Composition containing lactic acid bacteria for treating respiratory or inflammatory diseases caused by fine dust irritation

Abstract

The present invention provides a novel strain of Lactobacillus plantarum GCWB1001 deposited under accession number KCCM12698P, a novel strain of Pediococcus acidilactici GCWB1085 deposited under accession number KCCM12699P, or a novel strain of Lactobacillus rhamnosus GCWB1156 deposited under accession number KCCM12700P, which are excellent in treating or ameliorating respiratory diseases. In addition, the present invention provides pharmaceutical compositions, health functional food compositions, and probiotics that contain any one of the novel strains and have the effect of treating or improving inflammatory diseases or respiratory diseases.

Description

[Technical Field]

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0061851 filed on May 22, 2020, and all contents disclosed in the documents of that Korean patent application are incorporated herein by reference.

[0002] The present invention relates to novel lactic acid bacteria having therapeutic or preventive effects on respiratory diseases or inflammatory diseases, and pharmaceutical compositions, functional health food compositions, and probiotics containing the same. [Background technology]

[0003] Fine dust is particulate matter of 10μm or less that floats or scatters in the air. Dust is classified according to its diameter, with PM10 being particles smaller than 10 / 1000mm and PM2.5 being particles smaller than 2.5 / 1000mm.

[0004] Fine dust is generated by direct emissions from anthropogenic sources such as combustion in industrial plants and automobile fuel combustion, as well as sulfur oxides (SO x ), nitrogen oxides (NO x It is produced by a secondary generation process in which substances such as toluene (O2), ammonia (NH3), and volatile organic compounds (VOCs) react with water vapor in the atmosphere.

[0005] Fine dust particles are known to be so small that they can penetrate directly into the alveoli or brain when inhaled without passing through the nasal mucosa, which is known to increase the prevalence of asthma, lung disease, and early mortality.

[0006] In particular, in 2013 the International Agency for Research on Cancer (IARC), an organization under the World Health Organization (WHO), classified fine dust as a Class 1 carcinogen, along with benzene and asbestos, which are confirmed to cause cancer in humans.

[0007] Probiotics are live microorganisms that improve the host's microbial environment in the gastrointestinal tract of animals, including humans, and have a beneficial effect on the host's health.

[0008] Lactic acid bacteria, a type of probiotic, live symbiotically in the human digestive system, breaking down fiber and complex proteins to produce important nutrients. Lactic acid bacteria are anaerobic bacteria that break down carbohydrates and use them to produce lactic acid, growing well in low-oxygen environments. Lactic acid bacteria can be broadly divided into five genera: Streptococcus, Lactobacillus, Leuconostoc, Bifidobacteria, and Pediococcus. Recently, the therapeutic effects of lactic acid bacteria on various diseases have been confirmed, and efforts are being made to develop therapeutic agents using this.

[0009] Unlike conventional respiratory and lung damage caused by bacteria, temporary poisoning, or other substances entering the respiratory tract, fine dust cannot be eliminated by the human immune system, and there is no way to forcibly expel fine dust once it enters the respiratory tract. Furthermore, since it is not known exactly when or what problems will occur due to such damage, there is a pressing need for new therapeutic agents that can prevent, mitigate, treat, or improve airway and lung damage induced by fine dust, and treat respiratory diseases induced thereby. Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention provides a novel Lactobacillus plantarum GCWB1001 strain deposited under accession number KCCM12698P, a Lactobacillus rhamnosus GCWB1156 strain deposited under accession number KCCM12700P, and a Pediococcus acidiclactici GCWB1085 strain deposited under accession number KCCM12699P, as well as compositions for preventing or treating inflammatory diseases or respiratory diseases, comprising the same. [Means for solving the problem]

[0011] In order to achieve the above purpose, The present invention provides the Lactobacillus plantarum GCWB1001 strain deposited under accession number KCCM12698P.

[0012] The present invention provides the Lactobacillus rhamnosus GCWB1156 strain deposited under accession number KCCM12700P.

[0013] The present invention provides the Pediococcus acidiclactici GCWB1085 strain deposited under accession number KCCM12699P.

[0014] The present invention also provides a pharmaceutical composition for preventing or treating inflammatory or respiratory diseases, comprising the Lactobacillus plantarum GCWB1001 strain deposited under accession number KCCM12698P, the Lactobacillus rhamnosus GCWB1156 strain deposited under accession number KCCM12700P, or the Pediococcus acidiclactici GCWB1085 strain deposited under accession number KCCM12699P, or a spray-dried, freeze-dried, vacuum-dried, drum-dried, or crushed strain of any of the strains, or a culture of any of the strains, or a concentrate, paste, or dilution of the culture.

[0015] The present invention also provides a functional health food composition for preventing or alleviating inflammatory or respiratory diseases, comprising the Lactobacillus plantarum GCWB1001 strain deposited under accession number KCCM12698P, the Lactobacillus rhamnosus GCWB1156 strain deposited under accession number KCCM12700P, or the Pediococcus acidiclactici GCWB1085 strain deposited under accession number KCCM12699P, or a spray-dried, freeze-dried, vacuum-dried, drum-dried, or crushed strain of any of the strains, or a culture of any of the strains, or a concentrate, paste, or dilution of the culture.

[0016] The present invention also provides probiotics comprising the Lactobacillus plantarum GCWB1001 strain deposited under accession number KCCM12698P, the Lactobacillus rhamnosus GCWB1156 strain deposited under accession number KCCM12700P, or the Pediococcus acidiclactici GCWB1085 strain deposited under accession number KCCM12699P. [Effects of the Invention]

[0017] According to one embodiment of the present invention, Lactobacillus plantarum GCWB1001 (KCCM12698P), Lactobacillus rhamnosus GCWB1156 (KCCM12700P), and Pediococcus acidilactici GCWB1085 (KCCM12699P) strains have anti-inflammatory effects and have been shown to have antitussive / expectorant effects and improve lung function in chronic respiratory diseases in animal models. According to one embodiment of the present invention, Lactobacillus plantarum GCWB1001 (KCCM12698P), Lactobacillus rhamnosus GCWB1156 (KCCM12700P), and Pediococcus acidilactici GCWB1085 (KCCM12699P) strains were isolated from kimchi, cheese, and infant feces, respectively, and each strain exhibits no cytotoxicity in mouse lung macrophages (MH-S cell line) and has the effect of reducing the secretion of cytokines TNF-alpha and TGF-beta.

[0018] According to one embodiment of the present invention, all of the strains of the present invention not only reduced the production of inflammatory factors NO (nitric oxide) and ROS (reactive oxygen species) increased by fine dust, but also reduced the promoter activity of inflammatory transcription factors NF-kB, iNOS, and COX2.

[0019] According to one embodiment of the present invention, all of the strains of the present invention not only alleviated cough symptoms and had antitussive / expectorant effects in actual animal models, but also showed the effect of alleviating various symptoms in animal models of chronic respiratory disease caused by fine dust.

