KR102434641B1 - Food composition for improving respiratory function using Pediococcus pentosaceus - Google Patents

Abstract

The present invention discloses a composition for improving lung diseases such as inflammation and chronic obstructive pulmonary disease using Pediococcus pentosaceus . Pediococcus pentosaceus ( Pediococcus pentosaceus ) significantly reduced the number of inflammatory cells, such as neutrophils and macrophages, which are involved in the main etiology in the lung lavage fluid in the COPD animal model, and IL-6, IL in the lung lavage fluid and lung tissue By inhibiting the expression of inflammatory cytokines and chemokines such as -1β, IL-17, TNF-α, IFN-γ, GM-CSF, MIP-2, KC, TARC, MCP-1, and MDC, it has a respiratory disease improving activity .

Description

Food composition for improving respiratory function using Pediococcus pentosaceus {Food composition for improving respiratory function using Pediococcus pentosaceus}

The present invention relates to a composition for improving inflammation or chronic obstructive pulmonary disease using Pediococcus pentosaceus .

Inflammation is a local defense reaction of the living body that appears in response to a pathological condition caused by physical trauma, harmful chemicals, infection by bacteria, fungi, viruses, or irritants in in vivo metabolites. Although various biochemical phenomena are involved in the inflammatory response, it is known that macrophages play an important role in the inflammatory response, especially by producing several inflammatory cytokines (IL-1β, IL-6, IL-8) (Curr Drug) Traget Inflamm Allergy, 2(3): 257-265, 2003).

On the other hand, chronic obstructive pulmonary disease (COPD) is a chronic airway disease that shows coughing, sputum, dyspnea, decreased expiratory flow rate, and gas exchange disorder. , is predicted to become the third leading cause of human death by 2020 (Am J Respir Crit Care Med, 2013, 187:347-365; Am J Respir Crit Care Med, 2009, 180:396-406).

COPD is caused by various causes such as smoking, air pollution, chemicals, occupational factors, and genetic predisposition. Pharm Bull, 2012, 35:1752-1760). The pathogenesis of COPD involves inflammation, protease activation in the lungs, oxidative stress, and the like, and cells involved in inflammation are mainly neutrophils, macrophages, and T lymphocytes. These inflammatory cells produce reactive oxygen species, various inflammatory cytokines, and various proteolytic enzymes that cause tissue damage (Korean Tuberculosis and Respiratory Research Society, Seoul: Gunja Publishing House; 2007, p 301-5; Am J Respir Crit Care Med, 1997 155, 1441-1447; Am J Physiol Lung Cell Mol Physiol, 2010, 298:L262-L269). In particular, neutrophils secrete substances such as elastase, collagenase, proteases such as MPO (myeloperoxidase), arachidonic acid metabolites (arachidonate), reactive oxygen free radicals, etc. It is known to play an important role in the pathogenesis of COPD by causing lung damage (Am J Respir Cell Mol Biol, 2013, 48:531-539; Eur Respir J, 1998, 12: 1200-1208).

There have been no reports of drugs that directly improve COPD, etc., and the current COPD treatment is to reduce symptoms and complications, such as bronchodilators (β2-agonists, anticholinergics, methylxanthines) and steroids (inhalation, oral). Roflumilast?, an inhibitor of the enzyme phosphodiesterase-4 (PDE-4) with anti-inflammatory activity, has recently been approved for marketing in the United States and Europe.

The present invention discloses the anti-inflammatory activity of Pediococcus pentosaceus , which was confirmed using the lung lavage solution of an experimental animal of the COPD model, and the COPD improvement activity based thereon.

An object of the present invention is Pediococcus pentosaceus ( Pediococcus pentosaceus ) Anti-inflammatory composition using; Food composition for enhancing respiratory function and preventing or improving respiratory diseases; Health functional food containing the food composition; feed composition; feed additive composition; Or to provide a pharmaceutical composition for preventing or treating respiratory diseases. Other objects or specific objects of the present invention will be set forth below.

