KR102165930B1 - Composition for prevention or treatment of respiratory diseases or inflammation induced by particulate matter comprising novel lactic acid bacteria - Google Patents

Abstract

The present invention relates to a Lactobacillus plantarum GCWB1001 strain deposited with a novel accession number KCCM12698P, a Pediococcus acidilactici GCWB1085 strain deposited with an accession number KCCM12699P, or a Lactobacillus rhamnosus GCWB1156 strain deposited with an accession number KCCM12700P with an excellent effect of treating or alleviating respiratory diseases. Additionally, provided are a pharmaceutical composition, a health functional food composition, and probiotics capable of providing an effect of treating or alleviating inflammation diseases or respiratory diseases, including any one of the novel strains.

Description

Composition for prevention or treatment of respiratory diseases or inflammation induced by particulate matter comprising novel lactic acid bacteria}

The present invention relates to novel lactic acid bacteria having a therapeutic or prophylactic effect for respiratory diseases or inflammatory diseases, and a pharmaceutical composition, a health functional food composition, and probiotics comprising the same.

Fine dust is a particulate matter of 10 μm or less that floats or scatters down in the air. PM10, which is classified according to dust diameter, is a particle smaller than 10/1,000 ㎜, and PM2.5 is a particle smaller than 2.5/1,000. it means.

The causes of fine dust are directly discharged from anthropogenic sources such as workplace combustion and automobile fuel combustion, or sulfur oxides (SO _x ), nitrogen oxides (NO _x ), ammonia (NH ₃ ), volatile organic compounds (VOCs), etc. Is produced by a secondary generation process that is produced by reacting with water vapor in the atmosphere.

Fine dust is known to have fine particles, so it is not filtered through the nasal mucosa and can penetrate directly into the alveoli or brain when inhaled, and is known to increase the prevalence of asthma and lung diseases and early mortality.

In particular, the International Cancer Research Institute (IARC) under the World Health Organization (WHO) classified fine dust as a group 1 carcinogen that was found to cause cancer in humans along with benzene and asbestos in 2013.

In the gastrointestinal tract of animals including humans, living microorganisms that have a beneficial effect on the health of the host by improving the intestinal microbial environment of the host are collectively referred to as probiotics.

Lactic acid bacteria, a kind of probiotics, play a role in the digestive system of the human body and break down fiber and complex proteins into important nutrients. Lactobacillus is a bacterium that breaks down carbohydrates and uses them to create lactic acid, and is an anaerobic bacteria that proliferate well in places with low oxygen. Lactobacillus can be classified into five genera, streptococcus , Lactobacillus , Leuconostoc , Bifidobacteria , and Pediococcus . Recently, the therapeutic effect of various diseases using lactic acid bacteria has been confirmed, and the development of therapeutic agents using the same has been attempted.

Unlike conventional bacteria, temporary poisoning, or damage to the respiratory tract and lungs caused by the influx of other substances into the airways, it cannot be eradicated by human immunity, and when fine dust is introduced through the respiratory tract, there is no way to forcibly discharge it. In addition, since we do not know exactly when and what problems will occur due to these damages, we can prevent, alleviate, treat, or improve the damage to the airways and lungs induced by fine dust, and through this, a new product that can treat the induced respiratory diseases. There is an urgent need for a treatment.

1. Republic of Korea Patent Registration No. 10-1912258 (2018.10.29.) A composition for protecting cells and tissues against the toxicity of fine dust containing Lactobacillus casei strain. 2. Republic of Korea Patent Registration No. 10-2021881 (2019.09.17.) Streptococcus thermophilus LM1012 strain and a composition for preventing or treating inflammatory diseases or metabolic diseases comprising the same.

The present invention is a novel Lactobacillus plantarum GCWB1001 ( Lactobacillus plantarum GCWB1001) strain deposited with accession number KCCM12698P, Lactobacillus rhamnosus GCWB1156 deposited with accession number KCCM12700P ( Lactobacillus rhamnosus GCWB1156 ( Lactobacillus rhamnosus GCWB1156) strain deposited with accession number KCCM12698P It is intended to provide a composition for the prevention or treatment of inflammatory diseases or respiratory diseases including the strain of Ococcus acidiclactici GCWB1085 ( Pediococcus acidiclactici GCWB1085).

To achieve the above objective

The present invention provides a Lactobacillus plantarum GCWB1001 ( Lactobacillus plantarum GCWB1001) strain deposited with accession number KCCM12698P.

The present invention provides a Lactobacillus rhamnosus GCWB1156 ( Lactobacillus rhamnosus GCWB1156) strain deposited with accession number KCCM12700P.

The present invention provides a Pediococcus acidiclactici GCWB1085 ( Pediococcus acidiclactici GCWB1085) strain deposited with accession number KCCM12699P.

In addition, the present invention is the Lactobacillus plantarum GCWB1001 ( Lactobacillus plantarum GCWB1001) strain deposited with the accession number KCCM12698P, Lactobacillus rhamnosus GCWB1156 ( Lactobacillus rhamnosus GCWB1156 deposited as the deposited number KCWB1156) deposited as KCCM12700P Pediococcus acidiclactici GCWB1085 ( Pediococcus acidiclactici GCWB1085) strain or its culture, concentrate, paste, spray dry, lyophilisate, vacuum dry, drum dry, liquid, dilution and crushed material It provides a pharmaceutical composition for prevention or treatment.

In addition, the present invention is the Lactobacillus plantarum GCWB1001 ( Lactobacillus plantarum GCWB1001) strain deposited with the accession number KCCM12698P, Lactobacillus rhamnosus GCWB1156 ( Lactobacillus rhamnosus GCWB1156 deposited as the deposited number KCWB1156) deposited as KCCM12700P Pediococcus acidiclactici GCWB1085 ( Pediococcus acidiclactici GCWB1085) strain or its culture, concentrate, paste, spray dry, lyophilisate, vacuum dry, drum dry, liquid, dilution and crushed material It provides a health functional food composition for prevention or alleviation.

In addition, the present invention is the Lactobacillus plantarum GCWB1001 ( Lactobacillus plantarum GCWB1001) strain deposited with the accession number KCCM12698P, Lactobacillus rhamnosus GCWB1156 ( Lactobacillus rhamnosus GCWB1156 deposited as the deposited number KCWB1156) deposited as KCCM12700P It provides probiotics comprising the strain of Pediococcus acidiclactici GCWB1085.

According to one embodiment of the present invention, Lactobacillus plantarum GCWB1001 (KCCM12698P), Lactobacillus rhamnosus GCWB1156 (KCCM12700P), and Pediococcus Ecidilactici GCWB1085 (KCCM12699P) strain has an anti-inflammatory effect, and a real animal model Shows the antitussive/expectant effect and the effect of improving lung function in chronic respiratory diseases.

According to an embodiment of the present invention, Lactobacillus plantarum GCWB1001 (KCCM12698P), Lactobacillus rhamnosus GCWB1156 (KCCM12700P), and Pediococcus Ecidilactici GCWB1085 (KCCM12699P) strains are kimchi, cheese and infant feces, respectively. Was isolated from, each strain does not show cytotoxicity in mouse lung macrophages (MH-S cell line), and has an effect of reducing the secretion of cytokines TNF-α and TGF-β.

According to an embodiment of the present invention, all strains of the present invention have reduced production of NO (nitrogen monoxide), ROS (reactive oxygen species), which are inflammatory factors increased by fine dust, as well as NF-, an inflammatory transcription factor. The promoter activities of κB, iNOS, and COX2 were also reduced.

According to one embodiment of the present invention, all strains of the present invention alleviate cough symptoms in an actual animal model, have antitussive/expectant effects, and alleviate various symptoms of chronic respiratory disease animal models caused by fine dust. Showed effect.

Accordingly, as a result, the Lactobacillus plantarum GCWB1001 (KCCM12698P), Lactobacillus rhamnosus GCWB1156 (KCCM12700P), and Pediococcus Ecidilactici GCWB1085 (KCCM12699P) strains of the present invention alleviate and prevent inflammatory diseases or respiratory diseases. Or it shows a therapeutic effect.

