JP5206134B2 - Immunomodulator and antiallergic agent containing the same - Google Patents

Description

  The present invention relates to an immunomodulator and an antiallergic agent containing the same.

  In recent years, various allergies such as hay fever, food allergies, and atopic dermatitis have become major problems in Japan. Among them, hay fever has increased to the point that it is said to be a national illness, and social and economic losses are regarded as a problem. Food allergies and the like are also increasing, and there are concerns about the effects of additives such as synthetic colorants, color formers, bleaches, synthetic fragrances, and preservatives. Therefore, in order to prevent and treat such allergies, attempts have been made to develop pharmaceuticals that are effective against allergies in parallel with measures such as environmental improvements and food ingredient labeling.

However, since pharmaceuticals are mainly organic compounds that are artificially designed and synthesized (see Non-Patent Document 1), for example, there are concerns about side effects, and some individuals cannot actually use them from side effects. In some cases. For this reason, there is a demand for provision of a substance with excellent safety that is effective in preventing and treating allergies.
"A case of anaphylactic shock caused by an antiallergic agent", Haruhiko Iwai, Monthly Clinical and Research, Vol. 75, No. 3, 115-116pp

  Then, this invention aims at provision of the new substance which can be used for the prevention and treatment of allergy, for example, has a food experience, and is excellent in safety.

  In order to achieve the above object, the immunomodulator of the present invention is characterized in that it contains a rice brew.

  The Th1 / Th2 balance regulator of the present invention and the antiallergic agent of the present invention are characterized by including the immunomodulator of the present invention.

  As a result of intensive studies, the present inventors have found that immunoregulation is possible with a brewed product made from rice, and have reached the present invention. According to the immunomodulator of the present invention containing such a rice raw brew, for example, it is possible to suppress the disruption of the Th1 / Th2 balance, which is a cause of allergic diseases, or to adjust to normalize. is there. Therefore, the immunomodulator of the present invention can be used as an antiallergic agent for various allergic diseases as described above. In particular, brewed rice ingredients are extremely useful because, for example, their safety is well supported by their long-term dietary history, for example, it is highly possible to avoid problems such as side effects of synthetic pharmaceuticals. .

<Immunomodulator>
As described above, the immunomodulator of the present invention is characterized by containing a rice raw brew. The immunomodulator of the present invention can adjust the Th1 / Th2 balance, for example, and can be used as a Th1 / Th2 balance regulator as described later. Moreover, since the immunomodulator of this invention shows an antiallergic effect by immunomodulation, it can be used as an antiallergic agent of this invention so that it may mention later.

  Moreover, since the immunomodulator of this invention has a function which suppresses cytokine production, for example, it can be used as a cytokine production inhibitor, for example. Examples of the cytokine include IL-4, IL-6, IL-10, and TNF-α. Moreover, since the immunomodulator of this invention has a function which suppresses IgE production, for example, it can be used also as an IgE production inhibitor, for example. In addition, it can be said that the rice raw material brewing product in this invention is very excellent in safety since it has a long history of food and its safety has been sufficiently proven.

  In this invention, the kind in particular of the said rice raw material brew is not restrict | limited, What is necessary is just a brew using rice as a raw material. Specific examples include sake, miso, miso, soy sauce, etc., and by-products such as koji and koji that are produced in the production process thereof. Of these, sake sake, which is a by-product of sake, is preferred, and examples thereof include general sake lees and liquefied liquors. The liquefied liquor is, for example, sake lees produced in sake production produced by alcohol fermentation of raw rice liquefied with a liquefaction enzyme (Satoshi Imayasu et al .: Journal of Japanese Agricultural Chemical Society, No. 63, page 971, 1989, JP 59-66875). The kind of rice that is the raw material of the brew is not limited. In this invention, the kind of rice raw material brew is not restrict | limited at all, One type may be sufficient and two or more types may be sufficient. In addition, the rice raw material brewed product in the present invention is, for example, the above-mentioned main products such as liquor, and by-products such as sake lees, and further processed main products and by-products, for example, as described below, Also included are products obtained by subjecting main products and by-products to further fermentation.