[0020] As a result, the Lactobacillus plantarum GCWB1001 (KCCM12698P), Lactobacillus rhamnosus GCWB1156 (KCCM12700P), and Pediococcus acidilactici GCWB1085 (KCCM12699P) strains of the present invention exhibit alleviating, preventive, or therapeutic effects on inflammatory diseases or respiratory diseases. [Brief explanation of the drawings]

[0021] [Figure 1] 1 is a graph showing the amount of TGF-β produced by seven bacterial strain samples measured in a mouse lung macrophage cell line (MH-S) (Experimental Example 1-2). [Figure 2] 1 is a graph showing the amounts of NO and ROS produced by seven bacterial strain samples measured in a mouse lung macrophage cell line (MH-S) (Experimental Examples 1-3 and 1-4). [Figure 3] 1 is a graph showing the amounts of NO and ROS produced by seven bacterial strain samples measured in a mouse lung macrophage cell line (MH-S) (Experimental Examples 1-3 and 1-4). [Figure 4] 1 is a graph showing the measurement (Experimental Example 1-5) of the activity of inflammatory transcriptional regulators (iNOS, COX2, and NF-kB promoter activity) of seven bacterial strain samples in a mouse lung macrophage cell line (MH-S). [Figure 5] 1 is a graph showing the measurement (Experimental Example 1-5) of the activity of inflammatory transcriptional regulators (iNOS, COX2, and NF-kB promoter activity) of seven bacterial strain samples in a mouse lung macrophage cell line (MH-S). [Figure 6] 1 is a graph showing the measurement (Experimental Example 1-5) of the activity of inflammatory transcriptional regulators (iNOS, COX2, and NF-kB promoter activity) of seven bacterial strain samples in a mouse lung macrophage cell line (MH-S). [Figure 7]1 is a graph showing the antitussive and expectorant effects of GCWB1001, GCWB1085, and GCWB1156 strains in a mouse cough model (Experimental Examples 2-2 and 2-3). [Figure 8] 1 is a graph showing the antitussive and expectorant effects of GCWB1001, GCWB1085, and GCWB1156 strains in a mouse cough model (Experimental Examples 2-2 and 2-3). [Figure 9] 1 is a graph showing the weights of body weight, liver, spleen, and lungs after treatment with GCWB1001, GCWB1085, and GCWB1156 strains in an animal model of chronic respiratory disease (Experimental Example 3-3). [Figure 10] 1 is a graph showing the measurement of the number of various immune cells in BALF (Broncoalveolar lavage fluid) after treatment of chronic respiratory disease animal models with GCWB1001, GCWB1085, and GCWB1156 strains (Experimental Example 3-4). [Figure 11] 1 is a graph showing the measurement of the amount of OVA-specific IgE in BALF (Broncoalveolar lavage fluid) after treatment of chronic respiratory disease animal models with GCWB1001, GCWB1085, and GCWB1156 strains (Experimental Example 3-5). [Figure 12] 1 is a graph showing the measurement of cytokine (TNF-alpha, IL-6, IL-1beta, IL-4, IL-13, MCP-1) levels in BALF (Broncoalveolar lavage fluid) after treatment with GCWB1001, GCWB1085, and GCWB1156 strains in an animal model of chronic respiratory disease (Experimental Example 3-6). [Figure 13] 1 shows photographs and graphs of lung tissue stained with inflammatory cells infiltrating into lung tissue after treatment with GCWB1001, GCWB1085, and GCWB1156 strains in an animal model of chronic respiratory disease (Experimental Example 3-7). [Figure 14] 1 shows photographs and graphs of lung tissue stained for mucus protein after treatment with GCWB1001, GCWB1085, and GCWB1156 strains in an animal model of chronic respiratory disease (Experimental Example 3-8). [Figure 15]1 is a graph showing caspase 3 activity and total collagen content in lung tissue after treatment with GCWB1001, GCWB1085, and GCWB1156 strains in an animal model of chronic respiratory disease (Experimental Examples 3-9). [Figure 16] 1 shows photographs of an analysis of MMP-9 activity in lung tissue (Experimental Examples 3-10) after treatment with GCWB1001, GCWB1085, and GCWB1156 strains in an animal model of chronic respiratory disease. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention will now be described in more detail.

[0023] Unless otherwise defined, all technical terms used in the present invention are used in the same manner as commonly understood by those skilled in the art. In addition, although preferred methods and samples are described in this specification, similar or equivalent methods and samples are also included within the scope of the present invention.

[0024] The present invention relates to the Lactobacillus plantarum GCWB1001 strain deposited under accession number KCCM12698P.

[0025] The novel strain, Lactobacillus plantarum GCWB1001, was named as above and deposited at the Korea Microorganism Collection on April 17, 2020. The accession number is KCCM12698P.

[0026] The strains can be isolated and identified from kimchi collected in various regions. The inventors of the present invention isolated and identified various novel strains from kimchi collected in various regions, and found that among the isolated and identified strains, Lactobacillus plantarum GCWB1001 strain exhibits significantly superior anti-inflammatory effects and respiratory disease prevention or treatment effects compared to conventionally known lactic acid bacteria, thereby completing the present invention.

[0027] The present invention relates to the Lactobacillus rhamnosus GCWB1156 strain deposited under accession number KCCM12700P.

[0028] The novel strain, Lactobacillus rhamnosus GCWB1156, was named as above and deposited at the Korea Microorganism Collection on April 17, 2020. The accession number is KCCM12700P.

[0029] The strains can be isolated and identified from cheese collected in various regions. The inventors of the present invention isolated and identified various novel strains from cheese collected in various regions, and confirmed that among the isolated and identified strains, Lactobacillus rhamnosus GCWB1156 strain exhibits significantly superior anti-inflammatory effects and respiratory disease prevention or treatment effects compared to conventionally known lactic acid bacteria, thereby completing the present invention. The present invention relates to the Pediococcus acidiclactici GCWB1085 strain deposited under accession number KCCM12699P.

[0030] The novel strain, Pediococcus acidiclactici GCWB1085, was named as above and deposited at the Korea Microorganism Collection on April 17, 2020. The accession number is KCCM12699P.

[0031] The strain can be isolated and identified from infant feces. The present inventors isolated and identified various novel strains from infant feces and found that among the isolated and identified strains, Pediococcus acidiclactici GCWB1085 strain exhibits significantly superior anti-inflammatory effects and respiratory disease prevention or treatment effects compared to conventionally known lactic acid bacteria, thereby completing the present invention.

[0032] That is, the Lactobacillus plantarum GCWB1001, Lactobacillus rhamnosus GCWB1156, and Pediococcus acidiclactici GCWB1085 strains suppressed NO, ROS, and the activity of inflammatory transcription factors (iNOS, COX2, and NF-kB promoter activity) in MH-S cells, a mouse lung macrophage cell line treated with inflammation inducers LPS and diesel exhaust particles (DEP).

[0033] In addition, the GCWB1001, GCWB1156 and GCWB1085 strains of the present invention were able to reduce cytokine (TNF-alpha) secretion induced by LPS and DEP in the MH-S cell line, confirming their anti-inflammatory effects. Oxidative stress refers to the phenomenon in which the production of reactive oxygen species or reactive nitrogen species in biological molecules, cells, and tissues in the body loses balance with the antioxidant defense mechanism, resulting in a relative excess of reactive oxygen species or reactive nitrogen species, which usually causes tissue damage.

[0034] These reactive oxygen species and nitrogen species are chemically unstable and highly reactive, causing extensive oxidative damage to enzyme catalytic reactions, mitochondrial electron transport, cell signaling systems, gene expression, transcription factor activation, and biomolecules, cells, and tissues in the body, thereby inducing inflammatory responses and becoming a major factor in tissue fibrosis. This oxidative damage can lead to various diseases in all tissues of the human body. Specifically, it is known to be involved in the development and progression of cancer in tissues such as the skin, kidneys, heart, joints, lungs, brain, blood vessels, intestines, and eyes, and is known to play an important role in almost all diseases, including cardiovascular disease, inflammation, fibrotic diseases, and diabetes.

[0035] When inflammatory substances such as LPS are processed by macrophages, inflammatory cytokines (IL-6, TNF-alpha, IL-1beta, etc.) and inflammatory mediators (oxidative stress) such as NO are produced, causing an inflammatory response. NO is produced by iNOS (induced nitric oxide synthase), and as an inflammatory response occurs, the NF-kB transcription factor is activated, and COX-2 (cyclooxygenase-2) produces prostaglandins (PGs) involved in the inflammatory response, accelerating the inflammatory response. Therefore, the GCWB1001, GCWB1156 and GCWB1085 strains of the present invention have anti-inflammatory effects that can suppress inflammation by suppressing the activity of inflammatory substances such as NO and ROS, inflammatory cytokine TNF-alpha, and inflammatory transcription factors (iNOS, COX2 and NF-kB promoter) induced by LPS and DEP.

[0036] The GCWB1001, GCWB1156 and GCWB1085 strains of the present invention suppressed cough and increased sputum production in a citric acid-induced cough model.