The present invention, as confirmed in the following Examples and Experimental Examples, Pediococcus pentosaceus ( Pediococcus pentosaceus ), particularly Pediococcus pentosaceus KF159 strain (Accession No. KCCM 11674P) lung in COPD animal model Reduces the number of inflammatory cells in the washing fluid and inflammatory cytokines and chemokines IL-6, IL-1β, TNF-α, GM-CSF, MIP-2, IFN-γ, IL-17, KC, TARK, MCP-1 . It was completed by confirming that it clearly inhibits the expression of MDC.

Considering the above, the present invention, in one aspect, Pediococcus pentosaceus ( Pediococcus pentosaceus ), in particular, Pediococcus pentosaceus KF159 strain (deposited) No. KCCM 11674P), its culture solution, its concentrate or its dried product can be identified as an anti-inflammatory composition comprising as an active ingredient, in another aspect, Pediococcus pentosaceus, in particular Pediococcus pentosaceus KF159 strain (Accession No. KCCM 11674P), its culture solution, its concentrate or its dried product as an active ingredient can be identified as a composition for prevention, treatment or improvement of respiratory diseases, particularly COPD, and in another aspect, Pediococcus pentosa It can also be understood as a composition for improving lung function or improving respiratory function, including, in particular, Pediococcus pentosaceus KF159 strain (Accession No. KCCM 11674P) as an active ingredient, and the present invention also provides Pediococcus pentosaceus Cough, sputum, dyspnea, airway hypersensitivity, airway obstruction, mucus hypersecretion, decrease in exhaled flow rate and/or gas exchange, especially comprising Pediococcus pentosaceus KF159 strain (Accession No. KCCM 11674P) as an active ingredient. It can also be identified as a composition for improving lung diseases accompanied by the symptoms of disorders.

Such lung diseases include not only the aforementioned asthma and COPD, but also diffuse interstitial lung disease, acute respiratory distress syndrome (ARDS), and acute lung injury. In addition to lung diseases caused by pollution, genetic predisposition, etc., in particular, it includes lung diseases caused by fine dust, which has become a problem recently. Fine dust containing various components such as carbon components such as soot and organic carbon, ionic components such as chlorine, nitric acid, ammonium, sodium, and calcium, metal components such as lead, arsenic and mercury, and polycyclic aromatic hydrocarbons such as benzopyrene It is known to cause various lung diseases such as asthma and COPD by depositing in the airways, bronchial tubes, small airways, and alveoli (J Korean Med Assoc, 2014;57:763-768).

In addition, the present invention in one aspect Pediococcus pentosaceus KF159 strain (Accession No. KCCM 11674P), its culture medium, its concentrate or its dry matter, comprising as an active ingredient, a feed for strengthening respiratory function and preventing or improving respiratory diseases It may be identified as a composition or a feed additive composition.

The Pediococcus pentosaceus KF159 strain of the present invention was deposited with the Korea Microorganism Conservation Center (KCCM), an international depository under the Budapest Treaty as of March 6, 2015, and was given an accession number KCCM 11674P.

As used herein, the term "culture medium" or "culture" refers to a medium containing by-products and strains generated through metabolism of a strain by culturing a strain in a medium, specifically pediococcus pen. It may mean a culture solution or culture of the Tosaceus KF159 strain. In addition, the concentrate or dilution of the medium, the dried product obtained by drying the medium, the prepared or purified product of the medium, or a mixture thereof may also be included as an active ingredient in the composition of the present invention.

In the present invention, the term "enhancement of respiratory function" refers to maintaining the original functions of respiratory organs such as nasal passages, pharynx, larynx, trachea, bronchi and lungs in a healthy state, or according to smoking, fine dust, neutrophil neutrophil activation or other respiratory diseases. It refers to any action that improves the function of the respiratory system, which has been deteriorated due to symptoms, to the original healthy state.

Also, in the present invention, the term "respiratory disease" refers to a disease occurring in the respiratory tract of an individual, such as the nasal cavity, pharynx, larynx, trachea, bronchi, and lung. Specifically, the respiratory disease is a respiratory disease caused by smoking or fine dust. ; Or it may mean a lung disease accompanied by netosis (Netosis), but is not particularly limited thereto. More specifically, the respiratory disease may refer to a pulmonary disease accompanied by symptoms of sputum, difficulty breathing, airway hypersensitivity, airway obstruction, mucus hypersecretion, decreased expiratory flow rate and/or impaired gas exchange, and more specifically asthma , chronic obstructive pulmonary disease (COPD), tracheitis, bronchitis, diffuse interstitial lung disease, acute respiratory distress syndrome (ARDS), acute lung injury, cystic fibrosis, capillary It may mean one or more selected from the group consisting of bronchitis, influenza virus infection, pneumonia, tuberculosis, and transfusion-related acute lung injury. , most specifically asthma or COPD.