1 is a graph showing the measurement (Experimental Example 1-2) of TGF-β production by 7 strain samples in a mouse lung macrophage cell line (MH-S).
2 and 3 are graphs showing NO and ROS production amounts measured by 7 strain samples (Experimental Examples 1-3 and 1-4) in a mouse lung macrophage cell line (MH-S).
Figures 4 to 6 are by measuring the inflammatory transcriptional regulator activity (iNOS, COX2 and NF-κB promoter activity) by 7 strain samples in a mouse lung macrophage cell line (MH-S) (Experimental Example 1-5) This is the graph shown.
7 and 8 are graphs showing antitussive and expectorant actions (Experimental Examples 2-2 and 2-3) of strains GCWB1001, GCWB1085 and GCWB1156 in a mouse cough model.
9 is a graph showing body weight, liver, spleen, and lung weight (Experimental Example 3-2) after treatment with strains GCWB1001, GCWB1085 and GCWB1156 in an animal model of chronic respiratory disease.
10 is a graph showing the number of immune cells measured in Broncoalveolar lavage fluid (BALF) after treatment with strains GCWB1001, GCWB1085 and GCWB1156 in a chronic respiratory disease animal model (Experimental Example 3-3).
11 is a graph of measuring the amount of OVA-specific IgE in Broncoalveolar lavage fluid (BALF) after treatment with strains GCWB1001, GCWB1085 and GCWB1156 in a chronic respiratory disease animal model (Experimental Example 3-4).
12 is a cytokine (TNF-α, IL-6, IL-1β, IL-4, IL-13, MCP- in BALF (Broncoalveolar lavage fluid) after treatment with strains GCWB1001, GCWB1085 and GCWB1156 in a chronic respiratory disease animal model. 1) A graph obtained by measuring the amount (Experimental Example 3-5)
13 is a photograph and graph of lung tissue obtained by staining inflammatory cells infiltrating lung tissue after treatment with strains GCWB1001, GCWB1085 and GCWB1156 in an animal model of chronic respiratory disease (Experimental Example 3-6).
14 is a photograph and graph of lung tissue stained with mucous proteins (Experimental Example 3-7) after treatment with strains GCWB1001, GCWB1085 and GCWB1156 in a chronic respiratory disease animal model.
15 is a graph showing caspase 3 activity and total collagen content in lung tissue after treatment with strains GCWB1001, GCWB1085 and GCWB1156 in a chronic respiratory disease animal model (Experimental Example 3-8).
16 is a photograph of analyzing MMP-9 activity (Experimental Example 3-9) in lung tissue after treatment with strains GCWB1001, GCWB1085 and GCWB1156 in an animal model of chronic respiratory disease.

Hereinafter, the present invention will be described in more detail.

All technical terms used in the present invention, unless otherwise defined, are used in the same sense as those of ordinary skill in the art generally understand in the related field of the present invention. In addition, although preferred methods or samples are described in the present specification, those similar or equivalent are included in the scope of the present invention.

The present invention relates to a Lactobacillus plantarum GCWB1001 ( Lactobacillus plantarum GCWB1001) strain deposited with accession number KCCM12698P.

The novel strain, Lactobacillus plantarum GCWB1001 ( Lactobacillus plantarum GCWB1001) strain, was named by the above name and deposited with the Korea Microbial Conservation Center on April 17, 2020. The accession number is KCCM12698P.

The strain can be isolated and identified from kimchi collected in each region. The inventors of the present invention isolate and identify various new strains from kimchi collected in each region, and among the isolated and identified strains, Lactobacillus plantarum GCWB1001 ( Lactobacillus plantarum GCWB1001) strain is significantly superior compared to conventional known lactic acid bacteria. The present invention was completed by confirming that it provides an anti-inflammatory effect and a respiratory disease prevention or treatment effect.

The present invention relates to a Lactobacillus rhamnosus GCWB1156 ( Lactobacillus rhamnosus GCWB1156) strain deposited under accession number KCCM12700P.

The novel strain, Lactobacillus rhamnosus GCWB1156 ( Lactobacillus rhamnosus GCWB1156) strain was named under the above name and deposited with the Korea Microbiological Conservation Center on April 17, 2020. The accession number is KCCM12700P.

The strain can be isolated and identified from cheese collected in each region. The inventors of the present invention isolate and identify various new strains from cheese collected in each region, and among the isolated and identified strains, Lactobacillus rhamnosus GCWB1156 ( Lactobacillus rhamnosus GCWB1156) strain is significantly superior to conventional known lactic acid bacteria. The present invention was completed by confirming that it provides an effect and a respiratory disease prevention or treatment effect.

The present invention relates to a Pediococcus acidiclactici GCWB1085 ( Pediococcus acidiclactici GCWB1085) strain deposited with accession number KCCM12699P.

The novel strain, Pediococcus acidiclactici GCWB1085 ( Pediococcus acidiclactici GCWB1085) strain was named under the above name and deposited with the Korea Microbiological Conservation Center on April 17, 2020. The accession number is KCCM12699P.

The strain can be isolated and identified from feces of infants. The inventors of the present invention isolate and identify various new strains from feces of infants, and among the isolated and identified strains, Pediococcus acidiclactici GCWB1085 ( Pediococcus acidiclactici GCWB1085) strain is significantly superior to conventional lactic acid bacteria. The present invention was completed by confirming that it provides an effect and a respiratory disease prevention or treatment effect.

That is, the Lactobacillus plantarum GCWB1001 ( Lactobacillus plantarum GCWB1001) strain, the Lactobacillus rhamnosus GCWB1156 ( Lactobacillus rhamnosus GCWB1156) strain, and the Pediococcus E. In the MH-S cell line, a mouse lung macrophage cell line treated with diesel exhaust particles (DEP), the activities of NO, ROS and inflammatory transcriptional regulators (iNOS, COX2 and NF-κB promoter activity) were inhibited.

In addition, the GCWB1001, GCWB1156 and GCWB1085 strains of the present invention were able to confirm the anti-inflammatory effect by reducing the secretion of cytokines (TNF-α) induced by LPS and DEP in the MH-S cell line.

Oxidative stress refers to a phenomenon in which the production of reactive oxygen species or reactive nitrogen species and antioxidant defense mechanisms are lost in balance and the production of reactive oxygen species or active nitrogen species is relatively excessive in a living body molecule, cell, or tissue. At this time, tissue damage is usually caused.

Since these reactive oxygen species or reactive nitrogen species are chemically very unstable and highly reactive, they are widely used in enzyme catalytic reactions in vivo, electron transfer in mitochondria, cell signaling system and gene expression, activation of transcription factors and biomolecules, cells, tissues, etc. By causing oxidative damage, it induces inflammatory reaction around and is involved as a major factor in tissue fibrosis. Such oxidative damage causes various diseases in all tissues of the human body. Specifically, it is known that it is involved in the development and progression of cancer in tissues such as skin, kidneys, heart, joints, lungs, brain, blood vessels, intestines, and eyes, as well as cardiovascular diseases, inflammation, fibrotic diseases, It is known to play an important role in almost all diseases such as diabetes.

When inflammatory substances such as LPS are treated on macrophages, inflammatory cytokines (IL-6, TNF-α, IL-1β, etc.) and inflammatory mediators (oxidative stress) such as NO are generated, resulting in an inflammatory reaction. NO is produced by iNOS (induced nitric oxide synthase), and NF-κB transcription factor is activated as the inflammatory reaction occurs, and prostaglandins (PGs) involved in the inflammatory reaction are produced by COX-2 (cyclooxygenase-2). It accelerates the inflammatory response.

Therefore, the GCWB1001, GCWB1156 and GCWB1085 strains of the present invention are inflammatory substances induced by LPS and DEP, NO, ROS, inflammatory cytokine TNF-α, and inflammatory transcriptional regulators (iNOS, COX2 and NF-κB promoters). By inhibiting all of the activity, the strains have an anti-inflammatory effect capable of suppressing inflammation.

The GCWB1001, GCWB1156 and GCWB1085 strains of the present invention inhibited cough and increased sputum excretion in a citric acid-induced cough model.

Antitussive drugs are drugs that relieve cough regardless of the cause, and can be divided into drugs that act on the central and peripheral drugs according to the mechanism of action, and drugs that act on the center can be divided into narcotic drugs, drug derivatives and non-drug drugs. Representative drugs of narcotic drugs are codein, hydrocodone, morphin, etc., which have proven limited cough suppression effect, but the results are not constant, and drowsiness, constipation, digestive disorders, abuse or dependence at an appropriate dose. There is a risk of it. Among the peripheral antitussive drugs, the most commonly used drug in Korea is Levodropropizine, which is thought to have an effect by controlling the level of sensory neuropeptides in the airways. In addition to this, theobromine corresponds to this.

The main component of sputum is composed of mucus, and bronchial mucus is secreted from mucous cells and serous cells constituting the mucous glands and submucosa that are normally distributed in the bronchial mucosa. The mucus is composed of 95% water and about 5% glycoproteins, lipids, and minerals. The glycoprotein structure is a gel with a linear polymeric dual structure, so it is sticky. Expectant agents that remove sputum are classified into drugs that increase the moisture content of sputum and mucolytic agents that reduce viscosity by breaking the S-S bond of sputum protein. As such an expectorant, cysteine derivatives such as N-acetylcysteine and carbocysteine are used, and these cysteine derivatives are concerned with side effects such as bronchial spasm when used for a long time. Therefore, development of an expectorant agent having low side effects and toxicity and excellent expectorant effect is required.

