JP4921499B2 - Antiallergic composition using new strains Lactobacillus crispatas KT-11, KT-23, and KT-25 - Google Patents

Description

  The present invention relates to an antiallergic composition containing a novel strain Lactobacillus crispatas KT-11, KT-23, and KT-25.

  Helper T cells (Th cells) secrete cytokines that regulate the growth and function of B cells and T cells to induce humoral immunity and cellular immunity. Helper T cells are classified into two types, type 1 helper T cells (Th1 cells) and type 2 helper T cells (Th2 cells), based on their cytokine production patterns. Th1 activates cellular immunity, and Th2 It is known to activate humoral immunity. The so-called immune balance generally refers to the balance between Th1 and Th2, and is widely used as an index indicating the allergic state of a patient, for example.

  Allergy is a disease caused by an excessive reaction of the immune system to a foreign antigen that is normally harmless when the Th1 / Th2 balance is disrupted and abnormally biased toward Th2. There are four types, type I to type IV, depending on the reaction mechanism. In Japan, the number of patients with type I allergies represented by perennial allergic rhinitis caused by house dust such as mites and dust and seasonal allergic rhinitis caused by cedar pollen is remarkable, and the number of patients will continue to increase in the future. There are concerns.

  Type I allergy is an allergy that develops mainly when mast cells are stimulated by IgE bound to an antigen and a chemical mediator such as histamine is released. Pollen and house dust that have entered the body are recognized as antigens and specific IgE antibodies against them are produced. Specific IgE antibodies become sensitized by binding to Fc receptors on the surface of basophils in mast cells and blood. Subsequently, when the antigen enters the body again, the antigen binds to the IgE antibody, forms an antigen-antibody complex, causes degranulation, and releases chemical transmitters such as histamine and leukotriene in the granule. Appears as an allergic symptom.

  Drugs used for such allergic symptoms include antihistamines, leukotriene antagonists, thromboxane antagonists, Th2 cytokine inhibitors, mediator release inhibitors, and steroids. However, for example, antihistamines may cause drowsiness and dry mouth as side effects, and side effects of leukotriene antagonists include cytopenia and gastrointestinal disorders, and side effects of mediator release inhibitors include gastrointestinal disorders and cystitis-like symptoms. These drugs are not always safe.

  Against this background, in recent years, there has been an active search for foods with abundant experience and confirmed safety, or materials that have anti-allergic effects from their ingredients, such as lactic acid bacteria, polyphenols, and nucleotides. It is attracting attention as an antiallergic material (see Patent Documents 1 and 2).

As foods that are said to have an antiallergic effect, yogurt and lactic acid bacteria beverages, and other health foods and pharmaceuticals that use dried and killed bacterial powder of lactic acid bacteria are also sold. The lactic acid bacteria used in these include Enterococcus faecalis (Enterococcus faecalis) is an animal lactobacilli, promotes cytokine production, such as interferon enhances Th1 immune response, reduce allergic symptoms by inhibiting the Th2 immune response It is already known (Patent Document 3), but there are problems such as longevity as a food, difficulty in preparation and handling, and inadequate antiallergic effect, which requires a large amount of intake. It was.

  In general, many bacteria can grow on simple nutrient media (only glucose and minerals), but lactic acid bacteria are particularly auxotrophic for amino acids, vitamins, minerals, and so on. As a medium generally used for culturing lactic acid bacteria, an MRS medium and a BL medium are generally widely known (Non-Patent Document 1 and Non-Patent Document 2).

  As an example using a culture substrate other than the above-mentioned general medium, there is a shochu fermented food that is fermented by mixing potato and lactic acid bacteria with distilled spirits of shochu shochu. Proliferation of microorganisms such as lactic acid bacteria, such as those that exhibit improvement effects on biological functions due to effects, antitumor effects, etc. (Patent Document 4) and those that are processed (purified, concentrated, powdered, etc.) of barley shochu distillation residue There is an example of culturing lactic acid bacteria using wine starch (Non-patent Document 3). Residues from alcohol fermentation as described above include sugars, amino acids, and peptides generated by the action of various enzymes secreted by microorganisms on saccharide raw materials (cereals such as barley and straw, fruits such as grapes). Are abundant, and these serve as nutrient sources for lactic acid bacteria during culture.

