Composition comprising lactic acid bacteria for treating respiratory or inflammatory diseases caused by fine dust stimulation

Abstract

The present disclosure provides a novel strain of Lactobacillus plantarum GCWB1001, Pediococcus acidilactici GCWB1085, or Lactobacillus rhamnosus GCWB1156, wherein the Lactobacillus plantarum GCWB1001, the Pediococcus acidilactici GCWB1085, the Pediococcus acidilactici GCWB1085, the Lactobacillus rhamnosus GCWB1156, the Lactobacillus plantarum GCWB1001, the Pediococcus acidilactici GCWB1085, the Lactobacillus rhamnosus GCWB1156 having a preservation number KCCM12698P, and the Furthermore, a pharmaceutical composition, a health-care functional food composition and a probiotic are provided, each comprising any one of the novel strains and having an effect of treating or alleviating inflammatory diseases or respiratory diseases.

Description

[0001] This application is a divisional application. The application number of the original application is "202180060898.X", the application date is April 23, 2021, and the name of the invention is "A composition containing lactic acid bacteria for treating respiratory diseases or inflammatory diseases caused by fine dust irritation." Technical Field

[0002] This application claims the benefit of Korean Patent Application No. 10-2020-0061851 filed in the Korean Intellectual Property Office on May 22, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to novel lactic acid bacteria having the effect of treating or preventing respiratory diseases or inflammatory diseases, and pharmaceutical compositions, health functional food compositions and probiotics comprising the lactic acid bacteria. Background Art

[0004] Fine dust is particulate matter of 10 μm or less that floats in the air or is blown off, and is classified according to the dust diameter, with PM10 representing particles smaller than 10 / 1,000mm and PM2.5 representing particles smaller than 2.5 / 1,000mm.

[0005] Fine dust is caused by direct emissions through anthropogenic sources such as workplace combustion and automobile fuel combustion, or is produced through secondary generation processes in which substances such as sulfur oxides (SOx), nitrogen oxides (NOx), ammonia (NH3) and volatile organic compounds (VOCs) react with water vapor in the air.

[0006] Fine dust is known to be able to penetrate directly into the alveoli or the brain when inhaled due to its fine particles without being filtered through the nasal mucosa, and is known to increase the morbidity and premature mortality of asthma and lung diseases.

[0007] In particular, in 2013, the International Agency for Research on Cancer (IARC) under the World Health Organization (WHO) classified fine dust as a Group 1 carcinogen, which is confirmed to cause human cancer as well as benzene and asbestos.

[0008] Live microorganisms that beneficially affect the host's health by improving the host's intestinal microbial environment in the gastrointestinal tract of animals, including humans, are collectively referred to as probiotics.

[0009] Lactic acid bacteria is a type of probiotics, which is used to decompose fiber and complex protein into important nutrients, and coexists in the digestive system of human body. Lactic acid bacteria decompose carbohydrates and utilize carbohydrates to produce lactic acid, and it is an anaerobic bacteria that is well propagated in a place with little oxygen. Lactic acid bacteria are mainly divided into five genera of Streptococcus, Lactobacillus, Leuconostoc, Bifidobacteria and Pediococcus. Recently, it has been confirmed that the therapeutic effect of using lactic acid bacteria to various diseases has been confirmed, and the therapeutic agent using lactic acid bacteria has been attempted to be developed.

[0010] Unlike respiratory and lung damage caused by conventional bacteria, temporary poisoning or other substances flowing into the airways, fine dust cannot be eradicated by human immunity, and there is no method of forcibly expelling fine dust when it enters through the respiratory tract. In addition, since it is not known exactly when and what problems will occur due to such damage, new therapeutic agents are urgently needed that can prevent, alleviate, treat or improve damage to the airways and lungs caused by fine dust, thereby treating respiratory diseases caused by fine dust. Summary of the invention

[0011] Technical issues

[0012] An object of the present disclosure is to provide a novel Lactobacillus plantarum GCWB1001 strain deposited with the accession number KCCM12698P, a Lactobacillus rhamnosus GCWB1156 strain deposited with the accession number KCCM12700P, a Pediococcus acidilactici GCWB1085 strain deposited with the accession number KCCM12699P, and a composition comprising the same for preventing or treating inflammatory diseases or respiratory diseases.

[0013] Technical Solution

[0014] In order to achieve the above objectives,

[0015] The present disclosure provides a Lactobacillus plantarum GCWB1001 strain deposited under the deposit number KCCM12698P.

[0016] The present disclosure provides a Lactobacillus rhamnosus GCWB1156 strain deposited with the deposit number KCCM12700P.

[0017] The present disclosure provides a Pediococcus acidilactici GCWB1085 strain deposited under the accession number KCCM12699P.

[0018] In addition, the present disclosure provides a pharmaceutical composition for preventing or treating inflammatory diseases or respiratory diseases, comprising: Lactobacillus plantarum GCWB1001 strain preserved with accession number KCCM12698P, Lactobacillus rhamnosus GCWB1156 strain preserved with accession number KCCM12700P, or Pediococcus acidilactici GCWB1085 strain preserved with accession number KCCM12699P; one selected from a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product, or a pulverized product of the strain; or a culture of the strain and any one of a concentrate, a paste, and a dilution of the culture.

[0019] In addition, the present disclosure provides a health functional food composition for preventing or alleviating inflammatory diseases or respiratory diseases, comprising: Lactobacillus plantarum GCWB1001 strain preserved with the preservation number KCCM12698P, Lactobacillus rhamnosus GCWB1156 strain preserved with the preservation number KCCM12700P or Pediococcus acidilactici GCWB1085 strain preserved with the preservation number KCCM12699P; one selected from a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product or a pulverized product of the strain; or a culture of the strain and any one of a concentrate, a paste and a dilution of the culture.

[0020] In addition, the present disclosure provides probiotics, including Lactobacillus plantarum GCWB1001 strain deposited with the accession number KCCM12698P, Lactobacillus rhamnosus GCWB1156 strain deposited with the accession number KCCM12700P, or Pediococcus acidilactici GCWB1085 strain deposited with the accession number KCCM12699P.

[0021] Beneficial Effects

[0022] According to one embodiment of the present disclosure, Lactobacillus plantarum GCWB1001 strain (KCCM12698P), Lactobacillus rhamnosus GCWB1156 strain (KCCM12700P) and Pediococcus acidilactici GCWB1085 strain (KCCM12699P) have anti-inflammatory effects and exhibit antitussive / expectorant effects and the effect of improving lung function in chronic respiratory diseases in actual animal models.

[0023] According to one embodiment of the present disclosure, Lactobacillus plantarum GCWB1001 strain (KCCM12698P), Lactobacillus rhamnosus GCWB1156 strain (KCCM12700P) and Pediococcus acidilactici GCWB1085 strain (KCCM12699P) were isolated from kimchi, cheese and infant feces, respectively, and each strain did not exhibit cytotoxicity in mouse lung macrophages (MH-S cell line) and had the effect of reducing the secretion of TNF-α and TGF-β as cytokines.

[0024] According to one embodiment of the present disclosure, all strains of the present disclosure not only reduce the production of inflammatory factors nitric oxide (NO) and reactive oxygen species (ROS) increased by fine dust, but also reduce the promoter activities of NF-kB, iNOS and COX2 which are inflammatory transcription factors.

[0025] According to one embodiment of the present disclosure, all strains of the present disclosure have the effects of relieving cough symptoms and antitussive / expectorant effects in actual animal models, and also show the effects of relieving various symptoms of an animal model of chronic respiratory diseases caused by fine dust.

[0026] Therefore, the Lactobacillus plantarum GCWB1001 strain (KCCM12698P), the Lactobacillus rhamnosus GCWB1156 strain (KCCM12700P) and the Pediococcus acidilactici GCWB1085 strain (KCCM12699P) of the present disclosure show the effects of alleviating, preventing or treating inflammatory diseases or respiratory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a graph showing the measurement of TGF-β production amounts of 7 types of strain samples in a mouse lung macrophage cell line (MH-S) (Experimental Example 1-2).

[0028] Figure 2 and Figure 3 It is a graph showing the measurement of NO and ROS production amounts of 7 types of strain samples in a mouse lung macrophage cell line (MH-S) (Experimental Example 1-3 and Experimental Example 1-4).

[0029] Figures 4 to 6 It is a graph showing the measurement of inflammatory transcription regulator activities (promoter activities of iNOS, COX2, and NF-kB) of 7 types of strain samples in a mouse lung macrophage cell line (MH-S) (Experimental Examples 1-5).

[0030] Figure 7 and Figure 8It is a figure which shows the antitussive and expectorant effects of the GCWB1001 strain, the GCWB1085 strain, and the GCWB1156 strain in the mouse cough model (Experimental Example 2-2 and Experimental Example 2-3).

[0031] Fig. 9 It is a graph showing body weight and the weights of the liver, spleen, and lung after treatment with the GCWB1001 strain, the GCWB1085 strain, and the GCWB1156 strain in the chronic respiratory disease animal model (Experimental Example 3-3).

