KR102264826B1 - a composition comprising a combination consisting of an extract of Veronicastrum sibiricum L. Pennell and Lactobacillus plantarum KC3 as an active ingredient for preventing or treating immune disorders, respiratory organ disease, allergy or asthma - Google Patents

Abstract

The present invention relates to a composition for preventing or treating immune disorder, respiratory disease, allergy or asthma comprising novel Lactobacillus plantarum KC3 strain (also referred to as CKDB-KC3) as an active ingredient. Through an experiment (experimental example 1) on a defense effect against respiratory damage caused by air pollutants such as fine dust, etc., with regard to the combination according to the present invention, an experiment (experimental example 2) on a therapeutic effect on chronic obstructive pulmonary disease (COPD) in blood, and the like, it was confirmed that administering the combination of the present invention exhibits a more synergistic effect on bronchial inflammation inhibition or treatment than a respiratory inflammatory disease treatment effect by a separate administration of each of veronicastrum sibiricum L. pennell extract and the novel Lactobacillus plantarum KC3 strain. Accordingly, the composition is useful as a pharmaceutical composition, a health functional food or a health supplement food for preventing or treating immune disorder, respiratory disease, allergy or asthma.

Description

A composition for the prevention and treatment of immune disorders, respiratory diseases, allergies or asthma, comprising a combination consisting of a cold herb extract and a novel Lactobacillus plantarum KC3 strain as an active ingredient {a composition comprising a combination consisting of an extract of Veronicastrum sibiricum L. Pennell and Lactobacillus plantarum KC3 as an active ingredient for preventing or treating immune disorders, respiratory organ disease, allergy or asthma}

The present invention relates to a composition for the prevention and treatment of immune disorders, respiratory diseases, allergies or asthma, comprising a combination consisting of a cold herb extract and a novel Lactobacillus plantarum KC3 strain as an active ingredient.

[Document 1] Minoguchi K and Adachi M. Pathophysiology of asthma. In: Cherniack NS, Altose MD, Homma I, editors. Rehabilitation of the patient with respiratory disease. New York: McGraw-Hill, 1999, pp97-104

[Document 2] Elias JA, et al., J. Clin. Invest., 111, pp291-297, 2003

[Document 3] Maggi E., Immunotechnology, 3, pp233-244, 1998;

[Document 4] Pawankar R., Curr. Opin. Allergy Clin. Immunol., 1, pp3-6, 2001

[Document 5] Barnes PJ, et al., Pharmacol Rev., 50, pp515-596, 1998

[Document 6] Hele DJ, Belvisi MG. 2003. Novel therapies for the treatment of inflammatory airway diseases. Expert Opin Investig Drugs 12: 5-18

[Document 7] Fox JC, Fitzgerald MF. 2009. The role of animal models in the pharmacological evaluation of emerging anti-inflammatory agents for the treatment of COPD. Curr Opin Pharmacol. 9: 231-242

[Document 8] Barnes PJ. 2000. Mechanisms in COPD: differences from asthma. Chest 117: 10S-14S

[Document 9] Seatta M, Turato G, Maestrelli P, Mapp CE, Fabbri LM. 2001. Cellular and structural base of chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 163: 1304-1309

[Document 10] Data from the National Biodiversity Information Sharing System of the National Center for Biodiversity, http://www.kbr.go.kr/home/rsc/rsc01002v.do?data_gbn_cd=BIO&ktsn_no=120000063371&menuKey=448).

[Document 11] Korean Patent Laid-Open No. 10-2006-0125489

[Document 12] Wenyuan GAO, et al., Chem. Pharm. Bull. 52(1) 136?137 (2004)

[Document 13] Korean Patent Publication No. 10-2013-046897

[Document 14] Korean Patent Registration No. 10-2011883

[Document 15] Korean Patent Publication No. 10-2018-0044245

[Document 16] Korean Patent Registration No. 10-1956867

[Document 17] Korean Patent Registration No. 10-2135879

[Document 18] Korean Patent Registration No. 10-1794567

[Document 19] Korean Patent Registration No. 10-1770766

[Document 20] Korean Patent Publication No. 10-2016-0021038

[Document 21] Korean Patent No. 10-2094338

[Document 22] PCT/KR2019/005466

[Document 23] Korean Patent Registration No. 10-1940042

[Document 24] PCT/WO2018/208094 A2

[Document 25] Korean Patent Registration No. 10-1940042

[Document 26] PCT/WO2018/208094 A2

[Document 27] Lim et al., Free Radic Biol Med. 25(6), 635-644. (1998)

[Document 28] Shin et al., Korean J. Medicinal Crop Sci 27(3), 218-231. (2019)

[Document 29] Schins et al., Toxicol Appl Pharmacol. 195(1), 1-11 (2004)

[Document 30] Smith et al., Toxicol Sci, 93(2), 390-399 (2006)

[Document 31] Takano et al., Am J Respir Crit Care Med, 156(1), 36-42. (1997)

[Document 32] Brandt EB et al., J. Allergy Clin. Immunol., 132(5):1194-1204, (2013)

[Document 33] Jeremy AS et al., Int. J. Mol. Sci., 15:6062-6071, (2014).

The present invention relates to a composition for the prevention and treatment of immune disorders, respiratory diseases, allergies or asthma, comprising a combination consisting of a cold herb extract and a novel Lactobacillus plantarum KC3 strain as an active ingredient.

In general, the inflammatory reaction is a defense reaction process of the living body that tries to repair and regenerate the damaged area when an invasion that causes any organic change is applied to the cells or tissues of the living body. Therefore, this series of reactions includes local blood vessels, various tissue cells of body fluids, and immune-related cells. Recently, with the development of molecular biology, understanding of inflammatory diseases at the molecular level called cytokines is being attempted, and factors affecting these diseases are being identified one by one.

Allergic reactions can be classified into four types: type I, type II, type III and type IV according to the type of reaction, or type I, II according to the time until onset after resensitization with an antigen Types and type III allergies are called immediate allergies, and type IV allergies can be classified as delayed type allergies.

Among these, type I allergy is a reaction involving the IgE antibody and is called anaphylactic type allergy, which includes bronchial asthma, atopic diseases (dermatitis, enteritis, etc.), allergic rhinitis such as hay fever, allergic conjunctivitis, food allergy, etc. This is included.

Asthma is a disease characterized by hypersensitivity of the airways to various stimuli. Clinical symptoms such as wheezing, dyspnea, and cough caused by extensive stenosis of the airways are reversibly improved spontaneously or by treatment. can be Most asthma is allergic and is characterized by chronic airway inflammation and bronchial hyperresponsiveness (Minoguchi K and Adachi M. Pathophysiology of asthma. In: Cherniack NS, Altose MD, Homma I, editors) Rehabilitation of the patient with respiratory disease (New York: McGraw-Hill, 1999, pp97-104).

Asthma can be divided into extrinsic asthma and intrinsic asthma according to the cause. Exogenous asthma refers to asthma in which symptoms appear when exposed to a causative antigen. Skin tests or bronchial provocation tests for the causative antigen show a positive result, and the age of onset is generally young. House dust and mites are the most common causative antigens, while pollen, animal epithelium, mold, etc. act as causative antigens. In the case of endogenous asthma, upper respiratory tract infection, exercise, emotional instability, and changes in cold climate and humidity cause or exacerbate asthma, which is commonly seen in adult asthma. Others include drug-induced asthma, exercise-induced asthma, and occupational asthma.

Asthma is also recognized as a chronic inflammatory disease because inflammatory cells proliferate, differentiate, and activate by interleukin-4, 5, and 13 generated by TH2 (T helper 2) type immune cells and migrate and infiltrate into the airways and surrounding tissues. (Elias JA, et al., J. Clin. Invest., 111, pp291-297, 2003). Inflammatory cells such as eosinophils, mast cells, and alveolar macrophages activated in the bronchi of patients with asthma secrete various inflammatory mediators (cysteine leukotrienes, prostaglandins, etc.) and are involved in strong bronchial contraction (Maggi E., Immunotechnology, 3, pp233-244, 1998; Pawankar R., Curr. Opin. Allergy Clin. Immunol., 1, pp3-6, 2001; Barnes PJ, et al., Pharmacol Rev., 50, pp515-596, 1998 ).