[0037] Cough suppressants are drugs that relieve coughs regardless of the cause. They can be divided into centrally acting and peripherally acting drugs based on their mechanism of action. Centrally acting drugs can be divided into narcotics, narcotic derivatives, and non-narcotics. Representative narcotics, such as codeine, hydrocodone, and morphine, have been shown to have limited cough suppression effects, but results are inconsistent, and at appropriate doses can cause drowsiness, constipation, digestive disorders, and the risk of abuse and dependence. The most commonly used peripherally acting cough suppressant in Korea is levodropropizine, which is believed to work by regulating the level of sensory neuropeptides in the airways. Theobromine is another example.

[0038] The main component of sputum is mucus, which is secreted by mucous and serous gland cells that make up the mucous and submucosal glands normally distributed in the bronchial mucosa. Mucus is 95% water, with the remaining 5% consisting of glycoproteins, lipids, and minerals. The glycoprotein structure is a gel-like structure consisting of a linear polymer double structure, resulting in a sticky appearance. Expectorants used to remove sputum are classified into drugs that increase the water content of sputum and mucolytic agents that reduce viscosity by cleaving disulfide bonds in sputum proteins. Cysteine ​​derivatives such as N-acetylcysteine ​​and carbocysteine ​​are commonly used, but these cysteine ​​derivatives can cause side effects such as bronchospasm with long-term use. Therefore, there is a need for expectorants with excellent expectorant efficacy and minimal side effects and toxicity.

[0039] In addition, ivy extract and sinetura, a compound of ivy and coptis extract, are widely used as natural antitussive and expectorant agents.

[0040] The GCWB1001, GCWB1156 and GCWB1085 strains of the present invention reduce the frequency of coughs in a citric acid-induced cough model and exhibit antitussive / expectorant effects, thereby improving the symptoms of respiratory diseases.

[0041] Furthermore, the strain of the present invention significantly reduced immune cells in bronchoalveolar fluid (BALF) in a chronic respiratory disease model using ovalbumin (OVA) and diesel exhaust particles (DEP), as well as immune cells that had infiltrated into lung tissue, reduced OVA-specific IgE, and reduced the inflammatory cytokines TNF-alpha, IL-6, IL-1beta, IL-4, MCP-1, and IL-13 in BALF, while significantly increasing the anti-inflammatory cytokine IFN-gamma.

[0042] Therefore, the GCWB1001, GCWB1156 and GCWB1085 strains of the present invention not only have antitussive / expectorant effects themselves, but also inhibit the activity of MMP9, preventing the infiltration of inflammatory cells into bronchoalveolar fluid and lung tissue, preventing the secretion of inflammatory cytokines, increasing the secretion of anti-inflammatory cytokines, and reducing the amount of IgE immunoglobulins that increase in allergic symptoms, thereby being understood to have the effect of reducing allergic and inflammatory reactions in the respiratory organs.

[0043] Representative respiratory diseases include asthma, pneumonia, chronic obstructive pulmonary disease, allergic rhinitis, acute chronic bronchitis, bronchiolitis, pharyngitis, tonsillitis, laryngitis, bronchiectasis, idiopathic pulmonary fibrosis, cystic fibrosis, emphysema, sequelae of pulmonary tuberculosis, lower respiratory tract infections, sinusitis, acute upper respiratory tract infections, and allergic lung disease. Asthma refers to chronic inflammation in the airways, particularly the bronchi. Inflammation caused by asthma can be exacerbated by a variety of factors, including smoke, allergens, cold winds, exercise, and respiratory infections, and persistent inflammation leads to airway deformation and hyperresponsiveness.

[0044] Respiratory diseases can also be caused by respiratory viruses, including adenovirus, vaccinia virus, herpes simplex virus, parainfluenza virus, rhinovirus, varicella zoster virus, measles virus, respiratory syncytial virus (RSV), 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).

[0045] The respiratory tract is largely made up of mucous membranes and bronchial smooth muscle. The mucous membrane contains many secretory glands that continuously secrete necessary secretions, and when the bronchial smooth muscle contracts, the respiratory tract narrows. When an inflammatory response occurs due to a wide variety of factors, such as smoke, allergens, cold wind, exercise, or respiratory infection, the secretions from the secretory glands increase. The secretions secreted at this time are due to an exudative response caused by inflammation, and are mostly sticky mucus secretions consisting of a mixture of inflammatory mediators and mucin.

[0046] Mucin normally plays a role in protecting the body, but in chronic inflammatory respiratory diseases such as asthma, COPD, and chronic bronchitis, excessive production or secretion of sticky secretions is observed. Abnormalities in the quantity and quality of these secretions act as pathological factors, causing the accumulation of secretions to block the lumen of organs and block airflow to the airways, resulting in severe paroxysmal coughing and difficulty breathing accompanied by wheezing, and during attacks, a dry cough and chest tightness. Lung damage caused by viral respiratory diseases is also thought to be caused by this mucus.

[0047] In animal models of chronic respiratory disease, the GCWB1001, GCWB1156, and GCWB1085 strains of the present invention inhibited the deposition of mucus proteins in lung tissue, reduced the activity of caspase 3, a cell death factor in the alveoli, decreased collagen deposition, and reduced MMP-9 activity. The reduction in MMP-9 activity prevents the deposition of inflammatory cells in lung tissue, ultimately preventing pulmonary fibrosis.

[0048] Pulmonary fibrosis is the final stage of respiratory disease and involves complex pathological and physiological processes. In the early stages, pulmonary inflammation is the focus, leading to chronic thickening of the alveolar walls due to the infiltration of large numbers of inflammatory cells. In the middle and late stages, excessive deposition of extracellular matrix elements such as collagen by fibroblasts causes overgrowth of lung tissue, resulting in alveolar deformation, hardening, and scarring, destroying normal lung tissue structure and causing loss of function.

[0049] Fibroblasts play a role in the recruitment of immune cells to sites of inflammation and tissue injury. Furthermore, fibroblasts produce and respond to numerous inflammatory cytokines. Therefore, fibroblasts can contribute to chronic inflammation, and conversely, inflammatory cytokines promote the transformation of fibroblasts into myofibroblasts, thereby promoting fibrosis. Therefore, injury and inflammation of lung tissue can lead to pulmonary fibrosis.

[0050] It has been reported that TGF-β stimulation in the lung tissue of patients with progressing pulmonary fibrosis causes increased ROS production, which increases the expression of collagen and α-SMA (a-smooth muscle actin), which are important in fibrosis, and that ROS exacerbates pulmonary fibrosis in the lung tissue of patients with idiopathic pulmonary fibrosis. Therefore, the strain of the present invention not only induces antitussive / expectorant effects in cough model mice, but also suppresses oxidative stress in animal models of chronic respiratory disease, thereby inhibiting the expression of inflammatory cytokines and MMP-9 expression, thereby preventing the deposition of immune cells involved in inflammatory responses in lung tissue and bronchoalveolar lavage fluid, suppressing mucus protein production and collagen production, and ultimately showing an effect of improving pulmonary fibrosis.

[0051] The present invention relates to a pharmaceutical composition for preventing or treating an inflammatory disease or a respiratory disease, comprising the Lactobacillus plantarum GCWB1001 strain deposited under accession number KCCM12698P, the Lactobacillus rhamnosus GCWB1156 strain deposited under accession number KCCM12700P, or the Pediococcus acidilactici GCWB1085 strain deposited under accession number KCCM12699P, or one selected from the group consisting of a spray-dried product, freeze-dried product, vacuum-dried product, drum-dried product, and crushed product of any of the strains, or a culture of any of the strains, or a concentrate, paste, or dilution of the culture.

