bacterium having a high antiallergic activity of the present invention as a lactic acid bacterium or a part thereof in the production of a food or beverage using such a lactic acid bacterium, or as an additive to a produced food or beverage, an antiallergic function can be imparted to the food or beverage.

In another embodiment of the present invention, when the effective component lactic acid bacteria is added to the food or drink, it is not necessary to consider fermentation of the lactic acid bacteria itself, and when it is desired to maintain the flavor of the food or drink itself, it is particularly preferable to sterilize the lactic acid bacteria by a treatment such as heat sterilization. In addition, the food or drink having a high antiallergic function of the present invention is preferably packed in a sealed container for preventing other microorganisms and foreign substances from being mixed and maintaining the quality of the contents.

The food of the present invention can be prepared into a health food, a functional food, a food for specified health care, or a food for patients, which retains an antiallergic function. The form of the food is not particularly limited, and the food may be a beverage having an antiallergic function. Since the active ingredient of the present invention has an antiallergic effect, it is possible to provide a food which can be continuously ingested while maintaining the function of preventing and treating allergy by incorporating the active ingredient of the present invention into a food to be ingested on a daily basis or a health food, a functional food, or the like to be ingested as a supplement. By incorporating the lactic acid bacterium having a high antiallergic activity of the present invention into a food or drink containing no allergy-inducing component, for example, a tea, a drink, a tablet or the like, a food or drink having an antiallergic function in which allergy problems are completely avoided can be provided. In the present invention, a beverage or food having a high antiallergic function can be obtained by partially or completely replacing lactic acid bacteria produced by lactic acid bacteria or contained in a beverage or food containing lactic acid bacteria with the lactic acid bacteria having a high antiallergic activity of the present invention.

(use in the form of food or drink preparations)

In the present invention, various examples of the health food and functional food containing the lactic acid bacteria having a high antiallergic activity of the present invention are given, and the health food and functional food can be used in the form of a food or drink preparation using an auxiliary agent such as an excipient, an extender, a binder, a disintegrator, a lubricant, a dispersant, a preservative, a moistening agent, a dissolution aid, a preservative, a stabilizing agent, and a capsule base, in addition to a usual food material and food additives in connection with the production of the health food and functional food. Specific examples of the auxiliary agent include lactose, fructose, glucose, starch, gelatin, magnesium carbonate, synthetic magnesium silicate, talc, magnesium stearate, calcium carbonate, methyl cellulose, carboxymethyl cellulose, or a salt thereof, gum arabic, polyethylene glycol, syrup, vaseline, glycerin, ethanol, propylene glycol, citric acid, sodium chloride, sodium sulfite, sodium phosphate, pullulan, carrageenan, dextrin, palatinose (palatinose), sorbitol, xylitol, stevia, synthetic sweetener, citric acid, vitamin C, sour agent, sodium bicarbonate, sucrose ester, vegetable hardened fat, potassium chloride, safflower oil, beeswax, soybean lecithin, perfume, and the like. In connection with the production of such health foods and functional foods, reference may be made to the reference books of pharmaceutical preparations, such as "Practical guide of Japanese Pharmacopoeia (General canal interference)" (Hirokawa Shoten), and the like.

In the present invention, the shape particularly suitable for the health food and the functional food includes a sheet, a capsule, a granule, a powder, a suspension, an emulsion and the like, and from the viewpoint of the problem to be solved by the present invention, it is preferable to use a combination of the health food and the functional food containing the lactic acid bacterium having a high antiallergic activity of the present invention and a raw material containing no specific allergen.

Specifically, the method for producing a sheet-like health food or functional food includes pressing a material containing the lactic acid bacterium having a high antiallergic activity of the present invention into a predetermined shape, or pouring a mixture wetted with a solvent such as water or ethanol into a mold having a predetermined shape, and molding the mixture. Specifically, a method for producing a capsule-like health food and a functional food, which comprises filling a substance containing the lactic acid bacterium having a high antiallergic activity of the present invention in the form of a liquid, suspension, paste, powder or granule in a capsule, or coating a capsule base such as a hard capsule, a soft capsule or the like, is exemplified.

(incorporation into food)

In the present invention, the food having an antiallergic function can be prepared by incorporating the lactic acid bacterium having a high antiallergic activity of the present invention into a food. Specifically, such foods are various and include western-style foods such as pudding, cookie, biscuit, French fries, snack, bread, cake, chocolate, doughnut, jelly and the like, japanese snacks such as pancake, lamb, yofu (daifuku), おはぎ, steamed bread, cake and the like, cold snack (candy and the like), bread such as chewing gum, noodles such as vegetable and cut noodles, buckwheat noodles, flat noodles and the like, and fish cakes such as fish cake, ham, fish sausage and the like, and meat foods and salts such as ham, sausage, beef cake, beef can and the like, pepper, bean paste, soybean sauce, soy sauce, dressing, mayonnaise, tomato sauce, sweet seasoning, seasoning such as spicy seasoning and soft octopus ball (akashiyaki), octopus ball (takoyaki), half-cooked black crepe-like pancake (mojayaki) appetizing pancake (okonomyyaki) and cheese, fermented dairy products such as hard bean cake and hard cake, and fermented dairy products such as yogurt, Various products such as pressed bean curd, yam paste, kimchi rice ball, fish paste, dumpling, steamed dumpling, fried wheat, fried meat cake, sandwich, pizza, hamburger, salad, and various foods such as powdered solid products and powdered drinks and foods (instant coffee, instant black tea, instant milk powder, instant soup powder, japanese bean paste soup, etc.) of various powders (animal products such as beef, pork, chicken, etc., shrimp, scallop, clam, kelp, etc., vegetables and fruits, plants, yeast, algae, etc.), oils and fats, spices (vanilla, citrus, bonito, etc.), and the like, but not limited thereto.

In particular, when the effective ingredient of the present invention is contained in a dairy product, the effective ingredient of the present invention is added to a milk raw material in the form of viable bacteria, and the bacteria are proliferated and fermented to prepare a fermented lactic acid bacteria food or drink such as yogurt.

(incorporation into beverages)

The highly antiallergic composition of the present invention, particularly in the form of a drink, can provide a drink having an antiallergic function which can be ingested continuously on a daily basis and is effective within the range of the possible intake amount for continuous ingestion. When the lactic acid bacterium having a high antiallergic activity of the present invention is incorporated into a beverage, it is preferable to use a heat-sterilized form of lactic acid bacterium in order to prepare a beverage which retains the flavor and stability of the beverage itself.

When the lactic acid bacterium having a high antiallergic activity of the present invention is blended in a beverage, the blending amount of the lactic acid bacterium can be appropriately determined, but the blending amount is generally adopted so that the antiallergic activity of the lactic acid bacterium can be sufficiently exhibited within an intake amount range in which the lactic acid bacterium can be continuously ingested as a beverage. The effective amount of the active ingredient of the present invention in foods and beverages is expressed by the number of lactic acid bacteria, and the daily intake is preferably 5 × 10⁹More than one, more preferably 1X 10 per day¹⁰More than one, most preferably 5X 10¹⁰More than one. Therefore, the number of lactic acid bacteria contained in each beverage can be determined according to the above index, usually according to the amount of beverage taken daily. For example, when 100g of a drink is taken daily, it is preferable to add 10 to 100g of the drink⁹More than one bacterium. On the other hand, the amount of lactic acid bacteria is preferably in the range of not impairing the flavor and appearance of the beverage10¹¹One below. More preferably 5X 10¹⁰One below. Therefore, as a beverage having an antiallergic function, good stability, and high taste and keeping quality, the concentration of lactic acid bacteria is particularly preferably 10 per 100g of the beverage⁹～10¹¹And (4) respectively. The relationship between the number of cells of lactic acid bacteria and the weight of dry bacteria is, for example, that the number of cells is 10 for Lactobacillus paracasei KW3110 strain¹²In this case, the amount of the microorganism corresponds to 1g of the weight of the dry microorganism.

In the present invention, various beverages containing the lactic acid bacterium having high antiallergic activity of the present invention can be prepared, and any of sugars, flavors, fruit juices, food additives and the like used in the design of general beverage formulations can be used. For the manufacture of beverages, reference may be made to existing reference books, such as "Revised new edition: soft rainks "(Kohrin), and the like.

