WO2024167307A1 - Composition containing bifidobacterium longum as active ingredient for prevention or treatment of allergic diseases - Google Patents

Abstract

The present invention relates to a composition containing Bifidobacterium longum as an active ingredient for the prevention or treatment of allergic diseases. According to an aspect, the strain can be effectively used for the prevention, alleviation, and/or treatment of allergic diseases, inflammatory respiratory diseases, asthma, allergic asthma, eosinophilic asthma, and/or eosinophilia through alleviating the influx of inflammatory cells, reducing cytokine production, mitigating antibody generation, and relieving inflammatory cell infiltration.

Description

Composition for preventing or treating allergic diseases containing Bifidobacterium longum as an active ingredient

It relates to the use of microorganisms for the prevention or treatment of allergic diseases.

Allergic diseases are diseases caused by allergies, which are antigen-antibody reactions that occur when an antigen or allergen (the cause of allergy) enters an organism and antibodies are produced against it, and then the same substance (antigen) enters the body again (Dictionary of Nutrition, March 15, 1998, Chae Beom-seok, Kim Eul-sang). The main diseases are respiratory diseases, dermatitis (including atopic dermatitis), drug allergies, drug allergies, and serum sickness, and various types of diseases appear depending on the type of allergen or the tissue causing the allergic reaction.

Allergic respiratory disease refers to respiratory diseases involving allergic reactions, and representative examples include bronchial asthma, bronchitis, and allergic rhinitis.

Bronchial asthma is a disease in which the bronchi in the lungs become very sensitive, sometimes narrowing the bronchi, causing shortness of breath and severe coughing. It is an allergic disease caused by an allergic inflammatory reaction in the bronchi. The prevalence of bronchial asthma is on the rise due to recent westernization and changes in eating habits.

Bronchitis is an inflammation of the bronchi in the lungs. Unlike acute bronchitis, which is caused by a viral infection in more than 90% of cases, chronic bronchitis is caused by chronic inhalation of highly irritating dust from occupations such as air pollution, coal mining, grain handling, textile manufacturing, livestock farming, and metal casting. It is characterized by wheezing and shortness of breath during exercise, low oxygen saturation, and persistent phlegm and cough for more than two years, lasting more than three months each year.

Allergic rhinitis is a disease in which the nasal mucosa shows a hypersensitivity reaction to a specific substance. When the nasal mucosa is exposed to an allergen or antigen that causes allergies, various types of inflammatory cells mediated by IgE antibodies, including mast cells and eosinophils, flock to the stimulated area, causing an inflammatory response through various mediators secreted by them.

Among these allergic diseases, bronchial asthma has various pathogenesis mechanisms, in which the Th2 (T helper type 2) type immune response is enhanced, which increases the secretion of interleukin-4, 5, and 13 (Liu et al., 2006; Williams et al., 2012), and in conjunction with this response, many inflammatory cells, including eosinophils, migrate and infiltrate into lung tissue (Zhou et al., 2011). In addition, inflammatory cells release various proinflammatory factors and chemotactic factors, which further aggravate the inflammatory response, increase mucus secretion by goblet cells in the airway, and cause airway hyperresponsiveness (Chibana et al., 2008). Due to this series of reactions, patients with bronchial asthma show clinical symptoms such as dyspnea, cyanosis, and chest pain.

Currently, drugs used to treat allergic diseases include steroid preparations, antihistamines, bronchodilators, and antibiotics. Steroid preparations and antibiotics are used to treat allergic diseases by suppressing immune responses and inflammatory responses, and bronchodilators are used to offset clinical symptoms such as dyspnea when they occur. However, these drugs have been found to have side effects such as immunosuppression, bone marrow suppression, antibiotic resistance, and side effects when used long-term, so their use as therapeutic agents is very limited.

Therefore, there is a need for a treatment that can replace the existing treatments for allergic diseases.

One aspect is to provide a pharmaceutical composition for preventing or treating allergic diseases, comprising as an active ingredient a Bifidobacterium longum strain belonging to the genus Bifidobacterium sp. , an endoplasmic reticulum derived from the strain, a lysate or culture medium of the strain, or a mixture thereof.

Another aspect is to provide a health functional food for preventing or improving allergic diseases, which contains as an effective ingredient a Bifidobacterium longum strain belonging to the genus Bifidobacterium sp. , an endoplasmic reticulum derived from the strain, a lysate or culture medium of the strain, or a mixture thereof.