[0032] Fig.10 It is a graph showing the results of measuring the number of various immune cells in bronchoalveolar lavage fluid (BALF) from an animal model of chronic respiratory disease after treatment with the GCWB1001 strain, the GCWB1085 strain, and the GCWB1156 strain (Experimental Example 3-4).

[0033] Fig.11 It is a graph measuring the amount of OVA-specific IgE in bronchoalveolar lavage fluid (BALF) after treatment with GCWB1001 strain, GCWB1085 strain, and GCWB1156 strain in a chronic respiratory disease animal model (Experimental Example 3-5).

[0034] Fig.12 It is a graph measuring the amount of cytokines (TNF-α, IL-6, IL-1β, IL-4, IL-13 and MCP-1) in bronchoalveolar lavage fluid (BALF) after treatment with GCWB1001 strain, GCWB1085 strain and GCWB1156 strain in a chronic respiratory disease animal model (Experimental Examples 3-6).

[0035] Fig.13 These are photos and diagrams of lung tissue obtained by staining inflammatory cells infiltrated into lung tissue after treatment with GCWB1001, GCWB1085, and GCWB1156 strains in a chronic respiratory disease animal model (Experimental Examples 3-7).

[0036] Fig.14 These are photos and diagrams of lung tissue obtained by staining for mucin after treatment with GCWB1001, GCWB1085, and GCWB1156 strains in an animal model of chronic respiratory disease (Experimental Examples 3-8).

[0037] Fig.15 It is a graph showing the caspase 3 activity and total collagen content in lung tissue after treatment with GCWB1001 strain, GCWB1085 strain and GCWB1156 strain in a chronic respiratory disease animal model (Experimental Example 3-9).

[0038] Fig.16 These are photographs of analyzing MMP-9 activity in lung tissues after treatment with GCWB1001, GCWB1085, and GCWB1156 strains in an animal model of chronic respiratory disease (Experimental Example 3-10). DETAILED DESCRIPTION

[0039] Hereinafter, the present disclosure will be described in detail.

[0040] Unless otherwise defined, all technical terms used in this disclosure have the meanings commonly understood by those skilled in the art of the disclosure. In addition, although preferred methods and samples are described herein, methods and samples similar or equivalent thereto are also included within the scope of this disclosure.

[0041] The present disclosure relates to a Lactobacillus plantarum GCWB1001 strain deposited under the accession number KCCM12698P.

[0042] Lactobacillus plantarum GCWB1001 is a novel strain, which was named as described above and deposited in the Korea Culture Center for Microorganisms on April 17, 2020. The deposit number is KCCM12698P.

[0043] The strain can be isolated and identified from kimchi collected from various regions. The inventors of the present disclosure have isolated and identified various novel strains from kimchi collected from various regions, and confirmed that, by comparison with conventionally known lactic acid bacteria, the Lactobacillus plantarum GCWB1001 strain provides significantly excellent anti-inflammatory effects and preventive or therapeutic effects on respiratory diseases among the isolated and identified strains, and then completed the present disclosure.

[0044] The present disclosure relates to a Lactobacillus rhamnosus GCWB1156 strain deposited under the deposit number KCCM12700P.

[0045] Lactobacillus rhamnosus GCWB1156 is a novel strain, which was named as described above and deposited in the Korea Culture Center for Microorganisms on April 17, 2020. The deposit number is KCCM12700P.

[0046] The strain can be isolated and identified from cheese collected from various regions. The inventors of the present disclosure have isolated and identified various novel strains from cheese collected from various regions, and confirmed that, by comparison with conventionally known lactic acid bacteria, the Lactobacillus rhamnosus GCWB1156 strain provides significantly excellent anti-inflammatory effects and preventive or therapeutic effects on respiratory diseases among the isolated and identified strains, and then completed the present disclosure.

[0047] The present disclosure relates to a Pediococcus acidilactici GCWB1085 strain deposited under the deposit number KCCM12699P.

[0048] Pediococcus acidilactici GCWB1085 is a novel strain, which was named as described above and deposited in the Korea Culture Center for Microorganisms on April 17, 2020. The deposit number is KCCM12699P.

[0049] The strain can be isolated and identified from infant feces. The inventors of the present disclosure have isolated and identified various novel strains from infant feces, and confirmed that, by comparison with conventionally known lactic acid bacteria, the Pediococcus acidilactici GCWB1085 strain provides significantly excellent anti-inflammatory effects and preventive or therapeutic effects on respiratory diseases among the isolated and identified strains, and then completed the present disclosure.

[0050] That is, Lactobacillus plantarum GCWB1001 strain, Lactobacillus rhamnosus GCWB1156 strain, and Pediococcus acidilactici GCWB1085 strain inhibited the activities of NO, ROS, and inflammatory transcription regulators (promoter activities of iNOS, COX2, and NF-kB) in the MH-S cell line, which is a mouse lung macrophage cell line treated with LPS and fine dust (DEP; diesel exhaust particles) as inflammation-inducing substances.

[0051] It can also be confirmed that the GCWB1001, GCWB1156, and GCWB1085 strains of the present disclosure have anti-inflammatory effects by reducing the secretion of cytokines (TNF-α) induced by LPS and DEP in the MH-S cell line.

[0052] Oxidative stress refers to an imbalance in the production of reactive oxygen or reactive nitrogen species and antioxidant defense mechanisms in biomolecules, cells, and tissues in the body, as well as a phenomenon in which the production of reactive oxygen and reactive nitrogen species becomes relatively excessive, which generally causes tissue damage.

[0053] Since these reactive oxygen or reactive nitrogen are chemically very unstable and highly reactive, they cause inflammatory reactions in their surroundings and participate in tissue fibrosis as a major factor by causing enzyme catalytic reactions, electron transfer in mitochondria, cell signaling systems and gene expression, activation of transcription factors, and extensive oxidative damage to biomolecules, cells, tissues, etc. in the body. This oxidative damage causes various diseases in all tissues of the human body. Specifically, oxidative damage is not only known to be related to the occurrence and progression of cancer in tissues such as skin, kidneys, heart, joints, lungs, brain, blood vessels, intestines and eyes, but also known to play an important role in almost all diseases such as cardiovascular disease, inflammation, fibrotic diseases and diabetes.

[0054] When macrophages are treated with inflammatory substances such as LPS, inflammatory mediators (oxidative stress) such as inflammatory cytokines (IL-6, TNF-α, IL-1β, etc.) and NO are produced to cause an inflammatory response. NO is produced by inducible nitric oxide synthase (iNOS), which activates the NF-kB transcription factor as the inflammatory response occurs, and accelerates the inflammatory response by producing prostaglandins (PGs) that participate in the inflammatory response of cyclooxygenase-2 (COX-2).

[0055] Therefore, since the GCWB1001 strain, GCWB1156 strain and GCWB1085 strain of the present disclosure inhibit all activities of NO and ROS, which are inflammatory substances induced by LPS and DEP, TNF-α as inflammatory cytokines, and inflammatory transcription regulators (iNOS, COX2 and NF-kB promoter), the strains have anti-inflammatory effects that inhibit inflammation.

[0056] The GCWB1001 strain, the GCWB1156 strain, and the GCWB1085 strain of the present disclosure inhibit cough and increase sputum production in a citric acid-induced cough model.

[0057] Cough suppressants are a kind of medicine for relieving cough that is unrelated to the cause of disease, and can be divided into central acting medicine and peripheral acting medicine according to the mechanism of action, wherein central acting medicine can be divided into narcotic drugs, narcotic derivatives and non-narcotic drugs. Representative narcotic drugs are codeine, hydrocodone, morphine, etc., which have been shown to have limited cough suppressant effects, but the results are variable, and at appropriate doses, there are risks of drowsiness, constipation, digestive problems and abuse or dependence. In peripheral acting cough suppressants, the most commonly used drug in Korea is levoderopropizine (Levodropropizine), which is considered to be effective by controlling the level of sensory neuropeptides in the airway. In addition, theobromine (theobromine) corresponds to it.

[0058] The main component of sputum includes mucus, and bronchial mucus is secreted by mucus cells and serous gland cells constituting mucus glands and submucosal glands conventionally distributed in bronchial mucosa. Mucus is composed of 95% water and the remaining 5% glycoprotein, lipid, minerals, etc., and exhibits viscosity due to the gel form of the double structure of the glycoprotein structure with linear polymers. Expectorants for removing sputum are divided into drugs that increase the water content of sputum and mucolytic agents that reduce viscosity by breaking the SS bond of sputum protein. As these expectorants, cysteine ​​derivatives such as N-acetylcysteine ​​and carboxymethylcysteine ​​are used, and when used for a long time, these cysteine ​​derivatives can produce side effects such as bronchospasm. Therefore, it is necessary to develop an expectorant with little side effects and toxicity and excellent expectorant effect.

[0059] In addition, as cough and expectorant drugs made from natural materials, ivy leaf extract and Synatura, which is a combination of ivy leaf and Coptis chinensis extracts, are widely used.