In the present invention, a beverage particularly suitable for blending is preferably a beverage containing no milk component from the viewpoint of an antiallergic effect and storage and stabilization of a product, that is, a beverage containing no milk component such as a fruit juice-containing refreshing beverage or a tea beverage.

Specific examples of the blended beverage include various beverages such as alcoholic beverages (whisky, bourbon, spirits, liqueur, wine, fruit wine, yellow wine (sake), chinese liquor, white spirit, beer, nonalcoholic beer with an alcohol content of 1% or less, sparkling liquor, carbonated white spirit, and the like) and nonalcoholic beverages (fruit juices such as drinking yogurt, apple, mandarin orange, grape, banana, pear, plum, and watermelon, vegetable juices such as potato, carrot, celery, and cucumber, refreshing beverages, milk, soybean milk, coffee, cocoa, black tea, green tea, barley tea, brown rice tea, roasted tea, yulu tea, roasted tea, oolong tea, ginger yellow tea, black tea, Rooibos tea, rose tea, chrysanthemum tea, peppermint tea, jasmine tea, various herb teas, sports beverages, mineral water, nutritional beverages, and the like).

Examples of the beverage include tea-based beverages such as green tea, oolong tea, black tea, barley tea, and mixed tea, and coffee, fruit juice beverages, vegetable beverages, sports beverages, and nutritional beverages.

In the present invention, when a beverage having an antiallergic function is produced, the beverage can be appropriately sterilized according to a method prescribed by food sanitation act. As the sterilization method, pasteurization, retort sterilization, UHT sterilization, retort sterilization, or the like can be used depending on the pH of the beverage.

Further, as for the product form, a form of a sealed container used in a conventional product form is particularly preferable, and as for the sealed container, any of a can, a bottle, PET, and a paper container can be used. The volume is not particularly limited, and is usually determined appropriately in consideration of the daily intake of the beverage by the consumer, the number of bacteria of lactic acid bacteria to be blended, and the number of bacteria necessary for 1 day.

The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.

Example 1

(lactic acid bacteria used in examples of the present invention)

The lactic acid bacteria used in the examples of the present invention and the results are shown in FIG. 1(No.1) and FIG. 2(No. 2). All strains in the present invention are identified by the serial number at the beginning of KW. Some of these test strains were isolated individually, some were used in commercial dairy products, and some were obtained from public institutions, and for convenience, their identification names were unified. The relationship between each KW strain and the source of the strain is shown in FIG. 1(No.1) and FIG. 2(No. 2). In "acquired place" of FIG. 1, "JCM" stands for Japan Collection of Microorganisms, and "IFO" stands for former institute of fermentation (now national institute of independent administrative sciences Bioresource center (NBRC)).

Example 2

(evaluation of the in vitro antiallergic Activity of the lactic acid bacteria group)

1. Sample (I)

Each strain of lactic acid bacteria was cultured in m.r.s. (de Man, Rogosa, sharp) medium (OXOID) for 48 hours, washed 3 times with sterilized water, suspended in sterilized water, treated at 100 ℃ for 30 minutes, and sterilized. This was freeze-dried and suspended in PBS. The lactic acid bacteria used are shown in FIGS. 1(No.1) and 2(No. 2).

2. Experimental animal and breeding

BALB/c mice (Charles River) 7-10 weeks old (day 0 and day 6) were injected intraperitoneally with Ovalbumin (OVA)1mg and adjuvant Alum2 mg. Spleen was dissected on day 13, isolated and lymphocytes were prepared. In the experiment, n is 6.

3. Determination of IL-4 and IL-12 in splenocytes

IL-4 and IL-12 were measured using an OptEIA ELISA Set (Becton Dickinson).

4. Conditions of the experiment

Spleen lymphocytes prepared in the method of 2. Experimental animals and Breeding were cultured in RPMI1640(SIGMA) + 10% FCS (Rosche) +1mg OVA Medium and suspended at 2.5X 10⁶Cell/ml concentration. Then 0.1 or 1. mu.g/ml of the lactic acid bacteria to be tested are added. After 1 week, the culture supernatant was collected and examined for Th1 type cytokine IL-12 and Th2 type cytokine IL-4, respectively.

5. Results of the experiment

The amounts of Th1 type cytokine IL-12 produced by each lactic acid bacterium are shown in FIG. 3 and Table 2. The addition amount of lactic acid bacteria was 1. mu.g/ml. It can be seen that the yield varies from strain to strain. Of about 100 strains, the strain of Lactobacillus paracasei KW3110 showing the strongest Th 1-inducing ability was identified. The data are all shown as the ratio of the amount of IL-12 produced by KW3110 strain to 100%. For convenience, the yield of KW3110 strain with a yield of 75% or more was rated A, 50% or more and less than 75% was rated B, 25% or more and less than 50% was rated C, 10% or more and less than 25% was rated D, and less than 10% was rated E.

The production amounts of Th2 type cytokine IL-4, which is responsible for allergy, by each lactic acid bacterium are shown in FIG. 4 and Table 2. The amount of lactic acid bacteria added was 0.1. mu.g/ml. The inhibitory effect varies depending on the strain. Of about 100 strains, Lactobacillus paracasei KW3110 strain was used as the most potent Th 2-inhibiting strain. All data are expressed as the ratio of the IL-4 production of the control (without addition of lactic acid bacteria) to 100%. For convenience, yield was rated as A for 30% less than the control, B for 30% or more and 50% or less, C for 50% or more and 70% or less, D for 70% or more and 100% or less, and E for 100% or more. The "results of evaluation of antiallergic lactic acid bacteria" are shown in tables 1 and 2, which summarize parameters a to E of Th1 and Th2, respectively.

(Table 1)

Th1

Th2

Th1

Th2

Th1

Th2

Th1

Th2

KW3118

C

B

KW4510

A

C

KW3910

D

E

KW3317

D

B

KW3110

A

A

KW4511

D

E

KW3914

C

B

KW3811

A

D

KW4210

E

E

KW3812

D

E

KW3918

E

D

KW3711

E

D

KW3510

E

E

KW3813

C

E

KW3917

C

B

KW4010

B

B

KW3310

E

E

KW3814

D

E

KW3918

E

D

KW3413

C

C

KW3411

C

C

KW3815

E

E

KW3919

D

C

KW4110

A

B

KW3211

D

C

KW3816

C

E

KW3920

C

C

KW3513

E

D

KW3820

C

E

KW3817

D

E

KW3921

E

C

KW3514

E

E

KW4211

C

D

KW3818

E

E

KW3922

E

C

KW4310

A

B

KW4810

B

C

KW3819

E

E

KW3923

C

B

KW3210

A

C

KW3515

E

D

KW3821

C

E

KW3924

C

C

KW3810

D

D

KW3822

C

D

KW3911

C

B

KW3812

E

E

KW3823

C

D

KW3114

C

B

KW3710

E

E

KW3824

C

D

KW3115

E

D

KW3511

E

E

KW3825

C

D

KW3116

E

C

KW3316

D

E

KW3826

C

D

KW3117

E

D

KW3613

D

E

KW3827

E

E

KW3913

C

B

KW3412

D

D

KW3828

C

D

KW3119

B

A

(Table 2)

Strain number	Th1 (% of KW3110)	Grade	Th2 corresponds to control (%)	Evaluation value
					KW 3111	42.1	C	36.3	B
KW 3112	43.6	C	48.8	B
					KW 3113	38.3	C	54.8	C
KW 3120	38.8	C	43.9	B
					KW 3311	19.4	D	66.9	C
KW 3315	7.4	E	90.4	D
					KW 3410	38.7	C	49.2	B
KW 3414	11.7	D	74.6	D
					KW 3712	18	D	70.7	D
KW 3927	53.7	B	45.3	B
					KW 3931	19.3	D	49	B
KW 3932	17.9	D	57	C
					KW 3933	17.1	D	50.1	C
KW 3934	8.1	E	48.7	B
					KW 3929	30.7	C	43	B
KW 3938	1.2	E	69.8	C
					KW 3930	19.5	D	59.7	C
KW 3935	5.5	E	63.7	C
					KW 3936	5.1	E	54	C
KW 3926	70.1	B	30.2	B
					KW 3928	39.2	C	59.8	C
KW 3925	77.1	A	45.2	B
					KW 3939	2.3	E	59.1	C
KW 3941	38.3	C	50.3	C
					KW 4511	21.4	D	134.4	E
KW 4611	1.3	E	55.4	C
					KW 4700	3.6	E	109	E
KW 4701	2.6	E	138.2	E
					KW 4702	2.1	E	96.5	D
KW 4703	0	E	103.5	E
					KW 4704	2.3	E	94.3	D
KW 3610	1	E	104	E
					KW 3212	19	D	67.1	C
KW 3213	65.3	B	54.8	C
					KW 3214	37.2	C	49.5	B
KW 3510	0.5	E	105.5	E
					KW 3515	0	E	79.3	D
KW 3511	0	E	80	D