Therefore, the production of cytokines such as IL-4, IL-5, IL-13, and immunoglobulin E, which are involved in the activation of inflammatory cells, and the biosynthesis of cysteine leukotriene secreted from inflammatory cells such as eosinophils due to their action, are responsible for inflammatory and allergic reactions and thus Because it is a major cause of asthma, many studies are being conducted to develop drugs to inhibit their production.

In addition, chronic obstructive pulmonary disease should be appropriately treated separately from asthma, which is characterized by reversible airflow obstruction and allergic bronchial inflammatory response, but current treatments only provide symptomatic relief, and recent treatments None of these have demonstrated clinical outcomes for the underlying therapeutic effect of chronic obstructive pulmonary disease (Hele DJ, Belvisi MG. 2003. Novel therapies for the treatment of inflammatory airway diseases. Expert Opin Investig Drugs 12: 5-18; Fox JC. , Fitzgerald MF. 2009. The role of animal models in the pharmacological evaluation of emerging anti-inflammatory agents for the treatment of COPD. Curr Opin Pharmacol. 9: 231-242)

Asthma and COPD show different pathological mechanisms in principle, for example, (1) in terms of inflammatory cells, asthma has mast cells, eosinophils, CD4+ cells (Th2), macrophages, etc. Whereas COPD mainly acts on neutrophils, CD8+ cells (Tc), etc. are different; (2) In terms of inflammatory mediators, asthma is leukotriene B, histamine, IL-4, IL-5, IL-13, eotaxin, RANTES, oxidative stress, etc. Whereas COPD is mainly involved, TNF-alpha, IL-8, GRO-alpha, etc. are mainly involved; (3) In terms of inflammation, asthma acts on the entire airway, with AHR (airway hyperresponsiveness), epithelial shedding, fibrosis, no parenchymal involvement, and mucus While muscus secretion, relatively reversible airflow disturbance, coughing, sneezing, and dyspnea mainly occur in childhood, COPD acts on the peripheral airways, leading to epithelial metaplasia and tissue parenchyma. It is known to have distinct pathological mechanisms such as parenchymal destruction, relatively irreversible airflow obstruction, chronic bronchitis, and emphysema, which mainly occur in adulthood (Barnes PJ. 2000. Mechanisms in COPD: differences from Asthma Chest 117: 10S-14S; Seatta M, Turato G, Maestrelli P, Mapp CE, Fabbri LM. 2001. Cellular and structural base of chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 163: 1304-1309).

On the other hand, Veronicastrum sibiricum L. Pennell is a plant of the genus Veronicastrum, and unlike plants of the genus Cauliflower, which are native to all parts of Korea, the Russian Far East, Japan, and northeastern China, the leaves are rotated and the flowers have a goblet shape. It is distinguished from Veronica genus plants of Veronica longifolia in that only the tip is shallowly divided into four parts in a cylindrical shape, and is also called Sumwinamul or Siberian cold herb. (Data from the National Biodiversity Information Sharing System of the National Center for Biodiversity, http://www.kbr.go.kr/home/rsc/rsc01002v.do?data_gbn_cd=BIO&ktsn_no=120000063371&menuKey=448).

Pharmaceutical composition containing a plant extract of the genus Fenugreek having anti-inflammatory, anti-allergic and anti-asthmatic activity (Korean Patent Application Laid-Open No. 10-2006-0125489); Immunosuppressive activity of diperpene compounds such as sibiriquinone A and B isolated from Veronicastrum sibiricum (Wenyuan GAO, et al., Chem. Pharm. Bull. 52(1) 136-137 (2004) )); Prior literatures such as a composition for treating inflammatory diseases containing Lactobacillus plantarium DRS CK10 or Lactobacillus plantarium DRS M2 as an active ingredient (Korean Patent Application Laid-Open No. 10-2013-046897) have been disclosed.

So far, the present inventors have focused on the development of therapeutic agents using various resources, especially natural resources including lactic acid strains whose safety and efficacy are already known, as therapeutic agents using antibodies to various types of cytokines and chemokines characteristic of respiratory inflammatory diseases. It has been developed as

The present inventors Lactobacillus plantarum KC3 strain having an anti-obesity effect and its use (Korea Patent Registration No. 10-2011883); A novel lactic acid bacterium having an effect of improving constipation and its use (Korean Patent Publication No. 10-2018-0044245); Lactobacillus rhamnosus strain CKDB009 having cholesterol lowering and obesity symptom alleviation effects and uses thereof (Korean Patent Registration No. 10-1956867); A composition for preventing or treating immune disorders, respiratory inflammatory diseases, allergies and asthma using a novel Lactobacillus plantarum KC3 strain and uses thereof (Korean Patent Registration No. 10-2135879); In addition, a composition for the prevention or treatment of respiratory inflammatory diseases comprising a specific crude extract or a purified fraction separated therefrom as an active ingredient (Korean Patent Registration No. 10-1794567); A composition for the prevention or treatment of respiratory inflammatory diseases comprising a specific extract of motherwort as an active ingredient (Korean Patent Registration No. 10-1770766); A composition for the prevention or treatment of respiratory inflammatory diseases comprising a specific extract of arborvitae or a compound isolated therefrom as an active ingredient (Korean Patent Application Laid-Open No. 10-2016-0021038); A composition for the prevention or treatment of inflammation, allergy and asthma comprising a cold herb extract as an active ingredient and use thereof (Korean Patent No. 10-2094338 = PCT/KR2019/005466); Inventions on a composition for the prevention and treatment of respiratory inflammatory diseases (Korean Patent Registration No. 10-1940042 = PCT/WO2018/208094 A2) containing a combination extract composed of cold herb and red ginseng as an active ingredient have been continuously performed.

However, there is no disclosure or teaching in any of the above documents for improving immune disorders and treating respiratory diseases including a combination consisting of a novel Lactobacillus plantarum KC3 strain isolated from Korean kimchi and a cold herb extract as an active ingredient.

Accordingly, the present inventors searched for a therapeutic agent using natural resources effective for respiratory inflammatory diseases and searched for a new combination. As a result, a combination of a novel Lactobacillus plantarum KC3 strain and cold extract extract was targeted for respiratory damage caused by air pollutants such as fine dust. defense effect test against (Experimental Example 1); When administering the combination of the present invention through the chronic obstructive pulmonary disease (COPD) treatment effect test in blood (Experimental Example 2), etc., the treatment effect of respiratory inflammatory diseases caused by the administration of individual cold herb extracts and novel Lactobacillus plantarum KC3 strains By confirming that it exhibits a synergistic bronchial inflammation inhibition or therapeutic effect, the present invention was completed.

An object of the present invention is that the present invention is Lactobacillus plantarum KC3 strain (also referred to as "CKDB-KC3", accession number:: KCTC13375BP), a culture thereof, at least one selected from the group consisting of a concentrate or a dry product of the culture, and It is to provide a pharmaceutical composition for the prevention or treatment of immune disorders, respiratory diseases, allergies or asthma comprising a combination consisting of a cold herb extract as an active ingredient.

In addition, the present invention is another aspect, the present invention is Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number:: KCTC13375BP), its culture, a group consisting of a concentrate or dried product of its culture It is to provide a health functional food for the prevention or improvement of immune disorders, respiratory diseases, allergies or asthma comprising a combination consisting of one or more selected from and cold herb extract as an active ingredient.

In addition, the present invention is another aspect, the present invention is Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number:: KCTC13375BP), its culture, a group consisting of a concentrate or dried product of its culture It is to provide a health supplement for the prevention or improvement of immune disorders, respiratory diseases, allergies or asthma comprising a combination consisting of one or more selected from and cold herb extract as an active ingredient.

In addition, the present invention is another aspect, the present invention is Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number:: KCTC13375BP), its culture, a group consisting of a concentrate or dried product of its culture It is to provide a probiotic preparation for the prevention or treatment of immune disorders, respiratory diseases, allergies or asthma comprising a combination consisting of one or more selected from and cold herb extract as an active ingredient.

As an aspect for achieving the above object, the present invention is Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number:: KCTC13375BP), its culture, consisting of a concentrate or a dry product of its culture It provides a pharmaceutical composition for the prevention and treatment of immune disorders, respiratory diseases, allergies or asthma comprising a combination consisting of one or more selected from the group and cold extract extract as an active ingredient.

