JP2010047504A - Atopic dermatitis mitigative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an excellent atopic dermatitis mitigative which has a high function to mitigate atopic dermatitis and is orally bioavailable. <P>SOLUTION: The atopic dermatitis mitigative contains a bacterial cell of Lactobacillus plantarum HSK201 strain (NITE P-589), a culture of the bacterial cell, and a treated product of the bacterial cell or an extract thereof as active ingredients. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a novel lactic acid bacterium having a useful function in the prevention and treatment of atopic dermatitis among allergic diseases, and an atopic dermatitis alleviating agent containing the lactic acid bacterium as an active ingredient.

Allergic diseases are counted as one of the most common diseases in developed countries. The pathogenesis of allergies is usually classified from type I to type IV. Typical allergic diseases such as hay fever, asthma, and urticaria involving IgE antibodies are classified as type I, and include anaphylactic shock. Type II allergy involves IgG and IgM antibodies, and is a cytotoxic allergy. Type III allergy damages surrounding tissues by an immune complex formed by combining antigens, antibodies, and complements. It is a reaction. Type IV allergy involves T cells, and is a delayed type allergy typified by tuberculin reaction and contact dermatitis. In the case of atopic dermatitis, there are aspects of type I reaction as well as hay fever, but there are also aspects of type IV, which is a delayed type allergy, and treatment is often difficult.
Type I allergies such as pollinosis are characterized by the induction of antigen-specific IgE antibodies and the release of chemical mediators such as histamine and leukotrienes. IgE antibodies produced by the action of Th2 cytokines (IL-4 and IL-5) released by helper T cells (Th2 cells) activated by allergens that have invaded the body can be detected in mast cells in the tissue and blood. Binds to the surface of the base sphere. Allergic symptoms are manifested by chemical mediator explosion from mast cells and blood basophils caused by recognizing the same allergen that IgE has re-entered.
On the other hand, the mechanism of the onset of atopic dermatitis has not been elucidated precisely, and it is thought that the onset is caused by the condition that the barrier function of the skin is reduced for some reason in addition to the overproduction of IgE.
In the prevention and treatment of atopic dermatitis, antihistamines, antiallergic agents and steroidal agents similar to the prevention and treatment of type I allergy are used, and moisturizers are used for the purpose of alleviating skin symptoms. However, none of them are regarded as problematic in terms of side effects such as sleepiness due to long-term use and burden on the liver, and none of them are decisively superior in terms of effects.
In recent years, certain bacteria such as Mycobacterium tuberculosis, Streptococcus, and Lactic acid bacteria can suppress Th2 immunity related to immediate allergic reaction and reduce IgE antibody by enhancing Th1 immunity mainly related to cellular immunity. Has been reported (Non-patent Document 1). Among them, lactic acid bacteria are easy to apply to foods from the viewpoint of safety, and are useful materials. Therefore, lactic acid bacteria are mainly used for anti-I type allergies such as hay fever. Lactic acid bacteria have been found, and Lactobacillus paracasei (Patent Document 1), Enterococcus faecalis and Lactobacillus reuteri (Patent Document 2) have been shown to exhibit certain effects on atopic dermatitis. ing.

Lactobacillus plantarum is a lactic acid bacterium mainly isolated from plants. This bacterium is resident in pickles and the like, and lactic acid bacteria beverages using this bacterium are already on the market, and the safety is a known microbial species. Therefore, if Lactobacillus plantarum can be used as an antiallergic substance / atopic dermatitis relief agent in medicines and foods, it is considered promising as being very effective in terms of safety, efficacy and economy. It is one of the lactic acid bacteria that have been used. Patent Document 3 describes that the Lactobacillus plantarum No. 14 strain belonging to the Lactobacillus plantarum No. 14 (Mikoken No. 11550) has a function of improving antiallergic activity. However, even with anti-allergic activity enhancing function, even for patients with hay fever of type I allergy, the symptoms of nasal congestion are only suppressed, and the remarkable effects of various symptoms in general are not shown. Furthermore, there is no knowledge about the symptoms of atopic dermatitis.
Int. Arch. Allergy Immunol. 115,278 (1998). Food and Development, Vol. 43, No. 4 (2008), p. 44 Japanese Patent No. 3585487 JP 2000-95697 A JP 2007-126365 A

  An object of the present invention is to provide an excellent atopic dermatitis alleviating agent that has a high atopic dermatitis alleviation function and can be administered orally.