Strains having a Th1 parameter of 60% or more of the strain KW3110 and a Th2 parameter of 50% or less of the control strain were used as antiallergic lactic acid bacteria, and 5 strains of the strains KW3110, T, NRIC1917, JCM8132 and JCM1149 were included in the strain. Further, as the immuno-activated lactic acid bacteria, there were strains exhibiting a strong Th 1-inducing ability of 75% or more of KW3110 strain, including 6 strains of KW3110 strain, KW4510 strain, JCM1149 strain, T strain, NRIC1917 strain and JCM1059 strain. Thus, an immune activating composition (medicine, food or drink) can be prepared by the method described in the present specification, wherein the lactic acid bacteria have an immune activating activity equivalent to that of KW3110 strain, or have an immune activating activity equivalent to or higher than that of KW4510 strain, JCM1149 strain, NRIC1917 strain, and JCM1059 strain. In addition, the yield of IL-12 of Lactobacillus acidophilus L-92 strain known as an antiallergic lactic acid bacterium was about 11.7% of that of KW3110 strain, and the IL-4 inhibitory activity was 26.4% of that of KW3110 strain (no addition), while the yield of L-92 strain was 74.6% of that of L-92 strain, indicating that either parameter is absolutely inferior to that of a strong antiallergic lactic acid bacterium such as KW3110 strain (FIG. 5). The results of measurement of the antiallergic activity of the lactic acid bacteria strain of the present invention and the control lactic acid bacteria strain are shown in table 3.

(Table 3)

Example 3

(selection of derivative Strain)

This example is an example of selecting a strain having an antiallergic activity from strains derived from the lactic acid bacterium strain KW3110 obtained by the present invention. The strain KW3110 is statically cultured in MRS medium at 37 ℃ until the OD600 value is 0.8-1.0.

After the culture medium was diluted to an OD600 of 0.5 with a fresh MRS medium, the cell suspension was suspended in PBS (a tablet manufactured by Dainippoin pharmaceutical) adjusted to pH3.0 with hydrochloric acid at a predetermined concentration at 10% and cultured at 37 ℃ for 3 hours. The suspension of acid-treated cells was diluted with PBS and transferred to MRS plate medium to form colonies. Among the colonies formed, a slightly colored colony was observed. The colonies were selected and cultured in MRS medium for 48 hours. This strain was designated as No.90 strain. The antiallergic activity of strain No.90 was measured by the method of example 2. The ability of No.90 strain to produce IL-12 was the same as compared with the wild-type strain (FIG. 6). In the figure, "specific activity (%)" is the IL-12-producing ability of the derivative strain (No.90) when the IL-12-producing ability of KW3110 strain was taken as 100. Strain No.90 was deposited as FERM BP-08635 at the International patent organism depositary, national institute of advanced Industrial science and technology.

Example 4

(in vivo evaluation of in vitro highly antiallergic Strain KW3110 Strain)

1. Application of samples

Culturing Lactobacillus casei KW3110 strain with MRS culture medium for 48 hr, washing with sterilized water for 3 times, suspending in sterilized water, treating at 100 deg.C for 30 min, and sterilizing. The resulting mixture was freeze-dried and mixed with AIN93 (standard composition of national center for Nutrition research) as a standard powder feed in an amount of 1mg per mouse taken 1 day to prepare a mixed feed.

2. Experimental animal and breeding

BALB/c was used at 8 weeks of age. Group 1, 6 BALB/c mice, which were allowed to freely take up CE-2 (CLEA in Japan) and water, were acclimatized and raised for 1 week. After the antigen sensitization on day 0, the control group was fed with AIN93 powdered feed prepared from purified raw materials, and KW3110 group was fed with mixed feed prepared by mixing KW3110 bacteria with AIN 93.

3. Measurement of IgE, splenocyte IL-4, and IL-12 in blood

IgE, IL-4, and IL-12 were assayed using an OptEIA ELISA Set (Becton Dickinson).

4. Conditions of the experiment

As experimental groups, a control group (AIN93) was set, and KW3110 strain was added to AIN93, and KW3110 group was prepared at 1 mg/mouse/day. The control group, KW3110 group were given 4g of pasty powder feed with water daily until the end of the experiment (day 98). Drinking water is freely ingested. Allergic sensitization with Ovalbumin (OVA) was achieved by intraperitoneal injection of 100. mu.g OVA together with 2mg adjuvant Alum on days 0, 14, 42, 70, and 94 for 5 times. During this time, blood was taken from the fundus vein every 1 week. At the end of the experiment whole blood was taken and spleen was removed and lymphocytes were prepared (FIG. 7). With RPMI1640(SIGMA) + 10% FCS (Ros)che) + 100. mu.g of OVA Medium, 5% CO at 37 ℃₂Spleen lymphocytes were cultured for 1 week under the conditions described above, and the culture supernatant was collected. The amount of IgE in blood samples was measured, and IL-4 and IL-12 were measured for spleen lymphocyte culture supernatant samples.

5. Results of the experiment

The change in blood IgE during the 98-day experiment period is shown in FIG. 8. The figure shows a significant reduction in IgE in blood at day 48 in KW3110 compared to control with p < 0.05. IgE in blood on day 48 and day 98 at dissection is shown as spots in fig. 9. The figure shows that IgE in blood of KW3110 group was also significantly reduced compared to the control group at day 98. IL-12 production in spleen cell culture supernatant at day 98 dissection of control group, KW3110 group is shown in FIG. 10, and IL-4 production is shown in FIG. 11. IL-12 production was significantly increased in KW3110 group compared to control group. There was no significant difference in IL-4, with a tendency to decrease compared to the control group. These results suggest that intake of KW3110 strain can shift the immune balance in vivo to Th1, thereby improving allergic conditions. In addition, as shown in FIG. 12, on day 30, no exception occurred around the nose in the control group, whereas no exception in KW3110 caused no depilation. Frequent nasal grasping was observed in the control group at this time. Before and after day 60, depilation and nasal grasping behavior around the nose were observed in KW3110 group as in control group. The observation of the nasal grasping behavior suggests that in fact KW3110 group suppressed allergic symptoms.

Example 5

(comparison with known antiallergic lactic acid bacteria, KW4610 strain (L. rhamnosus LGG strain))

1. Application of samples

Culturing KW3110 strain and KW4610 strain in MRS culture medium for 48 hr, washing with sterilized water for 3 times, suspending in sterilized water, treating at 100 deg.C for 30 min, and sterilizing. Freeze-drying, and mixing with standard powder feed AIN93 (according to standard composition of national center for Nutrition research) in an amount of 1-10 mg per mouse per day to obtain mixed feed.

2. Experimental animal and breeding

BALB/c was used at 8 weeks of age. Group 1, 6 BALB/c mice, which were allowed to freely take up CE-2 (CLEA in Japan) and water, were acclimatized and raised for 1 week. A mixed feed of KW3110 strain, KW4610 strain or AIN93 powdered feed was ingested 21 days before antigen sensitization.

3. Measurement of IgE in blood

IgE was assayed using an OptEIA ELISA Set (Becton Dickinson).

4. Conditions of the experiment

Experimental groups A control group (AIN93), a KW3110 group and a KW4610 group in which KW3110 or KW4610 strain was added to AIN93 at a dose of 10 mg/mouse/day were established. The control group, KW3110 group and KW4610 group were given water to prepare a pasty powdery feed 4g each day until the end of the experiment (day 133). Drinking water is freely ingested. Allergic sensitization with Ovalbumin (OVA) was achieved by intraperitoneal injection 6 times on days 0, 14, 42, 70, 98, 126, using 100. mu.g OVA together with adjuvant Alum2 mg. During this time, blood was taken from the fundus vein every 1 week (fig. 13). The amount of IgE was measured on the blood samples.