In addition, ivy leaf extract and Cinechura, which is a combination of ivy leaf and Coptis chinensis extract, are widely used as a natural substance 　jinhae expectorant.

GCWB1001, GCWB1156, and GCWB1085 strains of the present invention reduce the number of coughs in the citric acid-induced cough model and show antitussive/expectant efficacy, and thus have the effect of improving respiratory disease symptoms.

Additionally, the strain of the present invention not only significantly reduced immune cells in the bronchial alveolar cell fluid (BALF) in a chronic respiratory disease model using ovalbumin (OVA) and diesel exhaust particles (DEP), but also penetrated into lung tissue. Immune cells also decreased, OVA-specific IgE was reduced, and inflammatory cytokines TNF-α, IL-6, IL-1β, IL-4, MCP-1, and IL-13 were decreased in BALF, and anti-inflammatory cytokines Phosphorus IFN-γ was significantly increased.

Therefore, the GCWB1001, GCWB1156 and GCWB1085 strains of the present invention not only have antitussive/expectant functions themselves, but also inhibit the activity of MMP9 to prevent the penetration of bronchoalveolar cell fluid and inflammatory cells in lung tissue, and prevent the secretion of inflammatory cytokines. It is believed to have an effect of reducing allergic reactions and inflammatory reactions in respiratory organs by preventing, increasing the secretion of anti-inflammatory cytokines, and reducing the amount of IgE immunoglobulin that is increased in allergic symptoms.

Asthma, pneumonia, chronic obstructive pulmonary disease, allergic rhinitis, acute and chronic bronchitis, bronchiolitis, sore throat, tonsillitis, laryngitis, bronchiectasis, idiopathic pulmonary fibrosis, cystic fibrosis, emphysema, pulmonary tuberculosis sequelae, lower respiratory tract infection, sinusitis, acute upper respiratory infection, Allergic lung diseases, etc. are representative diseases of respiratory diseases. Asthma refers to chronic inflammation of the airways, especially the bronchi.　Inflammation caused by asthma can be aggravated by a wide variety of predispositions such as soot, allergens, cold wind, exercise, and respiratory infections, and persistent 　 inflammation causes airway deformity and airway hyper-responsiveness.

In addition, respiratory diseases can also be caused by respiratory infection viruses. Types of viruses that cause respiratory diseases include adenovirus, vaccinia virus, herpes simplex virus, parainfluenza virus, rhinovirus, and varicella virus. Zoster Virus), measle virus, respiratory syncytial virus, dengue virus, human immunodeficiency virus, influenza virus, coronavirus, severe acute respiratory syndrome virus ( severe acute respiratory syndrome associated virus (SARS-associated virus), middle east respiratory syndrome coronavirus (MERS-CoV), and the like.

The respiratory tract is largely composed of mucous membranes and muscles called bronchial smooth muscles, and there are many glands in the mucous membrane, which keep secreting necessary secretions. When the bronchial smooth muscle contracts, the respiratory tract narrows. When an inflammatory reaction occurs due to a wide variety of predispositions such as soot, allergens, cold wind, exercise, and respiratory infections, secretions from the glands are further increased. At this time, the secretion is caused by an exudative reaction due to inflammation, and is mostly a sticky, mucous secretion consisting of a mixture of an inflammatory mediator and mucin (mucin).

In general, mucin plays a role in protecting the living body, but in chronic inflammatory respiratory diseases such as asthma, COPD, and chronic bronchitis, overproduction or oversecretion of sticky secretions is observed. Abnormal quantity and quality of this secretion acts as a pathology factor, causing obstruction of the tracheal cavity and inhibition of air inflow to the airways due to the accumulation of secretions, resulting in severe seizure cough and shortness of breath accompanied by wheezing, and dry cough during seizures. It occurs and you feel a feeling of chest pressure. Lung damage caused by viral respiratory disease is also believed to be due to this mucus.

In an animal model of chronic respiratory disease, the strains GCWB1001, GCWB1156 and GCWB1085 of the present invention inhibit the deposition of mucous proteins in lung tissue, reduce the activity of caspase 3, a cell death factor in the alveoli, lower collagen deposition, and activate MMP-9. Decreased. The decrease in the activity of MMP-9 can prevent inflammatory cell deposition in lung tissue and ultimately prevent lung fibrosis.

Pulmonary fibrosis is the last stage of respiratory disease, and its pathological and physiological processes are complex.In the early stage, a large amount of inflammatory cells infiltrate centering on the lung inflammation, the alveolar wall is chronically thickened, and the extracellular matrix such as collagen in the middle/end stage. Normal lung tissue structure is destroyed and function is lost due to overgrowth, alveolar deformity, hardening and scarring of lung tissue caused by overdeposition of urea by fibroblasts.

Fibroblasts play a role in recruitment of immune cells to the site of inflammation and tissue damage. In addition, fibroblasts produce and respond to many inflammatory cytokines. Thus, fibroblasts can contribute to chronic inflammation, and conversely, inflammatory cytokines promote fibrosis by promoting the conversion of fibroblasts to myofibroblasts. Therefore, injury or inflammation of the lung tissue can lead to pulmonary fibrosis.

It has been reported that TGF-β stimulation in lung tissue of patients undergoing pulmonary fibrosis induces increased ROS production and increased expression of collagen and α-SMA (a-smooth muscle actin), which are important for fibrosis, and idiopathic pulmonary fibrosis. It has been reported that pulmonary fibrosis worsens due to ROS in the patient's lung tissue.

Therefore, the strain of the present invention not only induces antitussive/expectant action in cough model mice, but also inhibits oxidative stress in an animal model of chronic respiratory disease, thereby inhibiting the expression of inflammatory cytokines and inhibiting the expression of MMP-9. It prevented the deposition of immune cells involved in the reaction in the lung tissue and bronchoalveolar lavage fluid, inhibited the production of mucous protein and collagen, and ultimately showed pulmonary fibrosis improvement.

The present invention is the Lactobacillus plantarum GCWB1001 strain deposited with the accession number KCCM12698P, the Lactobacillus rhamnosus GCWB1156 strain deposited with the accession number KCCM12700P, or the Pediococcus escidylactis strain GCWB1085 deposited with the accession number KCCM12699P. It relates to a pharmaceutical composition for preventing or treating inflammatory diseases or respiratory diseases, including cultures, concentrates, pastes, spray dried products, lyophilisates, vacuum dried products, drum dried products, liquids, dilutions or crushed products.

The inflammatory diseases include chronic and acute rhinitis, chronic and acute gastritis, enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis, irritable bowel syndrome, inflammatory bowel disease, enteroenteritis, Inflammation in rheumatoid arthritis, osteoarthritis, pneumonia, hepatitis, glomerulonephritis, gastritis, vasculitis, pancreatitis, peritonitis, bronchitis, myocarditis, encephalitis, postischemic reperfusion injury, immunity after transplantation of tissues and organs Inflammation from rejection, burns, psoriasis, allergic contact dermatitis, various skin inflammations, back pain, fascia disease, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, It may be scleritis, uveitis, dermatitis, atopic dermatitis, eczema, diabetic inflammation, infectious inflammation caused by viral or bacterial infection, or autoimmune diseases such as lupus, psoriasis, and atherosclerosis.

The respiratory disease is caused by any one of fine dust, viral infection, and pneumonia. The respiratory diseases include respiratory inflammatory lung disease, asthma, bronchiectasis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, emphysema, pulmonary tuberculosis sequelae, chronic bronchitis, allergic rhinitis, antitussive expectorant, It is selected from the group consisting of lower respiratory tract infections, bronchitis, bronchiolitis, acute upper respiratory infections, allergic lung disease, bronchiectasis, pneumonia, acute chronic bronchitis, sinusitis, sore throat, tonsillitis, laryngitis and pulmonary fibrosis.

The viruses are adenovirus, vaccinia virus, herpes simplex virus, parainfluenza virus, rhinovirus, varicella zoster virus, measles virus (measle virus), respiratory syncytial virus, dengue virus, HIV (human immunodeficiency virus), influenza virus, coronavirus, severe acute respiratory syndrome virus (severe acute respiratory syndrome associated virus) ; SARS-associated virus), middle east respiratory syndrome coronavirus (MERS-CoV).

The strain of the present invention may be used having a viable cell content of 1x10 ¹ to 1x10 ¹³ CFU/g, but is not limited thereto.