As used herein, the term "active ingredient" refers to a component capable of exhibiting the desired activity by itself or in combination with a carrier having no activity by itself. In the composition of the present invention, the active ingredient may be used in any amount ( effective amount), a typical effective amount will be determined within the range of 0.001 weight % to 20.0 weight % based on the total weight of the composition. As used herein, the term "effective amount" refers to the intended functional and pharmacological effects such as anti-inflammatory effects and COPD improvement effects when the composition of the present invention is administered to a mammal, preferably a human, to which it is applied during the administration period as suggested by a medical professional. Refers to the amount of the active ingredient included in the composition of the present invention that can be represented. Such effective amounts can be determined empirically within the ordinary ability of one of ordinary skill in the art.

Subjects to which the composition of the present invention can be applied are mammals and humans, particularly preferably humans.

In addition to the active ingredient, the composition of the present invention has already been verified for safety in the art, and in order to enhance the convenience of administration or intake through the addition of similar activities such as anti-inflammatory activity, or anti-allergic activity, in addition to the active ingredient. It may further include any compound or natural extract known to have activity. Such compounds or extracts include compounds or extracts, drugs listed in compendial documents such as pharmacopeias of each country (“Korean Pharmacopoeia” in Korea) and health functional food regulations of each country (“Health Functional Food Standards and Specifications” announced by the Ministry of Food and Drug Safety in Korea). Each country's laws governing the manufacture and sale of compounds, extracts, and health functional foods that have been approved for items in accordance with the laws of each country (the "Pharmaceuticals Act" in Korea) governing the manufacture and sale of '), compounds or extracts whose functionality is recognized are included. For example, in accordance with the Korean 「Health Functional Food Act」, MSM (dimethylsulfonylmethane), N-acetylglucosamine, etc., which are recognized for their anti-inflammatory (“arthritis improvement”) function, and anti-allergic (“alleviation of hypersensitive immune response”) function Certified Enterococcus faecalis heat-treated dry powder, guava leaf extract, etc., Actinidia extract, leaf extract, picaopreto powder, etc., PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol), etc. such compounds or extracts. One or more of these compounds or natural extracts may be included in the composition of the present invention together with the active ingredient.

The composition of the present invention may be identified as a food composition in a specific embodiment, and the food may include health functional (sex) food.

In the present invention, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful for the human body. The "functionality" refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological actions with respect to the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the dosage form of the health functional food may also be manufactured without limitation as long as it is a dosage form recognized as a health functional food. The food composition of the present invention can be prepared in various types of dosage forms, and unlike general drugs, there are no side effects that may occur during long-term administration of drugs using food as a raw material, and excellent portability, Health functional food can be taken as a supplement to enhance asthma or COPD improvement effect, and further enhance respiratory function and respiratory disease improvement effect.

The food composition of the present invention may be prepared in any form, for example, beverages such as tea, juice, carbonated beverages, and ionic beverages, processed oils such as milk and yogurt, gums, rice cakes, Korean sweets, bread, Foods such as confectionery and noodles, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, etc. can be manufactured as health functional food preparations. The food composition of the present invention may have any product classification in terms of legal and functional classification as long as it conforms to the enforcement regulations at the time of manufacturing and distribution. For example, it is a health functional food according to the Korean 「Health Functional Food Act」, or confectionery, beans, tea, and beverages according to each food type according to the Food Ordinance of Korea 「Food Sanitation Act」 (Ministry of Food and Drug Safety Notification 「Food Standards and Specification」) , special purpose food, and the like.