[0060] The GCWB1001 strain, GCWB1156 strain and GCWB1085 strain of the present disclosure reduce the number of coughs in a citric acid-induced cough model and exhibit antitussive / expectorant effects, thereby improving the symptoms of respiratory diseases.

[0061] In addition, the strain disclosed in the present invention not only significantly reduces immune cells in bronchoalveolar lavage fluid (BALF) in a chronic respiratory disease model using ovalbumin (OVA) and diesel exhaust particles (DEP), but also reduces immune cells infiltrating into lung tissue, reduces OVA-specific IgE, reduces TNF-α, IL-6, IL-1β, IL-4, MCP-1 and IL-13 as inflammatory cytokines in BALF, and significantly increases the anti-inflammatory cytokine IFN-γ.

[0062] Therefore, the GCWB1001 strain, GCWB1156 strain and GCWB1085 strain disclosed in the present invention not only have an antitussive / expectorant effect themselves, but also inhibit the activity of MMP9 to block BALF and inflammatory cells in lung tissue, block the secretion of inflammatory cytokines, increase the secretion of anti-inflammatory cytokines, and reduce the amount of IgE immunoglobulins increased in allergic symptoms, thereby reducing allergic and inflammatory responses in respiratory organs.

[0063] Representative respiratory diseases include asthma, pneumonia, chronic obstructive pulmonary disease, allergic rhinitis, acute and chronic bronchitis, bronchiolitis, pharyngitis, tonsillitis, laryngitis, bronchiectasis, idiopathic pulmonary fibrosis, cystic fibrosis, emphysema, sequelae of pulmonary tuberculosis, lower respiratory tract infection, sinusitis, acute upper respiratory tract infection, allergic lung disease, etc. Asthma is a chronic inflammation of the airway, especially the bronchi. The inflammation caused by asthma can be aggravated by various factors, such as coal smoke, allergens, cold wind, exercise and respiratory tract infection, and the continuous inflammation causes airway deformation and airway hyperresponsiveness.

[0064] In addition, respiratory diseases may be caused by respiratory infectious viruses. Types of viruses that cause respiratory diseases include adenovirus, vaccinia virus, herpes simplex virus, parainfluenza virus, rhinovirus, varicella zoster virus, measles virus, respiratory syncytial virus, dengue virus, human immunodeficiency virus (HIV), influenza virus, coronavirus, severe acute respiratory syndrome-related virus (SARS-related virus), Middle East respiratory syndrome coronavirus (MERS-CoV), etc.

[0065] The respiratory tract is mainly composed of mucous membranes and muscles called bronchial smooth muscles, and the mucous membranes have many glands to continuously secrete necessary secretions. When the bronchial smooth muscles contract, the respiratory tract narrows. When an inflammatory reaction occurs due to a variety of factors such as soot, allergens, cold wind, exercise, and respiratory tract infections, the secretions from the glands increase further. The secretions secreted at this time are caused by an exudative reaction caused by inflammation, and most of the secretions are viscous mucus secretions composed of a mixture of inflammatory mediators and mucin.

[0066] Mucins usually play a role in protecting living bodies, but in chronic inflammatory respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and chronic bronchitis, excessive production or excessive secretion of viscous secretions is observed. Abnormalities in the quantity and quality of secretions act as pathological factors, causing obstruction in the trachea and obstruction of air flow into the airways due to accumulation of secretions, so that paroxysmal coughs and dyspnea accompanied by wheezing are severe, and during attacks, dry coughs occur and chest pressure is felt. Lung damage caused by viral respiratory diseases is also believed to be caused by this mucin.

[0067] In the animal model of chronic respiratory disease, the GCWB1001 strain, GCWB1156 strain and GCWB1085 strain disclosed in the present invention inhibit the deposition of mucin in lung tissue, reduce the activity of caspase3 as an apoptotic factor in alveoli, reduce collagen deposition, and reduce the activity of MMP-9. The reduced activity of MMP-9 can prevent the deposition of inflammatory cells in lung tissue, thereby ultimately preventing pulmonary fibrosis.

[0068] Pulmonary fibrosis is the final stage of respiratory disease, and its pathological and physiological processes are complex. In the early stage, a large number of inflammatory cells infiltrate the lungs due to inflammation, and the alveolar walls become chronically thickened. In the middle / late stage, due to the excessive deposition of extracellular matrix components such as collagen by fibroblasts, the lung tissue proliferates, the alveolar deforms, hardens and scars, thus destroying the normal lung tissue structure and leading to loss of function.

[0069] Fibroblasts play a role in attracting immune cells to sites of inflammation and tissue damage. In addition, fibroblasts produce and respond to many inflammatory cytokines. Therefore, fibroblasts contribute to chronic inflammation, and in turn, inflammatory cytokines promote the transformation of fibroblasts to myofibroblasts, thereby promoting fibrosis. Therefore, damage or inflammation of lung tissue can cause pulmonary fibrosis.

[0070] It has been reported that in lung tissues of patients with advanced pulmonary fibrosis, TGF-β stimulation induces an increase in ROS production and increases the expression of collagen and α-SMA (a-smooth muscle actin), which are important for fibrosis, and in lung tissues of patients with idiopathic pulmonary fibrosis, ROS have been reported to aggravate pulmonary fibrosis.

[0071] Therefore, the strain disclosed in the present invention not only induces antitussive / expectorant effects in cough model mice, but also inhibits oxidative stress in chronic respiratory disease animal models to inhibit the expression of inflammatory cytokines, inhibits the expression of MMP-9 to prevent the deposition of immune cells involved in inflammatory responses in lung tissue and bronchoalveolar lavage fluid, and inhibits the production of mucin and collagen, thereby ultimately showing an alleviating effect on pulmonary fibrosis.

[0072] The present disclosure relates to a pharmaceutical composition for preventing or treating inflammatory diseases or respiratory diseases, comprising: Lactobacillus plantarum GCWB1001 strain preserved with the preservation number KCCM12698P, Lactobacillus rhamnosus GCWB1156 strain preserved with the preservation number KCCM12700P or Pediococcus acidilactici GCWB1085 strain preserved with the preservation number KCCM12699P; one selected from a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product or a pulverized product of the strain; or a culture of the strain and any one of a concentrate, a paste and a dilution of the culture.

[0073] The inflammatory disease can be chronic and acute rhinitis, chronic and acute gastritis, enteritis, ulcerative gastritis, acute and chronic nephritis, acute and acute hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis, irritable bowel syndrome, inflammatory bowel disease, enterocolitis, rheumatoid arthritis, osteoarthritis, pneumonia, hepatitis, glomerulonephritis, gastritis, vasculitis, pancreatitis, peritonitis, bronchitis, myocarditis, encephalitis, ischemia-reperfusion injury inflammation, inflammation caused by immune rejection after tissue and organ transplantation, burns, psoriasis, various skin inflammations such as allergic contact dermatitis, back pain, myofascial diseases, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, 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.

[0074] Respiratory diseases are caused by any one of fine dust, viral infection and lung infection. Respiratory diseases are selected from respiratory inflammatory lung diseases, asthma, bronchiectasis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, emphysema, sequelae of pulmonary tuberculosis, chronic bronchitis, allergic rhinitis, cough suppressants and expectorants, lower respiratory tract infections, bronchitis, bronchiolitis, acute upper respiratory tract infections, allergic lung diseases, bronchiectasis, pneumonia, acute and chronic bronchitis, sinusitis, pharyngitis, tonsillitis, laryngitis and pulmonary fibrosis.

[0075] The virus can be selected from adenovirus, vaccinia virus, herpes simplex virus, parainfluenza virus, rhinovirus, varicella-zoster virus, measles virus, respiratory syncytial virus, dengue virus, human immunodeficiency virus (HIV), influenza virus, coronavirus, severe acute respiratory syndrome-related virus (SARS-related virus) and Middle East respiratory syndrome coronavirus (MERS-CoV).

[0076] The strains disclosed herein can be grown at 1×10 1 Up to 1×10 13 The viable cell content of CFU / g was used, but not limited thereto.

[0077] In addition, the present disclosure relates to a food composition for preventing inflammatory diseases or respiratory diseases, comprising: Lactobacillus plantarum GCWB1001 strain preserved with the preservation number KCCM12698P, Lactobacillus rhamnosus GCWB1156 strain preserved with the preservation number KCCM12700P, or Pediococcus acidilactici GCWB1085 strain preserved with the preservation number KCCM12699P; one selected from a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product, or a pulverized product of the strain; or a culture of the strain and any one of a concentrate, a paste, and a dilution of the culture.

[0078] In addition, the present disclosure relates to a health functional food composition for preventing inflammatory diseases or respiratory diseases, comprising: Lactobacillus plantarum GCWB1001 strain preserved with the preservation number KCCM12698P, Lactobacillus rhamnosus GCWB1156 strain preserved with the preservation number KCCM12700P or Pediococcus acidilactici GCWB1085 strain preserved with the preservation number KCCM12699P; one selected from a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product or a pulverized product of the strain; or a culture of the strain and any one of a concentrate, a paste and a dilution of the culture.