5. Results of the experiment

The variation in the amount of IgE in the blood during the test period is shown in FIG. 14, and the amount of IgE in the blood of each individual after administration of the OVA5 times and 6 times is shown by a dot, as shown in FIG. 15. It was confirmed that the blood IgE level increased in all groups with sensitization with the OVA antigen, but the blood IgE level significantly decreased in KW3110 group after 5 OVA administrations compared to the control group. On the other hand, no significant decrease in the amount of IgE in blood was observed in KW4610 group compared to the control group, suggesting that the antiallergic activity of KW3110 strain was stronger. In addition, it was suggested that the 10mg intake group had a stronger IgE reduction effect than the 1mg intake group.

Example 6

(acid resistance and bile acid resistance of lactic acid bacteria)

Since lactic acid bacteria live and reach the intestinal tract to exert their probiotic effect, it is necessary to tolerate digestive juices such as gastric acid and bile acid. To verify whether or not KW3110 strain satisfies such conditions, acid resistance and bile acid resistance were measured in vitro.

1. Method of producing a composite material

The acid resistance was measured by culturing lactic acid bacteria until the logarithmic growth phase, adjusting the OD600 to 0.5 with PBS (pH6.5), adding 1/10 amounts of MRS medium adjusted to pH3.0 with hydrochloric acid to the cells, and culturing at 37 ℃. Samples were taken 1 hour, 2 hours, and 3 hours after the start of the culture, and the viable cell rate was measured using MRS agar medium.

2. Results of the experiment

The Bile acid resistance was measured by adding a Bile acid (Bile Salts, Oxoid corporation) at a final concentration of 2% to an MRS liquid medium, inoculating 1% of a preculture solution of KW3110 strain, and measuring OD630 with a BIOPLOTTER from Oriental Instruments.

A standard strain of Lactobacillus delbrueckii KW3317 (JCM1012 strain) was used as a control in all experiments. The results suggest that about 50% of KW3110 strain survived at 3 hours in the acid resistance test (fig. 16). In addition, in the bile acid resistance test (FIG. 17), KW3110 strain grew sufficiently even when cultured in a medium containing 2% bile acid, suggesting that it was resistant to bile acid. On the other hand, it was suggested that KW3317 strain had low resistance to both acids and bile acids.

Example 7

(intestinal adhesion of lactic acid bacteria)

Since lactic acid bacteria live to reach the intestine to exert their probiotic effect, it is necessary to stay in the intestine. As an index for the lactic acid bacteria to stay in the intestinal tract and to exert their effects sufficiently, adhesion tests in vitro on a human intestinal tract-derived cell line Caco-2 are often applied. The ability of KW3110 strain to adhere to Caco-2 cells was then determined.

1. Method of producing a composite material

The culture of Caco-2 cells was carried out according to the method of Coconnier et al (Applied and environmental microbiology, Vol.58, p.2034, 1992). Incubation on glass slides 1.2X 10⁴Per cm²Caco-2 cells ofAbout 1-2 weeks until after the full. KW3110 was cultured in MRS medium at 37 ℃ for 2 nights, and the strain was collected, washed 1 time with PBS, and then adjusted to OD600 of 1.0 with PBS. In 2ml of the prepared KW3110 solution, a slide glass for culturing Caco-2 cells was immersed, contacted at 37 ℃ in the presence of 10% carbon dioxide for 1 hour, and thereafter the slide glass was washed 3 times with DMEM (Dulbecco's Modified eagleMedium). Next, the slide glass was fixed with methanol, and KW3110 strain was stained by gram staining (Medical Technology, Vol.23, p.205, 1995). The number of lactic acid bacteria adhering to Caco-2 cells was counted under a microscope, and the average value per 4 fields was taken as the adhesion number. In addition, 15 strains of lactobacillus casei were similarly tested, and lactobacillus paracasei including KW3913 strain (JCM8130 strain) and KW3115 strain (JCM1134 strain) were used as standard strains.

2. Results of the experiment

The results of the test for the adhesion ability of each lactic acid bacterium to Caco-2 cells are shown in Table 3. Ouwehand et al reported that Lactobacillus paracasei and Lactobacillus casei have low adhesion to Caco-2 cells (Food microbiology and safety, vol.66, p.856, 2001), and this experiment demonstrated that strains other than the KW3110 strain have low adhesion. However, KW3110 strain has a remarkably high Caco-2 cell adhesion ability among lactobacillus paracasei and lactobacillus casei, suggesting that it is a strain that can be expected to have a good probiotic effect.

(Table 4)

Example 8

(the Effect of KW3110 Strain on ameliorating pollinosis in pollen antigen and airway inflammation)

1. Experimental methods

(species of animal)

BDF1 female mice were purchased and aged 4 weeks (Charles River).

(Experimental group)

Experiment A: antigen sensitization by nasal instillation (pollinosis model)

Control group: group to which standard feed (AIN-76base) was applied

KW group: group to which KW1 mg/mouse (1mg KW3110/3gAIN-76base) was administered

Experiment B: antigen sensitization with nebulizer (airway inflammation model)

Control group: group to which standard feed (AIN-76base) was applied

KW group: group to which KW1 mg/mouse (1mg KW3110/3gAIN-76base) was administered

(reagent/apparatus)

Cedar pollen extract CP (LSL company)

ALUM (Sigma Co.)

Ultrasonic sprayer XE-U12(OMRON Co., Ltd.)

And (3) cell centrifuge: cytospin4(ThermoBioAnalysis Co., Ltd.)

(evaluation method)

Experiment A

BDF-1 mice were purchased and acclimated for 1 week, and blood plasma obtained by blood sampling of eyeballs was classified into groups according to total IgE concentration in blood (n ═ 7). Administration of experimental diets was started after group. Intraperitoneal administration of CP + ALUM solution (CP 10 μ g + ALUM2 mg/mouse) was started from 3 weeks after grouping, 1/3 weeks. Two nosedrops of CP solution (1mg/ml) were administered to mice starting at 2 weeks after the 3 rd administration of CP + ALUM solution, 10. mu.l each time. In addition, an unstimulated model (Basal) mouse was also set, and saline was used when the nose was spotted. The nose was applied for 5 days, and the number of sneezes (days 1, 3, and 5) and nasal scratches (days 3 and 5) 5 minutes after the nose was applied were counted.

Experiment B

BDF-1 mice were purchased and acclimated for 1 week, and blood plasma obtained by blood sampling of eyeballs was classified into groups according to total IgE concentration in blood (n ═ 7). Administration of experimental diets was started after group. Intraperitoneal administration of CP + ALUM solution (CP 10 μ g + ALUM2 mg/mouse) was started from week 3 after the grouping, 1/3 weeks per week. Mice were sprayed with CP solution (40. mu.g/ml) in a closed container for 15 minutes using a nebulizer starting 2 weeks after the 3 rd administration of CP + ALUM solution. In addition, an unstimulated model (Basal) was set, and the mice were sprayed with physiological saline. Spraying was carried out for 5 days. Blood sampling of the eyeball was performed at the time of intraperitoneal administration of CP (day 0), the start of nebulizer (day 35), and the time of dissection (day 40), and the total IgE concentration in blood was measured. Bronchoalveolar lavage fluid (BALF) was collected under anesthesia on day 5, cells in BALF were counted, cells in BALF were identified, and cytokines in BALF were measured. BALF was obtained by opening the neck of a mouse under anesthesia, performing tracheal intubation, and washing with Saline0.7ml × 3 times (total 2.1ml) of saline added with 0.1% BSA. After centrifugation, the supernatant and cells were collected, and the number of cells was counted. Picture specimens were also prepared and cells were identified by leu giemsa staining.

2. Results of the experiment

(experiment A)

The number of sneezes 5 minutes after nasal application was divided into 0 to 1 point and 1 to 5 points after nasal application in consideration of the influence of physical stimulation, and the total number of sneezes was evaluated. As a result, the control group showed an increased frequency of sneezing by repeated nasal instillation of antigen, as opposed to almost no sneezing symptoms by nasal instillation of physiological saline. On the other hand, the tendency to suppress sneezing symptoms was observed in KW group (fig. 18a, b, c).