In addition, the present invention is Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number:: KCTC13375BP), its culture, one or more selected from the group consisting of a concentrate or dried product of its culture and a cold herb extract It provides a health functional food for the prevention and improvement of immune disorders, respiratory diseases, allergies or asthma, comprising the composed combination as an active ingredient.

Lactobacillus plantarum KC3 strain as defined herein (also referred to as “CKDB-KC3”, accession number: KCTC13375BP), a culture thereof, and at least one selected from the group consisting of a concentrate or a dry product of the culture, and a cold herb extract. The combination is preferably in a dry weight relative blending ratio of 1: 0.01 to 100 (w/w) by weight, preferably 1: 0.5 to 50 (w/w) by weight, more preferably, 1: 0.1 to 10 (w/w) blended parts by weight, even more preferably 1: 0.3 to 5 (w/w) blended parts by weight, most preferably 1: 1 to 3 (w/w) Combinations that are parts by weight of the blend are included.

The Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”) as defined herein does not produce biogenic amines from one or more amino acid precursors selected from the group consisting of tyrosine, histidine, ornithine and lysine. do.

The Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”) as defined herein is a Lactobacillus plantarum KC3 strain comprising the nucleotide sequence set forth in SEQ ID NO: 1 as 16s rRNA (also referred to as “CKDB-KC3”) , Accession number: KCTC13375BP).

As one embodiment of the present invention, the present invention provides a combination consisting of a Lactobacillus plantarum KC3 strain, a novel lactic acid bacterium isolated from kimchi, and a cold herb extract, which has anti-inflammatory, particularly, anti-inflammatory, immune disorder improvement and respiratory disease treatment efficacy do.

"Immune disorder" as defined herein is a disease caused by a decrease in immune function by an anticancer therapy such as chemotherapy and radiotherapy or a decrease in immunity after bone marrow transplantation, AIDS due to immune system damage, cancer diseases caused by a decrease in immune function, etc. Immunodeficiency, preferably a disease caused by a decrease in immune function due to chemotherapy such as chemotherapy and radiotherapy or immunosuppression after bone marrow transplantation, more preferably, bacterial/viral infection in the elderly, chronic respiratory infection, chronic These include urinary tract infections, bedsores, flu, pneumonia, chickenpox in children, measles, rash, hand, foot and mouth disease, rubella, or Crohn's disease.

As defined herein, "respiratory diseases" are diseases of respiratory organs such as external nasal passages, nasal passages, pharynx, trachea, bronchial tubes, and lungs, specifically, rhinitis, otitis media, pharyngitis, It may be any one respiratory inflammatory disease selected from the group consisting of tonsillitis, pneumonia, asthma and chronic obstructive pulmonary disease, and more specifically, rhinitis caused by air pollutants or fine dust, otitis media, sore throat, tonsillitis, pneumonia, asthma and It includes any one respiratory inflammatory disease selected from the group consisting of chronic obstructive pulmonary disease, but is not limited thereto.

As defined herein, “culture of strain, culture concentrate and dried product of strain” is a term commonly used in the art, but is not limited thereto, but “contains the Lactobacillus plantarum KC3 strain of the present invention as an active ingredient” It includes a culture of a variety of strains, a culture concentrate and a dried product of a strain concentrated thereon.

The lysate may refer to a product obtained by crushing the strain itself by chemical or physical force.

The culture may refer to a material including some or all materials contained in the culture medium regardless of the form of the culture, for example, a material containing a metabolite or secretion resulting from the culture of the strain, or a lysate thereof may mean, and the strain itself may also be included in the culture. In addition, the culture may mean including a fermented product.

The extract may refer to a product obtained by extracting the strain itself, the lysate of the strain, the culture of the strain, or a mixture thereof regardless of the extraction method, the extraction solvent, the extracted component or the form of the extract, and fractionation, concentration after extraction It is a broad concept that includes all substances that can be obtained by processing or processing by other methods, such as.

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

The strain of the present invention can be obtained by the following preparation method.

For example, but not limited thereto, the microbial isolation and identification process for the Lactobacillus plantarum CKDB-CK3 strain of the present invention can be obtained by the following method.

The new Lactobacillus plantarum KC3 strain of the present invention (also referred to as “CKDB-KC3”) is kimchi of different regions and types in Korea, preferably commercially ^{available kimchi such as Jonggajip ®} , CJ bibigo ^® , or Gyeongsangbuk-do. , Chungcheongbuk-do, Gyeonggi-do, Jeollabuk-do, Jeollanam-do, etc., domestic Korean kimchi produced in restaurants, homes, temples, etc., more preferably, kimchi produced in Jeonju, Jeollabuk-do, more preferably, but not limited thereto, (a ) Prepare the Jeollabuk-do Chinese cabbage, remove the inedible part, cut it in half, and put 1/5 to 1/30 weight part (w/w) of the weight of the cabbage in a pickle container, preferably, 1/10 to 1/20 Dissolve salt in an amount of (w/w) by weight in water to soak Chinese cabbage, take it out, add salt one by one in cabbage leaves, pickle it for 3 to 8 hours, preferably, for about 5 to 6 hours, then pickle it 2 to 12 times, preferably A first step of washing about 3 to 5 times and drying it through a sieve; (b) 1/2 to 1/10 parts by weight (w/w), preferably, 1/3 to 1/6 parts by weight (w/w) of the weight of Chinese cabbage, prepare radish, remove radish, trim and wash After 3 to 6 cm, preferably, about 4 to 5 cm long shredded, each 1/5 to 1/30 parts by weight (w/w) of the weight of Chinese cabbage, preferably, 1/10 to 1/20 parts by weight ( w/w) the second step of trimming and washing sheep's green onions, chives, and caps, and then cutting them to a length similar to that of radishes; (c) 1/50 to 1/300 parts by weight (w/w) of the weight of Chinese cabbage, preferably, 1/80 to 1/120 parts by weight (w/w) of garlic, 1/100 to 1/100 of the weight of Chinese cabbage 1/1000 parts by weight (w/w), preferably 1/300 to 1/600 parts by weight (w/w), 1/10 to 1/100 parts by weight (w/w) of ginger and Chinese cabbage , Preferably, chopped shrimp in an amount of 1/20 to 1/40 parts by weight (w/w), 1/10 to 1/100 parts by weight (w/w), preferably, 1 A third step of preparing anchovy fish sauce in an amount of /20 to 1/40 parts by weight (w/w); (d) 1/10 to 1/200 parts by weight (w/w), preferably, 1/20 to 1/80 parts by weight (w/w) of the weight of Chinese cabbage, soaking glutinous rice in an amount and cooling the porridge, In the cooled glutinous rice porridge, 1/2 to 1/40 parts by weight (w/w), preferably 1/5 to 1/10 the weight of the salted shrimp, anchovy sauce, garlic, ginger and Chinese cabbage prepared in step 3 a fourth step of adding a part (w/w) of red pepper powder and tossing it evenly; (e) 1/2 to 1/40 parts by weight (w/w) of the weight of Chinese cabbage (w/w), preferably 1/5 to 1 1/2 to 1/40 parts by weight (w/w), preferably 1/5 to 1/10 parts by weight (w/w) of salt and Chinese cabbage in an amount of /10 parts by weight (w/w) a fifth step of preparing kimchi seasoning by seasoning with sugar; (f) Spread the kimchi seasoning evenly between the cabbage leaves and wrap it around the outer leaf so that the cut section of the cabbage is up and put it in a container step by step to maintain a temperature of 10 to -10℃, preferably 0 to -2℃ A first step of obtaining a raw material for kimchi seasoning prepared in a sixth step process of producing a raw material for cabbage kimchi by storing it in a low-temperature warehouse and aging it for 3 months to 5 years, preferably for 6 months to 2 years; The kimchi seasoning raw material is diluted with MRS medium, preferably peptone diluent, and a certain amount is inoculated into a modified MRS medium to which Bromcresol purple and sodium azide are added by flat plating. A second step of obtaining the inoculated medium; The inoculated medium of step 2 is cultured at 27 ° C. to 47 ° C., preferably 32 ° C. to 39 ° C. for 12 hours to 72 hours, preferably for 26 hours to 52 hours, more preferably for 33 hours to 46 hours. Third step to obtain colonies: In the modified MRS medium to which Bromcresol purple and sodium azide are added by diluting the colonies of step 3 with MRS medium, preferably peptone diluent. A fourth step of obtaining a colony that has turned yellow by separation of pure water; a fifth step of selecting the colony of step 4 as provisional lactic acid bacteria; An agent for purely isolating the Lactobacillus plantarum KC3 novel strain of the present invention, characterized in that it has the following characteristics by aerobic culture after plating the tentative strain selected in step 5 on MRS medium, preferably electro-modified MRS medium It is obtainable in a six-step process.