In order to solve the above problems, the present inventors have conducted intensive studies, and as a result, the inventors have previously confirmed the allergic symptom alleviation and IgE antibody lowering ability in experiments using nasal allergy model mice. It was found that Bacillus plantarum HSK201 strain (Non-patent Document 2) has not only a strong antiallergic activity improving function but also a remarkable atopic dermatitis alleviating function.
HSK201 strain of the present invention (NITE P-589) is determined to be a novel lactic acid bacterium belonging to Lactobacillus plantarum by its bacteriological properties (Table 1) and r-DNA sequence search, The anti-allergic activity enhancing function was found to be much better than the known Lactobacillus plantarum. And it confirmed that especially the effect in the atopic dermatitis alleviation function was remarkable, and completed this invention.

That is, the present invention is as follows.
[1] An atopic dermatitis alleviating agent containing as an active ingredient a cell, a cell culture, a cell-treated product or an extract thereof of Lactobacillus plantarum HSK201 strain (NITE P-589).
[2] A preparation for oral administration for the prevention and / or treatment of atopic dermatitis, comprising the atopic dermatitis alleviating agent according to [1].
[3] A topical skin preparation for preventing and / or treating atopic dermatitis, comprising the atopic dermatitis alleviating agent according to [1].
[4] A food and drink comprising the atopic dermatitis alleviating agent according to [1].
[5] A cosmetic comprising the atopic dermatitis alleviating agent according to [1].
[6] Lactobacillus plantarum HSK201 strain (NITE P-589) that can be used as an active ingredient of the atopic dermatitis alleviating agent described in [1].

  The Lactobacillus plantarum strain HSK201 (NITE P-589) in the present invention has a significant anti-allergic activity relieving function as well as a high anti-allergic activity improving function. Excellent mitigating effect can be exhibited against atopic dermatitis. In addition, since it is highly reachable to the intestine, it can be administered orally. By taking it as a food or drink, the symptoms of atopic dermatitis can be prevented and improved without side effects or burden on the body.

1. Microorganism according to the present invention The novel microorganism according to the present invention is Lactobacillus plantarum HSK201 strain, and this cell is a lactic acid bacterium belonging to Lactobacillus plantarum. Deposited at the Deposit Center (NITE) as deposit number NITE P-589.

<Mycological properties>
The mycological properties of Lactobacillus plantarum strain HSK201 according to the present invention are shown in Table 1.

This strain is different from the JCM1057 reference strain in assimilability to melibiose, raffinose, α-methyl-D glucoside and D-turanose, and is different from the JCM1149 reference strain in utilization to rhamnose, lactose and D-turanose. The utilization of rhamnose is different from Lactobacillus plantarum No. 14 (FERM P-11550), which has been reported to have antiallergic activity.
Table 2 compares the sugar assimilation properties of this strain with the reference strains JCM1057, JCM1149 and Lactobacillus plantarum No.14.

<Mycological identification>
As a result of sequencing the above mycological properties and bases from the 5 'end to the 1437th position of the 16s rDNA gene, 100% homology with the Lactobacillus plantarum standard strain was observed, and as shown in FIG. The strain of the present invention was identified as Lactobacillus plantarum because it showed a positive reaction with a Bacillus plantarum-specific primer (FIG. 1). The DNA sequence data of the Lactobacillus plantarum HSK201 strain (base sequence from the 5 ′ end to the 1437th position of the 16sDNA gene) is shown as (SEQ ID NO: 1).