In addition, as for nasal scratching behavior, continuous scratching behavior 5 minutes after stippling was regarded as 1 point (total number of points). Further, the point evaluation was performed in a manner divided into mild scratching (scratching behavior continued for 1 to 4 times) and severe scratching (scratching behavior continued for 5 or more times). The KW group inhibited the scratching behavior compared to the control group, and significant inhibition was seen in the number of mild scratching points on day 5 (fig. 19a, b).

(experiment B)

The KW group significantly suppressed the increase in total IgE concentration in blood before nebulization and at the time of dissection compared to the control group (fig. 20). The cell type in BALF is usually a monocyte, dominated by macrophages. However, after antigen stimulation, the control group had around 65% eosinophils in BALF and the proportion of monocytes decreased. On the other hand, a tendency to suppress the increase in% eosinophils was observed in the KW group compared to the control group (fig. 21A). In addition, a tendency to inhibit eosinophil local infiltration was also observed in KW group compared to the control group (fig. 21 b).

In addition, the measurement of cytokines in BALF supernatant revealed that IL-5 was lower in KW group than in control group, and IL-5 was a Th2 type cytokine and involved in the proliferation and differentiation of eosinophils (FIG. 22).

These results suggest that by ingesting KW3110, sneezing and scratching movements due to cedar pollen antigens can be suppressed, and these symptoms are close to clinical symptoms of pollinosis in immediate allergy. In addition, KW3110 was found to inhibit IL-5 production against airway inflammation caused by antigen exposure by inhibiting IgE elevation, and also inhibit infiltration of eosinophils. The relationship between IL-5 and airway inflammation and eosinophils is described in J.allergy Clin.Immunol88(6)935-942, 1991, which suggests that administration of KW3110 is effective in allergic asthma with airway inflammation as a major symptom.

Example 9

(efficacy of KW3110 Strain on atopic dermatitis model mice)

The atopic dermatitis model coated with trinitrochlorobenzene was used to verify the improvement effect of the strain KW3110 on the atopic dermatitis model mouse. (citation 59(6)123-

1. Experimental methods

(species of animal)

Purchasing NC/Ng a TndCyj mice Male 5 weeks old (Charles River)

(Experimental group)

Control group: administering standard feed (AIN-76base) for 11 weeks

KW1mg-11wk group: 1mg KW3110/3g AIN-76base11 was administered for weeks

KW 10mg-11wk group: administration of 10mg KW3110/3g AIN-76base11 for weeks

KW1mg-8wk group: 3 weeks after standard feed administration, 1mg KW3110/3g AIN-76base8 weeks

(reagent)

Trinitrochlorobenzene: PCI (Tokyo chemical industry)

Solution for sensitization: 5% solution (ethanol: acetone ═ 4: 1)

Abdomen 100. mu.l, hind limbs 25. mu.l

Solution for challenge: 0.8% solution (olive oil)

External ear 10. mu.l each (40. mu.l total) and dorsal skin 50. mu.l each

(evaluation method)

NC/Ng a mice were purchased and acclimated for 1 week, and the mice were divided into groups according to the total IgE concentration in serum obtained by blood sampling from eyeballs (n is 10). The test food was divided and applied for 3 weeks, the PC1 sensitizing solution was applied to both sides of the unhaired abdomen and hind limbs, and the challenge was performed after 1 week, and the challenge solution was applied to both outer ears, both sides, and the unhaired back skin. Challenge was performed every 2 weeks for a total of 77 times. In addition, in order to confirm that the attack solution solvent did not cause thickening of the ear, the thickness of the ear was measured with a dial thickness gauge (dial thickness gauge) at 0, 1, 4, 24, 72, 96, 120, 144, and 168 hours after the olive oil application. After the 1 st and 3 rd attacks, the thickness of the ear mass was measured with a scale thickness meter in the same manner as 0, 1, 4, 24, 72, 96, 120, 144 and 168h, and the edema degree was evaluated. Blood was collected from the eyeball every week after sensitization, and clinical score evaluation was performed 2 times a week after sensitization. Finally, 1 week after challenge 7, slaughter, serum and external ear tissues were collected. The serum was measured for total IgE concentration, and HE staining and toluidine blue staining were performed on auricle tissues.

(clinical score)

The evaluation scores were obtained by scoring 5 items such as pruritus, redness, bleeding, erosion, edema, abrasion, tissue defect, formation of crust, and dryness of the external ear, and 8 items such as depilation, redness, bleeding, erosion, formation of crust, and dryness of the scalp, and adding the scores of no symptom (score 0), mild symptom (score 1), moderate symptom (score 2), and severe symptom (score 3).

(statistics)

Statistics scatter analysis and Dunnett's multiple comparisons against the control group were used, and the risk rates were significantly different below 5% in each analysis.

2. Results of the experiment

Total IgE concentration in blood: the control group showed a large increase from 4 weeks after PCI challenge, whereas all KW3110 intake groups suppressed this increase, with KW 10mg-11wk and KW1mg-8wk groups starting from the 4 th challenge and KW1mg-11wk groups significantly suppressing the increase from the 6 th challenge (fig. 23).

Clinical score: in the control group, the disease deterioration of the ear and scalp was observed from an early stage, and the increase of the score was confirmed. On the other hand, the increase was suppressed in all groups ingested with KW3110, in groups KW1mg-11wk and KW 10mg-11wk, starting from challenge 2, and in groups KW1mg-8wk, starting after challenge 3, the increase was significantly suppressed. Significant differences were also seen in all KW3110 intake groups for the external ear only clinical scores and the scalp only clinical scores (fig. 24, 25, 26). In addition, from the photographs of the head, it was confirmed that erosion of the outer ear, tissue defects, and the like were suppressed in the KW ingestion group (fig. 27).

Regarding auricular hypertrophy due to PCI attack: the solvent applied in the sensitization stage did not cause thickening of the ear, but thickening, redness, and edema of the ear due to PCI attack were observed. In the control group, stage 1 external ear thickening associated with an immediate response was observed 1 to 4 hours after the 1 st challenge, stage 2 external ear thickening associated with a delayed response occurred 48 hours, and stage 3 external ear thickening associated with an immediate response occurred further 120 to 144 hours.

On the other hand, the group 3 ingested with KW3110 was found to significantly inhibit the development of hypertrophy at 1 hour and 4 hours associated with the immediate response, the group KW 10mg-11wk also significantly inhibited the development of hypertrophy at 144 hours, and the group KW1mg-8wk significantly inhibited the development of hypertrophy at 24 hours and 144 hours. After the 3 rd challenge, the control group continued thickening after application, whereas the KW3110 intake group showed a tendency to inhibit thickening as a whole, significant inhibition was confirmed in KW1mg-11wk and KW 10mg-11wk (fig. 28 and 29).

As a result of pathological evaluation of the outer ear, inflammation such as thickening and edema of the dermis and epidermis was observed in the control group; in all KW intake groups, it was confirmed that the thickening was suppressed overall compared to the control group (fig. 30). Further, evaluation of pathological tissues by toluidine blue staining revealed that the number of KW taken in the mast cell group was smaller than that in the control group (fig. 31).

The above results confirmed that the increase of total IgE concentration in blood was suppressed by KW ingestion, the hypertrophy and hypertrophy of ear induced by hapten stimulation was suppressed, and the symptom was suppressed at the clinical level. Therefore, it was suggested that the symptoms of hapten-induced atopic dermatitis in NC/Ng a mice could be improved by KW3110 intake.

Example 10

(Effect of KW3110 Strain on pollinosis in human body)

To verify the effect of KW3110 strain in humans, pollen disease volunteers were subjected to the following tests.

1. Test method

(test sample)

Original yogurt was prepared using Lactobacillus delbrueckii B strain or Lactobacillus paracasei KW3110 strain evaluated for both parameters Th1 and Th2 in example 2. The number of bacteria contained is 2X 10⁸cfu/ml。

(test object)

28 volunteers of pollen disease patients who worked in the enterprise. The test was carried out in accordance with the declaration of helsinki, and all the test subjects were fully described in advance with respect to the contents of the test and the method, and informed consent was obtained.

(test group)

28 subjects were randomly divided into 2 groups. The yogurt of Lactobacillus delbrueckii B strain was ingested in group 1 as a control yogurt, and the yogurt of Lactobacillus casei KW3110 strain was ingested in group 1 as a group 1 at a daily dose of 200ml (equivalent to 40mg of dried cells of KW3110 strain). As for the grouping result, a double-blind trial was performed by the 3 rd person of the management information until the end of the trial.