[0079] In the present disclosure, in addition to the active ingredients, the above-mentioned food composition or health functional food composition may include ingredients that are usually added during food production, and may include, for example, proteins, carbohydrates, fats, nutrients, seasonings, sweeteners and flavoring agents, but are not limited thereto. Examples of carbohydrates may include monosaccharides, for example, glucose, fructose, etc.; disaccharides, for example, maltose, sucrose, oligosaccharides, etc.; and polysaccharides, for example, conventional sugars, such as dextrin, cyclodextrin, etc.; and sugar alcohols, such as xylitol, sorbitol, erythritol, etc. As sweeteners, natural sweeteners (taumatin, stevia extract, rebaudioside A, glycyrrhizin, etc.) and synthetic sweeteners (saccharin, aspartame, etc.) may be used. In addition, oligosaccharides from plants, such as fructans, galactans, resistant starch, pectin, β-glucans and xylo-oligosaccharides (which would be feed for the strain) may be included as probiotics.

[0080] However, the present disclosure is not limited thereto, and any component that does not impair the effects of the present disclosure as a component known in the art may be used.

[0081] Examples of the food composition or health functional food composition may include patient nutritional food, meat, cereals, caffeinated beverages, regular beverages, dairy products, chocolate, bread, snacks, candy, pizza, jelly, noodles, chewing gum, ice cream, alcoholic beverages, wine, vitamin complexes and other health supplements, but are not limited thereto. When prepared in the form of a food composition or health functional food composition as described above, it is preferred that the composition can be easily and conveniently administered.

[0082] In the present disclosure, the health functional food composition and the pharmaceutical composition can be prepared in the form of granules, lemon juice, powder, syrup, liquid and solution, extracts, elixirs, fluid extracts, suspensions, decoctions, infusions, tablets, spirits, capsules, lozenges, pills, and soft or hard gelatin capsules, but are not limited thereto.

[0083] The pharmaceutical composition can also include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present disclosure is commonly used in preparations, and includes lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but is not limited thereto.

[0084] In addition to the above-mentioned ingredients, the pharmaceutical composition of the present disclosure may also include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like.

[0085] The pharmaceutical compositions of the present disclosure may be administered orally or parenterally.

[0086] The pharmaceutical composition of the present disclosure can be formulated by using pharmaceutically acceptable carriers and / or excipients according to methods that can be easily performed by those skilled in the art so as to be prepared in a unit dosage form or by introducing into a multi-dose container.

[0087] In addition, the present disclosure relates to a probiotic, comprising: Lactobacillus plantarum GCWB1001 strain preserved under the preservation number KCCM12698P, Lactobacillus rhamnosus GCWB1156 strain preserved under the preservation number KCCM12700P, or Pediococcus acidilactici GCWB1085 strain preserved under the preservation number KCCM12699P.

[0088] The probiotics may include one or more selected from the three types of deposited strains disclosed herein, may also include one or more selected from the seven types of lactic acid bacteria isolated in the examples, and may also include known strains that can be used for the purpose of the present disclosure.

[0089] The probiotics can be used at 1×10 1 Up to 1×10 13 The probiotics can be used with a live cell content of CFU / g, and the probiotics can be effectively used for anti-inflammation or prevention or relief of respiratory diseases.

[0090] In the present disclosure, it is preferred to determine the dosage of the GCWB1001 strain, the GCWB1156 strain, and the GCWB1085 strain in consideration of the administration method, the age, sex, and weight of the user, and the severity of the disease.

[0091] For example, GCWB1001 strain, GCWB1156 strain and GCWB1085 strain can be administered once a day or twice or more separately, each time at 1×10 1 Up to 1×10 13 The live cell content of CFU / g was used for dosing.

[0092] In addition, based on the active ingredients, the pharmaceutical composition, food composition, health functional food composition and probiotics containing the ingredients can be taken once a day or twice or more dividedly, with 1×10 1 Up to 1×10 13 The live cell content of CFU / g was used for dosing.

[0093] However, the above dosage is just an example and may be changed by a doctor's prescription according to the condition of the user.

[0094] Hereinafter, the present disclosure will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the present disclosure, and the scope of the present disclosure is not limited thereto.

[0095] Example 1: Isolation and identification of Lactobacillus plantarum GCWB1001 strain

[0096] (1) Isolation of strains

[0097] Kimchi collected from various regions was placed in a sterile saline solution equal to 10 times the amount of kimchi and homogenized. The homogenized sample was diluted 10 steps in sterile saline and the strain was isolated by the dilution plate method. The diluted strain sample was spread on MRS medium (MRS broth agar; BD Difco) and then cultured anaerobically at 37°C for 72 hours. The colonies appearing on the MRS agar plate were inoculated for the second time in PCA medium (MBcell, South Korea) containing 0.005% bromocresol purple (BCP) as a pH indicator, and the colonies in which the purple medium turned yellow were inoculated for the third time on the MRS agar plate to isolate the probiotics in pure form.

[0098] (2) Identification of Lactobacillus plantarum GCWB1001 strain

[0099] DNA extraction and purification were performed on the strains completely isolated in (1) above. After 16s rRNA gene amplification using two universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'), sequence analysis was performed on the amplified 16s rRNA gene. Using the analyzed 16s rRNA sequence data and the EzTaxon server (http: / / www.ezbiocloud.net), only two types of strains corresponding to Generally Recognized as Safe (GRAS) were selected and shown in Table 1 below.

[0100] [Table 1]

[0101] Identified strains Lactobacillus plantarum GCWB1001 Leuconostoc mesenteroides GCWB1031

[0102] The 16S rRNA sequence results of Lactobacillus plantarum GCWB1001 are as follows.

[0103] <16S rRNA sequence of Lactobacillus plantarum GCWB1001>

[0104] SEQ ID NO:1

[0105] GGTCGTACGA ACTCTGTGTA TTGATTGGTG CTTGCATCAT GATTTACATT TGCAGTGAGT

[0106] GGCGAACTGG TGAGTAACAC GTGGGAAACC TGCCCAGAAG CGGGGGATAACACCTGGAA

[0107] CAGATGCTAA TACCGCATAA CAACTTGGAC CGCATGGTCC GAGTTTGAAA GATGGCTTCG

[0108] GCTATCACTT TTGGATGGTC CCGCGGCGTA TTAGCTAGAT GGTGGGGTAA CGGCTCACCA

[0109] TGGCAATGAT ACGTAGCCGA CCTGAGAGGG TAATCGGCCA CATTGGGACT GAGACACGGC

[0110] CCAAACTCCT ACGGGAGGCA GCAGTAGGGA ATCTTCCACA ATGGACGAAA GTCTGATGGA

[0111] GCAACGCCGC GTGAGTGAAG AAGGGTTTCG GCTCGTAAAA CTCTGTTGTT GAAGAAGAAC

[0112] ATATCTGAGA GTAACTGTTC AGGTATTGAC GGTATTTAAC CAGAAAGCCA CGGCTAACTA

[0113] CGTGCCAGCA GCCGCGGTAA TACGTAGGTG GCAAGCGTTG TCCGGATTTA TTGGGCGTAA

[0114] AGCGAGCGCA GGCGGTTTTT TAAGTCTGAT GTGAAAGCCT TCGGCTCAAC CGAAGAGTG

[0115] CATCGGAAAC TGGGAAACTT GAGTGCAGAA GAGGACAGTG GAACTCCATG TGTAGCGGTG

[0116] AAATGCGTAG ATATATGGAA GAACACCAGT GGCGAAGGCG GCTGTCTGGT CTGTAACTGA

[0117] CGCTGAGGCT CGAAAGTATG GGTAGCAAAC AGGATTAGAT ACCCTGGTAG TCCATACCGT

[0118] AAACGATGAA TGCTAAGTGT TGGAGGGTTT CCGCCCTTCA GTGCTGCAGC TAACGCATTA

[0119] AGCATTCCGC CTGGGGAGTA CGGCCGCAAG GCTGAAACTC AAAGGAATTG ACGGGGGCCC

[0120] GCACAAGCGG TGGAGCATGT GGTTTAATTC GAAGCTACGC GAAGAACCTT ACCAGGTCTT

[0121] GACATACTAT GCAAATCTAA GAGATTAGAC GTTCCCTTCG GGGACATGGA TACAGGTGGT

[0122] GCATGGTTGT CGTCAGCTCG TGTCGTGAGA TGTTGGGTTA AGTCCCGCAA CGAGCGCAAC

[0123] CCTTATTATC AGTTGCCAGC ATTAAGTTGG GCACTCTGGT GAGACTGCCG GTGACAAACC

[0124] GGAGGAAGGT GGGGATGACG TCAAATCATC ATGCCCCTTA TGACCTGGGC TACACACGTG

[0125] CTACAATGGA TGGTACAACG AGTTGCGAAC TCGCGAGAGT AAGCTAATCT CTTAAAGCCA

[0126] TTCTCAGTTC GGATTGTAGG CTGCAACTCG CCTACATGAA GTCGGAATCG CTAGTAATCG

[0127] CGGATCAGCA TGCCGCGGTG AATACGTTCC CGGGCCTTGT ACACACCGCC CGTCACACCA

[0128] TGAGAGTTTG TAACACCCAA AGTCGGTGGG GTAACCTTTT AGGAACCAGC CGCT

[0129] Example 2: Isolation and identification of Pediococcus acidilactici GCWB1085 strain

[0130] (1) Isolation of strains

[0131] The cheese collected from various regions was placed in a sterile saline solution equal to 10 times the amount of cheese and homogenized. The homogenized sample was diluted 10 steps in sterile saline and the strain was isolated by the dilution plate method. The diluted strain sample was spread on MRS medium (MRS broth agar; BD Difco) and then cultured anaerobically at 37°C for 72 hours. The colonies appearing on the MRS agar plate were inoculated for a second time in a PCA medium (MBcell, South Korea) containing 0.005% bromocresol purple (BCP) as a pH indicator, and the colonies in which the purple medium turned yellow were inoculated for a third time on the MRS agar plate to isolate the probiotics in pure form.