In addition, the present invention is the Lactobacillus plantarum GCWB1001 strain deposited with the accession number KCCM12698P, the Lactobacillus rhamnosus GCWB1156 strain deposited with the accession number KCCM12700P, or the Pediococcus Ecidilactici GCWB1085 strain deposited with the accession number KCCM12699P It relates to a food composition for preventing inflammatory diseases or respiratory diseases, including a culture product, a concentrate, a paste product, a spray dried product, a freeze dried product, a vacuum dried product, a drum dried product, a liquid product, a dilution product or a crushed product thereof.

In addition, the present invention is the Lactobacillus plantarum GCWB1001 strain deposited with the accession number KCCM12698P, the Lactobacillus rhamnosus GCWB1156 strain deposited with the accession number KCCM12700P, or the Pediococcus Escidialactis GCWB1085 strain deposited with the accession number KCCM12699P, It relates to a health functional food composition for preventing inflammatory diseases or respiratory diseases, including a culture product, a concentrate, a paste product, a spray dried product, a freeze dried product, a vacuum dried product, a drum dried product, a liquid product, a dilution product or a crushed product.

In the present invention, the above-described food composition or health functional food composition may include, in addition to the active ingredient, ingredients commonly added during food production, for example, protein, carbohydrate, fat, nutrient, seasoning agent , A sweetening agent and a flavoring agent may be included, but are not limited thereto. Examples of the carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides such as dextrin, cyclodextrin, and the like; And sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetening agent, natural sweeteners (taumatin, stevia extract, rebaudioside A, glycyrrhizin, etc.) and synthetic sweeteners (saccharin, aspartame, etc.) can be used. In addition, plant-derived oligosaccharides such as fructan, galactan, low-top starch, pectin, beta glucan, and xylooligosaccharide, which may be food for the strain, may be included as prebiotics.

However, the present invention is not limited thereto, and any components known in the art that do not impair the effects of the present invention may be used.

Examples of the food composition or health functional food composition include patient nutrition, meat, grains, caffeine beverages, general beverages, dairy products, chocolate, breads, snacks, confectionery, pizza, jelly, noodles, gums, ice cream, alcoholic beverages, alcohol, Vitamin complexes, other health supplements, and the like, but are not limited thereto. When prepared in the form of a food composition or a health functional food composition as described above, it is preferable because it can be conveniently and easily taken.

In the present invention, the health functional food composition and pharmaceutical composition are granules, limonades, powders, syrups, liquids and solutions, extracts, and elixirs. (elixirs), fluidxtracts, suspensions, decoctions, infusions, tablets, spirits, capsules, troches, pills (pills), may be prepared in the form of soft or hard gelatin capsules, but are not limited thereto.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and are lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, silicic acid. Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It is not.

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.

The pharmaceutical composition of the present invention can be administered orally or parenterally.

The pharmaceutical composition of the present invention is prepared in a unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art Alternatively, it may be manufactured by placing it in a multi-volume container.

In addition, the present invention is the Lactobacillus plantarum GCWB1001 strain deposited with the accession number KCCM12698P, the Lactobacillus rhamnosus GCWB1156 strain deposited with the accession number KCCM12700P, or the Pediococcus esidilactissi GCWB1085 strain deposited with the accession number KCCM12699P. It relates to containing probiotics.

The probiotics may include one or more selected from the following three deposited strains, and may further include one or more selected from the seven lactic acid bacteria strains isolated in the examples, and in addition to the purpose of the present invention It may further include a known strain.

The probiotics may be used having a viable cell content of 1Х10 ¹ to 1Х10 ¹³ CFU/g, and the probiotics may be usefully used for preventing or alleviating anti-inflammatory or respiratory diseases.

In the present invention, the dosage of the strains GCWB1001, GCWB1156 and GCWB1085 is preferably determined in consideration of the administration method, the age, sex, weight, and severity of the disease of the recipient.

For example, the GCWB1001, GCWB1156, and GCWB1085 strains may be administered by dividing the viable cell content of 1x10 ¹ to 1x10 ¹³ CFU/g per day into one or two or more times.

In addition, pharmaceutical compositions, food compositions, health functional food compositions, and probiotics containing the above ingredients are divided into 1 or 2 or more times of viable bacteria content of 1x10 ¹ to 1x10 ¹³ CFU/g per day based on the active ingredient. Can be administered.

However, the above dosage is only an example, and may be changed by a doctor's prescription depending on the state of the taker.

Hereinafter, the present invention will be described in detail based on the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Lactobacillus plantarum ( Lactobacillus plantarum ) Isolation and identification of GCWB1001 strain

(1) Isolation of strain

Kimchi collected from each region was added to 10 times the amount of sterilized physiological saline and homogenized. The homogenized sample was diluted 10 steps in sterilized physiological saline, and the strain was isolated by a dilution plating method. The diluted strain sample was inoculated with MRS medium (MRS broth agar; BD Difco), followed by anaerobic culture at 37°C for 72 hours. Colonies appearing on the MRS agar plate were secondarily inoculated in PCA medium (MBcell, South Korea) containing 0.005% BCP (bromocresol purple) as a pH indicator, and colonies whose purple medium turned yellow were third inoculated on the MRS agar plate to obtain probiotics. Pure separation.

(2) Identification of Lactobacillus plantarum GCWB1001 strain

DNA extraction and purification were performed on the purely isolated strain in (1). After performing 16s rRNA gene amplification using two universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'), sequencing analysis of the amplified 16s rRNA gene Was performed. Using the analyzed 16s rRNA sequence data and EzTaxon server (http://www.ezbiocloud.net), only two strains corresponding to GRAS (Generally Recognized as Safe) were selected and shown in Table 1 below.

Identified strains Lactobacillus plantarum GCWB1001 Leuconostoc mesenteroides GCWB1031

The 16S rRNA sequencing results of the Lactobacillus plantarum GCWB1001 are as follows.

<16S rRNA sequence of Lactobacillus plantarum GCWB1001>

Example 2: Pediococcus esidilactici ( Pediococcus acidilactici ) Isolation and identification of GCWB1085 strain

(1) Isolation of strain

The cheese collected from each region was added to 10 times the amount of sterilized physiological saline and homogenized. The homogenized sample was diluted 10 steps in sterilized physiological saline, and the strain was isolated by a dilution plating method. The diluted strain sample was inoculated with MRS medium (MRS broth agar; BD Difco), followed by anaerobic culture at 37°C for 72 hours. Colonies appearing on the MRS agar plate were secondarily inoculated in PCA medium (MBcell, South Korea) containing 0.005% BCP (bromocresol purple) as a pH indicator, and colonies whose purple medium turned yellow were third inoculated on the MRS agar plate to obtain probiotics. Pure separation.

(2) Identification of the strain of Pediococcus Escidilactis GCWB1085

Chromosomal DNA extraction and purification were performed on the purely isolated strain in (1). After performing 16s rRNA gene amplification using two universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'), sequencing analysis of the amplified 16s rRNA gene Was performed. Using the analyzed 16s rRNA sequence data and EzTaxon server (http://www.ezbiocloud.net), only 4 strains corresponding to GRAS (Generally Recognized as Safe) were selected and shown in Table 2 below.

Identified strains Pediococcus acidilactici GCWB1085 Lactobacillus paracasei GCWB1084 Bifidobacterium longum GCWB1136

The 16S rRNA sequencing results of the Pediococcus acidilactici GCWB1085 are as follows.

<16S rRNA sequence of Pediococcus acidilactici GCWB1085>

Example 3: Lactobacillus rhamnosus ( Lactobacillus rhamnosus) Isolation and identification of strain GCWB1156

(1) Isolation of strain

The feces of healthy infants born with natural delivery were diluted 10 steps in sterilized physiological saline, and the strain was isolated by the dilution plating method. The diluted fecal sample was inoculated with BSM agar medium (Bifidus Selective Medium Agar; Sigma, USA), and then anaerobic cultured at 37°C for 72 hours. Colonies from BSM agar plate were secondarily inoculated into PCA medium (MBcell, South Korea) containing 0.005% bromocresol purple (BCP) as a pH indicator, and colonies whose purple medium turned yellow were then added to BL agar medium (MBcell, South Korea). Probiotics were purely separated by the third inoculation.

(2) Identification of Lactobacillus rhamnosus GCWB1156 strain

Chromosomal DNA extraction and purification were performed on the purely isolated strain in Example 1. After performing 16s rRNA gene amplification using two universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'), sequencing analysis of the amplified 16s rRNA gene Was performed. Using the analyzed 16s rRNA sequence data and EzTaxon server (http://www.ezbiocloud.net), only one strain corresponding to GRAS (Generally Recognized as Safe) was selected and shown in Table 3 below.

Identified strains Lactobacillus rhamnosus GCWB1156 Bifidobacterium breve GCWB1144

The 16S rRNA sequencing results of the Lactobacillus rhamnosus GCWB1156 are as follows.