[0052] The inflammatory diseases include chronic and acute rhinitis, chronic and acute gastritis, colitis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis, irritable bowel syndrome, inflammatory bowel disease, intravenous colitis, rheumatoid arthritis, osteoarthritis, pneumonia, hepatitis, glomerulonephritis, gastritis, vasculitis, pancreatic colitis, peritonitis, bronchitis, myocardial infarction, encephalitis, and postischemic reperfusion injury. Infectious diseases include inflammation in skin injuries, inflammation caused by immune rejection after tissue and organ transplants, burns, various inflammations of the skin such as psoriasis and allergic contact dermatitis, lower back pain, fascial diseases, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatic colitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis, atopic dermatitis, eczema, diabetic inflammation, infectious inflammation caused by viral or bacterial infection, lupus, psoriasis, and atherosclerosis. The respiratory disease is caused by any one of fine dust, viral infection, and pneumonia, and is selected from the group consisting of respiratory inflammatory lung disease, 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 disease, bronchiectasis, pneumonia, acute and chronic bronchitis, sinusitis, pharyngitis, tonsillitis, laryngitis, and pulmonary fibrosis.

[0053] The virus may be selected from adenovirus, vaccinia virus, herpes simplex virus, parainfluenza virus, rhinovirus, varicella zoster virus, measles virus, respiratory syncytial virus (RS 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).

[0054] The strain of the present invention is 1 × 10 1 ~1×10 13 Those having a viable bacterial content of CFU / g can be used, but are not limited thereto. The present invention also relates to a food composition for preventing inflammatory or respiratory diseases, comprising the Lactobacillus plantarum GCWB1001 strain deposited under accession number KCCM12698P, the Lactobacillus rhamnosus GCWB1156 strain deposited under accession number KCCM12700P, or the Pediococcus acidilactici GCWB1085 strain deposited under accession number KCCM12699P, or one selected from the group consisting of a spray-dried product, freeze-dried product, vacuum-dried product, drum-dried product, and crushed product of any of the strains, or a culture of any of the strains, or a concentrate, paste, or dilution of the culture.

[0055] The present invention also relates to a functional health food composition for preventing inflammatory diseases or respiratory diseases, comprising the Lactobacillus plantarum GCWB1001 strain deposited under accession number KCCM12698P, the Lactobacillus rhamnosus GCWB1156 strain deposited under accession number KCCM12700P, or the Pediococcus acidilactici GCWB1085 strain deposited under accession number KCCM12699P, or one selected from the group consisting of a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product, and a crushed product of any of the strains, or a culture of any of the strains, or a concentrate, a paste, or a dilution of the culture.

[0056] In addition to the active ingredients, the food compositions or health functional food compositions of the present invention may contain ingredients commonly added during food production, such as, but not limited to, proteins, carbohydrates, fats, nutrients, seasonings, sweeteners, and flavorings. Examples of carbohydrates include common sugars such as monosaccharides (e.g., glucose, fructose, etc.); disaccharides (e.g., maltose, sucrose, oligosaccharides, etc.); and polysaccharides (e.g., dextrin, cyclodextrin, etc.), as well as sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of sweeteners include natural sweeteners (e.g., thaumatin, stevia extract, rebaudioside A, glycyrrhizin, etc.) and synthetic sweeteners (e.g., saccharin, aspartame, etc.). Prebiotics may also be included, such as plant-derived oligosaccharides (e.g., fructan, galactan, resistant starch, pectin, beta-glucan, and xylooligosaccharides) that can serve as food for the bacterial strain.

[0057] However, the present invention is not limited to these, and any component known in the art that does not impair the effects of the present invention may be used.

[0058] Examples of the food composition or health functional food composition include, but are not limited to, patient nutritional foods, meat, grains, caffeinated drinks, regular drinks, dairy products, chocolate, bread, snacks, sweets, pizza, jelly, noodles, gum, ice cream, alcoholic drinks, alcohol, vitamin complexes, and other health supplements. When prepared in the form of such a food composition or health functional food composition, it is preferable because it is easy and convenient to take.

[0059] In the present invention, the health functional food composition and pharmaceutical composition may be prepared in the form of, but not limited to, granules, lemonades, powders, syrups, liquids and solutions, extracts, elixirs, fluid extracts, suspensions, decoctions, infusions, tablets, spirits, capsules, troches, pills, and soft or hard gelatin capsules.

[0060] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention include those commonly used in formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginic acid, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto.

[0061] The pharmaceutical composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like in addition to the above ingredients.

[0062] The pharmaceutical compositions of the present invention can be administered orally or parenterally.

[0063] The pharmaceutical composition of the present invention may be prepared in a unit dose form or in a multi-dose container by formulating it using pharmaceutically acceptable carriers and / or excipients in a manner that can be easily carried out by a person skilled in the art to which the invention pertains.

[0064] The present invention also relates to probiotics comprising the Lactobacillus plantarum GCWB1001 strain deposited under accession number KCCM12698P, the Lactobacillus rhamnosus GCWB1156 strain deposited under accession number KCCM12700P, or the Pediococcus acidilactici GCWB1085 strain deposited under accession number KCCM12699P.

[0065] The probiotics may include one or more strains selected from the three strains deposited in the present invention, and may further include one or more strains selected from the seven lactic acid bacteria strains isolated in the Examples, and may further include known strains useful for the purposes of the present invention.

[0066] The probiotics are 1 x 10 1 ~1×10 13 Those having a viable bacterial content of CFU / g can be used, and the probiotics can be useful for preventing or alleviating anti-inflammatory or respiratory diseases.

[0067] In the present invention, the dosage of the GCWB1001, GCWB1156 and GCWB1085 strains should be determined taking into consideration the administration method, age, sex, weight and severity of the disease of the recipient.

[0068] As an example, the GCWB1001, GCWB1156 and GCWB1085 strains were cultured at 1×10 per day. 1 ~1×10 13The viable CFU / g content can be administered in one or more divided doses.

[0069] In addition, the pharmaceutical compositions, food compositions, health functional food compositions, and probiotics containing the above ingredients are administered at a dose of 1 x 10 per day based on the active ingredient. 1 ~1×10 13 The viable CFU / g content can be administered in one or more divided doses.

[0070] However, the above dosage is merely an example and may be changed by a doctor's prescription depending on the patient's condition.

[0071] The present invention will be described in detail below with reference to the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0072] Example 1: Isolation and identification of Lactobacillus plantarum GCWB1001 strain (1) Isolation of strains Kimchi collected from each region was homogenized in a 10-fold volume of sterile saline. The homogenized sample was serially diluted 10 times in sterile saline, and bacterial strains were isolated by the dilution plating method. The diluted bacterial strain samples were smeared onto MRS broth agar (BD Difco) and anaerobically cultured at 37°C for 72 hours. Colonies detected on the MRS agar plate were then inoculated onto PCA medium (MBcell, South Korea) containing 0.005% BCP (bromocresol purple), a pH indicator, for a second time. Colonies that turned the purple medium yellow were then inoculated onto MRS agar plates for a third time to isolate the probiotics.

[0073] (2) Identification of Lactobacillus plantarum GCWB1001 strain DNA extraction and isolation were performed on the purely isolated strains in (1). Two universal primers, 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'), were used to amplify the 16s rRNA gene, followed by sequencing analysis of the amplified 16s rRNA gene. Using the analyzed 16s rRNA sequence data and the EzTaxon server (http: / / www.ezbiocloud.net), only two strains that were GRAS (Generally Recognized as Safe) were selected, as shown in Table 1 below.