[0132] (2) Identification of Pediococcus acidilactici GCWB1085 strain

[0133] The strain completely isolated in (1) above was subjected to chromosomal DNA extraction and purification. After 16s rRNA gene amplification using two universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'), the amplified 16s rRNA gene was sequenced. Using the analyzed 16s rRNA sequence data and the EzTaxon server (http: / / www.ezbiocloud.net), only four types of strains corresponding to Generally Recognized as Safe (GRAS) were selected and shown in Table 2 below.

[0134] [Table 2]

[0135] Identified strains Pediococcus acidilactici GCWB1085 Lactobacillus paracasei GCWB1084 Bifidobacterium longum GCWB1136

[0136] The 16S rRNA sequence results of Pediococcus acidilactici GCWB1085 are as follows.

[0137] <16S rRNA sequence of Pediococcus acidilactici GCWB1085>

[0138] SEQ ID NO:2

[0139] CTCAGGATGA ACGCTGGCGG CGTGCCTAAT ACATGCAAGT CGAACGAACT TCCGTTAATT

[0140] GATCAGGACG TGCTTGCACT GAATGAGATT TTAACACGAA GTGAGTGGCG GACGGGTGAG

[0141] TAACACGTGG GTAACCTGCC CAGAAGCAGG GGATAACACC TGGAAACAGATGCTAATACC

[0142] GTATAACAGA GAAAACCGCC TGGTTTTCTT TTAAAAGATG GCTCTGCTAT CACTTCTGGA

[0143] TGGACCCGCG GCGCATTAGC TAGTTGGTGA GGTAACGGCT CACCAAGGCG ATGATGCGTA

[0144] GCCGACCTGA GAGGGTAATC GGCCACATTG GGACTGAGAC ACGGCCCAGA CTCCTACGGG

[0145] AGGCAGCAGT AGGGAATCTT CCACAATGGA CGCAAGTCTG ATGGAGCAAC GCCGCGTGAG

[0146] TGAAGAAGGG TTTCGGCTCG TAAAGCTCTG TTGTTAAAGA AGAACGTGGG TGAGAGTAAC

[0147] TGTTCACCCA GTGACGGTAT TTAACCAGAA AGCCACGGCT AACTACGTGC CAGCAGCCGC

[0148] GGTAATACGT AGGTGGCAAG CGTTATCCGG ATTTATTGGG CGTAAAGCGA GCGCAGGCGG

[0149] TCTTTTAAGT CTAATGTGAA AGCCTTCGGC TCAACCGAAG AAGTGCATTG GAAACTGGGA

[0150] GACTTGAGTG CAGAAGAGGA CAGTGGAACT CCATGTGTAG CGGTGAAATG CGTAGATATA

[0151] TGGAAGAACA CCAGTGGCGA AGGCGGCTGT CTGGTCTGTA ACTGACGCTG AGGCTCGAAA

[0152] GCATGGGTAG CGAACAGGAT TAGATACCCT GGTAGTCCAT GCCGTAAACG ATGATTACTA

[0153] AGTGTTGGAG GGTTTCCGCC CTTCAGTGCT GCAGCTAACG CATTAAGTAA TCCGCCTGGG

[0154] GAGTACGACC GCAAGGTTGA AACTCAAAAG AATTGACGGG GGCCCGCACA AGCGGTGGAG

[0155] CATGTGGTTT AATTCGAAGC TACGCGAAGA ACCTTACCAG GTCTTGACAT CTTCTGCCAA

[0156] CCTAAGAGAT TAGGCGTTCC CTTCGGGGAC AGAATGACAG GTGGTGCATG GTTGTCGTCA

[0157] GCTCGTGTCG TGAGATGTTG GGTTAAGTCC CGCAACGAGC GCAACCCTTA TTACTAGTTG

[0158] CCAGCATTCA GTTGGGCACT CTAGTGAGAC TGCCGGTGAC AAACCGGAGG AAGGTGGGGA

[0159] CGACGTCAAA TCATCATGCC CCTTATGACC TGGGCTACAC ACGTGCTACA ATGGATGGTA

[0160] CAACGAGTTG CGAAACCGCG AGGTTTAGCT AATCTCTTAA AACCATTCTC AGTTCGGACT

[0161] GTAGGCTGCA ACTCGCCTAC ACGAAGTCGG AATCGCTAGT AATCGCGGAT CAGCATGCCG

[0162] CGGTGAATAC GTTCCCGGGC CTTGTACACA CCGCCCGTCA CACCATGAGA GTTTGTAACA

[0163] CCCAAAGCCG GTGGGGTAAC CTTTTAGGAG CTAGCCGTCT AAGGTGGGAC AGATGATTA

[0164] Example 3: Isolation and identification of Lactobacillus rhamnosus GCWB1156 strain

[0165] (1) Isolation of strains

[0166] The feces of healthy infants born by natural delivery were diluted in sterile saline for 10 steps, and the strains were isolated by dilution plate method. The diluted fecal samples were smeared on BSM agar medium (Bifidobacterium selective medium agar; Sigma, USA), and then cultured anaerobically at 37°C for 72 hours. The colonies appearing on the BSM agar plate were inoculated for the second time in PCA medium (MBcell, South Korea) containing 0.005% bromocresol purple (BCP) as a pH indicator, and the colonies in which the purple medium turned yellow were inoculated for the third time on BL agar medium (MBcell, South Korea) to isolate the probiotics in pure form.

[0167] (2) Identification of Lactobacillus rhamnosus GCWB1156 strain

[0168] The strain completely separated in (1) above was subjected to chromosomal DNA extraction and purification. After 16s rRNA gene amplification using two universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'), the amplified 16s rRNA gene was subjected to sequence analysis. Using the analyzed 16s rRNA sequence data and the EzTaxon server (http: / / www.ezbiocloud.net), only one type of strain corresponding to Generally Recognized as Safe (GRAS) was selected and shown in Table 3 below.

[0169] [Table 3]

[0170] Identified strains Lactobacillus rhamnosus GCWB1156 Bifidobacterium breve GCWB1144

[0171] The 16S rRNA sequence results of Lactobacillus rhamnosus GCWB1156 are as follows.

[0172] <16S rRNA sequence of Lactobacillus rhamnosus GCWB1156>

[0173] SEQ ID NO:3

[0174] GTTGATCGGC CACATTGGGA CTGAGACACG GCCCAAACTC CTACGGGAGG CAGCAGTAGG

[0175] GAATCTTCCA CAATGGACGC AAGTCTGATG GAGCAACGCC GCGTGAGTGA AGAAGGCTTT

[0176] CGGGTCGTAA AACTCTGTTG TTGGAGAAGA ATGGTCGGCA GAGTAACTGT TGTCGGCGTG

[0177] ACGGTATCCA ACCAGAAAGC CACGGCTAAC TACGTGCCAG CAGCCGCGGT AATACGTAGG

[0178] TGGCAAGCGT TATCCGGATT TATTGGGCGT AAAGCGAGCG CAGGCGGTTT TTTAAGTCTG

[0179] ATGTGAAAGC CCTCGGCTTA ACCGAGGAAG TGCATCGGAA ACTGGGAAAC TTGAGTGCAG

[0180] AAGAGGACAG TGGAACTCCA TGTGTAGCGG TGAAATGCGT AGATATATGG AAGAACACCA

[0181] GTGGCGAAGG CGGCTGTCTG GTCTGTAACT GACGCTGAGG CTCGAAAGCA TGGGTAGCGA

[0182] ACAGGATTAG ATACCCTGGT AGTCCATGCC GTAAACGATG AATGCTAGGT GTTGGAGGGT

[0183] TTCCGCCCTT CAGTGCCGCA GCTAACGCAT TAAGCATTCC GCCTGGGGAG TACGACCGCA

[0184] AGGTTGAAAC TCAAAGGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT GTGGTTTAAT