  The immunomodulator in the present invention preferably contains, for example, γ-aminobutyric acid (hereinafter also referred to as “GABA”). Thereby, the Th1 / Th2 balance can be adjusted with higher efficiency. The γ-aminobutyric acid is generally known to have four isomers. In the present invention, any of these isomers may be used, and a derivative of γ-aminobutyric acid may be used as long as the same effect is obtained. There may be. The γ-aminobutyric acid contained in the immunomodulator of the present invention may be, for example, a chemically synthesized substance, a substance produced by genetic engineering, or a natural product. In the present invention, for example, as described later, it is particularly preferable to biochemically contain γ-aminobutyric acid in the rice raw brewed product by further subjecting the rice raw brewed product to fermentation. . In this case, for example, γ-aminobutyric acid produced by fermentation treatment may be added to the rice raw brewed product in the present invention, but the cost and labor can be reduced, and γ-aminobutyric acid can be efficiently contained. It is preferable to produce γ-aminobutyric acid in the brewed product by subjecting the rice brewed product to the fermentation treatment.

  The content of γ-aminobutyric acid in the immunomodulator of the present invention is not particularly limited, but is preferably, for example, 1 mg or more, more preferably 5 mg or more, particularly preferably 10 mg or more, per 1 g of rice raw brew. . The upper limit of the content of γ-aminobutyric acid is not particularly limited.

In the present invention, the rice raw brewed product is preferably a brewed product that has been further subjected to a fermentation treatment, for the reason described above. Here, the further fermentation treatment means, for example, a fermentation treatment further applied to the sake or sake lees obtained in the production process of sake or the like after the production process. The fermentation treatment is not particularly limited, and examples thereof include fermentation treatment using lactic acid bacteria and yeast, and among them, fermentation treatment using lactic acid bacteria is preferable. The lactic acid bacteria are not particularly limited, but for example, those having the ability to produce γ-aminobutyric acid are preferable, and examples include bacterial bodies such as Lactobacillus genus, Leuconostoc genus, Streptococcus genus, Pediococcus genus, and Bifidobacterium genus. Among them, preferably the genus Lactobacillus bacteria, specific examples, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus delbrueckii, Lactobacillus leichmannii, Lactobacillus plantarum, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus casei, and, Lactobacillus fermentum, and the like. Among these, a cell belonging to Lactobacillus brevis is particularly preferable. The lactic acid bacteria and yeast used in the fermentation treatment may be one kind or two or more kinds, respectively, or only lactic acid or yeast may be used, or both lactic acid and yeast may be used. Also good.

  The conditions for the fermentation treatment of the rice raw brewed material are not particularly limited as long as lactic acid bacteria, yeast, and the like can grow. For example, the fermentation temperature is preferably 15 to 45 ° C, more preferably 30 to 40 ° C, and the fermentation time is preferably 16 to 72 hours, and more preferably 20 to 50 hours. Moreover, although the form of the said rice raw material brewed product at the time of a fermentation process may be either solid and a liquid, since a fermentation process can be performed efficiently, a liquid state is preferable. When the rice raw brew is solid like the above-mentioned sake lees, for example, it is preferable to perform the fermentation treatment after suspending the rice raw brew in a solvent. The ratio of the solvent with respect to the volume of the rice brewed product is not particularly limited. For example, it is preferable to add 0.5 to 10 times the amount of the solvent with respect to the volume of the rice brewed product, more preferably 1 ~ 5 times. The type of the solvent is not particularly limited, and for example, water, a buffer solution, an acid aqueous solution, an alkaline aqueous solution, and the like are preferable. Moreover, it is preferable that pH of the suspension in which the said rice raw material brew was suspended is pH 3-9, for example, More preferably, it is pH 5-7. Moreover, also about a liquid rice raw material brewing, you may perform a fermentation process, for example, after mixing with a solvent as needed.

For the fermentation treatment, it is preferable to use, for example, lactic acid bacteria or yeast that have been pre-cultured in advance. When the subject of the fermentation treatment is, for example, a liquid rice brew or a suspension, the amount of lactic acid bacteria or yeast added is not particularly limited, but is preferably, for example, 10 ⁵ to 10 ⁹ cells / ml. More preferably, it is 10 ⁶ to 10 ⁸ cells / ml.

  In the fermentation treatment, for example, in order to promote the growth of lactic acid bacteria and yeast, it is preferable to add sugars such as glucose to the rice raw material brewed product. In the case where the rice raw brew is a solid such as the above-mentioned sake lees, for example, after the rice raw brew is suspended in a solvent, the saccharide may be added to the suspension. It is preferable that the saccharide is added to the rice raw brewed product, pulverized and mixed using a mixer or a grinder, and then suspended in a liquid. The amount of the saccharide added to the rice raw brewed product (for example, sake lees) is not particularly limited, but is, for example, 0 to 10% by weight, preferably 0 to 100% by weight of the rice raw brewed product. 0.5 to 5% by weight.