(test time)

20 days to 4 days in month 1 to 14 days in 2003.

(measurement items)

The test subjects were collected at 0 week (at the start of the test), 4, 8, and 12 weeks (at the end of the test). The following items were measured on blood by Falco Biosystem: NK cell activity, Th1 and Th2 cell ratios (Th1/Th2 ratio), eosinophil number, ECP value, total IgE level in blood, and pollen Pini specific IgE level in blood.

After the test is finished, statistical processing is carried out on the data to convert the data into data.

In addition, the questionnaire shown in the test subject answer sheet during blood sampling was divided into 5 levels (3 +: height; 2 +: middle degree; +: mild; slight; none). 3+ is 4 minutes, 2+ is 3 minutes, + is 2 minutes, + is 1 minute, -is 0 minute, carry on statistical processing after scoring.

(Table 5)

(Table 6)

2. Results of the experiment

As shown in fig. 32, when the test was compared between the start and the end of the test, the ratio of Th1/Th2 was significantly decreased in the yogurt group (control group) of the B strain of lactobacillus delbrueckii, and the ECP value was significantly increased as shown in fig. 33. On the other hand, in the lactobacillus casei KW3110 strain yogurt group (KW group), no significant change was found before and after the test regardless of the value. As shown in fig. 34, as for the score of subjective symptoms, the KW group tended to be lower than the control group in terms of sore throat, eye pain, and heavy eyelid. The above results indicate that the strain KW3110 also shows effectiveness against human pollinosis.

Hereinafter, examples in which the lactic acid bacterium having an antiallergic activity of the present invention is used for beverage production will be described.

Example 11

(method for producing Lactobacillus cells as an additive raw Material for beverages)

Preparation example 1 preparation of microbial cells of Lactobacillus KW3110 Strain

The culture medium (glucose 1%, yeast extract S (Takeda-Kirin Co.) 1%, MgSO 2) was used₄·7H₂O 50ppm、MnSO₄·5H₂O50 ppm) was cultured. The strain KW3110 is inoculated into 10ml of culture medium for culture, and is kept still at 37 ℃ for 20-24 Hr for pre-culture. For the culture, 0.6ml of the preculture solution was inoculated into 120ml of the culture medium and cultured at 28 ℃ in a 200ml fermenter (model BMJ-25, Able Co.). The aeration rate was 0.12L/min and the stirring speed was 500 rpm. The pH was controlled with 25% NaOH solution and adjusted to pH 4.5 or less. The culture was carried out for 48 hours.

After the completion of the culture, the cells were centrifuged at 8500Xg for 10 minutes to collect the cells. Suspended in 30ml of sterilized distilled water, centrifuged at 8500Xg for 10 minutes, and the cells were collected. Washing was repeated 3 times to remove components derived from the medium. The washed cells were suspended in 10ml of sterilized water, treated in an autoclave at 100 ℃ for 30 minutes, and then lyophilized. And finally, obtaining 50-70 mg of lactobacillus dried thalli. The antiallergic activity of the obtained lactic acid bacteria was confirmed.

Example 12

(production of tea beverage)

Deionized water was added to the extracts of oolong tea, black tea, green tea, roasted tea, and jasmine tea extracted with hot water at 85 ℃ to make the usage rate of tea 0.8 wt%. At this time, vitamin C was added to 0.025 wt%, and then the pH was adjusted to be drinkable with sodium bicarbonate. Adding lactobacillus into the mixed solution, wherein the number of strains of lactobacillus KW3110 strain added per 100g of the mixed solution is 1.5 × 10¹⁰2, 3 x 10¹⁰Then, UHT-sterilized by a conventional method and filled into 350ml PET bottles.

Sensory evaluation of these 10 examples (5 teas x 2 lactic acid bacteria concentrations) was performed by a panel of specialists. As a result, the flavor of any of the beverages was satisfactory and was highly evaluated because it was continuously drunk daily. But due to the addition of 3X 10¹⁰Since the appearance of the sample is precipitated, it is more preferable to add 1.5X 10¹⁰The beverage of the individual.

Example 13

(preparation of malt tea)

250g of barley tea was boiled in 10kg of hot water for 30 minutes, filtered and centrifuged. Adding Lactobacillus KW3110 strain to the extractive solution, and adding 8 × 10 strain per 100g product¹⁰、1.5×10¹⁰、3×10¹⁰Then, vitamin C and sodium bicarbonate are added for seasoning, and deionized water is added again to make the weight reach 10 kg. Then, UHT sterilization was carried out in a conventional manner, and aseptically filled into 500ml PET bottles.

The sensory evaluation was carried out with satisfactory flavor, and the evaluation was high because the tea was drunk daily. But due to the addition of 3×10¹⁰Samples of individuals appeared to be visually precipitated, so beverages below this number were more preferred.

Example 14

(production of fruit juice beverage)

A prescribed amount of lactobacillus cells was added to 900g of turbid apple juice and 1g of flavor, and deionized water was added thereto to a total of 1kg, and then the mixture was hot-packed into a 180ml glass bottle by a conventional method to prepare an apple juice beverage. The number of bacteria of the strain KW3110 added to 100g of the mixed solution is 2 × 10¹⁰Per, or 4 x 10¹⁰And (4) respectively.

As a result of sensory evaluation, any of the samples had no abnormal flavor due to the incorporation of lactic acid bacteria, and had no problem in appearance.

Example 15

(production of sports drink)

0.0143 wt% (1.5X 10 per 100g product) of a blending solution containing 4.5 wt% of granulated sugar, 0.2 wt% of citric acid, 0.05 wt% of sodium citrate, and 0.1 wt% of flavor¹⁰Pieces) of lactobacillus KW3110 strain. Heating the solution to 90 deg.C for sterilization, and hot packaging in PET bottle to obtain sports beverage. As a result of sensory evaluation, both appearance and flavor were satisfactory. The lactic acid bacteria cells in the beverage were centrifuged, washed with pure water 2 times, and then freeze-dried to obtain dried cells. The finished preparations were evaluated in an in vitro system using mouse lymphocytes, and the defined activity was maintained (FIG. 35).

Example 16

(preparation of Potato juice)

A predetermined amount of lactic acid bacteria cells were added to potato juice, and the mixture was filled in a 190g jar and sterilized in a retort according to a conventional method. The strain KW3110 is added into the mixed solution at a ratio of 2.5 × 10 per 100g¹⁰Or 5X 10¹⁰And (4) respectively.

As a result of sensory evaluation, no abnormal feeling was observed in any of the samples by the incorporation of lactic acid bacteria, and no problem was observed in appearance.

Example 17

(production of coffee)

Extracting pulverized roasted coffee beans (Columbia) with 95 deg.C hot water to obtain coffee extract. Deionized water was added thereto to dilute the soluble solid component to 1.4%. The number of strains of Lactobacillus strain KW3110 added per 100g of the mixed solution was 2X 10¹⁰4 x 10 pieces¹⁰Then, the resulting mixture was filled into a 190g jar and the retort was sterilized by a conventional method.

As a result of sensory evaluation, no abnormal feeling was observed in any of the samples by the incorporation of lactic acid bacteria. However, when pouring into the cup, add 4 × 10¹⁰The appearance of the sample is that the lactic acid bacteria appear to precipitate, more preferably 1.5X 10¹⁰And (4) respectively. But there is no problem when drinking directly from the can.

Hereinafter, examples of the lactic acid bacteria having an antiallergic activity of the present invention used in food production will be described.

Example 18

(preparation of yogurt)

Mixing raw materials (milk, skimmed milk powder, butter, granulated sugar liquid, stabilizer, spice, and water) including milk, heating at 128 deg.C for 15 s for sterilization, cooling to below 40 deg.C, adding lactobacillus starter W3110, and fermenting at 37 deg.C in fermenter. Lactic acid bacterium KW3110 decomposed lactose in the mixture to give lactic acid, the pH was 4.6 for about 18 hours, and the lactic acid bacterium was stirred and cooled when it reached a stable point of gelation by isoelectric point aggregation of milk protein. Filling the stirred paste yogurt into a paper container, sealing, and refrigerating at below 10 deg.C. After cooling, the appropriate food taste and characteristics of the yogurt can be confirmed by sensory inspection. The blending ratio of the yogurt materials is shown in table 7. The proportions of fat components and SNF (fat-free solid content) in the raw materials and the sweetness are shown in table 8. The finished preparations were evaluated in an in vitro system using mouse lymphocytes, and the defined activity was maintained (FIG. 36).