[0185] TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCTT TTGATCACCT GAGAGATCAG

[0186] GTTTCCCCTT CGGGGGCAAA ATGACAGGTG GTGCATGGTT GTCGTCAGCT CGTGTCGTGA

[0187] GATGTTGGGT TAAGTCCCGC AACGAGCGCA ACCCTTATGA CTAGTTGCCA GCATTTAGTT

[0188] GGGCACTCTA GTAAGACTGC CGGTGACAAA CCGGAGGAAG GTGGGGATGA CGTCAAATCA

[0189] TCATGCCCCT TATGACCTGG GCTACACACG TGCTACAATG GATGGTACAA CGAGTTGCGA

[0190] GACCGCGAGG TCAAGCTAAT CTCTTAAAGC CATTCTCAGT TCGGACTGTA GGCTGCAACT

[0191] CGCCTACACG AAGTCGGAAT CGCTAGTAAT CGCGGATCAG CACGCCGCGG TGAATACGTT

[0192] CCCGGGCCTT GTACACACCG CCCGTCACAC CATGAGAGTT TGTAACACCC GAAGCCGGTG

[0193] GCGTAACCCT TTTAGGGAGC GAGCCGTCTA AGGTGGGACA AATGATTA

[0194] Experimental Example 1: Analysis of anti-inflammatory effects in mouse lung macrophages

[0195] 1-1) Evaluation of cytotoxicity of strains (LDH leakage and CCK-8 assay)

[0196] Mouse alveolar macrophage cell line MH-S was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and suspended to a cell concentration of 5 × 10 5 cells / ml, dispensed into a 96-well plate at 100 μl, treated with each sample at each concentration, and then cultured for 48 hours. Cytotoxicity was measured using an MTT assay kit and a cytotoxicity LDH assay kit, and the results are shown in Table 4 below.

[0197] [Table 4]

[0198]

[0199]

[0200] *P<0.001 vs. conventional group. **P<0.01 vs. conventional group.

[0201] (1) LDH test results

[0202] The 7 types of lactic acid bacteria in Table 4 above were treated in mouse MH-S lung macrophages at concentrations of 1 to 1,000 μg / ml for 24 hours, and then LDH detection was performed. As a result, no cytotoxicity was observed for all 7 types. However, it was confirmed that in the case of GCWB1001 strain, GCWB1085 strain, GCWB1176 strain, and GCWB1156 strain at a concentration of 1,000 μg / ml, the LDH value was significantly reduced, and therefore, all strains were non-cytotoxic.

[0203] (2) MTT test results

[0204] The 7 types of lactic acid bacteria in Table 4 above were treated in mouse MH-S lung macrophages at concentrations of 1 to 1,000 μg / ml for 24 hours, and then the MTT assay was performed. As a result, no cytotoxicity was observed for all 7 types at concentrations below 100 μg / ml. However, cytotoxicity was confirmed in the case of the GCWB1085 strain at a concentration of 100 μg / ml.

[0205] 1-2) Measurement of cytokine (TNF-α, TGF-β) secretion

[0206] MH-S cell lines were pretreated with the samples for 1 hour, and then treated with LPS (10 ng / ml) or ConA (10 μg / ml) and DEP (200 μg / ml) for 3 hours and 26 hours, respectively, and the amount of cytokines (TNF-α and TGF-β) secreted into the culture medium was measured using an ELISA kit (R&D system, USA). The results are shown in Tables 5 and 6 below. Figure 1 middle.

[0207] [Table 5]

[0208]

[0209]

[0210] ##P<0.01 vs. conventional group. *P<0.001 vs. LPS 10 ng / ml + DEP 200 μg / ml, **P<0.01 vs. LPS 10 ng / ml + DEP 200 μg / ml. ***P<0.05 vs. LPS 10 ng / ml + DEP 200 μg / ml.

[0211] (1) TNF-α measurement results

[0212] As shown in Table 5, the secretion of the cytokine TNF-α was reduced by LPS and DEP treatment in MH-S cells at most treatment concentrations of all strains of GCWB1001, GCWB1084, and GCWB1156. Thus, the strains of the present disclosure reduced the secretion of the proinflammatory cytokine TNF-α caused by fine dust in a concentration-dependent manner.

[0213] (2) TGF-β measurement results

[0214] like Figure 1 As shown in , the secretion of TGF-β induced by fine dust was decreased when GCWB1001, GCWB1085, and GCWB1156 strains were treated at 1 μg / ml and 10 μg / ml.

[0215] 1-3) Measurement of nitric oxide (NO)

[0216] Mouse lung macrophage cell line NH-S was suspended to a cell concentration of 5 × 10 5Cells / ml, and dispersed into 96-well plates at 100 μl. The 7 types of strains in Table 6 were used as samples, pretreated at concentrations of 1 μg / ml and 10 μg / ml for 1 hour, and then treated with 100 ng / ml of LPS for 24 hours. Then, 50 μl of the culture solution was transferred to a 96-well plate, mixed with Griess reagent I (NED solution) and Griess reagent II (sulfonamide solution) in equal amounts, reacted in a dark room for 10 minutes, and then measured at 540 nm using a microplate reader within 30 minutes.

[0217] [Table 6]

[0218]

[0219]

[0220] #P<0.001 vs. conventional group. *P<0.001 vs. LPS 10 ng / ml + DEP 200 μg / ml treated group. **P<0.01 vs. LPS 10 ng / ml + DEP 200 μg / ml treated group. ***P<0.05 vs. LPS 10 ng / ml + DEP 200 μg / ml treated group.

[0221] The experimental results are shown in the figure Figure 2 From Table 6 and Figure 2 As can be seen, it can be confirmed that the GCWB1001 strain, GCWB1085 strain and GCWB1156 strain of the present disclosure all significantly reduce the amount of NO produced by fine dust at a concentration of 1 μg / ml. However, at a concentration of 10 μg / ml, the strain did not significantly reduce the amount of NO produced by fine dust.

[0222] 1-4) Measurement of reactive oxygen species (ROS)

[0223] Mouse lung macrophage cell line NH-S was suspended to a cell concentration of 5 × 10 5 cells / ml, and dispersed into 96-well plates at 100 μl. The 7 types of strains in Table 6 above were used as samples, pretreated at concentrations of 1 μg / ml and 10 μg / ml for 1 hour, then treated with 100 ng / ml of LPS for 6 hours, washed with HBSS, and treated the culture solution with DCF-DA (2',7'-dichlorofluorescein diacetate) / HBSS at 25 μM per well for 30 minutes, and then measured the fluorescence value at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

[0224] The experimental results are shown in the figure Figure 3It can be confirmed that, similar to the NO analysis, the GCWB1001 strain, the GCWB1085 strain, and the GCWB1156 strain of the present disclosure all significantly reduced the amount of ROS generated by fine dust at a concentration of 1 μg / ml.

[0225] 1-5) Measurement of inflammatory transcriptional regulator activity (iNOS-Luc, COX2-Luc, NF-kB-Luc; luciferase promoter activity analysis)

[0226] MH-S cells were cultured at 1×10 6 After distribution at 10 cells / ml, plasmid vectors inserted with iNOS-Luc, COX2-Luc and NF-kB-Luc were introduced into cells using LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA). After the samples were treated at the concentrations for 24 hours, the cells were collected and the luciferase activity was measured.

[0227] according to Figures 4 to 6 , when GCWB1001, 1085 and 1156 strains were treated with 1 μg / ml, the promoter activity of inflammatory transcription factors iNOS, COX2 and NF-kB caused by fine dust was reduced. In general, the strains disclosed herein can inhibit the expression of inflammation-related genes by inhibiting the promoter activity of NF-kB transcription factors that induce the expression of inflammation-related genes and iNOS and COX genes known as inflammatory genes.

[0228] Experimental Example 2: Study on Antitussive / Expectorant Effects in Animal Models (In Vivo Test)

[0229] 2-1) Experimental conditions and rearing conditions

[0230] The experimental animals were provided by 6-week-old male BALB / c mice (weight: 20 ± 2g) from Samtako Co., Ltd., and adapted to more than 7 days in an animal breeding room with a temperature of 23 ± 1 ° C, a relative humidity of 55 ± 15%, an illumination of 300 to 500 Lux, and brightness adjusted at intervals of 12 hours. Afterwards, visual symptoms were observed, only normal animals were used for experiments, and solid feed (Samtako Co., Ltd.) and water were freely fed to experimental animals. All animal experiments were carried out with the approval of the Experimental Animal Ethics Committee of the International University of Korea, and were carried out according to the guidelines of the National Institutes of Health (NIH Publication No. 86-23, revised in 1985).

[0231] 2-2) Research on antitussive effect

[0232] The experimental group consisted of 4 groups: a group given 1M citric acid alone (control), a group given citric acid + Synatura 200 mg / kg (positive control), a group given citric acid + sample treatment (1×10 7 ) and citric acid + sample treatment group (10×10 9 ), the number of animals in each experimental group was 8 (n=8) (Table 7).