Another aspect provides a method for preventing or treating an allergic disease, comprising administering to a subject in need thereof an effective amount of a strain of Bifidobacterium longum belonging to the genus Bifidobacterium sp. , vesicles derived from the strain, a lysate or culture medium of the strain, or a mixture thereof.

Another aspect provides the use of a strain of Bifidobacterium longum belonging to the genus Bifidobacterium sp. , an endoplasmic reticulum derived from the strain, a lysate or culture medium of the strain or a mixture thereof, for use in the manufacture of a pharmaceutical preparation for preventing or treating allergic diseases.

One aspect provides a pharmaceutical composition for preventing or treating allergic diseases, comprising as an active ingredient a Bifidobacterium longum strain belonging to the genus Bifidobacterium sp. , an endoplasmic reticulum derived from the strain, a lysate or culture medium of the strain, or a mixture thereof.

The above Bifidobacterium longum strain may be a strain deposited under the deposit number KCTC 14523BP.

The strain may have an effect of preventing, improving or treating allergic diseases. The allergic diseases may be inflammatory respiratory diseases, asthma, allergic asthma, allergic rhinitis, eosinophilic asthma or eosinophilia.

The above inflammatory respiratory disease may be any one selected from the group consisting of chronic obstructive pulmonary disease (COPD), acute lung injury, empyema, lung abscess, pneumonia, pulmonary tuberculosis, cough, sputum, bronchitis, pharyngitis, tonsillitis, sinusitis, rhinitis, obliterative bronchiolitis, and laryngitis.

The strain may have an effect of alleviating the influx of inflammatory cells. In addition, the strain may have an effect of alleviating the influx of inflammatory cells in eosinophilic airway inflammation. In addition, the strain may have a great effect of alleviating the influx of eosinophils among inflammatory cells.

The strain may have an effect of alleviating cytokine production. In addition, the strain may have an effect of reducing IL-4, IL-5, IL-13 and Eotaxin among cytokines. IL-4 and IL-13 are cytokines important for Th2 cell differentiation and immune response. IL-5 and Eotaxin are cytokines important for eosinophil generation and migration. In addition, the strain may reduce IL-4 and/or IL-13 or effectively reduce Th2 cell differentiation. In addition, the strain may effectively reduce at least one selected from the group consisting of IL-5, Eotaxin and eosinophils.

The strain may have an effect of alleviating antibody production in serum. The antibody may be a specific antibody for an allergen. The antibody may be an IgE antibody. The effect of alleviating antibody production may be alleviating the production of a specific antibody for an allergen without affecting the total antibody production and change amount.

The strain may have an effect of alleviating inflammatory cell infiltration in lung tissue. In addition, the strain may have an effect of alleviating inflammatory cell infiltration in the basal part of blood vessels and alveoli.

The term "treat" in this specification may mean that allergic diseases, etc. are cured in a shorter period of time compared to natural healing. The treatment may include improvement and/or alleviation of allergic diseases, etc. In addition, the treatment may mean cure and/or recovery of symptoms caused by allergic diseases, etc.

The term "vesicle" as used herein refers to a particle secreted from a cell and released into the extracellular space, and may include a number of different species, such as exosomes, ectosomes, microvesicles, microparticles, and exosome-like vesicles. Extracellular vesicles can reflect the state of the secreting source cell (donor cell), exhibit various biological activities depending on which cell they are secreted from, and play an important role in cell-to-cell interaction while transferring genetic material and proteins between cells. In addition, cell-derived substances including the vesicles cause diseases or stimulate immune cells to fight diseases, and have the effect of helping humans decompose and absorb substances that they cannot digest through the metabolic process of microorganisms. The above endoplasmic reticulum is a membrane-structured endoplasmic reticulum, which is divided into an inside and an outside, and contains plasma membrane lipids, plasma membrane proteins, nucleic acids, and cytoplasmic components of the cell, and may be smaller than the original cell.

In one specific example, the vesicles may be isolated from a cell lysate of a culture medium of a Bifidobacterium longum strain.

In one specific example, the extracellular vesicles may have a diameter of 10 nm to 400 nm. For example, 10 nm to 400 nm, 10 nm to 350 nm, 10 nm to 300 nm, 10 nm to 250 nm.