[0074] [Table 1]

[0075] The results of analyzing the 16S rRNA base sequence of Lactobacillus plantarum GCWB1001 are as follows. <16S rRNA sequence of Lactobacillus plantarum GCWB1001> GGTCGTACGA ACTCTGTGTA TTGATTGGTG CTTGCATCAT GATTTACATT TGCAGTGAGT GGCGAACTGG TGAGTAACAC GTGGGAAACC TGCCCAGAAG CGGGGGATAA CACCTGGAAA CAGATGCTAA TACCGCATAA CAACTTGGAC CGCATGGTCC GAGTTTGAAA GATGGCTTCG GCTATCACTT TTGGATGGTC CCGCGGCGTA TTAGCTAGAT GGTGGGGTAA CGGCTCACCA TGGCAATGAT ACGTAGCCGA CCTGAGAGGG TAATCGGCCA CATTGGGACT GAGACACGGC CCAAACTCCT ACGGGAGGCA GCAGTAGGGA ATCTTCCACA ATGGACGAAA GTCTGATGGA GCAACGCCGC GTGAGTGAAG AAGGGTTTCG GCTCGTAAAA CTCTGTTGTT GAAGAAGAAC ATATCTGAGA GTAACTGTTC AGGTATTGAC GGTATTTAAC CAGAAAGCCA CGGCTAACTA CGTGCCAGCA GCCGCGGTAA TACGTAGGTG GCAAGCGTTG TCCGGATTTA TTGGGCGTAA AGCGAGCGCA GGCGGTTTTT TAAGTCTGAT GTGAAAGCCT TCGGCTCAAC CGAAGAGTG CATCGGAAAC TGGGAAACTT GAGTGCAGAA GAGGACAGTG GAACTCCATG TGTAGCGGTG AAATGCGTAG ATATATGGAA GAACACCAGT GGCGAAGGCG GCTGTCTGGT CTGTAACTGA CGCTGAGGCT CGAAAGTATG GGTAGCAAAC AGGATTAGAT ACCCTGGTAG TCCATACCGT AAACGATGAA TGCTAAGTGT TGGAGGGTTT CCGCCCTTCA GTGCTGCAGC TAACGCATTA AGCATTCCGC CTGGGGAGTA CGGCCGCAAG GCTGAAACTC AAAGGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GAAGCTACGC GAAGAACCTT ACCAGGTCTT GACATACTAT GCAAATCTAA GAGATTAGAC GTTCCCTTCG GGGACATGGA TACAGGTGGT GCATGGTTGT CGTCAGCTCG TGTCGTGAGA TGTTGGGTTA AGTCCCGCAA CGAGCGCAAC CCTTATTATC AGTTGCCAGC ATTAAGTTGG GCACTCTGGT GAGACTGCCG GTGACAAACC GGAGGAAGGT GGGGATGACG TCAAATCATC ATGCCCCTTA TGACCTGGGC TACACACGTG CTACAATGGA TGGTACAACG AGTTGCGAAC TCGCGAGAGT AAGCTAATCT CTTAAAGCCA TTCTCAGTTC GGATTGTAGG CTGCAACTCG CCTACATGAA GTCGGAATCG CTAGTAATCG CGGATCAGCA TGCCGCGGTG AATACGTTCC CGGGCCTTGT ACACACCGCC CGTCACACCA TGAGAGTTTG TAACACCCAA AGTCGGTGGG GTAACCTTTT AGGAACCAGC CGCT

[0076] Example 2: Isolation and identification of Pediococcus acidilactici GCWB1085 strain (1) Isolation of strains Cheese collected from each region was homogenized in a 10-fold volume of sterile saline. The homogenized sample was serially diluted 10 times in sterile saline, and bacterial strains were isolated using the dilution plating method. The diluted bacterial strain samples were smeared onto MRS broth agar (BD Difco) and anaerobically cultured at 37°C for 72 hours. Colonies detected on the MRS agar plate were then inoculated onto PCA medium (MBcell, South Korea) containing 0.005% BCP (bromocresol purple), a pH indicator, for a second time. Colonies that turned from purple to yellow were then inoculated onto MRS agar plates for a third time to isolate the probiotics.

[0077] (2) Identification of Pediococcus acidilactici GCWB1085 strain Chromosomal DNA extraction and preparation were performed for the purely isolated strains in (1). Two universal primers, 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'), were used to amplify the 16s rRNA gene, followed by sequencing analysis of the amplified 16s rRNA gene. Using the analyzed 16s rRNA sequence data and the EzTaxon server (http: / / www.ezbiocloud.net), only four strains that were GRAS (Generally Recognized as Safe) were selected, as shown in Table 2 below.

[0078] [Table 2]

[0079] The results of analyzing the 16S rRNA base sequence of Pediococcus acidilactici GCWB1085 are as follows. <16S rRNA sequence of Pediococcus acidilactici GCWB1085> CTCAGGATGA ACGCTGGCGG CGTGCCTAAT ACATGCAAGT CGAACGAACT TCCGTTAATT GATCAGGACG TGCTTGCACT GAATGAGATT TTAACACGAA GTGAGTGGCG GACGGGTGAG TAACACGTGG GTAACCTGCC CAGAAGCAGG GGATAACACC TGGAAACAGA TGCTAATACC GTATAACAGA GAAAACCGCC TGGTTTTCTT TTAAAAGATG GCTCTGCTAT CACTTCTGGA TGGACCCGCG GCGCATTAGC TAGTTGGTGA GGTAACGGCT CACCAAGGCG ATGATGCGTA GCCGACCTGA GAGGGTAATC GGCCACATTG GGACTGAGAC ACGGCCCAGA CTCCTACGGG AGGCAGCAGT AGGGAATCTT CCACAATGGA CGCAAGTCTG ATGGAGCAAC GCCGCGTGAG TGAAGAAGGG TTTCGGCTCG TAAAGCTCTG TTGTTAAAGA AGAACGTGGG TGAGAGTAAC TGTTCACCCA GTGACGGTAT TTAACCAGAA AGCCACGGCT AACTACGTGC CAGCAGCCGC GGTAATACGT AGGTGGCAAG CGTTATCCGG ATTTATTGGG CGTAAAGCGA GCGCAGGCGG TCTTTTAAGT CTAATGTGAA AGCCTTCGGC TCAACCGAAG AAGTGCATTG GAAACTGGGA GACTTGAGTG CAGAAGAGGA CAGTGGAACT CCATGTGTAG CGGTGAAATG CGTAGATATA TGGAAGAACA CCAGTGGCGA AGGCGGCTGT CTGGTCTGTA ACTGACGCTG AGGCTCGAAA GCATGGGTAG CGAACAGGAT TAGATACCCT GGTAGTCCAT GCCGTAAACG ATGATTACTA AGTGTTGGAG GGTTTCCGCC CTTCAGTGCT GCAGCTAACG CATTAAGTAA TCCGCCTGGG GAGTACGACC GCAAGGTTGA AACTCAAAAG AATTGACGGG GGCCCGCACA AGCGGTGGAG CATGTGGTTT AATTCGAAGC TACGCGAAGA ACCTTACCAG GTCTTGACAT CTTCTGCCAA CCTAAGAGAT TAGGCGTTCC CTTCGGGGAC AGAATGACAG GTGGTGCATG GTTGTCGTCA GCTCGTGTCG TGAGATGTTG GGTTAAGTCC CGCAACGAGC GCAACCCTTA TTACTAGTTG CCAGCATTCA GTTGGGCACT CTAGTGAGAC TGCCGGTGAC AAACCGGAGG AAGGTGGGGA CGACGTCAAA TCATCATGCC CCTTATGACC TGGGCTACAC ACGTGCTACA ATGGATGGTA CAACGAGTTG CGAAACCGCG AGGTTTAGCT AATCTCTTAA AACCATTCTC AGTTCGGACT GTAGGCTGCA ACTCGCCTAC ACGAAGTCGG AATCGCTAGT AATCGCGGAT CAGCATGCCG CGGTGAATAC GTTCCCGGGC CTTGTACACA CCGCCCGTCA CACCATGAGA GTTTGTAACA CCCAAAGCCG GTGGGGTAAC CTTTTAGGAG CTAGCCGTCT AAGGTGGGAC AGATGATTA

[0080] Example 3: Isolation and identification of Lactobacillus rhamnosus GCWB1156 strain (1) Isolation of strains Feces from healthy infants born naturally were diluted 10 times in sterile saline and the bacterial strains were isolated by the dilution plating method. The diluted fecal samples were smeared onto BSM agar medium (Bifidus Selective Medium Agar; Sigma, USA) and anaerobically cultured at 37°C for 72 hours. Colonies detected on the BSM agar plate were then inoculated onto PCA medium (MBcell, South Korea) containing 0.005% BCP (bromocresol purple), a pH indicator, for a second time. Colonies that turned from purple to yellow were then inoculated onto BL agar medium (MBcell, South Korea) for a third time to isolate the probiotics.