<16S rRNA sequence of Lactobacillus rhamnosus GCW1156 >

Experimental Example 1: Analysis of anti-inflammatory effect in mouse lung macrophages

1-1) Cytotoxicity evaluation of strain (LDH leakage and CCK-8 assay)

Mouse lung macrophage cell line MH-S cell line (mouse alveolar macrophage cell line) was pre-sold from the American Cell Line Bank (ATCC, Manassas, VA, USA) and suspended so that the cell concentration was ⁵ to 10 ⁵ cells/ml, and 100 in a 96-well plate. Each µl was dispensed, each sample was treated by concentration, and cultured for 48 hours.

Cytotoxicity was measured using the MTT assay kit and Cytotoxicity LDH Assay Kit, and the results are shown in Table 4 below.

division Volume
(㎍/ml) LDH(fold of control)
(24hr) MTT(% of control)
(24hr) Normal group (No treatment) 0 1.00±0.04 100.0±2.12 Excipient (100% maltodextrin) 1000 0.99±0.02 99.6±3.13 Lactobacillus plantarum GCWB1001 10 0.99±0.03 101.7±3.4 100 1.01±0.03 102.3±4.17 1000 0.60±0.02* 101.5±3.2 GCWB1031 10 1.00±0.02 104.8±8.9 100 1.03±0.06 100.9±2.88 1000 0.85±0.02* 98.2±3.8 GCWB1084 10 1.01±0.03 96.4±1.94 100 1.18±0.26 100.0±1.57 1000 1.20±0.03 93.4±0.4 Pediococcus acidilactici GCWB1085 10 0.98±0.02 108.95±5.31 100 0.95±0.03 113.06±4.63** 1000 0.58±0.03* 102.03±1.39 Lactobacillus rhamnosus GCWB1156 10 0.87±0.03 99.9±1.26 100 0.82±0.02* 98.3±0.63 1000 0.90±0.01 93.5±4.8 GCWB1136 10 1.01±0.00 101.4±3.08 100 0.87±0.02 99.1±3.14 1000 0.81±0.02* 97.1±3.92 GCWB1144 10 0.87±0.04 102.3±4.03 100 0.82±0.02** 96.4±1.56 1000 0.79±0.02* 95.7±2.88

*P <0.001 vs. normal group. **P <0.01 vs. normal group.

(1) LDH assay result

As a result of performing LDH assay after 24 hours treatment of the 7 types of lactic acid bacteria of Table 3 at a concentration of 1 to 1,000 μg/ml in mouse MH-S lung macrophages, no cytotoxicity was observed in all 7 types. However, it was found that the LDH value was significantly decreased at the concentration of 1,000 μg/ml of the strains GCWB1001, GCWB1085, GCWB1176, and GCWB1156, and all strains were confirmed to have no cytotoxicity.

(2) MTT assay result

As a result of performing MTT assay after treatment of the 7 types of lactic acid bacteria in Table 4 at a concentration of 1 to 1,000 μg/ml in mouse MH-S lung macrophages for 24 hours, all 7 types were cytotoxicity at a concentration of 100 μg/ml Did not appear. However, cytotoxicity was confirmed at the concentration of 100㎍/㎖ of the GCWB1085 strain.

1-2) Measurement of cytokine secretion ability (TNF-α, TGF-β)

After pretreatment of the sample in MH-S cell line for 1 hour, LPS (10 ng/ml) or ConA (10 µg/ml) and DEP (200 µg/ml) were treated for 3 hours and 26 hours, respectively, and then secreted into the culture medium. The amount of cytokines (TNF-α, TGF-β) was measured using an ELISA kit (R&D system, USA), and the results are shown in Table 5 and FIG. 1 below.

1-2) Measurement of cytokine secretion ability (TNF-α, TGF-β)

After pretreatment of the sample in MH-S cell line for 1 hour, LPS (10 ng/ml) or ConA (10 µg/ml) and DEP (200 µg/ml) were treated for 3 hours and 26 hours, respectively, and then secreted into the culture medium. The amount of cytokines (TNF-α, TGF-β) was measured using an ELISA kit (R&D system, USA), and the results are shown in Table 5 and FIG. 1 below.

division Dose (㎍/㎖) TNF-α inhibition rate (%) Normal group (No treatment) - - 52.4 Control
(LPS10 ng/mL +DEP 200 ㎍/mL) - - 100## LPS10 ng/ml + DEP 200 ㎍/ml Lactobacillus plantarum GCWB1001 10 58.1* 100 74.2* 1000 89.7** GCWB1031 10 86.8** 100 130.0 1000 157.4 Pediococcus acidilactici GCWB1085 10 90.2*** 100 60.0* 1000 54.8* Lactobacillus rhamnosus GCWB1156 10 66.7* 100 66.9** 1000 105.6 GCWB1136 10 304.7 100 370.4 1000 1270.8 GCWB1144 10 449.1 100 1573.7 1000 3401.6

##P <0.01 vs. normal group. *P<0.001 vs LPS 10ng/ml+DEP 200µg/ml, **P<0.01 vs. LPS 10ng/ml+DEP 200µg/ml. ***P <0.05 vs. LPS 10 ng/ml+DEP 200 μg/ml.

(1) TNF-α measurement result

As shown in Table 5, the cytokine TNF-α secretion by LPS and DEP treatment in MH-S cells was reduced at most treatment concentrations in all strains of GCWB1001, GCWB1084 and GCWB1156. Therefore, the strain of the present invention reduced the secretion of TNF-α, a proinflammatory cytokine caused by fine dust, in a concentration-dependent manner.

(2) TGF-β measurement result

As shown in FIG. 1, when the strains of GCWB1001, GCWB1085, and GCWB1156 were treated with 1 µg/ml and 10 µg/ml, the amount of TGF-β secretion by fine dust was reduced.

1-3) Nitric oxide (NO) measurement

Mouse lung macrophage cell line NH-S cell line was suspended so that the cell concentration was ⁵ to 10 ⁵ cells/ml, and 100 μl each was dispensed into a 96-well plate. The 7 strains of Table 6, which are samples, were pre-treated at concentrations of 1 µg/ml and 10 µg/ml, respectively, for 1 hour, and then LPS 100 ng/ml was treated for 24 hours. Thereafter, 50 µl of the culture solution was transferred to a 96-well plate, Griess reagent I (NED solution) and Griess reagent II (Sulfanilamide solution) were mixed in the same amount, reacted for 10 minutes in a dark room, and then used a microplate reader within 30 minutes. It was measured at 540 nm.

division Volume Dose (㎍/㎖) NO(μM) Normal group (No Treatment) - - 0.42±0.00 Control
(LPS 10ng/ml+ DEP 200 µg/ml) - - 4.88±0.32# LPS 10ng/ml+ DEP 200 μg/ml Lactobacillus plantarum GCWB1001 One 4.26±0.44 10 4.47±0.60 GCWB1031 One 3.91±0.24** 10 5.16±0.74 GCWB1084 One 2.90±0.36* 10 8.30±0.12* Pediococcus acidilactici GCWB1085 One 3.20±0.32* 10 5.51±1.058 Lactobacillus rhamnosus GCWB1156 One 4.19±0.21*** 10 4.47±0.12 GCWB1136 One 5.37±0.99 10 7.74±0.21 GCWB1144 One 5.09±0.48 10 8.44±0.53*

#P <0.001 vs. normal group. *P <0.001 vs. LPS 10ng/ml+DEP 200µg/ml treated group. **P <0.01 vs. LPS 10ng/ml+DEP 200µg/ml treated group. ***P <0.05 vs. LPS 10ng/ml+DEP 200µg/ml treated group.

The experimental results are shown in a graph in FIG. 2. As can be seen from Table 6 and FIG. 2, it was confirmed that all of the strains GCWB1001, GCWB1085 and GCWB1156 of the present invention significantly reduced the amount of NO produced by fine dust at a concentration of 1 μg/ml. However, at the concentration of 10 μg/ml, NO production by fine dust was not significantly reduced.

1-4) Reactive oxygen species (ROS) measurement

Mouse lung macrophage cell line NH-S cell line was suspended so that the cell concentration was ⁵ to 10 ⁵ cells/ml, and 100 μl each was dispensed into a 96-well plate. The 7 strains of Table 6, which are samples, were pre-treated at concentrations of 1 µg/ml and 10 µg/ml, respectively, for 1 hour, treated with 100 ng/ml of LPS for 6 hours, washed with HBSS, and then added DCF-DA ( 2′,7′-Dichlorofluorescein diacetate)/HBSS was treated with 25 μM per well, incubated for 30 minutes, and fluorescence values were measured at excitation wavelength 485 nm and emission wavelength 530 nm.