  However, the above-mentioned technology is intended to synergistically improve biological function by combining the health effects of dietary fiber and lactic acid bacteria originally contained in shochu, and components contained in distillation residue and wine starch. It focuses on promoting growth and is not a technique for enhancing the antiallergic effect itself of lactic acid bacteria. Moreover, if the microorganisms and saccharide raw materials used for fermentation differ, the components and content ratios of various nutrients contained in the residue are different and not uniform.

JP 5-252900 A Japanese Unexamined Patent Publication No. 9-2959 JP-A-7-228536 JP 2007-259742 A Japanese Patent Laid-Open No. 10-327852

Academic Publishing Center, Science and Technology of Lactic Acid Bacteria, p18, Lactic Acid Bacteria Research Meeting, 1996 Japan Food Sanitation Association, Food Sanitation Inspection Guidelines Microbiology, p354-355, supervised by the Ministry of Health, Labor and Welfare, 2004 Bustos, G., et al., 2004. Evalution of Vinification Lees as a General Medium for Lactobacillus Strains. J. Agric. Food Chem. 52 (16), 5233-9

  It is an object of the present invention to provide an antiallergic composition containing a novel lactic acid strain having a higher antiallergic effect than conventional lactic acid bacteria.

  The present invention has found that a novel Lactobacillus kristpatus strain isolated from feces has a high antiallergic effect and has been completed. That is, the present invention relates to an antiallergic composition having the following constitution.

(1) Lactobacillus crispatus (Lactobacillus crispatus) KT-11 strain (FERM P-21457), KT -23 strain (FERM P-21458), KT -25 strain (FERM P-21459) either one or two An antiallergic composition comprising at least one species.
(2) The antiallergic composition according to (1), which is cultured in a culture medium comprising a barley shochu distillation residue and a saccharide and an emulsifier added thereto.
(3) A pharmaceutical comprising the antiallergic composition according to (1) or (2).
(4) The pharmaceutical product according to (3), which is for mites.
(5) The pharmaceutical product according to (3), which is for allergic rhinitis.
(6) A food or drink or supplement containing the antiallergic composition according to (1) or (2).
(7) A pet food or pet supplement containing the antiallergic composition according to (1) or (2).
(8) A feed containing the antiallergic composition according to (1) or (2).
(9) Lactobacillus crispatus (Lactobacillus crispatus) KT-11 strain (FERM P-21457), KT -23 strain (FERM P-21458), any one of the KT-25 strain (FERM P-21459) or 2 A method for producing an antiallergic composition comprising mixing a seed or more and a pharmaceutical preparation.
(10) new strain Lactobacillus crispatus (Lactobacillus crispatus) KT-11 strain (FERM P-21457), KT -23 strain (FERM P-21458) or KT-25 strain (FERM P-21459).

  The present invention suppresses the action of Th2 type by inducing immune balance to Th1 type, and allergic diseases, especially food (milk / dairy products, eggs, wheat, buckwheat, peanuts, abalone, squid, how much, shrimp, orange , Crab, kiwi fruit, beef, walnut, salmon, mackerel, soy, chicken, banana, pork, matsutake, peach, yam, apple, gelatin, etc., house dust, animal hair (dog hair such as dogs and cats) , Wool, cotton, feathers, etc.), pollen of grass, weeds, trees, etc., asthma, dermatitis, rhinitis, hay fever, anaphylactic shock caused by insects (ticks, bees, mosquitoes, flies, fleas, cockroaches, etc.) It is possible to provide an antiallergic composition effective for improving type I allergic diseases.