In this specification, the term "culture medium" may be used interchangeably with "culture supernatant", "conditioned culture medium" or "conditioned medium", and may mean the entire medium including the strain, its metabolites, extra nutrients, etc., obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that Bifidobacterium longum can grow and survive in a test tube. In addition, the culture medium may mean a culture medium obtained by culturing the strain and removing the bacterial cells from the bacterial cell culture. Meanwhile, the liquid from the culture medium from which the bacterial cells have been removed is also called a "supernatant", and may be obtained by allowing the culture medium to stand still for a certain period of time and taking only the liquid in the upper layer excluding the portion that has settled to the lower layer, removing the bacterial cells through filtration, or centrifuging the culture medium to remove the sediment at the lower layer and taking only the liquid at the upper layer. The above "mycelia" refers to the strain of the present invention itself, and includes the strain itself selected by separating from a skin sample or the like, or the strain separated from the culture solution by culturing the strain. The mycelia can be obtained by centrifuging the culture solution and taking the portion that has settled to the lower layer, or by allowing the mycelia to settle to the lower layer of the culture solution by gravity and then leaving it alone for a certain period of time and then removing the upper liquid.

The above culture solution may include the culture solution itself, a concentrate thereof, or a lyophilized product obtained by culturing the strain, or a culture supernatant obtained by removing the strain from the culture solution, a concentrate thereof, or a lyophilized product thereof.

The above culture solution may be obtained by culturing Bifidobacterium longum in an appropriate medium (e.g., R2A medium or TSA medium) at a temperature of more than 10°C or less than 40°C for a certain period of time, for example, 4 to 50 hours.

In one specific example, the culture supernatant of the strain can be obtained by a step of removing the strain by centrifuging or filtering the strain culture solution.

In another specific embodiment, the concentrate can be obtained by a step of concentrating the strain culture itself, or the supernatant obtained after filtering the culture using centrifugation or a filter.

The culture medium and culture conditions for culturing the above Bifidobacterium longum can be appropriately selected or modified by a person of ordinary skill in the art.

The term "disruption solution" in this specification may mean a product obtained by disrupting the cell wall of the strain itself by chemical or physical force.

The term "culture solution extract" in this specification means an extract from the culture solution or a concentrate thereof, and may include an extract, a dilution or concentrate of the extract, a dried product obtained by drying the extract, or a adjusted or purified product thereof, or a fraction obtained by fractionating the same.

The composition is present in an amount of 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, 0.00001 wt% to 10 wt%, 0.00001 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 It may comprise from 0.1 wt % to 30 wt %, from 0.1 wt % to 20 wt %, from 0.1 wt % to 10 wt %, or from 0.1 wt % to 5 wt % of the strain, a lysate thereof, a culture medium, or an extract of the culture medium thereof.

The term, "including as an active ingredient" means that the strain of the present specification, vesicles derived from the strain, a lysate of the strain, a culture medium, or an extract of the culture medium thereof are added to an extent capable of exhibiting the effects mentioned above, and includes formulation in various forms by adding various components as auxiliary components for drug delivery and stabilization, etc.

In another specific embodiment, the composition may be a pharmaceutical composition.

The pharmaceutical composition may additionally comprise a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the glidant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a glidant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium dihydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The glidant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated as an oral or parenteral dosage form. The oral dosage form may be a granule, a powder, a liquid, a tablet, a capsule, a dry syrup, or a combination thereof. The parenteral dosage form may be an injection.

The above composition may be a health functional food composition.

The above health functional food composition can be used alone or in combination with the strain or its culture solution or with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the active ingredient mixture can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present specification can be added in an amount of 15 parts by weight or less to the raw material. There is no particular limitation on the type of the above health functional food. Among the types of health functional foods, the beverage composition can contain various flavoring agents or natural carbohydrates as additional ingredients, like a conventional beverage. The natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame can be used. The above health food composition may also contain nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, or a combination thereof. The above health functional food composition may also contain fruit pulp for the production of natural fruit juice, fruit juice drinks, vegetable drinks, or a combination thereof.

The above composition may be a cosmetic composition.

The above cosmetic composition may have, for example, a cosmetic formulation of a flexible toner, a nourishing toner, a massage cream, a nourishing cream, an essence, a pack, a gel, an ampoule or a skin adhesive type.

The ingredients included in the above cosmetic composition may include ingredients commonly used in cosmetic compositions in addition to the composition as an effective ingredient, and may include, for example, conventional auxiliary agents and carriers such as stabilizers, solubilizers, vitamins, pigments, and fragrances.