As a result of identifying the purely isolated strain by the above manufacturing process, it was confirmed that the strain was a Gram-positive bacillus, grew well regardless of the presence of oxygen and was negative for catalase and motility, and did not grow at 15°C and 45°C, It was confirmed that it belongs to the genus of Lactobacillus because it does not produce ammonia from gas and arginine from glucose.

In addition, the nucleotide sequence (SEQ ID NO: 1) obtained by collecting colonies grown in MRS medium and performing double-stranded DNA sequencing (Solgent, Korea) was searched with BLAST to identify the strain. It was confirmed that the novel microorganism of the present invention was a strain belonging to the Lactobacillus plantarum species by showing 99% homology with Lantarum.

The novel Lactobacillus plantarum KC3 (hereinafter also referred to as “CKDB-KC3”) according to the present invention is characterized by exhibiting the following characteristics:

(1) Form of bacteria: Form of bacteria when cultured on MRS agar plate at 37°C for 48 hours

①Cell type: Bacillus

② Mobility: None

③Spore-forming ability: None

④ Gram staining: positive

(2) Form of colony: Form of colony when cultured on MRS agar plate at 37°C for 48 hours

①Shape: Round

② ridge: convex

③Surface: smooth

④Color: milky white

(3) physiological properties

①Growth temperature

- Able to grow Growth temperature: 15~40℃

- Optimal growth temperature: 36~38℃

②Growth pH

- Growable Growth pH: 4.6~7.5

- Optimal pH: 6.0~7.0

③ Effect on oxygen: facultative anaerobic

(4) catalase: negative

(5) Gas generation: negative

(6) Indole production: negative

(7) lactic acid production: positive

(8) biogenic amine production: negative

A novel strain isolated from kimchi based on the microbial identification result and bacterial characteristics as described above was newly named Lactobacillus plantarum KC3 (also referred to as “CKDB-KC3”), and the existing Korean Patent Registration No. 10-2011883 As described in No., it was deposited with the Korea Research Institute of Bioscience and Biotechnology (Accession No.: KCTC13375BP) on October 20, 2017.

In addition, it is also possible to perform the conventional conventional culture thereof in the art, the concentrate and the dried process of the culture (Korean Patent Registration No. 10-1605516, increase the viability, storage stability, acid resistance or bile resistance of lactic acid bacteria) Refer to the invention of how to make

The extract as defined herein is water including purified water, alcohol, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably water, or a mixed solvent of water and a lower alcohol having 1 to 4 carbon atoms, more preferably water or extracts soluble in 10 to 100% (v/v) ethanol or spirits, even more preferably in water or 20 to 80% (v/v) ethanol or spirits.

The composition containing the extract of the present invention as an active ingredient contains 0.1 to 50% by weight of the extract based on the total weight of the composition.

As used herein, the term "prevention" refers to any action that suppresses or delays inflammation, allergy, or asthma by administration of a composition containing the extract.

In addition, the term "treatment" used in the present invention means any action in which the symptoms of a disease are improved or beneficially changed by administration of a composition containing the extract.

In addition, the combination extract of the present invention can be prepared as follows.

After washing and shredding the dried cold vinegar material, 1 to 20 times the weight of the dried material, preferably water containing about 4 to 8 times the volume of purified water, methanol, ethanol, butanol, etc. lower alcohols having 1 to 4 carbon atoms, After mixing several times with a solvent selected from alcohol or a mixture thereof, preferably a mixed solvent of water and ethanol, at 30 to 150° C., preferably 80 to 120° C. for 1 hour to 48 hours, preferably 8 hours to 14 hours a first step of repeatedly performing ultrasonic extraction, hot water extraction, room temperature extraction or reflux extraction, preferably reflux extraction, about 1 to 20 times, preferably 2 to 10 times; a second step of filtration, concentration under reduced pressure, and drying of the extract obtained in step 1 to obtain a dry cold extract extract; At least one selected from the group consisting of a Lactobacillus plantarum CKDB-CK3 strain, a culture thereof, a concentrate or a dried product of the Lactobacillus plantarum CKDB-CK3 strain prepared by the method disclosed herein in the dry state of the second step, and a dry weight The relative mixing ratio is 1: 0.01 to 100 (w/w) by weight, preferably 1: 0.5 to 50 (w/w) by weight, more preferably 1: 0.1 to 10 (w/w). w), more preferably 1: 0.3 to 5 (w/w), most preferably 1: 1 to 3 (w/w) to obtain a combination. Lactobacillus plantarum KC3 strain of the present invention through a three-step process (also referred to as “CKDB-KC3”, accession number:: KCTC13375BP), its culture, at least one selected from the group consisting of a concentrate or dried product of its culture, and Combinations consisting of cold herb extracts are obtainable.

The composition containing the combination of the present invention as an active ingredient contains 0.1 to 50% by weight of the combination based on the total weight of the composition.

Experimental Example 1: Defense effect test against respiratory damage caused by air pollutants such as fine dust (Experimental Example 1) for the combination prepared by the above manufacturing method; Experimental Example 2. Respiratory inflammation caused by administration of individual cold extracts and novel Lactobacillus plantarum KC3 strains when administering the combination of the present invention through a chronic obstructive pulmonary disease (COPD) treatment effect experiment in blood (Experimental Example 2), etc. By confirming that it has a synergistic inhibitory or therapeutic effect on bronchial inflammation rather than a disease therapeutic effect, it can be usefully used for the prevention or treatment of immune disorders, respiratory diseases, allergies or asthma by confirming a safe and strong therapeutic effect on respiratory inflammatory diseases.

Therefore, the present invention consists of the above manufacturing method and the Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number: KCTC13375BP), its culture, and a concentrate or dried product of the Lactobacillus plantarum KC3 strain obtained by the above manufacturing method. It provides a pharmaceutical composition and health functional food for the prevention or treatment of immune disorders, respiratory diseases, allergies or asthma containing a combination consisting of one or more selected from the group and cold extract extract as an active ingredient.

In addition, cold vinegar is a drug that has been eaten or used as a herbal medicine for a long time, and the Lactobacillus plantarum KC3 strain is a lactic acid strain isolated from kimchi, and the combination of the present invention does not have problems such as toxicity and side effects.

As another aspect, the Lactobacillus plantarum KC3 strain according to the present invention (also referred to as “CKDB-KC3”, accession number: KCTC13375BP), its culture, one selected from the group consisting of a concentrate or a dried product of its culture It provides a treatment method for treating an immune disorder, respiratory disease, allergy or asthma disease, comprising administering a combination consisting of the above and cold extracts to a patient with an immune disorder, respiratory disease, allergy or asthma.

As another aspect, the Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, Accession No.: KCTC13375BP) for the manufacture of a therapeutic agent for treating an immune disorder, respiratory disease, allergy or asthma disease patient, culture thereof It provides the use of a combination consisting of at least one selected from the group consisting of water, a concentrate or a dried product of a culture thereof, and a cold herb extract.

The pharmaceutical composition comprising the combination according to the present invention is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to a conventional method. can be used in combination. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulating, it is prepared by using diluents or excipients such as fillers, weights, binders, wetting agents, sealants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and fraction, for example, starch, calcium carbonate, sucrose (sucrose) or lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.

Non-aqueous solvents, suspending agents, propylene glycol (propylene glycol), polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate may be used in the above formulation. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition comprising the combination of the present invention varies depending on the patient's condition and weight, the degree of disease, the drug form, the route of administration, and the duration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the pharmaceutical composition comprising the combination of the present invention is preferably administered at 0.0001 to 100 mg/kg per day, preferably at 0.001 to 100 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

The combination of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The pharmaceutical composition of the present invention comprises 0.1 to 50% by weight of the above combination based on the total weight of the composition.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. Non-aqueous solvents and suspending agents include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, and glycerogelatin may be used.