In addition, the food composition of the present invention may contain food additives in addition to the active ingredient. Food additives can be generally understood as substances that are added to and mixed with or infiltrated into food in manufacturing, processing or preserving food. Food additives with guaranteed safety are limited in terms of ingredients or functions in the Food Additives Ordinance according to the laws of each country that regulates the manufacture and distribution of food (“Food Sanitation Act” in Korea). In the Korean Food Additives Code (“Food Additive Standards and Specifications” notified by the Ministry of Food and Drug Safety), food additives are classified into chemically synthetic products, natural additives, and mixed preparations in terms of ingredients. It is classified into an agent, a preservative, an emulsifier, an acidulant, and a thickener.

The sweetener is used to impart an appropriate sweetness to food, and may be natural or synthesized. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

The flavoring agent may be used to improve taste or flavor, and both natural and synthetic ones may be used. Preferably, it is a case where a natural thing is used. In the case of using a natural one, the purpose of nutritional enhancement in addition to flavor may be concurrently used. As a natural flavoring agent, it may be obtained from apple, lemon, tangerine, grape, strawberry, peach, etc., or it may be obtained from green tea leaf, dandelion, bamboo leaf, cinnamon, chrysanthemum leaf, jasmine, etc. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo biloba, etc. can be used. The natural flavoring agent may be a liquid concentrate or a solid extract. In some cases, a synthetic flavoring agent may be used, and the synthetic flavoring agent may include esters, alcohols, aldehydes, terpenes, and the like.

Calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid) and the like can be used as the preservative, and as the emulsifier, gum acacia, carboxymethyl cellulose, xanthan gum, Pectin may be used, and acidulant, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like may be used. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing the taste.

As the thickener, a suspending agent, a settling agent, a gel-forming agent, a bulking agent, and the like may be used.

The food composition of the present invention may contain, in addition to the food additives as described above, physiologically active substances or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutrition and guaranteed stability as food additives. Examples of the physiologically active substances include catechins contained in green tea and the like, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, and the like. Magnesium preparations, such as iron preparations, such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc, etc. are mentioned.

In the food composition of the present invention, the food additives as described above may be included in an appropriate amount to achieve the purpose of the addition according to the type of product, and with respect to other food additives that may be included in the food composition of the present invention, the food additives of each country Or you can refer to the Food Additives Ordinance.

The composition of the present invention in another specific embodiment, Pediococcus pentosaceus ( Pediococcus pentosaceus ), in particular, Pediococcus pentosaceus KF159 strain (Accession No. KCCM 11674P) that the applicant has isolated, identified and possessed , It can be identified as a pharmaceutical composition for preventing or treating respiratory diseases, including its culture solution, its concentrate or its dried product as an active ingredient.

The term "prevention" of the present invention refers to any action that suppresses or delays symptoms of respiratory diseases of an individual by administration of a composition comprising a culture medium of the Pediococcus pentosaceus KF159 strain according to the present invention.

As used herein, the term “treatment” refers to any action in which symptoms according to respiratory diseases of an individual are improved or cured by administration of a composition comprising a culture medium of the Pediococcus pentosaceus KF159 strain according to the present invention.

The pharmaceutical composition of the present invention may be prepared as an oral dosage form or parenteral dosage form according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the route of administration may be any suitable route including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of the combination of two or more routes is a case in which two or more formulations of drugs according to the route of administration are combined. For example, one drug is first administered by an intravenous route and the other drug is secondarily administered by a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specifically, reference may be made to the pharmacopoeia of each country including the "Korea Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers according to methods known in the art together with suitable carriers It can be prepared in a formulation such as Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Cellulose such as hydroxypropylmethylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease Serol etc. are mentioned. In the case of formulation activity, an appropriate binder, lubricant, disintegrant, colorant, diluent, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrist, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, and the like, and oleic acid as lubricant. sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc., and the disintegrant include starch, methyl cellulose , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt. Examples of the diluent include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, and the like.

When the pharmaceutical composition of the present invention is prepared for parenteral use, it may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories together with suitable carriers according to methods known in the art. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, an isotonic solution such as PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or 5% dextrose may be used. . When formulated for transdermal administration, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pasta agents, liniment agents, air rolls, and the like. In the case of nasal inhalants, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, and the like. witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters, and the like can be used.

Specific formulations of pharmaceutical compositions are known in the art, and reference may be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). This document is considered a part of this specification.

A preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, body weight, sex, age, patient's severity, and administration route. It can be in the range /kg. Administration may be performed once a day or divided into several times. Such dosages should not be construed as limiting the scope of the invention in any respect.

The composition of the present invention in another specific embodiment, Pediococcus pentosaceus ( Pediococcus pentosaceus ), in particular, Pediococcus pentosaceus KF159 strain (Accession No. KCCM 11674P) that the applicant has isolated, identified and possessed , It can be identified as a feed composition or feed additive composition for enhancing respiratory function and preventing or improving respiratory diseases, including its culture medium, its concentrate or its dried product as an active ingredient.

In the present invention, the term "feed" refers to any natural or artificial diet, one-meal meal, etc. or a component of the one-meal meal for livestock to eat, ingest and digest, or suitable for it, and the 　feed is a 　feed 　additive or auxiliary. It may contain feed. The type of 　 feed is not particularly limited, and 　 feed commonly used in the art may be used. Non-limiting examples of the 　 feed, such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourd or grain by-products, such as vegetable 　 feed; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

The present invention can provide a composition for preventing, treating or improving anti-inflammatory compositions or respiratory diseases, particularly COPD, using Pediococcus pentosaceus . The composition of the present invention is commercialized as a food product, particularly a health functional food or drug, and can be usefully used for anti-inflammatory effect, COPD improvement effect, and further lung function improvement or respiratory function improvement effect, cough, sputum, dyspnea , airway hypersensitivity, airway obstruction, and other lung diseases accompanied by symptoms such as mucus hypersecretion, such as diffuse interstitial lung disease, acute respiratory distress syndrome (ARDS), acute lung injury, etc. can be used

1 and 2, respectively, for the PPE/CSE-induced respiratory disease (COPD) mouse model, Pediococcus pentosaceus KF159 strain (PPKF159) and ROF as a positive control after administration of ROF from bronchoalveolar lavage (BALF) The total number of cells counted and the results of microscopic observation are shown.
3 to 6 show the results of analysis of immune cells (Macrophage, Lympocyte, Neutrophils and Eosinophil) in BALF.
7 shows Cytokine and Chemokine (IL-6, IL-1β, INF-γ, TNF-α, IL-17, MIP-2, KC, MCP-1, MDC, TARC in BALF of a respiratory disease (COPD) mouse model. , GM-CSF) analysis results are shown.
8 shows Cytokine and Chemokine (KC, MCP-1, MDC, MIP-2, TARC, TNF-α, GM-CSF, INF-γ, IL-1β, IL- 6, IL-10, IL-17) analysis results are shown.
9 shows the results of staining the lung tissue extracted from the respiratory disease (COPD) mouse model.
10 shows the results of evaluating MIP-2 inhibitory activity when treated with Pediococcus pentosaceus KF159 strain (PPKF159) for MH-S cells stimulated with CSE 1% and LPS 10ng/ml.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these Examples and Experimental Examples.

<Example> Sample preparation

For the sample, the Pediococcus pentosaceus KF159 strain (Accession No. KCCM 11674P) isolated, identified and possessed by the present applicant as a live cell preparation was used. For reference, this strain is MRS medium, and it can be cultured at a culture condition of pH 6.5 ± 0.2 and a temperature of 37°C. It can be obtained by suspending or lyophilizing a culture solution diluted with an appropriate solvent such as sterile saline).

<Experimental Example> Respiratory disease improvement activity test

1. Experimental method

1.1 Chronic obstructive pulmonary disease (COPD) induction and strain administration

For the animal experiment, a 5-week-old male, BALB/c mouse was purchased, and after a 7-day acclimatization period, the experiment was conducted by dividing the groups into a naive group, an induction group, a positive control group, and a test group. The disease was induced by intranasally administering CSE (Cigarette smoke extract) and PPE (Porcine pancreatic elatase) for 2 weeks. At this time, CSE was administered at a concentration of 20 μg/head 3 times a week, and PPE at a concentration of 1.2 unit/head once a week. In the naive group, PBS was intranasally administered instead of the inducer. The strain was administered daily at a concentration of 1*10 ⁸ CFU/head for a total of 3 weeks from 1 week before induction to 2 weeks after induction. To the positive control group, Roflumilast [ROF], a treatment for COPD, was administered daily after induction at a concentration of 10 mg/kg. After completion of the experiment period, the mouse was euthanized with isoflurane, a 600mm cutdown tube was put into the airway, the airway and tube were fixed with a surgical thread, and 1 mL of PBS was injected along the airway. μl was recovered and used for the experiment.