[0081] (2) Identification of Lactobacillus rhamnosus GCWB1156 strain Chromosomal DNA extraction and preparation were performed for the purely isolated strains described in (1). Two universal primers, 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'), were used to amplify the 16s rRNA gene, followed by sequencing analysis of the amplified 16s rRNA gene. Using the analyzed 16s rRNA sequence data and the EzTaxon server (http: / / www.ezbiocloud.net), only one strain was selected as GRAS (Generally Recognized as Safe), as shown in Table 3 below.

[0082] [Table 3]

[0083] The results of 16S rRNA base sequence analysis of Lactobacillus rhamnosus GCWB1156 are as follows. <16S rRNA sequence of Lactobacillus rhamnosus GCW1156> GTTGATCGGC CACATTGGGA CTGAGACACG GCCCAAACTC CTACGGGAGG CAGCAGTAGG GAATCTTCCA CAATGGACGC AAGTCTGATG GAGCAACGCC GCGTGAGTGA AGAAGGCTTT CGGGTCGTAA AACTCTGTTG TTGGAGAGA ATGGTCGGCA GAGTAACTGT TGTCGGCGTG ACGGTATCCA ACCAGAAAGC CACGGCTAAC TACGTGCCAG CAGCCGCGGT AATACGTAGG TGGCAAGCGT TATCCGGATT TATTGGGCGT AAAGCGAGCG CAGGCGGTTT TTTAAGTCTG ATGTGAAAGC CCTCGGCTTA ACCGAGGAAG TGCATCGGAA ACTGGGAAAC TTGAGTGCAG AAGAGGACAG TGGAACTCCA TGTGTAGCGG TGAAATGCGT AGATATATGG AAGAACACCA GTGGCGAAGG CGGCTGTCTG GTCTGTAACT GACGCTGAGG CTCGAAAGCA TGGGTAGCGA ACAGGATTAG ATACCCTGGT AGTCCATGCC GTAAACGATG AATGCTAGGT GTTGGAGGGT TTCCGCCCTT CAGTGCCGCA GCTAACGCAT TAAGCATTCC GCCTGGGGAG TACGACCGCA AGGTTGAAAC TCAAAGGAAT TGACGGGGGC CCGCACAAGC GGTGGAGCAT GTGGTTTAAT TCGAAGCAAC GCGAAGAACC TTACCAGGTC TTGACATCTT TTGATCACCT GAGAGATCAG GTTTCCCCTT CGGGGGCAAA ATGACAGGTG GTGCATGGTT GTCGTCAGCT CGTGTCGTGA GATGTTGGGT TAAGTCCCGC AACGAGCGCA ACCCTTATGA CTAGTTGCCA GCATTTAGTT GGGCACTCTA GTAAGACTGC CGGTGACAAA CCGGAGGAAG GTGGGGATGA CGTCAAATCA TCATGCCCCT TATGACCTGG GCTACACACG TGCTACAATG GATGGTACAA CGAGTTGCGA GACCGCGAGG TCAAGCTAAT CTCTTAAAGC CATTCTCAGT TCGGACTGTA GGCTGCAACT CGCCTACACG AAGTCGGAAT CGCTAGTAAT CGCGGATCAG CACGCCGCGG TGAATACGTT CCCGGGCCTT GTACACACCG CCCGTCACAC CATGAGAGTT TGTAACACCC GAAGCCGGTG GCGTAACCCT TTTAGGGAGC GAGCCGTCTA AGGTGGGACA AATGATTA

[0084] Experimental Example 1: Anti-inflammatory effect analysis in mouse lung macrophages 1-1) Cytotoxicity evaluation of strains (LDH leakage and CCK-8 assay) The mouse alveolar macrophage cell line, MH-S, was obtained from the American Cell Line Corporation (ATCC, Manassas, VA, USA) and grown at a cell density of 5 x 10 5 The cells were suspended at a concentration of 100 μl per well and placed in a 96-well plate. Each well was treated with a different concentration of each sample 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.

[0085] [Table 4-1] [Table 4-2]

[0086] (1) LDH assay results The seven strains of lactic acid bacteria listed in Table 3 were treated with mouse MH-S pulmonary macrophages at concentrations of 1 to 1,000 μg / ml for 24 hours, and then an LDH assay was performed. None of the seven strains exhibited cytotoxicity. However, at a concentration of 1,000 μg / ml, the LDH levels of GCWB1001, GCWB1085, GCWB1176, and GCWB1156 strains were significantly reduced, confirming that all strains were non-cytotoxic.

[0087] (2)MTT assay results The seven strains of lactic acid bacteria listed in Table 4 were treated with mouse MH-S lung macrophages at concentrations of 1 to 1,000 μg / ml for 24 hours, and then an MTT assay was performed. None of the seven strains exhibited cytotoxicity at concentrations below 100 μg / ml. However, GCWB1085 strain exhibited cytotoxicity at a concentration of 100 μg / ml.

[0088] 1-2) Cytokine secretion measurement (TNF-alpha, TGF-beta) The MH-S cell line was 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 and 26 hours, respectively. The amount of cytokines (TNF-alpha, TGF-beta) secreted into the culture medium was measured using an ELISA kit (R&D system, USA). The results are shown in Table 5 below and Figure 1.

[0089] [Table 5]

[0090] (1) TNF-alpha measurement results As shown in Table 5, the secretion of cytokine TNF-alpha by LPS and DEP treatment in MH-S cells was reduced at almost all treatment concentrations by all strains, GCWB1001, GCWB1084, and GCWB1156. Therefore, the strain of the present invention reduced the secretion of TNF-alpha, an infectious cytokine, by fine dust in a concentration-dependent manner.

[0091] (2) TGF-β measurement results As shown in Figure 1, the fine dust-induced TGF-beta secretion was reduced when GCWB1001, GCWB1085, and GCWB1156 strains were treated at 1 μg / ml and 10 μg / ml.

[0092] 1-3) Nitric oxide (NO) measurement The mouse lung macrophage cell line NH-S cell line was cultured at a cell density of 5 × 10 5 The cells were suspended at 100 μl / ml and columned in 96-well plates. The seven bacterial strains listed in Table 6 were pretreated for 1 hour at concentrations of 1 μg / ml and 10 μg / ml, respectively, and then treated with 100 ng / ml LPS for 24 hours. 50 μl of the culture medium was then transferred to a 96-well plate and mixed with equal amounts of Griess reagent I (NED solution) and Griess reagent II (sulfanilamide solution). After incubation in the dark for 10 minutes, the cells were measured at 540 nm using a microplate reader within 30 minutes.

[0093] [Table 6]

[0094] The experimental results are shown in the graph in Figure 2. As can be seen from Table 6 and Figure 2, the GCWB1001, GCWB1085, and GCWB1156 strains of the present invention all significantly reduced the amount of NO produced by fine dust at a concentration of 1 μg / ml, but did not significantly reduce the amount of NO produced by fine dust at a concentration of 10 μg / ml.

[0095] 1-4) Reactive oxygen species (ROS) measurement The mouse lung macrophage cell line NH-S cell line was cultured at a cell density of 5 × 10 5 The seven bacterial strains listed in Table 6 were pretreated at 1 μg / ml and 10 μg / ml for 1 hour, then treated with 100 ng / ml LPS for 6 hours, washed with HBSS, and the culture medium was treated with DCF-DA (2',7'-Dichlorofluorescein diacetate) / HBSS at 25 μM per well. After incubation for 30 minutes, fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

[0096] The experimental results are shown in the graph in Figure 3. It was confirmed that the GCWB1001, GCWB1085 and GCWB1156 strains of the present invention all significantly reduced the amount of ROS generated by fine dust at a concentration of 1 μg / ml, as in the NO analysis.