The preferred dosage of the combination of the present invention varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the combination of the present invention may be administered in an amount of 0.0001 to 100 mg/kg, preferably 0.001 to 100 mg/kg, divided once or several times a day. In the composition, the combination of the present invention may be formulated in an amount of 0.0001 to 50% by weight based on the total weight of the total composition.

The pharmaceutical composition of the present invention may be administered to mammals such as mice, mice, livestock, and humans by various routes. All modes of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural and intracerebroventricular injections.

As another aspect, the present invention provides at least one selected from the group consisting of Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number: KCTC13375BP), its culture, and its culture concentrate and dried product. Provided is a probiotic preparation for the prevention or treatment of immune disorders, respiratory diseases, allergies, or asthma diseases containing as an active ingredient.

In addition, the present invention is the Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number: KCTC13375BP), its culture, at least one selected from the group consisting of a concentrate or dried product of the culture, and cold extract extract It is also possible to provide a probiotic formulation comprising a combination consisting of

The Lactobacillus plantarum KC3 strain of the present invention can be isolated from kimchi.

The formulation may be prepared and administered in various formulations and methods according to methods known in the art. For example, the Lactobacillus plantarum KC3 strain of the present invention, a culture solution thereof, a concentrate of the culture solution, or a dried product thereof is mixed with a carrier commonly used in the pharmaceutical field to form a powder, a liquid (liquids and solutions), It may be prepared and administered in the form of tablets, capsules, syrups, suspensions, or granules. The carrier may be, for example, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant, a fragrance, and the like, but is not limited thereto. In addition, the administration dose can be appropriately selected according to the absorption of the active ingredient in the body, the inactivation rate, the excretion rate, the age, sex, breed, condition and severity of the disease of the recipient.

As another aspect, the present invention provides Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number: KCTC13375BP), a culture thereof, at least one selected from the group consisting of a concentrate or a dry product of the culture. And it provides a health functional food composition for the prevention or improvement of immune disorders, respiratory diseases, allergies or asthma diseases containing a combination consisting of a cold extract extract as an active ingredient.

As another aspect, the present invention provides Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number: KCTC13375BP), a culture thereof, at least one selected from the group consisting of a concentrate or a dry product of the culture. And it provides a health supplement for the prevention or improvement of immune disorders, respiratory diseases, allergies or asthma diseases containing a combination consisting of cold extract as an active ingredient.

"Health functional food" as defined herein means a food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 of the Health Functional Food Act. It refers to ingestion for the purpose of obtaining useful effects for health purposes such as regulating nutrients with respect to structure and function or physiological effects.

Health functional food for the prevention or improvement of immune disorders, respiratory diseases, allergies or asthma diseases of the present invention, 0.01 to 95% of the extract based on the total weight of the composition, preferably 1 to 80% by weight.

In addition, for the purpose of preventing or improving immune disorders, respiratory diseases, allergies or asthma diseases, pharmaceutical dosage forms such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, or tea bags, leaching tea, health drinks, etc. It can be manufactured and processed as a health functional food in the form of

In addition, the present invention is Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number: KCTC13375BP), its culture, and at least one selected from the group consisting of a concentrate or dried product of its culture and a cold herb extract. It provides a food or food additive for the prevention or improvement of immune disorders, respiratory diseases, allergies or asthma diseases containing the constituted combination as an active ingredient.

In addition, the health functional food may additionally contain food additives, and the suitability as a "food additive" is determined according to the general rules and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety, unless otherwise specified. It is judged according to the relevant standards and standards.

Items listed in the "Food Additives Code", for example, chemical synthetic products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as depigmentation, licorice extract, crystalline cellulose, and guar gum, L- Mixed preparations such as sodium glutamate preparations, noodles added alkali preparations, preservative preparations, and tar dye preparations may be mentioned.

Functional foods containing the strain of the present invention include bread, rice cakes, dried fruits, candies, chocolates, chewing gum, confectionery products such as jams, ice cream products, ice cream products, ice cream products such as ice cream powder milk, low-fat milk, lactose-decomposed milk , Processed milk, goat milk, fermented milk, buttermilk, concentrated milk, milk cream, butter oil, natural cheese, processed cheese, milk powder, milk products such as whey Processed meat products, processed eggs, meat products such as hamburgers Fish cakes, ham Fish and meat products such as processed fish products such as , sausages and bacon Ramen, dried noodles, raw noodles, fried noodles, luxurious dried noodles, improved soft noodles, frozen noodles, pastas, etc. Fruit drinks, vegetable drinks, carbonated drinks, soy milk , lactic acid bacteria drinks such as yogurt, beverages such as mixed drinks soy sauce, soybean paste, gochujang, chunjang, cheonggukjang, mixed soy sauce, vinegar, sauces, tomato ketchup, curry, seasoning foods such as dressings, margarine, shortening and pizza. It is not limited.

The health functional beverage composition of the present invention is not particularly limited in other ingredients except for containing the strain as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. Examples of the aforementioned natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); disaccharides, (eg maltose, sucrose, etc.); and conventional sugars such as polysaccharides (eg, dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts. salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the combination of the present invention may be added to food or beverage for the purpose of preventing the target disease. At this time, the amount of the strain in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, and the health drink composition may be added in a ratio of 0.02 to 5g, preferably 0.3 to 1g, based on 100ml. .

The content of the combination according to the present invention to be added to food including beverages in the process of manufacturing the health functional food may be appropriately increased or decreased as necessary.

Defense effect test against respiratory damage caused by air pollutants such as fine dust on the combination according to the present invention (Experimental Example 1); When administering the combination of the present invention through a chronic obstructive pulmonary disease (COPD) treatment effect experiment in blood (Experimental Example 2), etc., synergistic inhibition of bronchial inflammation or By confirming that it has a therapeutic effect, it can be usefully used for the prevention or treatment of immune disorders, respiratory diseases, allergies or asthma diseases.

1 shows the deposit of Lactobacillus plantarum KC3;
2 is a view showing the results of a biliary tolerance test for the Lactobacillus plantarum KC3 strain of the present invention (here, all numerical values (or values) are the mean ± standard deviation of 3 repetitions, * is with oxgall and without oxgall; liver p<0.05);
Figure 3 is a diagram showing the results of the pH resistance test against the Lactobacillus plantarum KC3 strain of the present invention;
4 is the experimental result of the effect on the total number of cells in the total bronchoalveolar lavage (BAL) of the sample of the present invention (here, PC: positive control group, KC3: KC3 lactic acid group alone, Ver: cold vinegar alone, KC3+ Ver: lactic acid bacteria, cold vinegar combined administration group);
5 is an experimental result showing the inhibition rate of the sample on the total number of cells in the total bronchoalveolar lavage (BAL) of the sample of the present invention (here, PC: positive control group, KC3: KC3 lactic acid group alone, Ver: cold vinegar Single, KC3+Ver: lactic acid bacteria, cold vinegar combined administration group).

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the content of the present invention is not limited by the examples.

Reference Example 1. Isolation of Lactobacillus plantarum KC3 new lactic acid bacteria (Korean Patent Registration No. 10-2135879)

1-1. Preparation of Kimchi Ingredients

Cabbage kimchi, the raw material of the present invention, was purchased from a local mart (Haro Mart, Wansan-gu, Jeonju) in the following process for household kimchi in Jeollabuk-do.

(1) Step 1: Prepare 5 napa cabbage (about 1kg each), remove the inedible part, and cut into 2 pieces. Dissolve 500g of salt in water in a pickle container to soak cabbage, then take it out, put salt in each cabbage leaf, pickle it for 5-6 hours, wash it 3-4 times, and drain it in a colander.

(2) step 2; Prepare 2 radishes (approx. 1.2 kg each), remove radish, trim and wash, then cut into 4-5cm lengths, 1/2 green onion (approx. 0.5 kg), 1/2 chives (approx. 0.5 kg), fresh mustard Trim and wash 1/2 tier (about 0.5 kg), and then cut into the same length as the radish.

(3) Step 3; Finely mince 50g of garlic, 10g of ginger, and 200g of salted shrimp, and prepare 300ml (about 200g) of anchovy sauce. Soak 150g of glutinous rice, cool it after serving the porridge, and put the prepared shrimp sauce, anchovy sauce, garlic, ginger and red pepper powder 500g into the cooled glutinous rice porridge and mix evenly.