1.2 Measurement of Inflammatory Cell Distribution in BALF

The recovered BALF was centrifuged, and the supernatant was stored frozen and used for ELISA measurement. The cells were treated with RBC to remove red blood cells. After dispensing the same amount of PBS as the previously recovered BALF, the total number of cells was collected using an automatic cell counter. was measured, and the distribution of macrophge, eosinophil, neutrophil, and lymphocytes in the BALF was confirmed through light microscopic observation after Diff quick staining with the same sample.

1.3 Analysis of Inflammatory Cytokines in BALF

After dissection, the recovered BALF was centrifuged to take the supernatant, and the cytokine was tested according to the manufacturer's protocol using an ELISA measurement kit. One day before the experiment, the capture antibody (Capture antibody) was diluted to an appropriate concentration in PBS, and 100 μl per well of a 96 well immune plate (SPL) was dispensed and left overnight. Then, after washing 3 times using a wash solution at 350 μl per well, 300 μl of a diluent solution was added, and the mixture was left at room temperature for 1 hour, and then washed twice. Thereafter, the standard and the supernatant sample were diluted to an appropriate concentration in the diluted solution, treated at 100 μl per well, incubated at room temperature for 2 hours, and washed twice. The detection antibody (Detection Antibody) was diluted to an appropriate concentration using a dilution solution, treated at 100 μl per well, and incubated at room temperature for 2 hours. After washing twice, 100 μl of a diluted solution containing streptavidin-HRP was treated, incubated at room temperature for 20 minutes, and washed twice. Finally, 50 μl of the substrate solution was treated per well, the plate was lightly tapped, and the absorbance was measured at a wavelength of 450 nm using a microplate reader.

1.4 Analysis of Inflammatory Cytokines in Lung Tissue

Respiratory disease (COPD) model After adding PBS corresponding to 10 times the weight of mouse lung tissue, and pulverizing with a homogenizer, it was centrifuged at 12,000 rpm and 20 min. The recovered supernatant was analyzed for cytokines and chemokines (KC, MCP-1, MDC, MIP-2, TARC, TNF-α, GM-CSF, INF-γ, IL-1β, IL-6, IL-10, IL-17 ) was used. After taking 30 uL of the supernatant for the analysis of cytokines and chemokines, the experiment was performed according to the protocol of the manufacturer (Quansis biosciences) of the Q-plex ELISA array kit.

1.5 Lung tissue analysis (Histological examination)

Lung tissue was removed from the dissected respiratory disease (COPD) model mouse, the lung mesenchyme was removed, and then fixed in 10% neutral buffered formalin for 3 days. The fixed tissue was dehydrated with ethyl alcohol and xylene, embedded in paraffin, trimmed, and sliced to a thickness of 5 μm. After attaching the prepared section to the slide, it was stained with H&E to check the general shape and observed under a microscope.

1.6 MIP-2 inhibitory activity in MH-S cell line

The MH-S cell line was purchased from ATCC, and the medium composition used for culture was RMPI-1640 (WELGENE, cat. LM 011-01), 10% FBS (WELGENE, cat. S 001-07), 1% Penicillin- Streptomycin (WELGENE, cat. LS 202-02), 14.3 mM 2-Mercptoethanol (SIGMA). MH-S cells were seeded in a cell culture 96 well plate (SPL, cat. 30096) at a concentration of 5*10 ⁴ /well and cultured for 24 hours. Thereafter, stimulants (CSE; final 1% and LPS; final 10 ng/mL) and Pediococcus pentosaceus KF159 (PPKF159) were treated with MH-S cell culture medium at a concentration of 10 ⁴ or 10 ⁵ CFU per well. . After culturing for 24 hours, the supernatant was recovered and MIP-2 was measured by ELISA according to the protocol of the manufacturer (R&D, DY452).