The lactic acid bacteria used in the present invention, Lactobacillus crispatus (Lactobacillus crispatus) KT-11 strain (FERM P-21457), KT -23 strain (FERM P-21458), KT -25 strain (FERM P-21459) is Can be mentioned. These strains are deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.

The bacteriological properties of the above strain are shown below.
[1] Morphological properties
In observation after culturing at 37 ° C. for 72 hours on MRS agar medium (Difco), the size of the cell is a gonococcus of 0.5-1 × 3-5 μm and does not move. There is no sporulation and Gram staining is positive.
[2] Culture characteristics
The colonies after culturing at 37 ° C. for 72 hours on MRS agar medium (Difco) have a diameter of 2 to 3 mm, are circular, and have full edges. The color of the colony is yellowish white and translucent.
[3] Physiological properties Gas production negative Glucose utilization positive Catalase activity negative Gelatin liquefaction negative Nitrate reduction negative Indole production negative Hydrogen sulfide production negative Attitude to oxygen Facultative anaerobic Optimal growth temperature 37-40 ° C
Optimum growth pH pH 5.5-5.8

Lactobacillus crispatus (Lactobacillus crispatus) KT-11, KT-23, KT-25 , respectively, as shown in Examples described later, as compared with Lactobacillus crispatus (Lactobacillus crispatus) JCM1185 and JCM2009 are standard strains, Th2 It is characterized by significantly increasing the number of Th1-type cells (IFN-γ-producing helper T cells) that suppress the function of the type.

The Lactobacillus crispatus (Lactobacillus crispatus) strains can be either live bacteria, killed bacteria, dried cells, wet cells, culture broth, the cell suspension, disrupted cells, cell components Any state may be used. Moreover, as a form of a composition, you may use any forms, such as a powder form, a granular form, a paste form, and a liquid form.

  The antiallergic composition of the present invention can be used by formulating the active ingredient according to the present invention by mixing it with a physiologically acceptable carrier, excipient, binder, diluent or the like. The antiallergic composition can be administered orally or parenterally, but is preferably administered orally. Oral preparations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. Examples of parenteral preparations include external preparations (eg, nasal preparation, transdermal preparation, ointment) and suppositories (eg, rectal suppository, vaginal suppository).

  Beverages such as cooked rice, confectionery, noodles, marine products such as kamaboko and chikuwa, processed livestock products such as ham and sausage, supplements, health functional foods, soft drinks, fruit drinks and tea drinks , Soups, mayonnaise, dressings, seasonings such as seasonings, and other foods, pet food, nutritional supplements (supplements), powders, paste products, pellets for livestock, poultry, other mammals or fish In addition, it can be used by blending it with a solid or flaky feed.

  The daily intake of the drug or food or drink of the antiallergic composition of the present invention varies depending on age, symptoms, body weight, use, etc., and thus cannot be determined in general, but as a temporary guide, 0.1 to 1000 mg / It is preferable to take kg body weight.

  The antiallergic composition of the present invention is an allergic disease, particularly food, house dust, animal hair, grass, weed, tree pollen, asthma caused by insects, dermatitis, rhinitis, hay fever, anaphylactic shock, etc. It is effective in improving type allergic diseases.

The figure which showed the relative value of the IFN-gamma and IL-2 production helper T cell number in the mouse | mouth spleen cell at the time of the microbial cell sample isolated from the stool. The figure which shows the result of having measured the thickness of the swelling of the right pinna site induced by the mite antigen. The figure which shows the allergy score which created based on Table 4 and evaluated the allergy symptom of the right auricle part. The figure which showed the total amount of IgE in a serum, and the amount of mite antigen-specific IgE. The figure which showed the number of T2 cytokine production helper T cells in a mouse | mouth spleen. Increase in the number of IFN-γ-producing helper T cells in mouse spleen cells when a cell sample cultured in a culture medium composed mainly of a barley shochu distillation residue (Barex from Sanwa Sake) FIG. It shows an increase in new Lactobacillus crispatus (Lactobacillus crispatus) KT-11 IFN -γ -producing helper T cell numbers in murine spleen cells upon culture powder addition containing a. The figure which showed the result which counted the frequency | count of the sneezing of allergic rhinitis induced by ovalbumin (OVA).