Additionally, the composition may be a composition for external use on the skin.

In the present specification, the skin external preparation may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof. The skin external preparation may be appropriately mixed with ingredients commonly used in skin external preparations such as cosmetics or medicines, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof, as needed. The above skin external preparation may also appropriately contain metal sequestrants such as sodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannin, bellapamil, licorice extract, glablidine, hot water extract of the fruit of Kalin; various crude drugs; tocopheryl acetate, glycyrrhizic acid, tranexamic acid and derivatives or salts thereof; and sugars such as vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

Another aspect provides the use of the composition described above for the manufacture of a pharmaceutical preparation for the prevention or treatment of allergic diseases.

Another aspect provides a method of preventing, ameliorating, or treating a condition of a subject comprising the step of treating or administering to a subject in need thereof an effective amount of the composition as described above.

The condition of the above entity may be related to an allergic disease. The allergic disease is as described above.

Administration can be by any method known in the art. Administration can be directly administered to the subject by any means, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. The administration can be systemic or local.

The subject may be a mammal, for example, a human, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat. The subject may be an individual in need of an effect of improving a condition related to an allergic disease. The allergic disease is as described above.

The above administration is in an amount of 0.00001 mg to 1,000 mg per subject per day, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, Or it may be administered 10 mg to 25 mg. However, the dosage may be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage considering these factors. The number of administrations can be once a day or twice or more within the range of clinically acceptable side effects, and the administration site can be administered to one or more sites, and the total number of administration days can be from 1 day to 30 days for one treatment, daily or at intervals of 2 to 5 days. If necessary, the same treatment can be repeated after an appropriate period. For animals other than humans, the same dosage as for humans per kg can be administered, or the amount converted from the above dosage can be administered based on the volume ratio (e.g., average value) of the organs (heart, etc.) of the target animal and the human.

According to the strain according to the daily aspect and the composition containing the strain as an effective ingredient, it can be effectively used for the prevention, improvement, and treatment of allergic diseases.

Figure 1 shows changes in immune cells upon strain treatment according to one specific example; PBS: negative control, Alum: positive control, Bifidobacterium longum: experimental group.

Figure 2 is a graph showing changes in cytokines in bronchoalveolar lavage fluid when treated with a strain according to one specific example; PBS: negative control, Alum: positive control, Bifidobacterium longum: experimental group.

Figure 3 is a graph showing the change in the total amount of three types of antibodies (IgE, IgG1, and IgG2c) and the amount of ovalbumin-specific antibody IgE when treating the strain according to one specific example in serum; PBS: negative control group, Alum: positive control group, Bifidobacterium longum: experimental group.

Figure 4 is a graph showing changes in inflammatory cell infiltration in lung tissue upon strain treatment according to one specific example; PBS: negative control group, Alum: positive control group, Bifidobacterium longum: experimental group.

The following examples are provided to illustrate in more detail. However, these examples are provided only to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of strains

Bifidobacterium longum deposited under the name KCTC14523BP was prepared as follows.

Specifically, to culture the strain, the strain was cultured in MRS broth (DeMan-Rogosa-Sharpe, MB Cell) at 37℃ under anaerobic conditions for 2 days. Afterwards, the culture solution was centrifuged at 5000 xg for 20 minutes to remove bacterial debris. Afterwards, it was dispensed into clean MRS broth at ^1x109 CFU/ml and stored in an ultra-low temperature freezer for use in the experiment.

Example 2. Induction of eosinophilic asthma animal model and administration of strains

To induce an animal model of eosinophilic asthma, 7-8 week old mice were used. On days 0 and 7, a mixture of LPS-free ovalbumin and aluminum hydroxide was administered intraperitoneally to induce immune memory to ovalbumin. On days 14, 15, 21, and 22, after anesthetizing the mice, ovalbumin alone was administered intranasally to induce airway inflammation.

After exposure to ovarian albumin in the nasal cavity, the strain of Example 1 was administered orally once a day for a total of 7 days on days 14 to 19, 21, and 22 to the experimental group. The strain administration dose was ^1X109 CFU/mouse per time.