(4) step 4; Put radish, green onion, chives, and mustard shredded in the same length as in step 2 and toss, then season with salt (about 0.5 kg) and sugar (about 0.5 kg) to make kimchi seasoning.

(5) Step 5; After putting the kimchi seasoning evenly between the cabbage leaves, wrap it around the outer leaf so that the cut section of the cabbage is facing up, put it in a container, and store it in a low-temperature warehouse (0 to -2℃) to ripen it for 1 year to produce raw materials for cabbage kimchi did.

1-2. Isolation and identification of a novel Lactobacillus plantarum KC3 strain

The microbial isolation and identification process for the Lactobacillus plantarum KC3 strain of the present invention is performed by diluting the raw material of cabbage kimchi of 1-1 with a dilution solution of peptone (MB-B2220, MB cell) and bromcresol purple (114375, Sigma) and Sodium azide (S2002, sigma) was added to MRS solid medium (MRS medium, DF0881-17-5, Difco), 1.5% agar (214010, Difco)) by 0.1 ㎖ by planar plating and then inoculated at 37°C for 48 After time anaerobic culture, a colony that turned yellow in the medium was selected as a provisional lactic acid bacteria.

As a result of identifying the purely isolated strain, it was confirmed that the strain was a Gram-positive facultative anaerobic bacillus, and negative for catalase and motility.

In addition, it did not grow at 15 ° C. and 45 ° C., and did not generate ammonia from gas and arginine from glucose, so it was confirmed that it belongs to the genus of Lactobacillus.

1-3. Identification of microorganisms (glucose availability analysis, 16s rRNA identification)

1-3-1. sugar availability analysis

For the lactic acid bacteria selected as described above, sugar availability was investigated using the API CHL50 kit (50300, bioMerieux). As a result of the analysis, as shown in Table 1 below, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, D-sorbitol, methyl-αD-mannopyranoside, amyglandin Sugar of arbutin, esculin ferric citrate, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-saccharose, D-trehalose, D-melechtose, D-raffinose was confirmed to be used.

Sugar solubility of Lactobacillus plantarum KC3 Party availability Party availability Control - Esculin ferric citrate + Glycerol - Salicin ± Erythritol - D-Celiobiose + D-Arabinose - D-Maltose + L-Arabinose - D-Lactose + D-Ribose + D-Melibiose + D-Xylose - D-Saccharose + L-Xylose - D-Trehalose + D-Adonitol - Inulin - Methyl-βD-Xylopyranoside - D-Melezitose + D-Galactose + D-Raffinose + D-Glucose + Amidon - D-Fructose + Glycogen - D-Mannose + Xylitol - L-Sorbose - Gentiobiose ± L-Rhamnose - D-Turanose - Dulcitol - D-Lyxose - Inositol - D-Tagatose - D-Mannitol + D-Fucose - D-Sorbitol + L-Fucose - Methyl-αD-Mannopyranoside + D-Arabitol - Methyl-αD-Glucopyranoside - L-Arabitol - N-AcetylGlucosamine ± potassium Gluconate - Amygdalin + potassium 2-KetoGluconate - Arbutin + potassium 5-KetoGluconate -

1-3-2. 16s rRNA Identification

Colonies grown on MRS solid medium (MRS medium (DF0881-17-5, Difco), 1.5% agar (214010, Difco)) were collected and double-stranded DNA sequencing was performed (Solgent, Korea). The obtained nucleotide sequence (SEQ ID NO: 1 in Table 2) was searched by BLAST to identify the strain, and showed 99% homology with Lactobacillus plantarum, so that the novel microorganism of the present invention is Lactobacillus plantarum species. It was confirmed that the strain belongs to (hereinafter also referred to as “KC3 new lactic acid bacteria” or “CKDB-KC3”).

16s rRNA sequence of Lactobacillus plantarum KC3 Lactobacillus plantarum KC3 16s rRNA sequence SEQ ID NO: TATGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTT GACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTACA One

1-3-3. Microbial properties

The characteristics of the novel Lactobacillus plantarum KC3 according to the present invention are as follows.

(1) the form of the fungus

: Form of bacteria when cultured on MRS agar plate at 37°C for 48 hours

①Cell type: Bacillus

② Mobility: None

③Spore-forming ability: None

④ Gram staining: positive

(2) the form of colony

: Form of colonies when cultured on MRS agar plate at 37°C for 48 hours

①Shape: Round

② ridge: convex

③Surface: smooth

④Color: milky white

(3) physiological properties

①Growth temperature

- Able to grow Growth temperature: 15~40℃

- Optimal growth temperature: 36~38℃

②Growth pH

- Growable Growth pH: 4.6~7.5

- Optimal pH: 6.0~7.0

③ Effect on oxygen: facultative anaerobic

(4) catalase: negative

(5) Gas generation: negative

(6) Indole production: negative

(7) lactic acid production: positive

(8) biogenic amine production: negative

: A novel strain isolated from kimchi was named Lactobacillus plantarum KC3 based on the microorganism identification results and characteristics of the bacteria, and was deposited at the Korea Research Institute of Bioscience and Biotechnology (Accession No.: KCTC13375BP) on October 20, 2017. (See Fig. 1)

2-1. Characteristics of Lactobacillus plantarum KC3 new lactic acid bacteria

2-1-1. Stomach acid and bile acid tolerance test

Gastric acid and bile acids secreted from gastric juice and pancreas are very important factors affecting the survival of microorganisms. Therefore, in order to confirm the gastric acid and bile acid resistance of the Lactobacillus plantarum KC3 novel lactic acid bacteria of the present invention, an experiment was performed as follows by applying the method described in the literature. (Kararli, TT, Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals. Biopharm Drug Dispos 1995 16(5):351-380. doi: 10.1002/bdd.2510160502)

It is a process of examining resistance to artificial gastric juice and bile, examining the potential as probiotics, and selecting and identifying strains with excellent activity and strong resistance among them.

2 is a view showing the results of the bile tolerance test for the Lactobacillus plantarum KC3 strain of the present invention.

More specifically, the Lactobacillus plantarum KC3 strain is a MRS medium (with oxgall) containing 0.03% of bile (oxgall) and 0.05% of L-cysteine (L-cysteine) and 0.05% of L-cysteine (L) -cysteine) containing MRS medium (without oxgall), all values (or values) are the mean ± standard deviation of 3 replicates, * indicates the case of p <0.05 between with and without oxgall .

3 is a view showing the results of the gastric acid pH tolerance test for the Lactobacillus plantarum KC3 strain of the present invention.

More specifically, it shows the survival rate of the Lactobacillus plantarum KC3 strain after 3 hours in hydrochloric acid service having a pH of 2.0, 3.0, 4.0 and 6.4, compared with the start time (or start time), * is p <0.05 case, ** denotes a case of p<0.01, and *** denotes p<0.001.

2-1-2. Antibacterial activity test

In order to confirm the antimicrobial activity of the Lactobacillus plantarum KC3 novel lactic acid bacteria of the present invention, an antibacterial activity test was performed.

The antibacterial activity test is an experiment to confirm the inhibitory power against Escherichia coli , Salmonella typhimurium , Staphylococcus aureus , Listeria monocytogenes. The stronger the deterrent, the better.

Table 3 below shows the results of the antibacterial activity test against the Lactobacillus plantarum KC3 strain, and the starting count of the Lactobacillus plantarum KC3 strain is 2.10±0.17 × 106 CFU/mL, and for 6 hours at 37° C. Results are shown, and all numerical values (or values) are the mean ± standard deviation of 3 replicates.