[0233] [Table 7]

[0234]

[0235]

[0236] One hour after the oral administration of the sample, 1 M of citric acid, a cough inducer, was intranasally administered, and then each experimental group was placed in a chamber and the number of coughs was measured over 10 minutes. Figure 7 As shown in Figure 2, GCWB1001 and GCWB1085 strains were given 1×10 9 cfu significantly reduced the number of coughs induced by citric acid.

[0237] 2-3) Research on expectorant effect

[0238] The experimental group consisted of 4 groups: a group given 1M phenol red (control), a group given phenol red + Synatura 200 mg / kg (positive control), a group given phenol red + sample treatment (1×10 7 ) and phenol red + sample treatment group (1×10 9 ), the number of animals in each experimental group was 8 (n=8) (Table 8).

[0239] [Table 8]

[0240] No Experimental Group concentration 1 Phenol red + saline 10mg / kg 2 Phenol Red + Synatura 200mg / kg 3 Phenol red + sample treatment group <![CDATA[1×10 7 cfu]]> 4 Phenol red + sample treatment group <![CDATA[1×10 9 cfu]]>

[0241] After the oral administration of the sample, 0.2 ml of phenol red (10 mg / ml) was intraperitoneally injected 1 hour later, and the experimental animals were killed 30 minutes later, and then the trachea was removed.

[0242] After measuring the weight of the removed organ, 0.5 ml of 0.9% physiological saline (w / v) was added and vortexed, and 100 μl of 1 M NaOH as a colorimetric substrate was added, and the absorbance at 550 nm was measured.

[0243] like Figure 8 As shown in Figure 2, GCWB1001 and GCWB1085 strains were treated with 1×10 9 cfu, the secretion of phenol red increased significantly.

[0244] In general, the strains of the present disclosure reduce the number of coughs and enhance expectoration, thereby improving the symptoms of respiratory diseases.

[0245] Experimental Example 3: Analysis of the effect of relieving lung function in an animal model of chronic respiratory disease (in vivo test)

[0246] 3-1) Experimental conditions and rearing conditions

[0247] The experimental animals were provided by 6-week-old male BALB / c mice (weight: 20 ± 2g) from Samtako Co., Ltd., and adapted to more than 7 days in an animal breeding room with a temperature of 23 ± 1 ° C, a relative humidity of 55 ± 15%, an illumination of 300 to 500 Lux, and brightness adjusted at intervals of 12 hours. Afterwards, visual symptoms were observed, only normal animals were used for experiments, and solid feed (Samtako Co., Ltd.) and water were freely fed to experimental animals. All animal experiments were carried out with the approval of the Experimental Animal Ethics Committee of the International University of Korea, and were carried out according to the guidelines of the National Institutes of Health (NIH Publication No. 86-23, revised in 1985).

[0248] 3-2) Establishment of animal model of chronic respiratory disease and administration of experimental substances

[0249] Male 6-week-old BALB / c mice were intraperitoneally administered 100 μg of ovalbumin (OVA) in 200 μl of a 1:1 mixture of aluminum hydroxide (Al(OH)3) and saline once on day 1 and day 12 (sensitization). On day 19 and day 20, at the start of the experiment, 50 μg of OVA was intranasally administered once (Challenge). Finally, after intranasal administration of OVA (day 20), 400 μg of diesel exhaust particles (DEP) were intranasally administered three times at 3-hour intervals, and the experiment was terminated on day 21.

[0250] Twenty-four hours after the last sample administration, urethane (Sigma Aldrich, UK, USA) (0.020 ml / g weight) was intraperitoneally administered for anesthesia, and then bronchoalveolar lavage fluid (BALF) was obtained. The strains of the present disclosure were orally administered once a day from day 0 to day 20. GCWB1001, GCWB1085, and GCWB1156 samples were administered at 1×10 7 cfu and 1×10 9 cfu were administered orally. Synatura (200 mg / kg) was used as a positive control, and the number of animals in each experimental group was 8 (n=8) (Table 9).

[0251] [Table 9]

[0252] No. Experimental group (n=8) concentration 1 NA (control) - 2 OVA (Ovalbumin) 100 μg 3 OVA+DEP (diesel exhaust particles) 400 μg 4 OVA+DEP+GCWB1001 <![CDATA[1×10 7 cfu]]> 5 OVA+DEP+GCWB1001 <![CDATA[1×10 9 cfu]]> 6 OVA+DEP+GCWB1085 <![CDATA[1×10 7 cfu]]> 7 OVA+DEP+GCWB1085 <![CDATA[1×10 9 cfu]]> 8 OVA+DEP+GCWB1156 <![CDATA[1×10 7 cfu]]> 9 OVA+DEP+GCWB1156 <![CDATA[1×10 9 cfu]]> 10 OVA+DEP+Synatura 200mg / kg

[0253] 3-3) Measurement of body weight and organ weight

[0254] 24 hours after the last sample administration, urethane (Sigma Aldrich, UK, USA) (0.020 ml / g weight) was intraperitoneally administered for anesthesia, bronchoalveolar lavage fluid (BALF) was obtained, and then the lungs were removed and weighed. In addition, body weight and liver and spleen weights were measured separately.

[0255] Body weights did not change in all groups, and lung weights increased in OVA and OVA+DEP treated groups compared with the untreated group, whereas lung weights increased in GCWB1001 (1×10 9 cfu), GCWB1085 (1×10 9 cfu, GCWB1156 (1×10 9 cfu) and Synatura-treated groups, the weight of lung tissue increased by OVA+DEP treatment was significantly reduced. Even in the spleen, the weight of lung tissue increased by OVA+DEP treatment was significantly reduced in the GCWB1001 (1×10 7 cfu), GCWB1085 (1×10 9 cfu) and GCWB1156 (1×10 9 cfu), the weights of spleen and lung tissues increased by OVA+DEP treatment were also significantly reduced.

[0256] 3-4) Evaluation of the strain disclosed herein in alleviating lung function in an animal model of chronic respiratory disease - analysis of bronchoalveolar cell fluid

[0257] 24 hours after the last sample administration, urethane (Sigma Aldrich, UK, USA) (0.020 ml / g weight) was intraperitoneally administered for anesthesia, and bronchoalveolar lavage fluid (BALF) was obtained, and then the immune cell profile (total cells, macrophages, eosinophils, neutrophils, lymphocytes) was measured in BALF.

[0258] according to Fig.10 , the number of all immune cells measured in the OVA-treated group increased significantly compared to the conventional group, and the number of immune cells including the total number of cells increased significantly even in the OVA+DEP-treated group compared to the OVA-treated group. However, in the samples of the groups treated with GCWB1001, GCWB1085, GCWB1156, and Synatura, the number of immune cells increased by OVA+DEP treatment was significantly reduced.

[0259] Specifically, when each strain was expressed at 1×10 9 When treated with 1×10 cfu, the cell numbers of all immune cells showed a decrease compared to the positive control, and even when 1×10 7 When treated with cfu, the number of cells other than lymphocytes (total cells, macrophages, eosinophils, neutrophils) was effectively reduced.

[0260] Therefore, because the strains of the present disclosure reduce the infiltration of immune cells into lung tissue, these strains can prevent the development of respiratory diseases.

[0261] 3-5) Evaluation of the strains disclosed herein in alleviating lung function in an animal model of chronic respiratory disease - measurement of OVA-specific IgE (BALF, serum)

[0262] 24 hours after the last sample administration, urethane (Sigma Aldrich, UK, USA) (0.020 ml / g weight) was intraperitoneally administered for anesthesia, bronchoalveolar lavage fluid (BALF) and serum were obtained, and then the amount of OVA-specific IgE, which is an indicator of allergic reaction as a type of inflammatory reaction, was measured.

[0263] like Fig.11 As shown in Figure , compared with the conventional group, the BALF ( Fig.11 A) and serum ( Fig.11 The OVA-specific IgE in the OVA+DEP treated group was significantly increased compared with the OVA treated group. The OVA-specific IgE increased by OVA+DEP treatment was significantly higher in sample GCWB1001 (1×10 7 , 1×10 9 cfu), GCWB1085 (1×10 7 , 1×10 9 cfu), GCWB1156 (1×10 7 , 1×10 9 cfu) and Synatura-treated groups. Therefore, the strains disclosed herein can reduce pulmonary and systemic allergic reactions, which are indicators of inflammatory reactions.

[0264] 3-6) Evaluation of the strains disclosed herein in alleviating lung function in an animal model of chronic respiratory disease - measurement of cytokines (TNF-α, IL-6, IL-1β, IL-4, IL-13, MCP-1 and IFN-γ) in BALF

[0265] Twenty-four hours after the last sample administration, urethane (Sigma Aldrich, UK, USA) (0.020 ml / g weight) was intraperitoneally administered for anesthesia, and then cytokines (TNF-α, IL-6, IL-1β, IL-4, IL-13, MCP-1, and IFN-γ) in BALF were measured using ELISA kits (R&D system USA).