On the 23rd, mice were dissected to obtain samples for the experiment. The obtained samples were bronchoalveolar lavage fluid, blood, and lung tissue. The mouse dissection was started by anesthetizing the mouse using an animal anesthetic, and then making an incision from the neck to the abdominal cavity. For blood, the diaphragm was removed and heart blood collection was performed. For bronchoalveolar lavage fluid, an IV catheter was connected to the trachea, inserted, and a 1 ml syringe containing cooled and sterile PBS was connected. By the method of injecting into the trachea and then recovering. Bronchoalveolar lavage fluid (BAL fluid) was obtained, washed twice with 1 ml, and this was repeated twice. For lung tissue, perfusion was performed from the heart using a 5 ml syringe and 1x PBS to remove any remaining blood in the lung tissue, and the lung tissue was excised, frozen, and fixed in 4% formaldehyde to obtain it. These were used in the following experimental examples.

In the experimental examples below, for statistical analysis, nonparametric statistical analysis methods such as the Kruskal-Wallis method and the Mann-Whitney U test method were used. A significant difference was determined when p < 0.05. PRISM 7 was used as the statistical program.

In the experimental examples below, the test was performed a total of three times, and each group consisted of 12 to 15 mice.

Experimental Example 1. Analysis of the effect of alleviating the influx of inflammatory cells into bronchoalveolar lavage fluid

The effect of the strain of Example 1 on alleviating the influx of inflammatory cells into the bronchoalveolar lavage fluid was analyzed. Through PBS-administered mice (negative control group), nasal sensitized mice with ovalbumin alone (positive control group), and strain-administered mice (experimental group), the immune cells influx into the bronchoalveolar lavage fluid during airway inflammation and the effect of the strain of Example 1 on reducing airway inflammation were analyzed.

Specifically, first, mice sensitized intranasally with ovamin alone (positive control group) and mice administered with strain (experimental group) were prepared using the method of Example 2.

Analysis of bronchoalveolar lavage fluid was conducted as follows. First, the obtained bronchoalveolar lavage fluid was centrifuged to separate cells and supernatant. After collecting cells, RBCs (red blood cells) were removed, and the number and type of cells were analyzed.

The test was performed a total of three times, and the results of each experiment were added together to perform a comparative analysis.

Figure 1 shows changes in immune cells upon strain treatment according to one specific example; PBS: negative control, Alum: positive control, Bifidobacterium longum: experimental group.

As shown in Figure 1, after inducing an asthma animal model using ovalbumin and Alum, it was confirmed that statistically significant airway inflammation occurred in ovalbumin-only nasal sensitized mice (positive control group) compared to PBS-administered mice (negative control group). The infused inflammatory cells were eosinophils, neutrophils, and macrophages, and it was confirmed that the proportion of eosinophils was the highest.

On the other hand, in the experimental group administered the strain according to one specific example, it was confirmed that a decrease in inflammatory cells was induced by approximately 37% compared to mice nasally sensitized with ovalbumin alone (positive control group). The effect of administering the strain according to one specific example was shown to be particularly notable in the inhibition of the influx of eosinophils among the introduced inflammatory cells.

The above results imply that the strain according to one specific example can be useful for preventing, improving, or treating allergic diseases, inflammatory respiratory diseases, asthma, allergic asthma, eosinophilic asthma, or eosinophilia.

Experimental Example 2. Analysis of the effect of alleviating cytokine production in bronchoalveolar lavage fluid

The effect of the strain of Example 1 on alleviating cytokine production in bronchoalveolar lavage fluid was analyzed. Through PBS-administered mice (negative control group), nasal sensitized mice with ovalbumin alone (positive control group), and strain-administered mice (experimental group), the cytokines that increase during airway inflammation in bronchoalveolar lavage fluid and the effect of the strain of Example 1 on alleviating cytokine production were analyzed.

Specifically, first, mice sensitized intranasally with ovamin alone (positive control group) and mice administered with strain (experimental group) were prepared using the method of Example 2.

The analysis of bronchoalveolar lavage fluid was conducted as follows. First, the obtained bronchoalveolar lavage fluid was centrifuged to separate cells and supernatant. After taking the supernatant, the amount of cytokine production and changes were evaluated through sandwich ELISA and colorimetric analysis. The evaluation items were IL-4, IL-5, IL-13, and Eotaxin, and the entire bronchoalveolar lavage fluid obtained after three repeated tests was analyzed.

Figure 2 is a graph showing changes in cytokines in bronchoalveolar lavage fluid when treated with a strain according to one specific example; PBS: negative control, Alum: positive control, Bifidobacterium longum: experimental group.