  The rice raw brewed product may further include rice bran such as rice bran, or glutamic acid or a salt thereof. Examples of the rice bran include red rice bran, medium rice bran, and white rice bran. Among them, rice red rice bran is preferable. In this invention, it is especially preferable that it is the said rice raw material brewed product in which the fermentation process as mentioned above was performed in the state containing these. By performing the fermentation treatment in the presence of these, for example, γ-aminobutyric acid is produced with better efficiency in the brewed product, and therefore γ-aminobutyric acid is produced with higher efficiency in the brewed product. It can be included. In addition, when the fermentation treatment is performed in the presence of these, for example, added rice bran, glutamic acid, or the like may or may not remain in the rice raw brewed product after the fermentation treatment. Good.

  Although the addition ratio of koji with respect to the said rice raw material brew is not restrict | limited, For example, it is 0-50 weight% with respect to 100 weight% of rice raw material brewing, Preferably, it is 2-20 weight%. Moreover, the addition amount of glutamic acid or its salt with respect to the said rice raw material brew is not specifically limited, For example, it is 0-30 weight%, Preferably, it is 1-15 weight%.

  The form of the rice raw brew used for the production of the immunomodulator of the present invention is not particularly limited, and may be, for example, liquid or solid. That is, when the rice brew is solid, it may be used as it is, or may be used as a powder by pulverization or lyophilization, or may be used by dissolving or suspending in a solvent. Good. Moreover, when the said rice raw material brew is a liquid, it may be used as it is, for example, may be used by concentrating, and may be used by making into powder by freeze-drying etc.

  The immunomodulator of the present invention only needs to contain the above-described rice brewed rice, but may further contain, for example, a pharmaceutically acceptable additive. Examples of the additive include conventional excipients, binders, disintegrants, lubricants, moisturizers, flavoring agents, pH adjusters, preservatives, isotonic agents, etc., depending on the dosage form described below. Known additives can be selected. The excipient is not particularly limited, and specific examples include, for example, sugars such as lactose, sucrose, and glucose; starches such as potato starch, wheat starch and corn starch; celluloses such as crystalline cellulose; Examples thereof include inorganic salts such as calcium oxyhydrogen and calcium carbonate. The binder is not particularly limited, and specific examples include crystalline cellulose, pullulan, gum arabic, sodium alginate, polyvinyl pyrrolidone, macrogol and the like. The disintegrant is not particularly limited, and examples thereof include carboxymethyl cellulose, carboxymethyl cellulose calcium, hydroxypropyl cellulose, hydroxypropyl starch, starch, and sodium alginate. The lubricant is not particularly limited, and examples thereof include magnesium stearate, talc, and hardened oil. The humectant is not particularly limited, and examples thereof include coconut oil, olive oil, sesame oil, peanut oil, soybean phospholipid, glycerin, sorbitol and the like. The flavoring agent is not particularly limited, and examples thereof include sweeteners, acidulants, and fragrances. Specific examples include sucrose, glucose, fructose, xylitol, sorbitol, mannitol, cinnamon oil, mint oil, Menthol and the like.

  The content of the rice raw brew in the immunomodulator of the present invention is not particularly limited, and can be appropriately determined according to, for example, the type and form of the rice raw brew. As a specific example, when the rice raw material brewed product is sake lees, for example, the daily intake is preferably set to be 100 mg to 100 g, and more preferably 1 g to 30 g.

  The form of the immunomodulator of the present invention is not particularly limited, and may be, for example, a solution, a solid, a powder, or a state in which the rice raw brew is dispersed in a solvent. Specific dosage forms include, for example, tablets, pills, fine granules (including powders), granules, capsules, liquids (including syrups) and the like, injections, depending on the administration method And parenteral agents such as drops, external preparations, and suppositories. According to each of these dosage forms, for example, it can be produced according to a usual method described in the Japanese Pharmacopoeia using suitable additives and base materials.

  The method of using the immunomodulator of the present invention is not limited at all. For example, a sample in which the immune system is unbalanced due to an allergic disease, a sample that may be unbalanced, a sample in which the Th1 / Th2 balance is unbalanced, or a sample that may be broken It is preferable to administer to sex specimens. As a result, for example, it is possible to prevent the occurrence of allergic symptoms in a specimen, to alleviate allergic symptoms that have occurred, and to improve or treat allergic constitutions.