[0097] 1-5) Activity measurement of inflammatory transcriptional regulators (iNOS-Luc, COX2-Luc, NF-kB-Luc; luciferase promoter activity analysis)

[0098] 1 × 10 MH‐S cells 6 After columning at 1000 cells / ml, the cells were transfected with plasmid vectors containing iNOS-Luc, COX2-Luc, and NF-kB-Luc using LipofectAMINE2000 reagent (Invitrogen, Carlsbad, CA). After 24 hours of treatment with the samples at different concentrations, the cells were harvested and luciferase activity was measured.

[0099] 4 to 6, treatment with GCWB1001, 1085, and 1156 strains at 1 μg / ml reduced the promoter activity of the inflammatory transcription factors iNOS, COX2, and NF-kB caused by fine dust. Overall, the bacterial strains of the present invention can suppress the expression of genes involved in inflammation by suppressing the promoter activity of the NF-kB transcription factor, which induces the expression of inflammation-related genes, and the iNOS and COX genes, which are known as inflammatory genes.

[0100] Experimental Example 2: Antitussive / expectorant efficacy study in animal models (in vivo assay) 2-1) Experimental and rearing conditions The experimental animals were 6-week-old male BALB / c mice (weight: 20±2g) supplied by Semtaco Co., Ltd. and kept in an animal breeding room with a temperature of 23±1°C, relative humidity of 55±15%, and illuminance of 300-500 lux, with a 12-hour light-dark cycle, for at least 7 days. After observing macroscopic symptoms, only healthy animals were used in the experiments. They were allowed free access to solid laboratory animal feed (Semtaco Co., Ltd.) and water. All animal experiments were approved by the Korea International University Animal Ethics Committee and conducted in accordance with the guidelines of the US National Institutes of Health (NIH publication No. 86-23, revised 1985).

[0101] 2-2) Antitussive efficacy research The experimental groups were a 1M citric acid only administration group (control), a citric acid + Synatura 200 mg / kg (positive control group), and a citric acid + sample treatment group (1 × 10 7 ), citric acid + sample treatment group (1 × 10 9 ) and each test group consisted of 8 animals (n=8) (Table 7).

[0102] [Table 7]

[0103] After oral administration of the sample for 1 hour, 1M citric acid, a cough inducer, was administered intranasally, and each experimental group was placed in a chamber to measure the number of coughs for 10 minutes. The results are shown in Figure 7. Both GCWB1001 and GCWB1085 strains had a cough rate of 1x10 9 After administration of cfu, the frequency of coughing caused by citric acid was significantly reduced.

[0104] 2-3) Study on expectorant efficacy The experimental groups were 1M phenol-red administration group, phenol-red + Synatura 200 mg / kg (positive control group), phenol-red + sample treatment group (1 × 10 7 ), citric acid + sample treatment group (1 × 10 9 ) and each test group consisted of 8 animals (n=8) (Table 8).

[0105] [Table 8]

[0106] 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 animals were sacrificed and their trachea were removed.

[0107] After weighing the excised organs, 0.5 ml of 0.9% saline (w / v) was added and vortexed. 100 μl of 1 M NaOH, a coloring substrate, was added, and the absorbance at 550 nm was measured.

[0108] As shown in Figure 8, both GCWB1001 and GCWB1085 strains were 1 × 10 9 After cfu administration, phenol red excretion increased significantly.

[0109] Overall, the strains of the present invention reduce the frequency of coughs and increase expectorant activity, thereby improving the symptoms of respiratory diseases.

[0110] 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 and rearing conditions Experimental animals were 6-week-old male BALB / c mice (weight: 20±2g) supplied by Semtaco Co., Ltd. and kept in an animal breeding room with a temperature of 23±1°C, relative humidity of 55±15%, and illuminance of 300-500 lux, with a 12-hour light-dark cycle, for at least 7 days. Only healthy animals were used in experiments after macroscopic observation. They were allowed free access to solid laboratory animal feed (Semtaco Co., Ltd.) and water. All animal experiments were approved by the Korea International University Animal Ethics Committee and conducted in accordance with the guidelines of the US National Institutes of Health (NIH publication No. 86-23, revised 1985).

[0111] 3-2) Establishment of an animal model of chronic respiratory disease and administration of experimental substances 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 on Day 1 and Day 12 (sensitization). On Days 19 and 20, 50 μg of OVA (challenge) was administered intranasally. After the final OVA administration (Day 20), 400 μg of diesel exhaust particles (DEP) were administered intranasally three times at 3-hour intervals. The experiment was terminated on Day 21.

[0112] 24 hours after the last sample administration, the mice were anesthetized by intraperitoneal administration of urethane (Sigma-Aldrich, UK, USA) (0.020 ml / g weight), and bronchoalveolar lavage fluid (BALF) was collected. The strain of the present invention was orally administered once daily from Day 0 to Day 20. Samples GCWB1001, GCWB1085, and GCWB1156 were each administered at 1 x 10 per mouse. 7 cfu, 1 × 10 9cfu were orally administered. Synatura (200 mg / kg) was used as a positive control, and the number of animals per experimental group was 8 (n=8) (Table 9).

[0113] [Table 9]

[0114] 3-3) Body weight and organ weight measurement Twenty-four hours after the last administration of the sample, the animals were anesthetized by intraperitoneal administration of urethane (Sigma-Aldrich, UK, USA) (0.020 ml / g weight), and after collecting bronchoalveolar lavage fluid (BALF), the lungs were excised and weighed. Body weight, liver weight, and spleen weight were also measured.

[0115] Body weight did not change in any group, and lung weight increased in the OVA and OVA+DEP treated groups compared with the untreated group. 9 cfu), GCWB1085 (1 × 10 9 cfu), GCWB1156 (1 × 10 9 cfu), and the weight of lung tissue increased by OVA+DEP treatment in the Synatura-treated group was significantly reduced. 7 cfu), GCWB1085 (1 × 10 9 cfu), GCWB1156 (1 × 10 9 The weights of spleen and lung tissues, which were increased by OVA+DEP treatment, were significantly reduced in the spleen and lung tissues.

[0116] 3-4) Evaluation of the improvement of pulmonary function by the present strain in an animal model of chronic respiratory disease - bronchoalveolar cell fluid analysis Twenty-four hours after the last sample administration, urethane (Sigma-Aldrich, UK, USA) was administered intraperitoneally (0.020 ml / g weight) to anesthetize the animals, and bronchoalveolar lavage fluid (BALF) was obtained. The immune cell profile (total cells, macrophages, eosinophils, neutrophils, and lymphocytes) was then measured in the BALF.

[0117] As shown in Figure 10, the number of all immune cells measured in the OVA-treated group was significantly increased compared to the normal group, and the number of immune cells, including the total number of cells, was also significantly increased in the OVA+DEP-treated group compared to the OVA-treated group. However, the number of immune cells increased by OVA+DEP treatment was significantly decreased in the GCWB1001, GCWB1085, GCWB1156, and Synatura-treated groups.

[0118] In particular, each strain was cultured at 1 × 10 9 When cfu was treated, the cell count of all the immune cells showed a decrease comparable to that of the positive control group, Synatura, with a decrease of 1 x 10 7 When cfu was treated, the number of cells other than lymphocytes (total cells, macrophages, eosinophils, neutrophils) was also effectively reduced.

[0119] Therefore, the strains of the present invention may reduce the infiltration of immune cells in lung tissue and thus prevent the progression of respiratory diseases.

[0120] 3-5) Evaluation of the improvement of pulmonary function by the present strain in an animal model of chronic respiratory disease - OVA-specific IgE measurement (BALF, serum) Twenty-four hours after the last sample administration, the animals were anesthetized by intraperitoneal administration of urethane (Sigma-Aldrich, UK, USA) (0.020 ml / g weight), and the bronchoalveolar lavage fluid (BALF) and serum were collected to measure the amount of OVA-specific IgE, an indicator of an allergic reaction, which is a type of inflammatory response.

[0121] As shown in Figure 11, OVA-specific IgE was significantly increased in BALF (Figure 11A) and serum (Figure 11B) of the OVA-treated group compared with the normal group, and OVA-specific IgE 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 OVA-specific IgE, which was increased by OVA+DEP treatment, was significantly reduced in the Synatura-treated group. Therefore, the strain of the present invention can reduce pulmonary and systemic allergic responses, which are indicators of inflammatory responses.