As shown in Figure 2, after inducing an asthma animal model using ovalbumin and Alum, the nasal sensitized mice only with ovalbumin (positive control group) showed a statistically significant increase in four cytokines (IL-4, IL-5, IL-13, Eotaxin) compared to the PBS-administered mice (negative control group).

The four cytokines that increased were IL-4 and IL-13, which are important cytokines for Th2 cell differentiation and immune response, and IL-5 and Eotaxin, which are important cytokines for eosinophil generation and migration.

On the other hand, in the experimental group administered the strain according to one specific example, it was confirmed that the production of IL-4 was suppressed by about 60%, the production of IL-5 was suppressed by about 50%, the production of IL-13 was suppressed by about 48%, and the production of Eotaxin was suppressed by about 55% compared to the nasal sensitized mice with ovalbumin alone (positive control group).

The above results indicate that the strain according to one specific example can effectively reduce the production of IL-4 or IL-13 or Th2 cell differentiation in allergic diseases, inflammatory respiratory diseases, asthma, allergic asthma, eosinophilic asthma, or eosinophilia.

In addition, it is shown that the strain according to one specific example can effectively reduce the production of IL-5, Eotaxin or eosinophils in allergic diseases, inflammatory respiratory diseases, asthma, allergic asthma, eosinophilic asthma or eosinophilia.

This means that the strain according to one specific example can be useful for preventing, improving, or treating allergic diseases, inflammatory respiratory diseases, asthma, allergic asthma, eosinophilic asthma, or eosinophilia.

Experimental Example 3. Analysis of the effect of alleviating antibody production in serum

The effect of the strain of Example 1 on alleviating antibody production in serum was analyzed. A comparative experiment was conducted using mice administered PBS (negative control group), mice intranasally sensitized with ovalbumin alone (positive control group), and mice administered the strain (experimental group).

Specifically, first, mice sensitized intranasally with ovamin alone (positive control group) and mice administered with strain (experimental group) were prepared using the method of Example 2.

Serum analysis was conducted as follows. Blood was obtained by the method of Example 1, and then centrifuged to separate the serum. The production and changes in total antibodies and ovalbumin-specific antibodies in the separated serum were analyzed using sandwich ELISA, indirect ELISA, and colorimetric analysis. The evaluation items were three types of antibodies (IgE, IgG1, and IgG2c), and all serum samples in the experiment were evaluated.

The change in the total amount of three types of antibodies (IgE, IgG1, and IgG2c) introduced into the serum during airway inflammation and the effect of the strain of Example 1 on reducing the production of the total amount of three types of antibodies were analyzed.

In addition, the change in ovalbumin-specific antibody IgE influx into the serum during airway inflammation and the effect of the strain of Example 1 on reducing ovalbumin-specific antibody IgE production were analyzed.

Figure 3 is a graph showing the change in the total amount of three types of antibodies (IgE, IgG1, and IgG2c) and the amount of ovalbumin-specific antibody IgE when treating the strain according to one specific example in serum; PBS: negative control group, Alum: positive control group, Bifidobacterium longum: experimental group.

As shown in Figure 3, after inducing an asthma animal model using ovalbumin and Alum, all three types of antibodies increased in nasal sensitized mice nasally sensitized with ovalbumin alone (positive control group) compared to mice administered PBS (negative control group).

On the other hand, in the experimental group administered the strain according to one specific example, it was confirmed that the production of IgE, IgG1, and IgG2c decreased compared to the mice nasally sensitized with ovalbumin alone (positive control group), and it was confirmed that the production of ovalbumin-specific IgE also decreased by approximately 40% with a statistically significant decrease.

The above results indicate that the strain according to one specific example can effectively reduce the total amount of antibodies and the production of antigen-specific IgE antibodies introduced into the serum during airway inflammation.

This means that the strain according to one specific example can be useful for preventing, improving, or treating allergic diseases, inflammatory respiratory diseases, asthma, allergic asthma, eosinophilic asthma, or eosinophilia.

Experimental Example 4. Evaluation of the effect of alleviating inflammatory cell infiltration in lung tissue

The effect of the strain of Example 1 on alleviating inflammatory cell infiltration in lung tissue was analyzed. Through mice administered PBS (negative control group), mice nasally sensitized with ovalbumin alone (positive control group), and mice administered the strain (experimental group), the infiltration of inflammatory cells around the airways and blood vessels that occurs during airway inflammation in lung tissue and the effect of the strain of Example 1 on alleviating inflammatory cell infiltration were analyzed.