  The method for administering the immunomodulating agent of the present invention to the specimen is not limited at all, and for example, it may be administered orally or parenterally as described above.

  Moreover, the immunomodulator of this invention can also be used as a foodstuff, a food additive, a nutritional supplement (supplement) etc., for example. In addition, when using an immunomodulator, for example as a food additive or a nutritional supplement, the form is not restrict | limited at all and can be used with the form similar to the above-mentioned. When used as the food additive, food to be added is not limited at all, for example, rice cake, gum, cake, pie, cookies, crackers, jelly, chocolate, pudding, ice cream, potato chips, sheep crab, rice cracker, Sweets such as buns, Chinese buns; beverages such as alcoholic beverages, teas, coffees, sports drinks, soft drinks, soups, dairy beverages; dairy products such as yogurt, butter, cheese; ham, sausage, rice cakes, Processed products such as bamboo rings; seasonings such as sauces, dressings, mayonnaise, soy sauce, miso, vinegar, miso, tomato processed products (ketchup, tomato paste, tomato puree), curry roux, sake lees, granulated stock; sprinkles, pickles , Cooked dishes such as boiled fish, salted kelp; prepared foods; staple foods such as noodles, cooked rice and rice cakes. Moreover, the said immunomodulator may be added to these foodstuffs beforehand as the said food additive, and may be added at the time of eating and drinking.

<Th1 / Th2 balance regulator>
The Th1 / Th2 balance regulator of the present invention includes the immunomodulator of the present invention as described above. The Th1 / Th2 balance regulator of the present invention only needs to contain the aforementioned rice raw material brewed product as an immunomodulator as described above, and other configurations and forms are not limited at all. Specifically, the Th1 / Th2 balance regulator of the present invention has the same configuration and form as the immunomodulator of the present invention, and can be used in the same manner.

  Th1 and Th2 belong to helper T cells (Th cells), respectively, and are functionally classified into Th1 type and Th2 type. Th1 (Th1-type helper T cells) mainly releases cytokines such as interleukin-2 (IL-2) and interferon (IFN) -γ to activate killer T cells and produce IgG2 and IgG3. It plays a role in promoting and activating cellular immunity. On the other hand, Th2 (Th2-type helper T cells) mainly releases cytokines such as IL-4 and IL-5 to promote mast cell activation, eosinophil migration, and IgE, IgG1 and IgA production. It plays a role in activating humoral immunity. The cytokine released by Th1 controls the activation of Th2, and the cytokine released by Th2 controls the activation of Th1. Thus, in the living body, these two helper T cells (Th1 and Th2) maintain a balance with each other to control the immune response. Such a balance between Th1 and Th2 is called “Th1 / Th2 balance”. However, it is known that when this Th1 / Th2 balance is tilted, it tends to fall into an allergic constitution. For example, when Th2 becomes dominant, the cytokine released by Th2 suppresses the growth of Th1, and the production of antibodies such as IgE by Th2 is excessively caused to cause an allergic reaction. According to the Th1 / Th2 balance regulator of the present invention, this balance inclination can be prevented or the balance can be adjusted to a normal balance. As a specific example, for example, a balance shifted to the Th2 side in an allergic patient can be adjusted to the Th1 side. Therefore, according to the Th1 / Th2 balance regulator of the present invention, for example, it can be used as an antiallergic agent effective for the prevention or treatment of allergy.

  The Th1 / Th2 balance regulator in the present invention, that is, the above-mentioned rice brew, for example, suppresses the production of various inflammatory cytokines that are increased by stimulation, and antibodies (for example, IgE) that increase by shifting to the Th2 side. Production suppression has been confirmed. From this, it became clear from this inventor that the Th1 / Th2 balance regulator of this invention can adjust the balance which Th1 / Th2 inclined.

<Antiallergic agent>
As described above, the antiallergic agent of the present invention is characterized by including the immunomodulator of the present invention. As described above, the antiallergic agent of the present invention only needs to contain the aforementioned rice raw material brewed product as an immunomodulator, and other configurations and forms are not limited at all. Specifically, the antiallergic agent of the present invention has the same configuration and form as the immunomodulator of the present invention, and can be used in the same manner.