(Table 7)

Raw materials	(wt%)
		Milk	50.00
Skimmed milk	4.63
		Cream	2.42
Sweet water	9.50
		Stabilizer	0.80
Perfume	0.08
		Lactic acid bacteria starter	5.00
Water (W)	27.57
		Total amount of	100.00

(Table 8)

Hereinafter, examples of the use of the lactic acid bacterium having antiallergic activity of the present invention in the production of a health food preparation will be described.

Example 19

(production of sheet-like health food)

Reducing palatinose, sorbitol, xylitol and Arabic gum are added into dried powder of 67g lactobacillus KW3110 strain, water is used as binder for heating granulation, citric acid, stevia, perfume, etc. are used for flavoring, powdered oil and fat and sucrose ester are added for easy molding to obtain 1000g of mixture, and the mixture is made into tablet by a tablet machine. Then, the mixture was sliced into 1.5g pieces, and the slices were prepared into a health food in the form of a sheet having superior flavor. In this case, the number of cells of the strain KW3110 was 5X 10 per granule¹⁰And (4) respectively.

Example 20

(hard capsule-like health food)

Adding dextrin 50g into prepared 100g lactobacillus KW3110 strain dry powder, mixing well, and filling into hard capsule raw material composed of pullulan, vegetable oil, carrageenan and potassium chloride to obtain hard capsule coated 1 capsule 213mg health food. In this case, the number of cells of the strain KW3110 was 5X 10 per 1 capsule¹⁰And (4) respectively.

Example 21

(hard capsule-like health food)

Adding dextrin 50g into 100g dried powder of Lactobacillus KW3110 strain, mixing, and filling into gelatin and glycerinIn the prepared hard capsule raw material, 213mg of health food of 1 capsule wrapped by the hard capsule is obtained. In this case, the number of cells of the strain KW3110 was 5X 10 per 1 capsule¹⁰And (4) respectively.

Example 22

(Soft capsule health food)

Mixing 100kg of dried powder of Lactobacillus KW3110 with 180kg of safflower oil, 20kg of beeswax and 5kg of soybean lecithin, suspending, coating with capsule raw materials containing carrageenan, starch and glycerol as main ingredients, and making into 1 capsule 450mg of elliptical soft capsule. In this case, the number of cells of the strain KW3110 was 5X 10 per 1 capsule¹⁰And (4) respectively.

Example 23

(Soft capsule health food)

Mixing 100kg of dried powder of Lactobacillus KW3110 with 180kg of safflower oil, 20kg of beeswax and 5kg of soybean lecithin, suspending, coating with capsule material containing gelatin and glycerol as main ingredients, and making into elliptical soft capsule with 1 capsule 450 mg. In this case, the number of cells of the strain KW3110 was 5X 10 per 1 capsule¹⁰And (4) respectively.

Example 24

(granular health food)

100kg of the prepared dried powder of lactic acid bacteria KW3110 strain was filled in a stick-shaped filling machine, and 1 stick was filled with 1g of the powder to obtain 5X 10 lactic acid bacteria per 1 stick¹¹A health food containing Lactobacillus KW3110 strain.

Example 25

(granular health food)

900kg of dextrin is added into 100kg of prepared dried powder of KW3110 lactic acid bacteria, water is used as a bonding agent, and a fluidized bed granulator is used for uniform mixing, heating and granulation to obtain 1000kg of granules. Thereafter, the granules were filled with a rod-shaped filler, 1 rod 1g, to obtain 5X 10 per 1 rod¹⁰A health food containing Lactobacillus KW3110 strain. Application of the inventionThe finished preparations were evaluated in vitro using mouse lymphocytes and retained the defined activity (FIG. 37).

Example 26

(powdery health food)

900kg of dextrin is added into 100kg of prepared dried powder of KW3110 lactic acid bacteria, water is used as a bonding agent, and a fluidized bed granulator is used for uniform mixing, heating and granulation to obtain 1000kg of granules. Thereafter, the granules were pulverized until the 18 mesh was completely passed, and packed by a rod packing machine to obtain 1 rod of 1g, 5X 10 per 1 rod¹⁰A powdery health food containing Lactobacillus KW3110 strain.

Example 27

(health food in suspension)

To 100g of the prepared dried powder of lactic acid bacteria KW3110 strain was added 10kg of squeezed juice of cabbage leaf, and the viscosity was adjusted with 10g of sodium alginate to facilitate dispersion of lactic acid bacteria in the liquid, thereby obtaining a suspension-like healthy food.

Example 28

(health food in suspension)

100g of the prepared dried powder of lactic acid bacteria KW3110 strain was mixed with 0.7kg of glucose, 5kg of distilled water and 5g of grape flavor, and sterilized by heating, and then 1 sealed container of 50ml was aseptically filled to obtain a healthy food in the form of a syrup suspension. The food is not unpleasant to children, so it is well appreciated.

Brief Description of Drawings

FIG. 1 is a list (No.1) of lactic acid bacteria used in examples of the present invention and their places of purchase.

FIG. 2 is a list (No.2) of lactic acid bacteria used in examples of the present invention and their places of purchase.

FIG. 3 shows the amounts of Th1 type cytokine IL-12 produced by various lactic acid bacteria in the examples of the present invention.

FIG. 4 shows the amounts of Th2 type cytokine IL-4 produced by various lactic acid bacteria in the examples of the present invention.

FIG. 5 shows the results of in vitro comparison of IL-12 and IL-4 production using the lactic acid bacterium strain of the present invention (KW3110) and a control strain (L-92 strain) in examples of the present invention.

FIG. 6 shows the results of measurement of antiallergic activity of No.90 strain derived from KW3110 strain selected in examples of the present invention.

FIG. 7 is a flowchart of an experiment for in vivo measurement of antiallergic ability of KW3110 strain, which is a lactic acid bacterium strain having high antiallergic activity of the present invention, in an example of the present invention.

FIG. 8 is a kinetic profile of IgE in blood between 98 days in an experiment for in vivo determination of antiallergic ability of KW3110 strain, which is a lactic acid bacterium strain of the present invention having high antiallergic activity, in an example of the present invention.

FIG. 9 shows the amount of IgE in blood on day 48 and day 98 of dissection in an experiment for measuring the antiallergic ability of KW3110 strain in vivo in the example of the present invention, wherein KW3110 strain is a lactic acid bacterium having a high antiallergic activity of the present invention.

FIG. 10 shows the IL-12 production in spleen cell culture supernatant at day 98 of dissection in experiments for in vivo determination of antiallergic ability of KW3110 strain, which is a lactic acid bacterium strain of the present invention having high antiallergic activity, in control group and KW3110 group in examples of the present invention.

FIG. 11 shows the IL-4 production in spleen cell culture supernatant at day 98 of dissection in experiments for in vivo determination of antiallergic activity of KW3110 strain, which is a lactic acid bacterium strain of the present invention having high antiallergic activity, in control group and KW3110 group in examples of the present invention.

FIG. 12 is a photograph showing the results of perinasal depilation in the control group and KW3110 group in the experiment for in vivo determination of antiallergic ability of KW3110 strain in the example of the present invention, KW3110 strain being a lactic acid bacterium strain of the present invention having high antiallergic activity.

FIG. 13 is a flow chart of experiments in which a preventive effect test was carried out using a mixed feed of lactic acid bacteria strain KW3110 having a high antiallergic activity of the present invention and KW4610 known as an antiallergic lactic acid bacterium in the examples of the present invention.

FIG. 14 shows the change in the amount of IgE in blood during the preventive effect test using the mixed feed of the lactic acid bacteria strain KW3110 having a high antiallergic activity of the present invention and KW4610 known as an antiallergic lactic acid bacterium in the example of the present invention.

FIG. 15 shows the amount of IgE in blood of each individual after applying OVA5 times and 6 times in the course of a preventive effect test using a mixed feed of the lactic acid bacteria strain KW3110 having a high antiallergic activity of the present invention and KW4610 known as an antiallergic lactic acid bacterium in comparison with each other in examples of the present invention.