Specifically, first, mice sensitized intranasally with ovamin alone (positive control group) and mice administered with strain (experimental group) were prepared using the method of Example 2.

Analysis of inflammatory cell infiltration in lung tissue was conducted as follows. First, fixed lung tissue was sectioned at 4 μcM and stained with hematoxylin and eosin. Using an optical microscope, inflammation in the tissue was identified (peri-vascular, peri-bronchial region) and quantified according to the degree, and then compared and analyzed to compare the degree of inflammatory cell infiltration.

Figure 4 is a graph showing changes in inflammatory cell infiltration in lung tissue upon strain treatment according to one specific example; PBS: negative control group, Alum: positive control group, Bifidobacterium longum: experimental group.

As shown in Figure 4, after inducing an asthma animal model using ovalbumin and Alum, it was confirmed that inflammatory cells infiltrated the airways and around blood vessels in ovalbumin-only nasal sensitized mice (positive control group) compared to PBS-administered mice (negative control group), and the same was observed in the alveoli.

On the other hand, in the experimental group administered the strain according to one specific example, it was confirmed that inflammatory cell infiltration was reduced compared to mice nasally sensitized with ovalbumin alone (positive control group), and it was confirmed that the cluster of inflammatory cells infiltrating the basal part of blood vessels and alveoli was reduced.

The above results imply that the strain according to one specific example can be useful for preventing, improving, or treating allergic diseases, inflammatory respiratory diseases, asthma, allergic asthma, eosinophilic asthma, or eosinophilia.

[Acceptance number]

Name of depositor: Korea Biological Resource Center (overseas)

Accession number: KCTC 14523BP

Date of acceptance: 20210408

Claims (13)

A pharmaceutical composition for preventing or treating allergic diseases, comprising as an active ingredient a Bifidobacterium longum strain belonging to the genus Bifidobacterium, an endoplasmic reticulum derived from the strain, a lysate or culture medium of the strain, or a mixture thereof. In claim 1, the strain is a pharmaceutical composition for preventing or treating allergic diseases, deposited under the deposit number KCTC 14523BP. A pharmaceutical composition according to claim 1, wherein the allergic disease is an inflammatory respiratory disease, asthma, allergic asthma, allergic rhinitis, eosinophilic asthma or eosinophilia. A pharmaceutical composition for preventing or treating allergic diseases, wherein the strain in claim 1 reduces at least one selected from the group consisting of IL-5, Eotaxin, and eosinophils. A pharmaceutical composition for preventing or treating allergic diseases, wherein in claim 1, the strain reduces at least one selected from the group consisting of IL-4, IL-13 and IgE or reduces Th2 cell differentiation. In claim 1, the pharmaceutical composition is a pharmaceutical composition for preventing or treating allergic diseases for oral administration. A health functional food for preventing or improving allergic diseases, comprising as an effective ingredient a Bifidobacterium longum strain belonging to the genus Bifidobacterium , an endoplasmic reticulum derived from the strain, a lysate or culture solution of the strain, or a mixture thereof. In claim 7, the strain is a health functional food for preventing or improving allergic diseases, deposited under the deposit number KCTC 14523BP. In claim 7, the health functional food is an inflammatory respiratory disease, asthma, allergic asthma, allergic rhinitis, eosinophilic asthma or eosinophilia. In claim 7, the strain is a health functional food for preventing or improving allergic diseases, which reduces at least one selected from the group consisting of IL-5, Eotaxin, and eosinophils. In claim 7, the strain is a health functional food for preventing or improving allergic diseases that reduces one or more selected from the group consisting of IL-4, IL-13 and IgE or reduces Th2 cell differentiation. A method for preventing or treating an allergic disease, comprising the step of administering to a subject in need thereof an effective amount of a strain of Bifidobacterium longum belonging to the genus Bifidobacterium, an endoplasmic reticulum derived from the strain, a lysate or culture medium of the strain, or a mixture thereof. Use of a strain of Bifidobacterium longum belonging to the genus Bifidobacterium sp. , an endoplasmic reticulum derived from said strain, a lysate or culture medium of said strain, or a mixture thereof, for use in the manufacture of a pharmaceutical preparation for preventing or treating allergic diseases.

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