The experimental results are shown in a graph in FIG. 3. It was confirmed that all of the GCWB1001, GCWB1085 and GCWB1156 strains of the present invention significantly reduced the amount of ROS produced by fine dust at a concentration of 1 μg/ml, as in the NO analysis.

1-5) Inflammatory transcriptional regulator activity measurement (iNOS-Luc, COX2-Luc, NF-κB-Luc; luciferase promoter activity analysis)

After dispensing MH-S cells at 1×10 ⁶ cells/ml, the plasmid vector into which iNOS-Luc, COX2-Luc, and NF-κB-Luc were respectively inserted was introduced into the cell using LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA). I did. After the samples were treated for 24 hours at each concentration, cells were harvested and the luciferase activity was measured.

4 to 6, the promoter activity of the inflammatory transcription factors iNOS, COX2, and NF-κB, which are inflammatory transcription factors caused by fine dust, was reduced when GCWB1001, 1085, and 1156 strains were treated with 1 μg/ml. Taken together, the strain of the present invention can suppress the expression of genes related to inflammation by inhibiting the promoter activity of the NF-κB transcription factor and iNOS and COX genes known as inflammation genes, which induce the expression of inflammation-related genes.

Experimental Example 2: Study on antitussive/expectant efficacy in an animal model ( in vivo assay)

2-1) Experimental conditions and breeding conditions

Experimental animals were supplied with 6-week-old male BALB/c mice (weight: 20±2 g) from Samtaco Co., Ltd. at a temperature of 23 ± 1 ^o C and a relative humidity of 55 ± 15% and an illuminance of 300-500 Lux every 12 hours. After acclimatizing for 7 days or more in an animal breeding room where the contrast is controlled, only normal animals are used for experiments by observing the visual symptoms, and solid feed for experimental animals (Samtaco, Inc.) and water are freely ingested. All animal experiments were conducted with the approval of the Korea International University Laboratory Animal Ethics Committee, and were performed according to the guidelines of the National Institutes of Health (NIH publication No. 86-23, revised 1985).

2-2) Research on antitussive effect

Experimental groups were 1M citric acid alone (control), citric acid + Synatura 200 mg/kg (positive control), citric acid + sample treatment group (1X10 ⁷ ), citric acid + sample treatment group (1X 10 ⁹ ), etc.4 groups, each test The number of animals per group was 8 (n=8) (Table 7).

No Experimental group (n=8) density One Citric acid (control) 1 M 2 Citric acid + Synatura 200 mg/kg 3 Citric acid + sample treatment group 1 x 10 ⁷ cfu 4 Citric acid + sample treatment group 1 x 10 ⁹ cfu

After oral administration of the sample for 1 hour, 1M citric acid, a cough inducing agent, was administered intranasal, and then each experimental group was placed in a chamber, and the number of coughing was measured for 10 minutes. As a result, as shown in FIG. 7, both GCWB1001 and GCWB1085 strains significantly reduced the number of coughs caused by citric acid after 1X10 ⁹ cfu administration.

2-3) Expectant effect research

Experimental group was 1M phenol red (phenol-red) administration group, phenol red + Synatura 200 mg/kg (positive control group), phenol red + sample treatment group (1X10 ⁷ ), citric acid + sample treatment group (1X10 ⁹ ) Etc. 4 groups were used, and the number of animals per each test group was 8 animals (n=8) (Table 8).

No Experimental group density One Phenol red+Saline 10mg/kg 2 Phenol red+Synatura 200 mg/kg 3 Phenol red+ sample treatment group 1x10 ⁷ cfu 4 Phenol red+ sample treatment group 1x10 ⁹ cfu

One hour after oral administration of the sample, 0.2 ml of phenol red (10 mg/ml) was injected intraperitoneally, and 30 minutes later, the experimental animal was sacrificed and the trachea was removed.

After measuring the weight of the extracted organs, 0.5 ml, 0.9% Saline (w/v) was added and vortexed. After 100 µl of 1M NaOH, which is a color developing substrate, was added, the absorbance at 550 nm was measured.

As shown in FIG. 8, both GCWB1001 and GCWB1085 strains significantly increased the amount of phenol red excreted after administration of 1X10 ⁹ cfu.

Taken together, the strain of the present invention reduces the number of coughs and increases expectorant action, so that symptoms of respiratory diseases can be improved.

Experimental Example 3: Analysis of the effect of improving lung function in an animal model of chronic respiratory disease ( in vivo assay)

3-1) Experimental conditions and breeding conditions

Experimental animals were supplied with 6-week-old male BALB/c mice (weight: 20±2 g) from Samtaco Co., Ltd. at a temperature of 23 ± 1 ^o C and a relative humidity of 55 ± 15% and an illuminance of 300-500 Lux every 12 hours. After acclimating for 7 days or more in an animal breeding room where the contrast is controlled, only normal animals were used for the experiment by observing the visual symptoms, and solid feed for experimental animals (Samtaco Co., Ltd.) and water were freely ingested. All animal experiments were conducted with the approval of the Korea International University Laboratory Animal Ethics Committee, and were performed according to the guidelines of the National Institutes of Health (NIH publication No. 86-23, revised 1985).

3-2) Establishment of chronic respiratory disease animal model and administration of experimental substances

Male 6-week-old BALB/c mice were treated on Day 1 and Day 12 (sensitization) once each 200 µl of aluminum hydroxide (Al(OH) ₃ ) and a 1:1 mixture of saline and 100 µg of ovalbumin; OVA) was administered intraperitoneally. On the 19th and 20th days of the start of the experiment, 50㎍ of OVA (Challenge) was administered intranasally once. After the last OVA was administered intranasally (20 days), 400 μg of diesel exhaust particles (DEP) were intranasally administered three times at 3 hour intervals, and the experiment was terminated on the 21st day.

24 hours after the last sample administration, urethane (Sigma-Aldrich, UK, USA) was administered intraperitoneally (0.020 ㎖/g weight) and anesthetized, and then bronchoalveolar lavage fluid (BALF) was obtained. The strain of the present invention was orally administered once a day for Day 0 to Day 20. Samples of GCWB1001, GCWB1085, and GCWB1156 were administered orally at 1× ^{10 7} cfu and 1× ^{10 9} cfu, respectively. Synatura (200 mg/kg) was used as a positive control, and the number of animals per each experimental group was 8 animals (n=8) (Table 9).

No. Experimental group (n=8) density One NA(control) - 2 Ovalbumin (OVA) 100 ㎍ 3 OVA+ DEP (Diesel exhaust particles) 400 ㎍ 4 OVA+DEP+GCWB1001 1x10 ⁷ cfu 5 OVA+DEP+GCWB1001 1x10 ⁹ cfu 6 OVA+DEP+GCWB1085 1x10 ⁷ cfu 7 OVA+DEP+GCWB1085 1x10 ⁹ cfu 8 OVA+DEP+GCWB1156 1x10 ⁷ cfu 9 OVA+DEP+GCWB1156 1x10 ⁹ cfu 10 OVA+DEP+Synatura 200 mg/kg

3-3) Weight and organ weight measurement

24 hours after the last sample administration, urethane (Sigma-Aldrich, UK, USA) was administered intraperitoneally (0.020 ㎖/g weight) and anesthetized. After securing bronchoalveolar lavage fluid (BALF), the lungs were excised and the weight was measured. In addition, body weight, liver and spleen weight were additionally measured.

Body weight There was no change in all groups, in the OVA and OVA + DEP-treated group was the weight of the waste increased as compared with the untreated ^{group, GCWB1001 (1X10 9 cfu),} GCWB1085 (1X10 9 cfu), GCWB1156 (1X10 9 cfu ), the weight of lung tissue increased by OVA+DEP treatment was significantly decreased in the Synatura treatment group. The spleen also significantly decreased the weight of spleen and lung tissue increased by OVA+DEP treatment in GCWB1001 (1X10 ⁷ cfu), GCWB1085 (1X10 ⁹ cfu), and GCWB1156 (1X10 ⁹ cfu).

3-4) Evaluation of lung function improvement of the strain of the present invention in an animal model of chronic respiratory disease-bronchoalveolar cell fluid analysis

24 hours after the last sample administration, urethane (Sigma-Aldrich, UK, USA) was intraperitoneally administered (0.020 ml/g weight) to obtain bronchoalveolar lavage fluid (BALF) after anesthesia, and then the immune cell profile ( Total cells, Macrophages, Eosinophils, Neutrophils, Lymphocytes) were measured.