<Culture method and separation method>
The strain Lactobacillus plantarum HSK201 according to the present invention is not particularly limited in the method for culturing and separating cells. The medium is not particularly limited as long as it is for the fungus, and can be cultured in a medium such as a natural medium, a synthetic medium, or a semi-synthetic medium. A medium containing a nitrogen source and a carbon source is used as the medium. Specific examples of the nitrogen source include meat extract, peptone, soybean flour, soybean hydrolysate, gluten, casein, yeast extract, amino acid, and the like. Specific examples of the carbon source include glucose, fructose, lactose, sorbitol, Examples include inositol, sucrose, starch syrup, rice bran juice, starch, bacus, bran, molasses, and the like. In addition, for example, ammonium sulfate, potassium phosphate, magnesium chloride, sodium acetate, sodium chloride, calcium carbonate, iron, manganese, molybdenum, and various vitamins can be added as inorganic substances.
The culture temperature is 10 to 50 ° C., more preferably 25 to 45 ° C., the culture time is about 6 to 36 hours, and aeration shaking and aeration stirring may be performed. The pH of the medium is 3 to 10, preferably 5 to 7.
According to the method for separating lactic acid bacteria according to the present invention, for example, after culturing, the cells are collected, the supernatant is removed by means such as centrifugation or membrane separation, distilled water or physiological saline is added, and this operation is repeated as necessary. Bacteria can be collected by separation or filtration.

2. About the preparation method of the Lactobacillus plantarum HSK201 strain lactic acid bacteria used for the atopic dermatitis alleviation agent of the present invention The atopic dermatitis alleviation agent of the present invention uses the Lactobacillus plantarum HSK201 strain lactic acid bacteria as a live cell or a fermented product The culture of lactic acid bacteria can be used as it is or concentrated together with the culture solution, and only the bacterial cells are isolated, and live cells, dead cells, or heated and frozen cells It can be used as a processed product that has been subjected to processing such as grinding, lysis, and extraction.
In addition, the separated cells or the treated product after the above treatment after separation can be used in the collected state or in a liquid state suspended in an appropriate liquid (for example, a branched dextrin solution). Further, it can be used after being dried. As the drying method, for example, a normal method such as a natural drying method, a heating method, a spray drying method, or a freeze drying method can be used.
In addition, the live cells isolated from the lactic acid bacteria of the present invention can be used as they are, or fermented products in a state where lactic acid fermentation is performed by adding the live cells to dairy products, fruits, cereals or processed products (food) thereof. It can be used as a processed product).
In addition, the lactic acid bacterium of the present invention can be used after the isolated microbial cells are inactivated by heating, ultraviolet irradiation, formalin treatment or the like and added to food or formulated. The separated live cells and dead cells can be further ground and crushed, and the obtained treated product can be heat-sterilized and aseptically filtered if necessary, and the filtrate can be freeze-dried for use. Examples of the processed product of the cells include the above-mentioned ground product, crushed product, extract from them, and freeze-dried product.

3. About formulation of atopic dermatitis alleviating agent The atopic dermatitis alleviating function is expected among the antiallergic activity improving functions, among others. And since it is a lactic acid bacterium which has no problem in taste and safety, and has high survival reach to the intestine, it is preferable to formulate for oral administration.
The atopic dermatitis alleviating agent of the present invention is an effective amount for the atopic dermatitis alleviating function alone or in combination with a known lactic acid bacteria preparation or other drug having an atopic dermatitis alleviating effect, It can be formulated using a pharmaceutically acceptable carrier such as a known excipient or bulking agent. The preparation at that time can be in various forms such as tablets, capsules, granules, powders, jellies, drinks, etc., for foods and drinks such as pickles, confectionery, yogurt, lactic acid bacteria beverages or supplements. It can be added directly or added to food additives and the like.
Moreover, it can be blended in an effective amount with respect to external preparations for skin such as ointments, sprays, patches, or cosmetics such as emulsions, creams, lotions, packs, shampoos, rinses and cleaning agents.
Specifically, the effective amount of the atopic dermatitis alleviating agent according to the present invention may be administered in an amount of 10 million to 1 trillion, preferably 1 billion to 100 billion equivalent per day. The content of the lactic acid bacteria of the present invention in the lactic acid bacteria preparation is set to 0.02 to 2000 mg, preferably 2 to 200 mg in terms of dry cell weight, taking into consideration the symptoms, age, sex, etc. of the human being administered, atopic skin What is necessary is just to determine suitably within the effective amount range for a flame relieving function.

EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited at all by these Examples.
(Example 1)
Lactobacillus plantarum HSK201 dry cells ingested MRS medium composition showing a manufacturing Lactobacillus plantarum HSK201 the following (viable) (bacteria count: 10 ⁴ / ml), 15-48 hours at 37 ° C. After culturing, a culture solution having a viable cell count of about 10 ⁸ cells / ml was obtained. The obtained culture broth was collected by centrifugation at 3,000 × g for 20 minutes, and washed twice with distilled water to obtain bacterial cells. The cells were suspended in distilled water and freeze-dried to obtain dry cells. (Hereinafter dry cells)
The composition of the MRS medium is shown (Difco Lactobacilli MRS Broth # 288130).
Proteose peptone no. 3 10g
10g beef extract
Yeast extract 5g
D-glucose 20g
Polysorbate 80 1g
2g of ammonium citrate
Sodium acetate 5g
Manganese sulfate 0.1g
Magnesium sulfate 0.05g
2g dipotassium phosphate
1000ml distilled water
pH 6.5
Sterilized at 121 ° C for 15 minutes

(Example 2)
Production of Lactobacillus plantarum HSK201 dry cells (dead cells) Live cells obtained by the same method as in Example 1 were suspended in distilled water, heated at 100 ° C. for 30 minutes, and freeze-dried. As a result, dried dead cells (hereinafter referred to as dried dead cells) were obtained.

Example 3
Promotion of Th1-type cytokine (IL-12) production from mouse spleen cells
BALB / c mice (7-week-old female) are administered intraperitoneally with 100 μg of ovalbumin (hereinafter abbreviated as OVA) together with an adjuvant (a general term for substances that increase antibody production and enhance immune responses by mixing or combining with antigen). did. Ten days later, the same amount of OVA was additionally sensitized. Seven days after the additional sensitization, spleen cells were collected and prepared at 2 × 10 ⁶ cells / mL using RPMI1640 medium containing 10% FBS. A 96-well plate was seeded, OVA having a final concentration of 1 mg / mL and 10 μg / mL of each lactic acid bacteria killed cell were added, and cultured for 48 hours. After collecting the culture supernatant, IL-12 (p70) was measured using a Quantikine Immunoassay kit (R & D Systems).
The results are shown in FIG. As shown in FIG. 2A, the Lactobacillus plantarum HSK201 strain of the present invention had the highest IL-12 production inducing ability when compared with the IL-12 production inducing ability of various lactic acid bacteria belonging to the genus Lactobacillus. . In comparison with lactic acid bacteria belonging to the same Lactobacillus plantarum, the IL-12 producing ability of HSK201 strain was also high (FIG. 2B).

Example 4
Inhibition of Th2-type cytokine (IL-4) production from mouse spleen cells In the same manner as in Example 3, spleen cells were cultured for 96 hours together with OVA and dead cells of each lactic acid bacterium, using a Quantikine Immunoassay kit (R & D Systems). IL-4 in the supernatant was measured.
The results are shown in FIG. As shown in FIG. 3, spleen cells of OVA-sensitized mice produced high IL-4 by antigen stimulation, but IL-4 production was suppressed by the addition of lactic acid bacteria. Among various lactic acid bacteria belonging to the genus Lactobacillus, HSK201 strain had the strongest ability to suppress IL-4 production (FIG. 3A), and was also suppressed more strongly in comparison with the same bacterial species (FIG. 3B).

(Example 5)
Effect of administration on atopic dermatitis model mice 35 NC / Nga mice (5 weeks old, male) known as atopic dermatitis model mice were used. Ten animals were fed with food containing 0.375% of HSK201 strain live cells, and 10 animals were fed with the same amount of dead heat cells until the end of the experiment. As a control, a control food group (10 animals) that was fed with food containing no lactic acid bacteria was provided. The abdomen and neck were shaved, and 2 weeks after switching to the test meal, 5% picryl chloride was applied to the abdomen and 120 μL to the foot pad. Then, 0.8% picryl chloride dissolved in olive oil and mite extract were applied once a week to the head and neck as onset-inducing reagents (first 4 weeks; 667 μg / mL, second 3 weeks; 1.6 mg / mL) )did. The symptom score was evaluated once a week in four grades (0: no symptoms, 1: mild, 2: moderate, 3: severe) of skin inflammation in the ear and neck (edema, dryness, bleeding, tissue loss). . The thickness of the auricle was measured once a week using a thickness gauge. In addition, blood was collected four times over time, and serum total IgE and tick-specific IgE values were measured.
The results are shown in FIG. Skin inflammation occurred due to continuous application of picryl chloride and mite extract. In the HSK201 strain intake group, the total IgE level in the blood after 8 weeks could be suppressed to 40%, and mite antigen-specific IgE could be significantly suppressed. At the same time, significant suppression of symptom scores and suppression of ear thickening were also observed. And this inhibitory effect was observed substantially in the dead cells as well as the live cells.
Thus, since HSK201 strain has an extremely high alleviating effect on atopic skin inflammation, it can be used as an active ingredient of an atopic dermatitis relieving agent, including for severe atopic diseases.