  Allergies are generally classified into types I to IV, but the type of allergy that the antiallergic agent of the present invention can deal with is not limited. The type of disease caused by allergy is not limited at all, for example, hay fever, allergic bronchial asthma, food allergy, atopic dermatitis, urticaria, food allergy, blistering, drug-induced hemolytic anemia, thrombocytopenia Examples include purpura, toxic epidermal necrosis, serum sickness, systemic lupus erythematosus, rheumatoid arthritis, and allergic contact dermatitis.

<Immunostimulator>
The immunostimulant of the present invention is characterized by including the immunomodulator of the present invention. Thus, the immunostimulant of this invention should just contain the above-mentioned rice raw material brew as an immunomodulator, and another structure, a form, etc. are not restrict | limited at all. Specifically, the immunostimulant of the present invention has the same configuration and form as the immunomodulator of the present invention, and can be used in the same manner.

  Moreover, since the immunostimulant of this invention has a function which accelerates | stimulates production of IgA antibody, for example, it can also be called IgA production promoter.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

A sample derived from sake lees was used as a rice brew, and its IL-6 production inhibitory ability was confirmed in vitro .

<Preparation of sample from sake lees>
Sample EK
Based on JP-A-59-66875, 120 g of liquefied sake lees (solid content 70%) was obtained from 1000 g of raw rice. This liquefied liquor was freeze-dried to obtain sample EK.

Sample SGK
100 g of liquefied liquor prior to lyophilization obtained in the preparation of sample EK was suspended in 200 ml of water, heated at 65 ° C. for 30 minutes, and then cooled to room temperature. The suspension after cooling was inoculated with Lactobacillus brevis that had been pre-cultured in advance so as to be 1 × 10 ⁶ cells / ml, and lactic acid fermentation was performed at 37 ° C. for 48 hours. The suspension after lactic acid fermentation was freeze-dried to obtain a freeze-dried sample SGK.

Sample GK1
100 g of liquefied liquor before freeze drying, 15 g of red koji, and 3 g of sodium glutamate obtained in the preparation of Sample EK were suspended in 200 ml of water, heated at 65 ° C. for 30 minutes, and then cooled to room temperature. The suspension after cooling was inoculated with Lactobacillus brevis that had been pre-cultured in advance so as to be 1 × 10 ⁶ cells / ml, and lactic acid fermentation was performed at 37 ° C. for 48 hours. The suspension after lactic acid fermentation was freeze-dried to obtain a freeze-dried sample GK1.

  After suspending 4 g of the above-mentioned various lyophilized samples in 30 ml of water, the supernatant was recovered by centrifugation. Each of the collected supernatants was freeze-dried, dissolved in PBS to a concentration of 100 mg / ml, and sterilized by filtration using a membrane (membrane pore diameter 0.22 μm). These liquids were used as liquid samples for measuring IgA (EK, SGK, GK1).

<Measurement of IL-6>
First, Peyer's patch cells were collected from Peyer's patches of 6 small intestines of BALB / c mice (14 weeks old, male, manufactured by Charles River Japan). Then, the collected cells are suspended at 2 × 10 ⁶ cells / ml using RPMI-1640 medium containing 10% fetal calf serum (FCS), and this is suspended at 5 × 10 ⁵ cells / 250 μl per well. 48-well cell culture plates. Furthermore, the above-mentioned various liquid samples (EK, SGK, GK1) and liposaccharide (LPS, E. coli origin, manufactured by Seikagaku Corporation) were added to the cell suspension in the well. As the liquid sample, a sample solution whose concentration was adjusted with the medium was prepared, and 50 μl / well was added so that the final concentration of the sample was 100 μg / ml per well. In addition, LPS was prepared by preparing an LPS solution whose concentration was adjusted with the above medium, and added at 200 μl / well so that the final concentration of LPS was a predetermined concentration (0 μg / ml or 0.5 μg / ml). Then, the cells were cultured for a predetermined time (48 hours, 72 hours) at 37 ° C. and 5% CO ₂ at a total of 500 μl / well. As a control, instead of the liquid sample, culture was carried out in the same manner except that PBS alone was diluted with the medium in the same manner as the liquid sample and added to the cell suspension. After completion of the culture, the cells were collected, and the IL-6 production amount was measured by ELISA.