Antibacterial activity of Lactobacillus plantarum KC3 Pathogens Growth
Inhibition
(%) pathogens ^a KC3+pathogens ^a CFU/mL pH CFU/mL pH Escherichia coli 3.23±0.25×10 ⁶ 5.98 8.50±0.05×10 ⁵ 4.84 73.98% Salmonella Typhimurium 6.46±0.35×10 ⁶ 6.10 4.00±0.26×10 ⁶ 5.25 38.14% Listeria monocytogenes 1.57±0.20×10 ⁵ 6.06 1.13±0.06×10 ⁵ 4.94 27.97% Staphyloccus aureus 3.46±0.87×10 ⁶ 6.08 2.83±0.61×10 ⁶ 4.9 18.27%

2-1-3. Antibiotic resistance test

In order to confirm the level of antibiotics susceptibility of the Lactobacillus plantarum KC3 novel lactic acid bacteria of the present invention, an experiment was performed as follows by applying the method described in the literature. ([1] Mathur, S. and R. Singh, Antibiotic resistance in food lactic acid bacteria - A review. Int. J. Food Microbiol 2005 105: 281-295); [2] European Food Safety Authority, Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance: EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP). The EFSA Journal 2012 2740:1-10. doi:10.2903/j.efsa.2012.2740)

MIC test is performed to measure the level of antibiotic resistance of the strain. LSM solid medium (90% iso sensitest broth (CM0473, Oxoid), 10% MRS medium (DF0881-17-5, Difco) was inoculated into MRS medium (DF0881-17-5, Difco) and cultured for 18 hours at 37°C. ), 1.5% agar (214010, Difco)). Amikacin (92018, Liofilchem srl), Gentamycin (92009, Liofilchem srl), Kanamycin (92034, Liofilchem srl), Streptomycin (92112, Liofilchem srl), Penicillin-G (92102, Liofilchem srl), Liofilchem srl), Oxacofillin (92015, Liofilchem srl) Ampicillin (920030, Liofilchem srl), Bacitracin (92019, Liofilchem srl), Rifampicin (92001, Liofilchem srl), Polymyxin B (92004, Liofilchem srl), Chloramphenicol (92075, Liofilchem srl), etc. each), Vancomycin (92057, Liofilchem srl), etc. After raising the strip for each type of antibiotic, incubating at 37°C for 24 hours, find the section where the clear zone disappears with the naked eye and measure the MIC.

Table 4 below shows the antibiotic resistance to the Lactobacillus plantarum KC3 strain. Here, R indicates resistance and means that the size of the inhibition zone is 0 mm, IS indicates intermediate resistance and means that the size of the inhibition zone is 1-5 mm, and S indicates resistance and the size of the inhibition zone means greater than 5mm.

Antibiotic resistance of Lactobacillus plantarum KC3 Anti-microbial agents antibiotic
resistance Anti-microbial agents antibiotic
resistance Aminoglycosides Gram-positive-spectrum IS (4mm) Amikacin IS (1mm) Bacitracin S (7mm) Gentamycin IS (3mm) Rifampicin S (7mm) Kanamycin R (0mm) Novabiocin S (7mm) Neomycin IS (3mm) Lincomycin S (10mm) Streptomycin R (0mm) Gram-negative spectrum β-lactams Polymyxin B R (0mm) Penicillin-G IS (5mm) Broad spectrum Oxacillin IS (2mm) Chloramphenicol S (10mm) Ampicillin S (14mm) Vancomycin R (0mm)

2-1-4. Bio amine production ability experiment

In order to confirm the biological amine-producing ability of the Lactobacillus plantarum KC3 novel lactic acid bacteria of the present invention, an experiment was performed as follows by applying the method described in the literature. (Bover-Cid S, Holzapfel W H, Improved screening procedure for biogenic amine production by lactic acid bacteria. Int J Food Microbiol 1999 53:33-41. doi:10.1016/S0168-1605(99)00152-X)

Biogenic amines are produced by fermentation of food, and may vary depending on the type of microorganism or chemical and physical conditions. Since biogenic amines produced in fermented foods can cause food poisoning or allergic reactions, it is an important criterion for selecting a safe strain for food engineering. ([1] Ladero V et al., Toxicological effects of dietary biogenic amines. Curr Nutr Food Sci. 2010 6:145-156. 10.2174/157340110791233256; [2] European Food Safety Authority, Scientific Opinion on risk based control of biogenic amine formation in fermented foods: EFSA Panel on Biological Hazards. The EFSA Journal 2011 9:1-93. doi:10.2903/j.efsa.2011.2393)

Accordingly, in order to check whether the strain of the present invention forms biological amines, MRS liquid medium (DF0881-17-5, Difco), a strain grown for 16 hours at 37 conditions, was prepared in the literature (Bover-Cid S, Holzapfel WH, Improved screening procedure for It was transferred to a special medium according to biogenic amine production by lactic acid bacteria.Int J Food Microbiol 1999 53:33-41.doi:10.1016/S0168-1605(99)00152-X) and cultured at 37°C for 48 hours.

Tyrosine (tyrosine, SIGMA, T1145)), histidine (histidine, SIGMA, H5659)), ornithine (ornithine, SIGMA, O2375)) and lysine (lysine, DAEJUNG, 5093-4105)) MRS with each amino acid precursor added A liquid medium (DF0881-17-5, Difco) is prepared, and the biological amines tyramine, histamine, putrescine and cadaverine) are produced by the strain in each medium. It was confirmed that Specifically, 1% of each Lactobacillus plantarum strain isolated in MRS liquid medium (DF0881-17-5, Difco) to which 0.1% of the amino acid precursor was added was inoculated and then subcultured 5 to 10 times.

Biological amine identification medium [Trypton 0.5%, yeast extract 0.5%, cocoon extract 0.5%, sodium chloride 0.5%, glucose 0.25%, Tween-80 0.05%, magnesium sulfate 0.02%, manganese sulfate 0.005%, iron sulfate 0.004%, citric acid salt 0.2%, thiamine 0.001%, K2PO4 0.2%, calcium carbonate 0.01%, pyridoxal-5-phosphate 0.005%, amino acid 1%, bromocresol purple 0.006% and agar After mixing 2% with distilled water, adjust the pH to 5.3, and then incubate for 24 to 48 hours at 37°C to determine whether the color changes to purple.

Bromocresol purple in decarboxylase medium is yellow at pH 5.2, but turns purple as the pH increases to 6.8. Therefore, bio-amine production can be confirmed by using the purple color when the pH is increased due to bio-amine production.

Table 5 below shows the results of analyzing the biogenic amine production ability for the Lactobacillus plantarum KC3 strain. As can be seen from Table 5, putrescine, tyramine, histamine and carda All were confirmed to be negative for cadaverine. From this, it was confirmed that the strain of the present invention has no biological amine-producing ability that can induce an overactive immune response.

Biogenic amine production ability of Lactobacillus plantarum KC3 strain Biogenic amines Putrescine Tyramine Histamine Cadaverine KC3 - - - -

3-1. Culture and production of Lactobacillus plantarum KC3 lactic acid bacteria

Lactobacillus plantarum KC3 culture isolated and identified above was performed in a flask containing MRS (MRS medium, DF0881-17-5, Difco) for lactic acid bacteria spawn at 37 ° C. for 24 hours.

Each culture was inoculated into an optimized medium (manufactured by ourselves) in a fermenter (Bio Control & Science, MARADO-05D-PS).

Fermentation was carried out at 37 °C for 18-20 hours with stirring at 120 rpm while maintaining the pH constant at 5.5 ~ 6.0 by automatically adding NaOH solution (25% w / v) to the medium.

Lyophilization of 40X enriched cells was performed according to the manual (Cooling & Heating System, Lab-Mast 10).

After freeze-drying, colony-forming units (CFU) per 1 g of each probiotic powder were measured by serial dilution.

Probiotics were suspended in 0.1 M PBS and adjusted to a density of ^{10 9 CFU/mL prior to use.}

Reference Example 2: Preparation of cold vinegar extract

Dried and crushed cold herb ( Veronicastrum sibiricum L. Pennell ; Veronicastrum : cold herb genus, cultivated (Ansan, Gyeonggi-do)) whole plant 500 g mixed with distilled water and 30% alcohol mixed with distilled water after primary extraction is completed at 80±2℃ for 4 hours. , The extract obtained by repeating the process of filtration with filter paper having a size of 1 μm was concentrated in a vacuum of 52.5±2.5° C. and 650±30 mmHg. After sterilization at 85.0±2.0℃ for 1 hour, it was cooled to 55℃, and dried using a spray dryer (KL-8, Sogang Engineering Co., Ltd., inlet temperature 190±10℃, outlet temperature 95±5℃) to prepare a cold vinegar 30% alcohol extract (74 g, hereinafter referred to as “VS”).