Example 6
Changes in Lactobacillus plantarum in feces
170 mL of HSK201 lactic acid bacteria beverage or placebo beverage was ingested daily for 2 months. The viable cell count in the HSK201 lactic acid bacteria beverage was 3.7 × 10 ⁸ cells / mL. Feces were collected before ingestion and after 2 consecutive months of ingestion. DNA was extracted from the stool using QIAGEN's QIA amp DNA stool Mini Kit, and real-time PCR was performed using L. plantarum-specific primers to quantify L. plantarum contained in the stool.
The results are shown in FIG. As shown in FIG. 5, the amount of Lactobacillus plantarum HSK201 strain in feces was significantly increased in the group ingesting a lactic acid bacteria beverage containing Lactobacillus plantarum HSK201 strain. This revealed that Lactobacillus plantarum strain HSK201 reaches the intestine alive by ingestion.

  As described above, since the atopic dermatitis alleviating agent of the present invention has a remarkable effect, it can be used as a pharmaceutical product for serious atopic diseases, and can be administered orally, so that it is routinely used as a health food and various foods and drinks. Since it can be ingested, it can also be used as a food for preventing atopic dermatitis.

It is the result of amplifying the HSK201 strain gene by PCR reaction using L.plamtarum-specific primers. It is the result which showed the IL-12 production induction ability between the lactic acid bacteria from which a bacterial species differs from the spleen cell of an OVA sensitized mouse | mouth by in vitro stimulation of various lactic acid bacteria. It is the result which showed IL-12 production induction ability between L. plantarum from the spleen cell of an OVA sensitized mouse | mouth by in vitro stimulation of various lactic acid bacteria. It is the result which showed the IL-4 production suppression ability between the lactic acid bacteria from which a bacterial species differs from the spleen cell of an OVA sensitized mouse | mouth by in vitro stimulation of various lactic acid bacteria. It is the result which showed the IL-4 production suppression ability between L.plantarum from the spleen cell of an OVA sensitized mouse | mouth by the in vitro stimulation of various lactic acid bacteria. It is the result which showed the change of the serum total IgE value by administration of HSK201 lactic acid bacteria with respect to the mouse | mouth which induced the atopic dermatitis like symptom. In the figure, ◆ indicates administration of live bacteria, ■ indicates administration of dead bacteria, × indicates a control food, and ○ indicates that the onset reagent is not applied. It is the result which showed the change of the serum mite antigen specific IgE value by the administration of HSK201 lactic acid bacteria with respect to the mouse | mouth which induced the atopic dermatitis like symptom. It is the result which showed the change of the symptom score by administration of HSK201 lactic acid bacteria with respect to the mouse | mouth which induced the atopic dermatitis like symptom. It is the result which showed the change of the auricle thickness by the administration of HSK201 lactic acid bacteria with respect to the mouse | mouth which induced the atopic dermatitis like symptom. It is the result shown about the bacterial count change of L. plantarum in feces before and after ingestion of HSK201 fermented milk.

Claims (6)

  An atopic dermatitis alleviating agent containing, as an active ingredient, a microbial cell, a microbial cell culture, a microbial cell treated product or an extract thereof of Lactobacillus plantarum HSK201 strain (NITE P-589).   The formulation for oral administration for the prevention and / or treatment of atopic dermatitis which mix | blended the atopic dermatitis alleviation agent of Claim 1.   A preparation for external use for the prevention and / or treatment of atopic dermatitis, comprising the atopic dermatitis alleviating agent according to claim 1.   A food and drink comprising the atopic dermatitis alleviating agent according to claim 1.   A cosmetic comprising the atopic dermatitis alleviating agent according to claim 1.   Lactobacillus plantarum HSK201 strain (NITE P-589) which can be used as an active ingredient of the atopic dermatitis alleviating agent according to claim 1.