  These results are shown in FIG. FIG. 1 is a graph showing the amount of IL-6 produced in mouse Peyer's patch primary culture cells in the presence of each sake lees-derived sample. In the figure, the vertical axis represents the IL-6 concentration (pg / ml). Each bar is the result of IL-6 production under the conditions of LPS addition (0 μg / ml) and no LPS addition (0.5 μg / ml), the white bar is the result of 48-hour culture, the black bar is It is a result of culture | cultivation for 72 hours. As shown in the 48-hour culture results (white bars) in the figure, the same tendency was confirmed in the 72-hour culture results (black bars) as compared to the control. Among them, it was found that SGK obtained by further fermenting sake lees can further suppress IL-6 production, and particularly, liquor sample GK1 fermented in the presence of koji can significantly suppress IL-6 production.

The freeze-dried sample GK1 prepared in Example 1 was subjected to a BALB / c mouse ingestion test, and its cytokine production inhibitory ability and antibody production inhibitory ability were confirmed in vivo and ex vivo .

  The freeze-dried sample GK1 of Example 1 was sterilized using ethylene oxide gas. The frozen sample GK1 after sterilization and powdered NMF (trade name, manufactured by Oriental Yeast Co., Ltd.) (gamma ray sterilized) were blended at a weight ratio of 5:95 to obtain a feed for the test group. In addition, the said powder NMF was used as a feed for control groups.

12 BALB / c mice (6 weeks old, male, manufactured by Charles River Japan Co., Ltd.) are divided into 2 groups, 6 test groups and 6 control groups. They were reared in the SPF environment for 28 days as free intake. There were no differences in body weight gain, food intake, or water intake during the intake test between the groups. After completion of the intake test, spleen cells were collected from the spleen of each mouse. Then, the collected cells are suspended at 4 × 10 ⁶ cells / ml using RPMI-1640 medium containing 10% fetal calf serum (FCS), and this is 1 × 10 ⁶ cells / 250 μl per well. 48-well cell culture plates. To the cell suspension in the well, E. LPS derived from Escherichia coli LPS (manufactured by Seikagaku Corporation) or LPS derived from Salmonella enterica serotype typhimuriumu (manufactured by SIGMA) was added. For each LPS, an LPS solution having a concentration adjusted with the above medium was prepared, and 250 μl / well was added so that the final concentration of LPS was a predetermined concentration (0 μg / ml or 0.5 μg / ml). Then, the cells were cultured for a predetermined time at 37 ° C. and 5% CO ₂ at a total of 500 μl / well. For various cytokines, supernatants after 48 hours of culture were used, and for various antibodies, supernatants after 1 week of culture were used, and the production amounts were measured by ELISA. In addition, serum was collected from each mouse before the start of administration of the sample for each group and at the end of administration, and various antibodies in the serum were measured by ELISA. These results are shown in the table below (Average ± SD, n = 6). In the following table, the control is the result of the control group to which the powder NMF was administered, and GK1 is the result of the test group to which the test group sample containing the sample GK1 was administered.

As shown in Table 1, regardless of whether E. coli- derived LPS or S. typhi- derived LPS was stimulated to the spleen primary cells, inflammatory cytokines IL-6 and TNF- α production could be suppressed. Further, as shown in Table 2, IgE production could be suppressed in the mouse serum.

The freeze-dried sample GK1 prepared in Example 1 was subjected to a DO11.10 mouse intake test, and its cytokine production inhibitory ability and antibody production inhibitory ability were confirmed in vivo and ex vivo . DO11.10 mice are transgenic mice that express only ovalbumin (OVA) -specific T cell receptors, and are experimental animals suitable for measuring changes in antigen-specific responses of T cells.

  The freeze-dried sample GK1 of Example 1 was sterilized using ethylene oxide gas. The frozen sample GK1 after sterilization and powdered NMF (gamma ray sterilized, manufactured by Oriental Yeast Co., Ltd.) were blended at a weight ratio of 1: 9 to obtain a test group feed. In addition, the said powder NMF was used as a feed for control groups.