According to Figure 10, compared to the normal group, the number of cells of all the immune cells measured in the OVA-treated group was significantly increased, and compared to the OVA-treated group, the number of immune cells including the total number of cells was It increased significantly. However, the number of immunized cells increased by OVA+DEP treatment significantly decreased in the samples GCWB1001, GCWB1085, GCWB1156, and Synatura treatment groups.

In particular, when each strain was treated with 1X10 ⁹ cfu, the number of cells of all immune cells decreased comparable to that of the positive control synatura.Even when treated with 1X10 ⁷ cfu, the number of cells excluding Lymphocytes (Total cells, Macrophages, Eosinophils, Neutrophils) were effectively reduced.

Therefore, the strain of the present invention reduces the penetration of immune cells in the lung tissue, and thus may prevent the progression of respiratory diseases.

3-5) Evaluation of lung function improvement of the strain of the present invention in an animal model of chronic respiratory disease-OVA-specific IgE measurement (BALF, serum)

24 hours after the last sample administration, urethane (Sigma-Aldrich, UK, USA) was administered intraperitoneally (0.020 ㎖/g weight) and anesthetized, and then bronchoalveolar lavage fluid (BALF) and serum were secured. The amount of the indicator OVA-specific-IgE was measured.

As shown in FIG. 11, OVA-specific-IgE was significantly increased in BALF (FIG. 11A) and serum (FIG. 11B) of the OVA-treated group compared to the normal group, and the OVA+DEP-treated group was compared with the OVA-treated group. OVA-specific-IgE was significantly increased in. Sample of ^{GCWB1001 (1X10 7, 1X10 9 cfu} ), the OVA- specific -IgE increased by OVA + DEP treated significantly in ^{GCWB1085 (1X10 7, 1X10 9 cfu} ), GCWB1156 (1X10 7, 1X10 9 cfu), Synatura treated group Decreased significantly. Accordingly, the strain of the present invention can reduce pulmonary and systemic allergic reactions, which are indicators of inflammatory reactions.

3-6) Evaluation of lung function improvement of the strain of the present invention in an animal model of chronic respiratory disease-cytokine (TNF-α, IL-6, IL-1β, IL-4, IL-13, MCP-1, IFN-gamma in BALF) ) Measure

, IL-4, IL-13, MCP-1, IFN-gamma)의 ELISA kit(R&D system USA)을 이용하여 측정하였다.24 hours after the last sample administration, urethane (Sigma-Aldrich, UK, USA) was administered intraperitoneally (0.020 ㎖/g weight) and anesthetized, followed by cytokines (TNF-α, IL-6, IL-1) in BALF.

, IL-4, IL-13, MCP-1, IFN-gamma) was measured using an ELISA kit (R&D system USA).

Compared to the normal group, TNF-α, IL-6, IL-1β, IL-4, IL-13, and MCP-1 were significantly increased in the OVA-treated group, compared to the OVA-treated group, and in the OVA+DEP-treated group. TNF-α, IL-6, IL-1β, IL-13, and MCP-1 were significantly increased. The strains of the present invention GCWB1001 (1X10 ⁷ , 1X10 ⁹ cfu), GCWB1085 (1X10 ⁷ , 1X10 ⁹ cfu), GCWB1156 (1X10 ⁷ , 1X10 ⁹ cfu), TNF-α increased by OVA+DEP treatment in the Synatura treatment group, IL-6, IL-1β, IL-4, IL-13, MCP-1 significantly decreased (Fig. 12A ~ Fig. 12D).

In addition, OVA + DEP decreased the amount of IFN-γ in the treatment ^{group, GCWB1001 (1X10 7, 1X10 9} cfu), GCWB1085 (1X10 7, 1X10 9 cfu), GCWB1156 (1X10 7, 1X10 9 cfu), Synatura treated group Significantly increased in (Fig. 12G).

Therefore, the strains of the present invention reduce pro-inflammatory cytokines in the lung and increase anti-inflammatory cytokines, so that the inflammatory response in the lung tissue may be reduced, thereby preventing the progression of respiratory diseases.

3-7) Evaluation of the improvement of lung function of the strain of the present invention in an animal model of chronic respiratory disease-Histological examination (H&E staining)

After fixing the left lobe lung tissue in 10% formalin for 24 hours, a paraffin block was prepared, cut to a thickness of 4 μm, and H&E staining was performed. After obtaining an image with an optical microscope, the degree of inflammation was measured.

Compared to the normal group, the inflammatory cell penetration was significantly increased in the OVA treatment group, and the inflammatory cell penetration was significantly increased in the OVA+DEP treatment group compared to the OVA treatment group.

Infiltration of inflammatory cells increased by OVA+DEP treatment in the strains of the present invention, GCWB1001 (1X10 ⁷ , 1X10 ⁹ cfu), GCWB1085 (1X10 ⁷ , 1X10 ⁹ cfu), GCWB1156 (1X10 ⁷ , 1X10 ⁹ cfu), Synatura treatment group Was significantly decreased (Fig. 13).

Therefore, the strains of the present invention prevent the penetration of inflammatory cells in the lung tissue, and thus reduce the inflammatory response in the lung tissue, thereby preventing the progression of respiratory diseases.

3-8) Evaluation of the improvement of lung function of the strain of the present invention in an animal model of chronic respiratory disease-histological examination (alcian blue-PAS stain)

In order to confirm whether the strain of the present invention actually inhibits the production of mucous proteins in lung tissue, lung tissue was stained with alcian blue-PAS staining method for staining glycogen and mucin.

14 is a photograph of the actual stained tissue. The production of mucous protein in the alveoli was significantly increased in the OVA-treated group compared to the normal group, and the mucous protein was produced in the OVA+DEP-treated group significantly compared to the OVA-treated group. Increased. Sample of ^{GCWB1001 (1X10 7, 1X10 9 cfu} ), GCWB1085 (1X10 7, 1X10 9 cfu), GCWB1156 (1X10 7, 1X10 9 cfu), the production of mucin protein, up by OVA + DEP treated in Synatura treated group significantly Decreased (Fig. 14).

Therefore, since the strain of the present invention reduces the mucous quality in the alveoli, it will be possible to prevent exacerbation of the stage of respiratory disease such as pulmonary fibrosis.

3-9) Evaluation of the improvement of lung function of the strain of the present invention in an animal model of chronic respiratory disease-caspase 3 activity and total collagen content

In order to confirm whether the strain of the present invention actually reduced apoptosis in lung tissue and improved lung damage, caspase 3 activity and total collagen content in lung tissue were measured.

According to FIG. 15, caspase-3 activity and collagen content were significantly increased in the OVA-treated group compared to the normal group, and caspase-3 activity and collagen content were significantly increased in the OVA+DEP-treated group compared to the OVA-treated group. . In all of the strains of the present invention GCWB1001 (1X10 ⁷ , 1X10 ⁹ cfu), GCWB1085 (1X10 ⁷ , 1X10 ⁹ cfu), GCWB1156 (1X10 ⁷ , 1X10 ⁹ cfu), Synatura treatment group, caspase-3 increased by OVA+DEP treatment Both activity and collagen content were significantly reduced (FIG. 15).

Accordingly, the strain of the present invention not only reduces apoptosis factor, but also reduces collagen content, thereby preventing pulmonary fibrosis, which is a stage of respiratory disease.

3-10) Evaluation of lung function improvement of strain of the present invention in animal model of chronic respiratory disease-MMP9 activity analysis

Protein isolated by 10% SDS-PAGE electrophoresis method containing 0.2% gelatin in BALF (bronchoalveolar lavage fluid) was stained with 0.25% Coomassie Brilliant Blue G250 (Sigma Chemical Co., St. Louis, MO, USA) to MMP. -9 Enzyme activity was measured.

According to FIG. 16, MMP-9 activity was significantly increased in the OVA-treated group compared to the normal group, and MMP-9 activity was significantly increased in the OVA+DEP-treated group compared to the OVA-treated group. In the strains of the present invention, GCWB1001 (1X10 ⁹ cfu), GCWB1085 (1X10 ⁹ cfu), GCWB1156 (1X10 ⁹ cfu), and Synatura treatment group, the MMP-9 activity increased by OVA+DEP treatment was significantly reduced.

Therefore, the strain of the present invention can prevent the deposition of inflammatory cells in the lung tissue, and thus can prevent inflammation and various complications due to inflammation in the lung tissue.