  Hereinafter, the present invention will be described using examples, but the present invention is not limited to the description of the examples.

(Isolation and identification of bacterial cells)
Below, the isolation and identification method of the microbial cell used by this invention are shown.

  Using 0.85% NaCl aqueous solution, stool refrigerated immediately after collection from an infant (3 months to 1.5 years old) was suspended and cultured on a commercially available MRS agar medium at 37 ° C. for 72 hours by the pour plate method. After culturing, colonies with different morphologies were fished and isolated. All the isolated bacteria were Gram positive and Catalase negative.

The isolated bacteria were inoculated into the MRS liquid medium shown in Table 1 and cultured at 37 ° C. for 72 hours. After culturing, the cells were collected by centrifugation (2,000 rpm, 10 minutes) and washed three times with 0.85% NaCl aqueous solution. The washed cells were freeze-dried, heat-treated (65 ° C., 30 minutes), and suspended in sterile 0.15 M sodium chloride-0.01 M phosphate buffer (PBS, pH 7.2) to obtain cell samples.

Spleens were aseptically collected from 6-week-old male C3H / HeN mice and suspended in 5 ml of RPMI 1640 medium containing penicillin 100 IU / ml and streptomycin 100 μg / ml. The suspension was subjected to centrifugal washing (4 ° C., 1200 rpm, 5 minutes) twice with the above RPMI1640 medium and once with the above RPMI 1640 medium containing 5% FBS. The washed cells were prepared at 1.0 × 10 ⁶ cells / ml and used as a mouse spleen cell suspension.

1,000 μl of the mouse spleen cell suspension prepared above was dispensed into a flat-bottomed 48-well microplate, and 100 μl of the cell sample was further added, followed by culturing at 37 ° C. in the presence of 5% CO ₂ for 48 hours. The final concentration of lactic acid bacteria is 0 and 100 μg / ml.

The spleen cell suspension cultured above was centrifuged and washed with Hank's Balanced Salt Solution (HBSS) containing 1 mM EDTA and 5% FBS (4 ° C., 2,000 rpm, 3 minutes). Suspend 1.0 x 10 ⁶ washed mouse spleen cells in 1,000 μl of activation medium (RPMI 1640 medium containing 10% FBS containing brefeldin A 20 μg / ml, ionomycin 2 μg / ml and PMA 20 ng / ml). The cells were cultured at 37 ° C. in the presence of 5% CO ₂ for 4 hours to produce and accumulate cytokines in the cells.

The cultured mouse spleen cells were centrifuged and washed with HBSS (4 ° C., 2,000 rpm, 3 minutes), 1 μl of biotin-labeled anti-mouse CD4 was added, and reacted at 4 ° C. for 15 minutes. Further, 1 μl of streptavidin-PE / Cy5 was added and left at 4 ° C. under light shielding for 15 minutes. The reaction solution was washed with HBSS (4 ° C., 2000 rpm, 3 minutes), 100 μl of IntraPrep ^™ Reagent 1 was added, and the cells were fixed by allowing to stand for 15 minutes in the dark at room temperature. After washing with HBSS (4 ° C., 2,000 rpm, 3 minutes), 100 μl of IntraPrep ^™ Reagent 2 was added and allowed to react for 15 minutes in the dark at room temperature for membrane permeation treatment. Next, 1 μl of PE-labeled anti-mouse IL-2 or PE-labeled anti-mouse IFN-γ was added to measure Th1 cytokine IL-2 or IFN-γ, and the mixture was allowed to stand at room temperature for 15 minutes. Thereafter, it was again washed with HBSS (4 ° C., 2,000 rpm, 3 minutes). After washing, the numbers of IFN-γ producing helper T cells and IL-2 producing helper T cells in the cultured cells were measured using Guava ^(R) Personal Cell Function Analyzer (PCA).