Seven DO11.10 mice (12 weeks old, female) were divided into two groups, 4 test groups and 3 control groups. Each group was fed in the same manner as in Example 2 above. Were kept for 17 days under free intake conditions. There were no differences in body weight gain, food intake, or water intake during the intake test between the groups. After completion of the intake test, spleen cells were collected from the spleen of each mouse and suspended in 4 × 10 ⁶ cells / ml using RPMI-1640 medium containing 10% FCS, and this was suspended at 1 × 10 ⁶ cells per well. Inoculated into a 48-well cell culture plate at a volume of 250 μl. OVA was added to the cell suspension in the well. For the OVA, an OVA solution adjusted in concentration in the medium was prepared, and 250 μl / well was added so that the final concentration of OVA was a predetermined concentration (0 μM, 5 μM, or 10 μM). Then, the cells were cultured for a predetermined time at 37 ° C. and 5% CO ₂ at a total of 500 μl / well. Then, cell proliferation activity after 72 hours of culture was measured by the BrdU labeling method. Cell proliferation activity was determined by measuring the amount of BrdU incorporated into cells by chemiluminescence ELISA using a cell proliferation ELISA, BrdU chemiluminescence kit (Roche Diagnostics), and measuring the luminescence intensity obtained. Judgment was made based on a value (CPS) measured by a predetermined method using a multiwell luminometer (Perkin Elmer). IL-2 and IL-4 use supernatants after 48 hours of culture, IL-10 and IFN-γ use supernatants after 72 hours of culture, and antibodies are cultured after 1 week of culture. Using the supernatant, the production amount was measured by the ELISA method.

  In FIG. 2, the graph of the measurement result of the number of cells 72 hours after culture | cultivation is shown. In the graph, the vertical axis represents the measured value (CPS) of luminescence intensity indicating the amount of BrdU taken into the cells. Each bar is a result of luminescence intensity under each OVA addition amount condition, that is, a result of cell proliferation activity. A white bar (Control) is a control, and a black bar (GK1) is a test group feed containing GK1. It is a result. As shown in the figure, the proliferation activity of the cells by OVA stimulation was suppressed by adding GK1 as compared with the control.

  FIG. 3 shows a graph of measurement results of IL-4 and IL-10 production amounts in cultured cells. The figure (A) is a result of IL-4, and the figure (B) is a result of IL-10. In both figures, the vertical axis represents the IL-4 concentration (pg / ml) or IL-10 concentration (pg / ml). Each bar is the result of IL-4 production or IL-10 under each OVA addition amount condition, the white bar (Control) is the control, and the black bar (GK1) is the test group feed containing GK1. It is a result. As shown in the figure, compared to the control, the addition of GK1 suppressed the production of IL-4 and IL-10, which are Th2-type cytokines, by OVA stimulation. On the other hand, when the Th1-type cytokines IL-2 and INF-γ were confirmed, there was no difference between the groups.

  The IgE concentration in the spleen cell culture supernatant was 4.3 ± 1.6 ng / ml for the control and 3.1 ± 0.4 ng / ml for the addition of GK1 when the final concentration of OVA was 5 μM. It was. Thus, compared to the control, the addition of GK1 suppressed the production of IgE by OVA stimulation.

  As described above, according to the sample derived from sake lees, for example, by having an inhibitory action on IL-6 and TNF-α, which are inflammatory cytokines, it is intended to improve or prevent inflammatory autoimmune diseases such as rheumatoid arthritis. Can do. In addition, for example, by having an inhibitory action on the production of IL-2, IL-6, and IL-10, which are Th2 type cytokines, the Th1 / Th2 balance that is inclined to the Th2 side due to allergic diseases can be adjusted to the Th1 side. it can. In addition, for example, in allergic diseases involving IgE, the allergic reaction can be suppressed by suppressing the promotion of IgE antibody production. Therefore, according to the present invention, for example, allergy can be prevented or treated by suppressing production of inflammatory cytokines, adjusting Th1 / Th2 balance, and suppressing antibody-dependent allergic reactions.

A sake lees-derived sample was used as a rice brew, and its ability to promote IgA antibody production was confirmed in vitro .

<Preparation of sample from sake lees>
In the same manner as in Example 1, freeze-dried samples EK, SGK and GK1 were prepared. After suspending 4 g of the various freeze-dried samples in 30 ml of water, the supernatant was collected by centrifugation. Each of the collected supernatants was freeze-dried, dissolved in PBS to a concentration of 100 mg / ml, and sterilized by filtration using a membrane (membrane pore diameter 0.22 μm). These liquids were used as liquid samples for measuring IgA (EK, SGK, GK1).