FIG. 16 shows the results of acid resistance test of the lactic acid bacterium strain KW3110 having high antiallergic activity of the present invention in examples of the present invention.

FIG. 17 shows the results of bile acid resistance test of the lactic acid bacterium strain KW3110 having high antiallergic activity of the present invention in examples of the present invention.

FIG. 18 shows the improvement effect of KW3110 strain on pollinosis and airway inflammation induced by pollen antigen in example of the present invention, and FIGS. 16a, b and c (FIGS. 18a, b and c) show the results of the test on sneezing behavior of model mice.

FIG. 19 shows the effect of KW3110 strain on the improvement of pollinosis and airway inflammation induced by application of pollen antigen in example of the present invention, and FIGS. 17a and b (FIGS. 19a and b) show the results of the test on scratching behavior of model mice.

FIG. 20 shows the results of experiments conducted to show the effect of KW3110 strain on the amelioration of pollinosis and airway inflammation induced by application of pollen antigen, and the inhibition of increase in total IgE concentration in blood of model mice in the examples of the present invention.

FIG. 21 shows the effect of KW3110 strain on amelioration of pollinosis and airway inflammation induced by application of pollen antigen in examples of the present invention, and is a result of experiments on inhibition of eosinophil elevation (FIG. 19a) and inhibition of local infiltration of eosinophils (FIG. 19b) in model mice.

FIG. 22 shows the effect of KW3110 strain on amelioration of pollinosis airway inflammation induced by application of pollen antigen in example of the present invention, and the results of the test on IL-5 value in model mice.

FIG. 23 shows the effect of KW3110 strain on the improvement of atopic dermatitis model mice in the example of the present invention, and the result of the experiment on the increase of total IgE concentration in blood.

FIG. 24 shows the improvement effect of KW3110 strain on atopic dermatitis model mice in the example of the present invention, and is the result of measuring the total clinical score of model mice.

FIG. 25 shows the effect of KW3110 strain on the improvement of atopic dermatitis model mice in examples of the present invention, and is a result of measuring clinical scores (auricles) of the model mice.

FIG. 26 shows the effect of KW3110 strain on the improvement of atopic dermatitis model mice in examples of the present invention, and is a result of measuring clinical scores (scalp) of model mice.

FIG. 27 is a photograph showing the effect of KW3110 strain on the improvement of atopic dermatitis model mice in example of the present invention, which shows the inhibition of erosion of the auricle and head and the tissue defect-like sinking of the model mice.

FIG. 28 shows the effect of KW3110 strain on improvement in atopic dermatitis model mice in examples of the present invention, and is a result of testing the tendency of inhibition of ear hypertrophy (FIG. 26b) after model mice were challenged 1 time (FIG. 26a) and model mice.

FIG. 29 shows the effect of KW3110 strain on improvement in atopic dermatitis model mice in examples of the present invention, and is a result of testing the tendency of inhibition of ear hypertrophy after model mice were challenged 3 times.

FIG. 30 shows the improvement effect of KW3110 strain on atopic dermatitis model mice in example of the present invention, and is the result of pathological evaluation of auricle with HE staining for inhibition of hypertrophy of auricle in model mice.

FIG. 31 shows the improvement effect of KW3110 strain on atopic dermatitis model mice in example of the present invention, and is the result of evaluation of auricle pathology by toluidine blue staining for inhibition of hypertrophy of ear in model mice.

FIG. 32 shows the effect of KW3110 strain on pollinosis in humans in the examples of the present invention, as a result of measuring the change of the ratio Th1/Th2 in a test using yogurt.

FIG. 33 shows the effect of KW3110 strain on pollinosis in human in the present example, and the change in ECP between before and after the start of the test was measured in the test using yogurt.

FIG. 34 shows the effect of KW3110 strain on pollinosis in human in the example of the present invention, and the change in subjective symptoms of pollinosis before and after the start of the test was observed in the test using yogurt.

FIG. 35 shows the results of a test for maintaining antiallergic activity when KW3110 strain was used in examples of the present invention and processed into products, and the results of a test for IL-12 production when water was used as a refreshing beverage.

FIG. 36 shows the results of a test for maintaining antiallergic activity when KW3110 strain was used in the examples of the present invention and was produced into a product by various processing treatments, and the results of a test for IL-12 production when incorporated into yogurt.

FIG. 37 shows the results of a test for the maintenance of antiallergic activity when KW3110 strain was used in examples of the present invention and a test for the yields of IL-12 and IL-4 when it was tableted.

Possibility of industrial utilization

According to the present invention, a lactic acid bacterium having a high antiallergic activity can be obtained, and a composition having the lactic acid bacterium as an active ingredient and exhibiting a high antiallergic effect can be obtained. The antiallergic composition of the present invention is particularly effective against environmental allergies such as pollinosis, atopic dermatitis, bronchial asthma, allergic rhinitis, allergic conjunctivitis, and the like. In particular, as shown in the data of pollinosis and experimental data of asthma treatment using pollen as antigen, it has remarkable effect in preventing and treating mucosal allergic diseases such as pollinosis, bronchial asthma, allergic rhinitis and allergic conjunctivitis. Since lactobacillus paracasei KW3110, which is one of the active ingredients of the antiallergic composition of the present invention, has not only strong antiallergic and immunostimulating activities but also excellent intestinal cell adhesion, gastric acid resistance, and bile acid resistance, oral administration of the lactic acid bacteria or products using the lactic acid bacteria is expected to be utilized as a medicament or a food or drink that can simultaneously exhibit conventionally known probiotic functions such as an antiallergic function, an intestinal function-regulating function, a cholesterol-lowering function, and a blood pressure-lowering function. The composition having a high antiallergic function of the present invention can be continuously ingested daily, particularly when applied as a drink, and can exert its effect in an amount of ingestion that can be continuously ingested, so that a drink having a good taste and a high keeping quality can be provided while maintaining the antiallergic function.

Admission official entrance board

International recognition of the Budapest treaty on the deposit of microorganisms for patent procedures

International depository issuing according to treaty 7.1

Proof of original deposit

Depositor's name (name)

Kirin Wheat Wine Co., Ltd.

Vital xue Kangyilang man

Address104-8288

Tokyo Central area Xinchuan No.2 Ding 10 Bi 1

International recognition of the Budapest treaty on the deposit of microorganisms for patent procedures

International depository issuing according to treaty 7.1

Proof of original deposit

Depositor's name (name)

Kirin Wheat Wine Co., Ltd.

Vital xue Kangyilang man

Address104-8288

Tokyo Central area Xinchuan No.2 Ding 10 Bi 1

Claims (7)

1. A method for producing a food or drink having a high antiallergic activity and having a flavor and taste inherent thereto by adding a lactic acid bacterium, wherein Lactobacillus paracasei (Lactobacillus paracasei) KW3110 strain having a accession number FERM BP-08634 or a modified strain NO.90 strain KW3110 strain having a accession number FERM BP-08635 is added in the production process, wherein said lactic acid bacterium KW3110 strain or said modified strain NO.90 strain thereof is subjected to a sterilization treatment under a heating temperature and time of at least 100 ℃ for 30 minutes.

2. The method for producing a food or drink having a high antiallergic activity and having an inherent flavor and taste according to claim 1, wherein the heat sterilization of the food or drink is performed by pasteurization, retort sterilization, UHT sterilization, or retort sterilization.

3. The method for producing a food or drink having a high antiallergic activity and having an inherent flavor and taste as claimed in claim 1, wherein the food or drink is sealed in a container.

4. The method for producing a food or drink having a high antiallergic activity and having an inherent flavor and taste as claimed in claim 1, wherein the food or drink is prepared as a healthy food or drink in a closed container.

5. The method for producing a food or drink having a high antiallergic activity and having an inherent flavor and taste according to claim 1, wherein the food or drink is prepared as a fruit juice-containing drink or a tea drink packed in a closed container.

6. The method for producing a food or drink having a high antiallergic activity and having an inherent flavor and taste as claimed in claim 1, wherein the drink is a milk component-free drink filled in a sealed container.

7. The method for producing a food or drink having a high antiallergic activity and having an inherent flavor and taste according to claim 1, wherein the amount of lactic acid bacteria added to the food or drink in a sealed container is 10 per 100g of the food or drink⁹～10¹¹Range of individual lactic acid bacteria.