  Figure 1 shows the number of IFN-γ-producing helper T cells and the number of IL-2 producing helper T cells in mouse spleen cells when each bacterial cell sample was added. It is shown. When KT-11, KT-23, and KT-25 were added, the number of IFN-γ producing helper T cells and the number of IL-2 producing helper T cells were remarkably increased.

Species of KT-11, KT-23 and KT-25 were identified by homology search of 16Sr DNA base sequence. A result of the identification, it was found that all of the above 3 strains are Lactobacillus crispatus (Lactobacillus crispatus). From the above results, the three strains, it was fit shown is a novel Lactobacillus crispatus to induce strong Th1 immune responses (Lactobacillus crispatus).

(Measure superiority of antiallergic effect)
The superiority of the anti-allergic effect of the new Lactobacillus crispatus (Lactobacillus crispatus) are shown below.

Lactic acid bacteria shown in Table 2 were prepared in the same manner as in Example 1, and used as lactic acid bacteria samples. In addition, Lactobacillus crispatus (Lactobacillus crispatus) JCM1185 and JCM2009 is a standard strain of Lactobacillus crispatus.

1,000 μl of a mouse spleen cell suspension prepared in the same manner as in Example 1 was dispensed into a flat-bottomed 48-well microplate, and the lactic acid bacteria sample was further added, followed by culturing at 37 ° C. in the presence of 5% CO ₂ for 48 hours. After culture, the number of IFN-γ-producing helper T cells was measured in the same manner as in Example 1. Significance of the results was determined by Dunnett's test analysis with reference to JCM1185 and JCM2009, which are lactic acid bacteria-free or Lactobacillus crispata standard strains. The test results were all p <0.001, and there was a significant difference at a risk rate of 0.1%.

Table 3 shows the number of IFN-γ producing helper T cells in mouse spleen cells when each lactic acid bacterium was added. Any newly isolated Lactobacillus crispatus (Lactobacillus crispatus) KT-11, KT-23, 3 strains of KT-25, from the standard strains JCM1185 and JCM2009 of Lactobacillus crispatus (Lactobacillus crispatus) IFN- The number of γ-producing helper T cells was significantly increased. In addition, it showed stronger activity than the other two animal lactic acid bacteria. Therefore, Lactobacillus crispatus (Lactobacillus crispatus) KT-11, KT-23, KT-25 was shown from the anti-allergic effective than conventional lactic acid bacteria.

(Effect on tick-induced allergic symptoms)
Effect of the new Lactobacillus crispatus (Lactobacillus crispatus) mite-induced allergic symptoms by oral administration to mice as follows.

As subjects, 4-week-old male and NC / Nga mice were used. Feed (commercial mouse feed) and water were ingested ad libitum, maintaining a 12-hour / 12-hour light-dark cycle and a breeding environment of 22 ± 2 ° C. New Lactobacillus crispatus (Lactobacillus crispatus) KT-11, KT-23, KT-25 was prepared in a similar manner as in Example 1, was lactobacillus sample. The lactic acid bacteria sample was administered once a day from 6 weeks of age using a stomach tube. Sensitization with mite antigen is induced by intradermal injection of 5 μg of commercial mite antigen dissolved in 10 μl of physiological saline once a week into the right auricle. The thickness was measured once a week. Allergic symptoms were evaluated based on the allergy scores shown in Table 4.

  For the above items, the allergic symptoms in the right auricular region were scored as "0 points ... no symptoms", "1 points ... mild symptoms", "2 points ... severe symptoms" The total score was scored.

FIG. 2 shows the result of measuring the thickness of swelling of the right auricle site induced by the mite antigen. FIG. 3 is an allergy score that was prepared based on Table 4 and evaluated for allergic symptoms in the right auricle. Lactobacillus sample as compared to mice not administered, Lactobacillus crispatus (Lactobacillus crispatus) when administered the KT-11, KT-23 and KT-25 thin swelling right auricle site, also significant allergic score Has decreased. From the above results, oral administration of the new Lactobacillus crispatus (Lactobacillus crispatus), it was found that allergic symptoms induced by mite is reduced.