<IgA measurement>
First, spleen cells were collected from three spleens of BALB / c mice (8 weeks old, male, manufactured by Charles River Japan). Then, the collected cells are suspended at 4 × 10 ⁶ cells / ml using RPMI-1640 medium containing 10% fetal calf serum (FCS), and this is 1 × 10 ⁶ cells / 450 μl per well. 48-well cell culture plates. Subsequently, the various liquid samples (EK, SGK, GK1) were added to the cell suspension in the well. For each of the various liquid samples, a sample solution whose concentration was adjusted with the medium was prepared, and 50 μl / well was added so that the final concentration of the sample was a predetermined concentration (50, 150, 200 μg / ml). Then, it was added a total 500 [mu] l / well, 37 ° C., and cultured for one week in 5% CO _2. As a control, instead of the liquid sample, culture was performed in the same manner except that PBS alone was diluted with the medium in the same manner as the sample and added to the cell suspension. After completion of the culture, the cells were collected, and the amount of IgA produced was measured by ELISA.

  These results are shown in FIG. FIG. 4 is a graph showing IgA production in primary mouse spleen cultured cells in the presence of each sake lees-derived sample. In the figure, the vertical axis represents the IgA concentration (ng / ml), and each bar represents the concentration of the added sample (μg / ml). As shown in the figure, the addition of samples EK, SGK and GK1 promoted IgA production. In addition, SGK and GK1 subjected to fermentation treatment further promoted IgA production by increasing the addition amount. Especially, GK1 which gave the fermentation process in presence of koji showed remarkable IgA production promotion ability. From the above results, it was found that according to the sake lees-derived sample of this example, the production of IgA can be promoted, so that it can be used as an immunostimulator.

As described above, rice raw material brewed products, especially fermented rice raw material brewed products, particularly rice raw material brewed products fermented in the presence of rice bran, are subjected to IgA by spleen cells, which are systemic immune cells, in an in vitro test. It has been found that it has an action of promoting production and an action of suppressing IL-6 production by Peyer's patch cells, which are intestinal immune cells. Recently, IgA is an important antibody involved in biological defense in the mucosal surface such as the intestinal tract, and IL-6 is a very important cytokine for induction of IL-17 that induces pathological inflammation. It has been found. Therefore, it can be said that the rice raw material brewed product in the present invention is a food material that has improved biological defense ability and can be used for prevention, improvement, and treatment of inflammatory bowel diseases such as Crohn's disease and ulcerative bowel disease. In addition, as a result of evaluating rice brews by in vitro and ex vivo tests, it shows a function of suppressing antigen-specific production of IL-2 and IL-10, which are typical Th2-type cytokines, by immune cells I understood it. Therefore, the rice brewed product according to the present invention can prevent or improve various allergies including, for example, type I allergies (for example, food allergies, hay fever, etc.) that are thought to be caused by excessive production of Th2-type cytokines. It can be said that it is a food material that can be used for treatment.

  As described above, according to the immunomodulator of the present invention, for example, it is possible to adjust the Th1 / Th2 balance, which is a cause of allergic diseases, to suppress or normalize. Therefore, the immunomodulator of the present invention can be used as an antiallergic agent for various allergic diseases as described above. In particular, the rice brewed product is extremely useful because it can sufficiently avoid problems such as side effects of synthetic drugs and the like because the safety of the rice brewing is well supported by a long history of food.

FIG. 1 is a graph showing IL-6 production in mouse Peyer's patch primary culture cells in the presence of each sake lees sample in one example of the present invention. FIG. 2 is a graph showing cell proliferation activity after 72 hours of primary culture of mouse spleen cells in other examples of the present invention. FIG. 3 is a graph showing the measurement results of IL-4 and IL-10 production levels in mouse spleen primary cultured cells in the Example, and FIG. 3 (A) shows the results of IL-4. (B) is the result of IL-10. FIG. 4 is a graph showing measurement results of IgA production in mouse spleen primary cells in still another example of the present invention.

Claims (5)

A method for producing an immunomodulator comprising the step of subjecting a rice raw brew to fermentation in the presence of koji , wherein the rice raw brew is liquefied liquor . The fermentation process is a fermentation process using at least one of lactic acid bacteria and yeast process according to claim 1, wherein the immunomodulator. The method for producing an immunomodulator according to claim 1 or 2 , wherein the rice raw brew further contains γ-aminobutyric acid. The method for producing an immunomodulator according to any one of claims 1 to 3 , wherein the rice brewed product further contains glutamic acid or a salt thereof . The method for producing an immunomodulator according to any one of claims 1 to 4 , wherein the cocoon is red cocoon.

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