[0122] 3-6) Evaluation of the improvement of pulmonary function by the present strain in an animal model of chronic respiratory disease - Measurement of cytokines (TNF-alpha, IL-6, IL-1beta, IL-4, IL-13, MCP-1, IFN-gamma) in BALF Twenty-four hours after the last sample administration, the mice were anesthetized by intraperitoneal administration of urethane (Sigma-Aldrich, UK, USA) (0.020 ml / g weight), and cytokines (TNF-alpha, IL-6, IL-1beta, IL-4, IL-13, MCP-1, and IFN-gamma) in the BALF were measured using an ELISA kit (R&D system, USA).

[0123] TNF-alpha, IL-6, IL-1beta, IL-4, IL-13, and MCP-1 were significantly increased in the OVA-treated group compared with the normal group, and TNF-alpha, IL-6, IL-1beta, 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 TNF-alpha, IL-6, IL-1beta, IL-4, IL-13, and MCP-1, which were increased by OVA+DEP treatment, were significantly reduced in the Synatura-treated group (Figures 12A to 12D).

[0124] In addition, the amount of IFN-gamma was reduced in the OVA+DEP treatment group, and GCWB1001 (1 × 10 7 , 1×10 9 cfu), GCWB1085 (1 × 10 7 , 1×10 9 cfu), GCWB1156 (1 × 10 7 , 1×10 9 cfu), significantly increased in the Synatura-treated group (Fig. 12G).

[0125] Therefore, the strain of the present invention reduces infectious cytokines in the lungs and increases anti-inflammatory cytokines, thereby reducing the inflammatory response in lung tissue and preventing the progression of respiratory diseases.

[0126] 3-7) Evaluation of pulmonary function improvement by the present strain in an animal model of chronic respiratory disease - histological examination (H&E staining)

[0127] Left lobe lung tissue was fixed in 10% formalin for 24 hours, and paraffin blocks were prepared and cut at 4 μm thickness. H&E staining was performed. Images were obtained under a light microscope, and the degree of inflammation was measured.

[0128] Inflammatory cell infiltration was significantly increased in the OVA-treated group compared with the normal group, and in the OVA+DEP-treated group compared with the OVA-treated group.

[0129] The strain of the present invention, GCWB1001 (1 × 10 7 , 1×10 9 cfu), GCWB1085 (1 × 10 7 , 1×10 9 cfu), GCWB1156 (1 × 10 7 , 1×10 9 cfu), the infiltration of inflammatory cells, which was increased by OVA+DEP treatment, was significantly reduced in the Synatura-treated group (Fig. 13).

[0130] Therefore, the strain of the present invention prevents the penetration of inflammatory cells into lung tissue, thereby reducing the inflammatory response in lung tissue and preventing the progression of respiratory diseases.

[0131] 3-8) Evaluation of pulmonary function improvement by the present strain in an animal model of chronic respiratory disease - histological examination (alcian blue-PAS stain) To confirm whether the strain of the present invention actually inhibits the production of mucus proteins in lung tissue, lung tissue was stained with alcian blue-PAS staining method to stain glycogen and mucin.

[0132] Figure 14 shows the actual stained tissue photographs, showing that the production of mucus protein in the alveoli was significantly increased in the OVA-treated group compared to the normal group, and that the production of mucus protein was significantly increased in the OVA+DEP-treated group compared to 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 the production of mucus protein, which was increased by OVA+DEP treatment, was significantly reduced in the Synatura-treated group (Fig. 14).

[0133] Therefore, the strain of the present invention reduces mucus in the alveoli, thereby preventing respiratory diseases from worsening to pulmonary fibrosis and the like.

[0134] 3-9) Evaluation of lung function improvement by the present strain in an animal model of chronic respiratory disease - caspase 3 activity and total collagen content To confirm whether the strain of the present invention actually reduces apoptosis in lung tissue and improves lung damage, caspase 3 activity and total collagen content in lung tissue were measured.

[0135] As shown in Figure 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. 7 , 1×10 9 cfu), GCWB1085 (1 × 10 7 , 1×10 9 cfu), GCWB1156 (1 × 10 7 , 1×10 9 In both the OVA+DEP treatment group and the Synatura treatment group, caspase-3 activity and collagen content, which were increased by OVA+DEP treatment, were significantly reduced (Fig. 15).

[0136] Therefore, the strain of the present invention can not only reduce cell death factors but also reduce collagen content, thereby preventing pulmonary fibrosis, a worsening of respiratory diseases.

[0137] 3-10) Evaluation of the pulmonary function improvement of the present strain in an animal model of chronic respiratory disease - MMP9 activity analysis Proteins separated from bronchoalveolar lavage fluid (BALF) by 10% SDS-PAGE electrophoresis containing 0.2% gelatin were stained with 0.25% Coomassie Brilliant Blue G250 (Sigma Chemical Co., St. Louis, MO, USA) to measure MMP-9 enzyme activity.

[0138] As shown in Figure 16, MMP-9 activity was significantly increased in the OVA-treated group compared to the normal group, and in the OVA+DEP-treated group compared to the OVA-treated group. 9 cfu), GCWB1085 (1 × 10 9 cfu), GCWB1156 (1 × 10 9 cfu), and MMP-9 activity, which was increased by OVA+DEP treatment, was significantly reduced in the Synatura-treated group.

[0139] Therefore, the bacterial strain of the present invention can prevent the deposition of inflammatory cells in lung tissue, thereby preventing inflammation in lung tissue and various complications caused by inflammation.

Claims

1. A healthy functional food composition for preventing or improving respiratory diseases caused by fine dust, comprising the Lactobacillus plantarum GCWB1001 strain deposited under accession number KCCM12698P, one selected from the group consisting of a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product, and a crushed product of the strain, or any one of a culture of the strain, a concentrate, a paste, and a dilution of the culture.

2. The healthy functional food composition according to claim 1, wherein the Lactobacillus plantarum GCWB1001 strain contains 16S rRNA consisting of the base sequence of SEQ ID NO:

1.

3. The health functional food composition according to claim 1, further comprising one or more selected from the group consisting of proteins, carbohydrates, fats, nutrients, seasonings, sweeteners and flavoring agents.

4. 4. The health functional food composition according to claim 3, wherein the health functional food composition is any one selected from the group consisting of granules, lemonades, powders, syrups, liquids and solutions, extracts, elixirs, fluid extracts, suspensions, decoctions, infusions, tablets, spirits, capsules, troches, pills, and soft or hard gelatin capsules.

5. A pharmaceutical composition for treating or preventing respiratory diseases caused by fine dust, comprising the Lactobacillus plantarum GCWB1001 strain deposited under accession number KCCM12698P, one selected from spray-dried, freeze-dried, vacuum-dried, drum-dried or crushed forms of said strain, or one of a culture of said strain, a concentrate, a paste or a dilution of said culture.

6. The pharmaceutical composition of claim 5, further comprising a pharmaceutically acceptable excipient or carrier.

7. 6. The pharmaceutical composition according to claim 5, wherein the respiratory disease is at least one selected from the group consisting of respiratory inflammatory lung disease, asthma, bronchiectasis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, emphysema, sequelae of pulmonary 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.

8. A live bacteria-containing composition for preventing or ameliorating respiratory diseases caused by fine dust, comprising the Lactobacillus plantarum GCWB1001 strain deposited under accession number KCCM12698P.

9. The composition is 1×10 １ ~1 x 10 １３ The viable bacteria-containing composition according to claim 8, having a viable bacteria content of CFU / g.

10. The live bacteria-containing composition according to claim 8, wherein the composition has an antitussive, expectorant or cough-reducing effect.

11. The live bacteria-containing composition of claim 8, wherein the composition reduces inflammatory responses in lung tissue.

12. The live bacteria-containing composition of claim 8, wherein the composition reduces pulmonary or systemic allergic reactions.

13. The live bacteria-containing composition according to claim 8, wherein the composition inhibits pulmonary fibrosis.

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