(Changes in total IgE and mite antigen-specific IgE levels in mouse serum)
Changes of the new Lactobacillus crispatus (Lactobacillus crispatus) total IgE and mite antigen-specific IgE level in mouse serum by oral administration are shown below.

Blood was collected from the abdominal division of the mice in each breeding test group shown in Table 5, and immediately centrifuged (4 ° C., 3,000 rpm, 30 minutes) to collect serum. The collected serum was stored at −30 ° C. until immediately before use.

  The total amount of IgE in the serum was measured by sandwich ELISA. First, 100 μl of goat anti-mouse IgE (10 μg / ml) dissolved in 0.05 M carbonate buffer (pH 9.6) was dispensed into each well of a 96-well microplate and allowed to stand overnight at 4 ° C. After washing 3 times with PBS-Tween, 300 μl of 0.4% BSA solution dissolved in 0.05 M carbonate buffer was added to each well and allowed to stand at 25 ° C. for 120 minutes. After washing again with PBS-Tween three times, 100 μl of mouse serum diluted 500-fold with PBS-Tween containing 2% polyvinylpyrrolidone (PVP) was dispensed into each well and reacted at 25 ° C. for 120 minutes. After completion of the reaction, the plate was again washed three times with PBS-Tween, and 100 μl of HRP-labeled goat anti-mouse IgE (0.2 μg / ml) diluted with PBS-Tween containing 2% PVP was dispensed into each well. The reaction was allowed to proceed at 60 ° C. for 60 minutes. After washing the plate 5 times with PBS-Tween, 100 μl of TMB substrate solution was dispensed into each well in the dark and allowed to react at 25 ° C. for 15 minutes, completely protected from light. After the reaction, 100 μl of 4N sulfuric acid was dispensed into each well to stop the reaction, and immediately the absorbance at 450 nm was measured using a model 550 microplate reader.

  The amount of mite antigen-specific IgE was measured by ELISA. First, 100 μl of commercially available mite antigen (100 μg / ml) dissolved in 0.1 M carbonate buffer (pH 9.6) was dispensed into each well of a 96-well microplate and allowed to stand overnight at 4 ° C. After washing 3 times with PBS-Tween, 300 μl of 0.4% BSA solution dissolved in 0.1 M carbonate buffer was added and allowed to stand at 25 ° C. for 120 minutes. After washing again with PBS-Tween three times, 100 μl of mouse serum diluted 10-fold with PBS-Tween containing 2% polyvinylpyrrolidone (PVP) was dispensed into each well and reacted at 25 ° C. for 120 minutes. After completion of the reaction, the reaction was processed in the same manner as described above, and the absorbance at 450 nm was measured using a model 550 microplate reader.

  FIG. 4 shows the total amount of IgE in the serum and the amount of mite antigen-specific IgE. The significance of the measurement results was determined by Dunnett's test analysis with reference to the control group. It was found that in the mice group administered with KT-11, KT-23, and KT-25, the total IgE amount and the mite antigen-specific IgE amount in the serum were decreased compared to the control group.

(Change in the number of Th2 cytokine-producing helper T cells)
From new Lactobacillus crispatus (Lactobacillus crispatus) oral administration, the following for changes in Th2 cytokine production helper T cell numbers mouse spleen cells.

  The spleen was aseptically collected from the mouse of Example 4, a spleen cell suspension was prepared in the same manner as in Example 1, and the number of IL-4 producing helper T cells, which are Th2 cytokine producing helper T cells, was measured. The significance of the measurement results was determined by Dunnett's test analysis with reference to the control group.

  FIG. 5 shows the number of IL-4 producing helper T cells in the mouse spleen. It was found that the number of Th2 cytokine-producing helper T cells in the spleen was significantly decreased in the mice group orally administered with the new Lactobacillus crispatas KT-11, KT-23, and KT-25 compared to the control group .