<110> GREEN CROSS WellBeing Corporation <120> Composition for prevention or treatment of respiratory diseases or inflammation induced by particulate matter comprising novel lactic acid bacteria <130> P2020-0050 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1434 <212> RNA <213> Lactobacillus plantarum <400> 1 ggtcgtacga actctgtgta ttgattggtg cttgcatcat gatttacatt tgcagtgagt 60 ggcgaactgg tgagtaacac gtgggaaacc tgcccagaag cgggggataa cacctggaaa 120 cagatgctaa taccgcataa caacttggac cgcatggtcc gagtttgaaa gatggcttcg 180 gctatcactt ttggatggtc ccgcggcgta ttagctagat ggtggggtaa cggctcacca 240 tggcaatgat acgtagccga cctgagaggg taatcggcca cattgggact gagacacggc 300 ccaaactcct acgggaggca gcagtaggga atcttccaca atggacgaaa gtctgatgga 360 gcaacgccgc gtgagtgaag aagggtttcg gctcgtaaaa ctctgttgtt gaagaagaac 420 atatctgaga gtaactgttc aggtattgac ggtatttaac cagaaagcca cggctaacta 480 cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggattta ttgggcgtaa 540 agcgagcgca ggcggttttt taagtctgat gtgaaagcct tcggctcaac cgaagaagtg 600 catcggaaac tgggaaactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg 660 aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgtaactga 720 cgctgaggct cgaaagtatg ggtagcaaac aggattagat accctggtag tccataccgt 780 aaacgatgaa tgctaagtgt tggagggttt ccgcccttca gtgctgcagc taacgcatta 840 agcattccgc ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc 900 gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaagaacctt accaggtctt 960 gacatactat gcaaatctaa gagattagac gttcccttcg gggacatgga tacaggtggt 1020 gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080 ccttattatc agttgccagc attaagttgg gcactctggt gagactgccg gtgacaaacc 1140 ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200 ctacaatgga tggtacaacg agttgcgaac tcgcgagagt aagctaatct cttaaagcca 1260 ttctcagttc ggattgtagg ctgcaactcg cctacatgaa gtcggaatcg ctagtaatcg 1320 cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380 tgagagtttg taacacccaa agtcggtggg gtaacctttt aggaaccagc cgct 1434 <210> 2 <211> 1499 <212> RNA <213> Pediococcus acidilactici <400> 2 ctcaggatga acgctggcgg cgtgcctaat acatgcaagt cgaacgaact tccgttaatt 60 gatcaggacg tgcttgcact gaatgagatt ttaacacgaa gtgagtggcg gacgggtgag 120 taacacgtgg gtaacctgcc cagaagcagg ggataacacc tggaaacaga tgctaatacc 180 gtataacaga gaaaaccgcc tggttttctt ttaaaagatg gctctgctat cacttctgga 240 tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggcg atgatgcgta 300 gccgacctga gagggtaatc ggccacattg ggactgagac acggcccaga ctcctacggg 360 aggcagcagt agggaatctt ccacaatgga cgcaagtctg atggagcaac gccgcgtgag 420 tgaagaaggg tttcggctcg taaagctctg ttgttaaaga agaacgtggg tgagagtaac 480 tgttcaccca gtgacggtat ttaaccagaa agccacggct aactacgtgc cagcagccgc 540 ggtaatacgt aggtggcaag cgttatccgg atttattggg cgtaaagcga gcgcaggcgg 600 tcttttaagt ctaatgtgaa agccttcggc tcaaccgaag aagtgcattg gaaactggga 660 gacttgagtg cagaagagga cagtggaact ccatgtgtag cggtgaaatg cgtagatata 720 tggaagaaca ccagtggcga aggcggctgt ctggtctgta actgacgctg aggctcgaaa 780 gcatgggtag cgaacaggat tagataccct ggtagtccat gccgtaaacg atgattacta 840 agtgttggag ggtttccgcc cttcagtgct gcagctaacg cattaagtaa tccgcctggg 900 gagtacgacc gcaaggttga aactcaaaag aattgacggg ggcccgcaca agcggtggag 960 catgtggttt aattcgaagc tacgcgaaga accttaccag gtcttgacat cttctgccaa 1020 cctaagagat taggcgttcc cttcggggac agaatgacag gtggtgcatg gttgtcgtca 1080 gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta ttactagttg 1140 ccagcattca gttgggcact ctagtgagac tgccggtgac aaaccggagg aaggtgggga 1200 cgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggatggta 1260 caacgagttg cgaaaccgcg aggtttagct aatctcttaa aaccattctc agttcggact 1320 gtaggctgca actcgcctac acgaagtcgg aatcgctagt aatcgcggat cagcatgccg 1380 cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatgaga gtttgtaaca 1440 cccaaagccg gtggggtaac cttttaggag ctagccgtct aaggtgggac agatgatta 1499 <210> 3 <211> 1188 <212> RNA <213> Lactobacillus rhamnosus <400> 3 gttgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtagg 60 gaatcttcca caatggacgc aagtctgatg gagcaacgcc gcgtgagtga agaaggcttt 120 cgggtcgtaa aactctgttg ttggagaaga atggtcggca gagtaactgt tgtcggcgtg 180 acggtatcca accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg 240 tggcaagcgt tatccggatt tattgggcgt aaagcgagcg caggcggttt tttaagtctg 300 atgtgaaagc cctcggctta accgaggaag tgcatcggaa actgggaaac ttgagtgcag 360 aagaggacag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca 420 gtggcgaagg cggctgtctg gtctgtaact gacgctgagg ctcgaaagca tgggtagcga 480 acaggattag ataccctggt agtccatgcc gtaaacgatg aatgctaggt gttggagggt 540 ttccgccctt cagtgccgca gctaacgcat taagcattcc gcctggggag tacgaccgca 600 aggttgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 660 tcgaagcaac gcgaagaacc ttaccaggtc ttgacatctt ttgatcacct gagagatcag 720 gtttcccctt cgggggcaaa atgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 780 gatgttgggt taagtcccgc aacgagcgca acccttatga ctagttgcca gcatttagtt 840 gggcactcta gtaagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca 900 tcatgcccct tatgacctgg gctacacacg tgctacaatg gatggtacaa cgagttgcga 960 gaccgcgagg tcaagctaat ctcttaaagc cattctcagt tcggactgta ggctgcaact 1020 cgcctacacg aagtcggaat cgctagtaat cgcggatcag cacgccgcgg tgaatacgtt 1080 cccgggcctt gtacacaccg cccgtcacac catgagagtt tgtaacaccc gaagccggtg 1140 gcgtaaccct tttagggagc gagccgtcta aggtgggaca aatgatta 1188

Claims (14)

Lactobacillus rhamnosus GCWB1156 strain deposited with accession number KCCM12700P, a culture of the strain, a concentrate of the culture, a paste product, a health functional food composition for preventing or improving respiratory diseases by fine dust comprising any one of a dilution. The method of claim 1,
The strain is comprising a 16S rRNA consisting of the nucleotide sequence of SEQ ID NO: 3, health functional food composition. The method according to claim 1 or 2,
The strain is a spray-dried product, a freeze-dried product, a vacuum dried product, a drum dried product, or a crushed product, health functional food composition. The method of claim 3,
The health functional food composition further comprises at least one selected from the group consisting of a seasoning agent, a sweetening agent, and a flavoring agent. The method of claim 4,
The health functional food composition includes granules, powders, tablets, and capsules. Any one selected from among, health functional food composition. Lactobacillus rhamnosus GCWB1156 strain deposited with accession number KCCM12700P, a pharmaceutical composition for preventing or treating respiratory diseases by fine dust comprising any one of a culture of the strain, a concentrate of the culture, a paste product, or a dilution,
The respiratory diseases include asthma, bronchiectasis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, emphysema, allergic rhinitis, lower respiratory tract infection, bronchiolitis, acute upper respiratory tract infection, bronchiectasis, pneumonia, Acute chronic bronchitis, sinusitis, sore throat, tonsillitis and laryngitis, which is selected from the group consisting of, a pharmaceutical composition for preventing or treating respiratory diseases. A pharmaceutical composition for Jinhae or Geodam comprising any one of the Lactobacillus rhamnosus GCWB1156 strain deposited with accession number KCCM12700P, a culture of the strain, a concentrate of the culture, a paste product, or a dilution. The method according to claim 6 or 7,
The strain is a spray-dried product, a freeze-dried product, a vacuum dried product, a drum dried product, or a crushed product. The method according to claim 6 or 7,
The pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier. Probiotics for the prevention or improvement of respiratory diseases caused by fine dust containing Lactobacillus rhamnosus GCWB1156 strain deposited as accession number KCCM12700P. The method of claim 10,
The probiotics will have a viable cell content of 1Х10 ¹ ~ 1Х10 ¹³ CFU / g, probiotics. The method of claim 6,
The pharmaceutical composition is to reduce the inflammatory response in lung tissue, pharmaceutical composition. The method of claim 6,
The pharmaceutical composition is to reduce allergic reactions, pharmaceutical composition. The method of claim 6,
The pharmaceutical composition is to inhibit lung fibrosis, the pharmaceutical composition.

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