2. Experimental results

2.1 Measurement result of inflammatory cell distribution in BALF

The results of analysis of the total number of infiltrated cells in the BALF are shown in FIGS. 1 and 2, and the measurement results of macrophge, eosinophil, neutrophil, and lymphocyte, which are inflammatory cells in the BALF, are shown in FIGS. 3 to 6, respectively. As a result, it was confirmed that the total number of cells infiltrated during the treatment of the sample was significantly reduced, and the number of the four inflammatory cells was also significantly reduced compared to the COPD-induced group (PPE/CSE).

2.2 Analysis of inflammatory cytokines in BALF

The analysis results of inflammatory cytokines in BALF and lung tissue are shown in FIGS. 7 and 8, respectively. As a result, the expression of Cytokine and Chemokine (IL-6, IL-1β, INF-γ, TNF-α, IL-17, MIP-2, KC, MCP-1, MDC, TARC, GM-CSF) in BALF samples was When administered, it was confirmed that it was significantly reduced compared to the COPD induction group (PPE/CSE), and Cytokine and Chemokine (KC, MCP-1, MDC, MIP-2, TARC, TNF-α, GM-CSF, INF-γ, IL-1β, IL-6, IL-10, IL-17) was also confirmed to be decreased compared to the COPD-induced group (PPE/CSE).

2.3 Lung tissue analysis results

The degree of lung tissue damage according to COPD induction and Pediococcus pentosaceus KF159 (PPKF159) treatment was analyzed and shown in FIG. 9 . As a result of H&E staining, in the COPD-induced group, damage to the epithelial cell layer in the bronchi, inflammatory cell infiltration around the bronchioles and alveoli, and bronchiolar obstruction with narrowing of the bronchiolar area were observed, but when Pediococcus pentosaceus KF159 (PPKF159) was administered was confirmed to improve these symptoms. In addition, as a result of PAS staining, hyperplasia of goblet cells was observed in the epithelial cell layer in the bronchioles in the COPD-induced group compared to the normal group, but it was confirmed that these symptoms were improved when Pediococcus pentosaceus KF159 (PPKF159) was administered.

2.4 Evaluation of MIP-2 Inhibition in MH-S Cell Line

For MH-S cells stimulated with CES and LPS, when the Pediococcus pentosaceus KF159 (PPKF159) strain was treated at a concentration of 10 ⁴ or 10 ⁵ CFU, the MIP-2 concentration was significantly higher in both compared to the case without treatment, respectively. It was confirmed that the Pediococcus pentosaceus KF159 (PPKF159) strain had excellent MIP-2 inhibitory activity.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Korea Microorganism Conservation Center (Overseas) KCCM11674P 20150306

Claims (16)

A food composition for improving chronic obstructive pulmonary disease (COPD), comprising the Pediococcus pentosaceus strain, its culture medium, its concentrate or its dried product as an active ingredient.
According to claim 1,
The Pediococcus pentosaceus is characterized in that the accession number is KCCM 11674P Pediococcus pentosaceus KF159 strain, the food composition.
delete A health functional food for improving chronic obstructive pulmonary disease (COPD), comprising the food composition of claim 1 or 2.
A pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease (COPD), comprising the Pediococcus pentosaceus strain, its culture medium, its concentrate or its dried product as an active ingredient.
6. The method of claim 5,
The Pediococcus pentosaceus is characterized in that the Pediococcus pentosaceus KF159 strain with an accession number KCCM 11674P, a pharmaceutical composition.
delete A feed composition for improving chronic obstructive pulmonary disease (COPD), comprising the Pediococcus pentosaceus strain, its culture medium, its concentrate or its dried product as an active ingredient.
9. The method of claim 8,
The Pediococcus pentosaceus is a feed composition, characterized in that the Pediococcus pentosaceus KF159 strain with an accession number KCCM 11674P.
A feed additive composition for improving chronic obstructive pulmonary disease (COPD), comprising the Pediococcus pentosaceus strain, its culture medium, its concentrate or its dried product as an active ingredient.
11. The method of claim 10,
The Pediococcus pentosaceus is a feed additive composition, characterized in that the Pediococcus pentosaceus KF159 strain of which the accession number is KCCM 11674P. delete delete delete delete delete

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