From the above results, compositions containing a novel Lactobacillus crispatus (Lactobacillus crispatus) KT-11, KT-23 and KT-25, is, IgE amount is reduced by suppressing the Th2 immune response, It has been shown to reduce allergic symptoms.

(Improvement of anti-allergic effect of lactic acid bacteria by culture substrate consisting of barley shochu distillation residue)
Below effects on Th1 immune response culture substrate made of barley shochu stillage (e.g. Sanwa type (bar Rex strains)) new Lactobacillus crispatus which were cultured in a medium containing as a main component (Lactobacillus crispatus) Show.

The medium shown in Table 6 was inoculated with Lactobacillus crispatus (Lactobacillus crispatus) KT-11, for 24 hours at 37 ° C.. After culturing, the cells were collected by centrifugation (2,000 rpm, 10 minutes) and washed with distilled water by centrifugation. The washed cells were heat-treated (65 ° C., 30 minutes) and then lyophilized. Furthermore, it was suspended in a sterilized 0.15M sodium chloride-0.01M phosphate buffer (PBS, pH 7.2) at a concentration of 1.1 × 10 ⁸ CFU / ml to obtain a lactic acid bacteria sample.

1,000 μl of a mouse spleen cell suspension prepared in the same manner as in Example 1 was dispensed into a flat-bottomed 48-well microplate, and the lactic acid bacteria sample was further added, followed by culturing at 37 ° C. in the presence of 5% CO ₂ for 48 hours. The final concentration of lactic acid bacteria is 0 and 1.0 × 10 ⁷ CFU / ml.

After culture, the number of IFN-γ-producing helper T cells was measured in the same manner as in Example 1. Significance of results, based on the Lactobacillus crispatus (Lactobacillus crispatus) KT-11 cultured in lactic acid bacteria without addition or MRS medium was determined by test analysis of Dunnett. When p <0.001, the risk rate is 0.1%, and when p <0.05, the risk rate is 5%.

FIG. 6 shows the number of Th1 cells in the mouse spleen. Culture substrate made of barley shochu stillage in (bar Rex) Lactobacillus crispatus cultured in a medium whose main component (Lactobacillus crispatus) mouse spleen cells with the addition of KT-11, IFN than when cultured in MRS medium -Number of γ-producing helper T cells increased. Accordingly, it was shown to improve the anti-allergic effect of barley shochu stillage culture substrate which comprises culturing in a medium composed mainly of at Lactobacillus crispatus (Lactobacillus crispatus) KT-11.

(Verification of anti-allergic effect of culture powder)
Culture substrate made of barley shochu stillage (the bar Rex) culturing new Lactobacillus crispatus (Lactobacillus crispatus) KT-11 in a medium whose main component, a culture solution was powdered. The effect of the culture powder on the Th1 immune response is shown below.

Example 6 and adjusted to new Lactobacillus crispatus (Lactobacillus crispatus) KT-11 culture solution 30 ° C. cultured similarly, dextrin powder (Pinedex # 100 of 7.4% as a powder base (w / w), Matsutani Chemical Industries) was added and dissolved. After dissolution, a spray dryer (L-12 type spray drier, manufactured by Ohkawara Kakohki Co., Ltd.) was spray dried to obtain a culture powder containing a novel Lactobacillus crispatus (Lactobacillus crispatus) KT-11.

1,000 μl of a mouse spleen cell suspension prepared in the same manner as in Example 1 was dispensed into a flat-bottomed 48-well microplate, and the above powder sample was added to a final concentration of 100 μg / ml, followed by 37 ° C., 5% CO _2. Cultured for 48 hours in the presence. As a control, a powder of only a medium mainly composed of a culture substrate (Barrex) composed of a barley shochu distillation residue was treated in the same manner.