[0266] Compared with the conventional group, TNF-α, IL-6, IL-1β, IL-4, IL-13 and MCP-1 were significantly increased in the OVA-treated group, and TNF-α, IL-6, IL-1β, IL-13 and MCP-1 were significantly increased in the OVA+DEP-treated group compared with the OVA-treated group. 7 , 1×10 9 cfu), GCWB1085 (1×10 7 , 1×10 9 cfu), GCWB1156 (1×10 7 , 1×10 9 cfu) and Synatura-treated groups, the increased TNF-α, IL-6, IL-1β, IL-4, IL-13 and MCP-1 levels in the OVA+DEP-treated group were significantly decreased ( Fig.12 A to Fig.12 D).

[0267] In addition, the amount of IFN-γ decreased in the OVA+DEP-treated group, but in the GCWB1001 (1×10 7 , 1×10 9 cfu), GCWB1085 (1×10 7 , 1×10 9 cfu), GCWB1156 (1×10 7 , 1×10 9 cfu) and Synatura-treated groups ( Fig.12 G).

[0268] Therefore, since the strains of the present disclosure reduce pro-inflammatory cytokines and increase anti-inflammatory cytokines in the lung, the inflammatory response in lung tissue can be reduced, which can prevent the development of respiratory diseases.

[0269] 3-7) Evaluation of the strain disclosed herein in alleviating lung function in an animal model of chronic respiratory disease - histological examination (H&E staining)

[0270] The left lobe lung tissue was fixed in 10% formalin for 24 hours, and then paraffin blocks were prepared, cut into 4 μm thickness, and then stained with H & E. After obtaining images with an optical microscope, the degree of inflammation was measured.

[0271] The infiltration of inflammatory cells was significantly increased in the OVA-treated group compared with the conventional group, and the infiltration of inflammatory cells was significantly increased in the OVA+DEP-treated group compared with the OVA-treated group.

[0272] In the strain GCWB1001 (1×10 7 , 1×10 9 cfu), GCWB1085 (1×10 7 , 1×10 9 cfu), GCWB1156 (1×10 7 , 1×10 9 cfu) and Synatura-treated groups, the infiltration of inflammatory cells increased by OVA+DEP treatment was significantly reduced ( Fig.13 ).

[0273] Therefore, since the strains of the present disclosure block the infiltration of inflammatory cells into lung tissue and thereby reduce the inflammatory response in lung tissue, these strains can prevent the development of respiratory diseases.

[0274] 3-8) Evaluation of the strain disclosed herein in alleviating lung function in an animal model of chronic respiratory disease - histological examination (Alcian blue-PAS staining)

[0275] To confirm whether the strains of the present disclosure actually inhibit the production of mucin in lung tissue, lung tissue was stained by Alcian blue-PAS staining to stain glycogen and mucin.

[0276] Fig.14 is a photograph of actual stained tissue, in which the production of mucin in the alveoli was significantly increased in the OVA-treated group compared with the conventional group, and the production of mucin was significantly increased in the OVA+DEP-treated group compared with the OVA-treated group. 7 , 1×10 9 cfu), GCWB1085 (1×10 7 , 1×10 9 cfu), GCWB1156 (1×10 7 , 1×10 9 cfu) and Synatura-treated groups, the production of mucins increased by OVA+DEP treatment was significantly reduced ( Fig.14 ).

[0277] Therefore, since the strain of the present disclosure reduces mucus in the alveoli, it can prevent the advanced stage of respiratory diseases from deteriorating into pulmonary fibrosis and the like.

[0278] 3-9) Evaluation of the strain disclosed herein in alleviating lung function in an animal model of chronic respiratory disease - caspase 3 activity and total collagen content

[0279] To confirm whether the strains of the present disclosure actually reduce apoptosis in lung tissue and improve lung injury, caspase 3 activity and total collagen content in lung tissue were measured.

[0280] according to Fig.15 Compared with the conventional group, the caspase-3 activity and collagen content in the OVA-treated group were significantly increased, and compared with the OVA-treated group, the caspase-3 activity and collagen content in the OVA+DEP-treated group were significantly increased. 7 , 1×10 9 cfu), GCWB1085 (1×10 7 , 1×10 9 cfu), GCWB1156 (1×10 7 , 1×10 9 cfu) and Synatura-treated groups, the caspase-3 activity and collagen content increased by OVA+DEP treatment were significantly reduced ( Fig.15 ).

[0281] Therefore, the strain of the present disclosure can prevent the deterioration of pulmonary fibrosis, the late stage of respiratory diseases, by reducing not only apoptotic factors but also collagen content.

[0282] 3-10) Evaluation of the strain disclosed herein in alleviating lung function in an animal model of chronic respiratory disease - Analysis of MMP9 activity

[0283] Proteins separated by 10% SDS-PAGE electrophoresis containing 0.2% gelatin in bronchoalveolar lavage fluid (BALF) were stained with 0.25% Coomassie Brilliant Blue G250 (Sigma Chemical Co., St. Louis, MO, USA), and then MMP-9 enzyme activity was measured.

[0284] according to Fig.16 Compared with the conventional group, the MMP-9 activity in the OVA-treated group was significantly increased, and compared with the OVA-treated group, the MMP-9 activity in the OVA+DEP-treated group was significantly increased. 9 cfu), GCWB1085 (1×109 cfu), GCWB1156 (1×10 9 cfu) and Synatura-treated groups, the MMP-9 activity increased by OVA+DEP treatment was significantly reduced.

[0285] Therefore, since the strain of the present disclosure can prevent the deposition of inflammatory cells in lung tissue, it can prevent inflammation in lung tissue and various complications caused by inflammation.

Claims

1. A Lactobacillus plantarum GCWB1001 strain deposited with the accession number KCCM12698P.

2. The plant lactobacillus GCWB1001 strain according to claim 1, wherein The strain contains 16S rRNA consisting of the nucleotide sequence of SEQ ID NO:

1.

3. A health functional food composition for preventing or alleviating inflammatory diseases or respiratory diseases, comprising: the strain according to any one of claims 1 to 2; one selected from a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product or a pulverized product of the strain; or a culture of the strain and any one of a concentrate, a paste and a dilution of the culture.

4. The health functional food composition according to claim 3, further comprising one or more selected from the group consisting of protein, carbohydrate, fat, nutrients, seasoning, sweetener and flavor enhancer.

5. The health functional food composition according to claim 4, wherein The health functional food composition is any one selected from granules, lemon juice, powder, syrup, liquid and solution, extract, elixir, fluid extract, suspension, decoction, infusion, tablet, spirit, capsule, lozenge, pill and soft or hard gelatin capsule.

6. The health functional food composition according to claim 3, wherein: The respiratory disease is caused by any one or more of fine dust, viral infection and inflammation.

7. A pharmaceutical composition for treating or preventing inflammatory diseases or respiratory diseases, comprising: the strain according to any one of claims 1 to 2; one selected from a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product or a pulverized product of the strain; or a culture of the strain and any one of a concentrate, a paste and a dilution of the culture.

8. The pharmaceutical composition according to claim 7, further comprising a pharmaceutically acceptable excipient or carrier.

9. The pharmaceutical composition according to claim 7, wherein The respiratory disease is caused by any one or more of fine dust, viral infection and inflammation.

10. The pharmaceutical composition according to claim 7, wherein The respiratory disease is selected from any one or more of respiratory inflammatory lung disease, asthma, bronchiectasis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, emphysema, sequelae of tuberculosis, allergic rhinitis, lower respiratory tract infection, bronchiolitis, acute upper respiratory tract infection, allergic lung disease, bronchiectasis, pneumonia, acute and chronic bronchitis, sinusitis, pharyngitis, tonsillitis and laryngitis.

11. The pharmaceutical composition according to claim 9, wherein The virus is selected from adenovirus, vaccinia virus, herpes simplex virus, parainfluenza virus, rhinovirus, varicella-zoster virus, measles virus, respiratory syncytial virus, dengue virus, human immunodeficiency virus (HIV), influenza virus, coronavirus, severe acute respiratory syndrome-associated virus (SARS-associated virus) and Middle East respiratory syndrome coronavirus (MERS-CoV).

12. A probiotic comprising the Lactobacillus plantarum GCWB1001 strain deposited under the accession number KCCM12698P.

13. The probiotic according to claim 12, wherein The viable cell content of the probiotics is 1×10 1 Up to 1×10 13 CFU / g.

14. The probiotic according to claim 13, wherein The probiotics are used for preventing or alleviating inflammatory diseases or respiratory diseases.

15. The probiotic according to claim 14, wherein The respiratory disease is caused by any one of fine dust, viral infection and pneumonia.

16. The probiotic according to claim 14, wherein The probiotics have an antitussive and expectorant effect or an effect of reducing cough.

17. The probiotic according to claim 14, wherein The probiotics reduce inflammatory responses in lung tissue.

18. The probiotic according to claim 14, wherein The probiotics reduce lung tissue or systemic allergic reactions.

19. The probiotic according to claim 14, wherein The probiotics inhibit pulmonary fibrosis.

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