  After culture, the number of IFN-γ-producing helper T cells was measured in the same manner as in Example 1. The significance of the results was determined by Dunnett's test analysis based on powders containing no lactic acid bacteria or medium only. p <0.01 indicates a significant difference with a 1% risk rate.

FIG. 7 shows the number of Th1 cells in the mouse spleen. The new Lactobacillus crispatus (Lactobacillus crispatus) mouse spleen cells with the addition of culture medium powder containing the KT-11, Lactobacillus additive-free and medium only powder when added from the IFN-gamma producing T-helper cell numbers significantly the Increased. In addition, the medium-only powder did not show an increase in the number of IFN-γ-producing helper T cells. Therefore, anti-allergic effects were shown to be improved by culture powder containing a novel Lactobacillus crispatus (Lactobacillus crispatus) KT-11.

(Effective for allergic rhinitis)
MRS medium or culture substrate made of barley shochu stillage Influence on (bar Rex) Lactobacillus were cultured in a medium mainly composed of crispatus (Lactobacillus crispatus) KT-11 administration by allergic rhinitis below.

1 week after preliminarily fed for 5 weeks old BALB / c mice commercial diet, Lactobacillus crispatus cultured in MRS medium (Lactobacillus crispatus) KT-11 or culture substrate made of barley shochu stillage, a (bar Rex) Lactobacillus crispatus cultured in a medium containing as a main component (Lactobacillus crispatus) KT-11 to 5 × 10 ⁷ CFU / g including commercial diet: were housed in (powder MF Oriental yeast). Only the commercial feed was given to the allergy negative group and the control group. Once at 6 and 8 weeks of age, 20 μg ovalbumin (OVA) and 2 mg aluminum hydroxide gel (Sigma, USA) prepared to 200 μl were administered intraperitoneally. 200 μl of physiological saline was administered to allergy negative mice. Also, 10 μl of 10 mg / ml OVA aqueous solution was administered into the mouse nasal cavity three times a week from 9 weeks of age. For allergic symptoms, the number of sneezes for 10 minutes was measured after 1 minute from the intranasal administration of OVA. In all groups, drinking water and feed were freely consumed.

  The significance of the results was determined by Dunnett's test analysis based on the absence of lactic acid bacteria. When p <0.05, there is a significant difference with a 5% risk rate.

FIG. 8 shows the number of allergic rhinitis in mice induced by OVA. MRS medium or Lactobacillus crispatus the bar Rex were cultured in a medium containing as a main component (Lactobacillus crispatus) mice given KT-11 is the number of sneezes was decreased significantly. These results were found to reduce the allergic rhinitis symptoms by administration of the novel Lactobacillus crispatus (Lactobacillus crispatus) KT-11.

Claims (10)

Lactobacillus crispatus (Lactobacillus crispatus) KT-11 strain (FERM P-21457), KT -23 strain (FERM P-21458), containing any one or more of the KT-25 strain (FERM P-21459) An antiallergic composition characterized by comprising:   2. The antiallergic composition according to claim 1, which is cultured in a culture medium comprising a barley shochu distillation residue and a saccharide and an emulsifier added thereto. An antiallergic agent comprising the antiallergic composition according to claim 1 or 2. The antiallergic agent according to claim 3, which is used for mites. Antiallergic agent according to claim 3, wherein it is for allergic rhinitis.   A food or drink or supplement containing the antiallergic composition according to claim 1 or 2.   A pet food or pet supplement containing the antiallergic composition according to claim 1 or 2.   A feed containing the antiallergic composition according to claim 1 or 2. Lactobacillus crispatus (Lactobacillus crispatus) KT-11 strain (FERM P-21457), KT -23 strain (FERM P-21458), KT -25 strain (FERM P-21459) any one or more of the A method for producing an antiallergic composition comprising mixing a pharmaceutical preparation. New strains Lactobacillus crispatus (Lactobacillus crispatus) KT-11 strain (FERM P-21457), KT -23 strain (FERM P-21458) or KT-25 strain (FERM P-21459).

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