Example 1: mixture preparation

6.7 mg/mL of KC3 lactic acid bacteria of Reference Example 1 (=1×10 ⁹ CFU/animal) and 8.3 mg/mL of cold vinegar extract solution of Reference Example 2 (=100 mg/kg BW) were mixed in a 1:1 ratio to obtain “lactic acid bacteria + Cold vinegar” complex was prepared (hereinafter referred to as “VS”).

Experimental Example 1: Protective effect against respiratory damage caused by air pollutants such as fine dust

In order to confirm the protective effect against respiratory damage caused by air pollutants of the sample of the above example, the method described in the literature was applied as follows.

1-1. Respiratory Injury Mouse Model Experimental Methods

BALB/c male mouse (7 weeks old, 20∼25g, male, Orient Bio) was assigned to 6 mice in each group, and 10 mg/mL coal combustible, a component of air pollutants, 10 mg/mL in all groups except the normal group. Dilute the fly ash, 5 mg/mL diesel exhaust particle (DEP) so that the final concentration of Alum is 1%, and the final concentration of each is a mixture of coal-fired fuel/flyash (1.5 mg/mL) and DEP (5 mg/mL). Intra-Nazal-Trachea (INT) injection method described in literature into the respiratory tract and nose of laboratory animals (Lim et al., Free Radic Biol Med. 25(6), 635-644. (1998), Shin et al., Korean) Using J. Medicinal Crop Sci 27(3), 218-231. (2019)), 50 μL of each was directly injected on the 4th, 7th, and 10th days of the start of the experiment.

The positive control group (dexamethasone, Sigma D2915) was 3 mg/kg BW, the lactic acid bacteria ( Lactobacillus plantarum KC3, KC3) was 1×10 ⁹ CFU/animal (about 2 mg/kg BW), and the cold herb extract was 100 mg/kg BW It was diluted in distilled water and administered orally every day (11 days) at a dose of 300 μL. The “lactic acid bacteria + cold vinegar” combination was diluted 1:1 with lactic acid bacteria and cold vinegar extract solution (KC3 dose: 0.5×10 ⁹ CFU/animal). , Cold herb extract dose: 50 mg/kg BW) was orally administered daily.

It was orally administered at a dose of 300 μl per mouse, and an autopsy was performed on the 12th day after the start of the experiment to recover BAL fluid.

1-2. Determination of the total number of cells in total bronchoalveolar lavage (BAL)

Experimental method

In order to confirm the effect on the total number of cells in the total bronchoalveolar lavage (BAL; bronchoalveolar lavage) of the Example sample, the method described in the literature was applied as follows (Schins et al., Toxicol Appl Pharmacol . 195 (Schins et al., Toxicol Appl Pharmacol. 195 ( 1), 1-11 (2004) and Smith et al., Toxicol Sci , 93(2), 390-399 (2006)).

Experiment result

Results of measuring the effect on the total number of cells in the total bronchoalveolar lavage (BAL; bronchoalveolar lavage) for the sample are shown in Table 6 and FIGS. 4 to 5 below. Compared to the bronchial damage induced group by air pollutants, the total number of BAL cells was significantly reduced by the administration of the “Lactobacillus Lactobacillus plantarum KC3(KC3)+Cold extract” complex, confirming that they had bronchial inflammation inhibitory activity, respectively. It was confirmed that the combination drug administration showed a synergic effect on respiratory damage protection compared to the first administration.

Effect of the total number of cells in total bronchoalveolar lavage (BAL; bronchoalveolar lavage) division Total BAL cells (×10 ⁴ cells/ml) Inhibition rate (based on trigger group) normal group 27.4 ± 3.6 Yu-Gi-Oh 160.1 ± 17.8 positive control 79.9 ± 8.5 50% KC3 lactic acid bacteria sweetener 83.4 ± 7.6 48% cold vinegar sweetener 70.4 ± 7.8 56% KC3+Cold Vinegar Complex 60.3 ± 7.1 62%

1-3. Measurement of the ratio of neutrophil cells to the total number of cells in total bronchoalveolar lavage (BAL)

Experimental method

In order to confirm the effect on the number of neutrophil cells compared to the total number of cells in the total bronchoalveolar lavage (BAL; Bronchoalveolar lavage) of the Example sample, an experiment was applied by applying the method described in the literature as follows (Schins et al., Toxicol Appl) (Schins et al., Toxicol Appl) Pharmacol . 195(1), 1-11 (2004) and Smith et al., Toxicol Sci , 93(2), 390-399 (2006)).

It proceeded in the same manner as in Experimental Example 1-2. Diff-Qick staining of recovered bronchoalveolar lavage (BAL; bronchoalveolar lavage) (Takano et al., Am J Respir Crit Care Med , 156(1), 36-42. (1997), Hemacolor Rapid staining of blood smear, 1.11661) .0001, Merck) was stained with neutrophils and observed.

Experiment result

Table 7 shows the results of measuring the effect on the ratio of neutrophils, which are inflammatory immune cells, to the total number of cells in the total bronchoalveolar lavage (BAL) for the sample. Neutrophil cells increased by air pollutants were significantly reduced by administration of the “Lactobacillus Lactobacillus plantarum KC3(KC3)+Cold Herb extract” complex, confirming that they had bronchial inflammation inhibitory activity, compared to administration of the single agent, respectively. It was confirmed that a synergic effect on respiratory damage defense was exhibited by administration of the combination drug.

Effect of total bronchoalveolar lavage (BAL) on the ratio of neutrophil cells to the total number of cells division Neutrophile No of BAL cel (400X) Inhibition rate (based on trigger group) normal group 1.0 ± 0.1 Yu-Gi-Oh 73.1 ± 9.5 positive control 39.3 ± 7.3 46% KC3 lactic acid bacteria sweetener 37.2 ± 4.7 49% cold vinegar sweetener 33.4 ± 3.5 54% KC3+Cold Vinegar Complex 21.3 ± 3.7 71%

1-4. Measurement of the expression of inflammatory factors in BAL fluid

Experimental method

In order to confirm the effect on the expression level of the inflammatory factor in the bronchoalveolar lavage fluid (BAL fluid) of the Example sample, an experiment was conducted by applying the method described in the literature as follows (Brandt EB et al., J. Allergy Clin. Immunol. , 132(5):1194-1204, (2013)).

In order to measure the expression of inflammatory factors such as IL-17A, TNF-α and CXCL-1 in bronchoalveolar lavage fluid (BAL fluid), an evaluation test was performed using ELISA.

Except for measuring the number of cells in bronchoalveolar lavage (BAL; bronchoalveolar lavage), the same procedure as in Experimental Example 1-3 was performed. The levels of IL-17A, TNF-α MIP2, and CXCL-1 in bronchoalveolar lavage (BAL fluid) were measured by ELISA.

IL-17A antibody (M1700, R&D Systems, Minneapolis, USA), TNF-α antibody (MTA00B, R&D Systems, Minneapolis, USA) CXCL-1 antibody (MKC00B, R&D Systems, Minneapolis, USA) was diluted in a buffer solution and diluted into microwells. After coating (micro well), it was incubated at 4°C for 16 hours. After washing each well with a buffer solution 3 times, 100 μl of serum diluted 10 times was dispensed.

After leaving it at room temperature for 1 hour, it was washed twice, and 100 μl of Avidin-HRP-conjugated antibody (DY007, R&D Systems, Minneapolis, USA) was treated and left at room temperature for 1 hour, and then washed again.

100 μl of TMB (Tetramethylbenzidine) substrate (DY007, R&D Systems, Minneapolis, USA) was dispensed at a time, left in the dark for 30 minutes, treated with 50 μl of stop solution (DY007, R&D Systems, Minneapolis, USA), and then at 450 nm. Absorbance was measured.

Experiment result

The results of measuring the contents of inflammatory biomarkers IL-17A, TNF-α, CXCL-1, etc. in bronchoalveolar lavage (BAL) for the sample are shown in Table 8 below. Respiratory damage biomarkers (IL-17A, TNF-α, CXCL-1) increased by air pollutants were significantly reduced by administration of the “Lactobacillus Lactobacillus plantarum KC3(KC3)+Cold Vinegar extract” complex, thereby reducing bronchial inflammation inhibitory activity It could be confirmed that the drug had a synergistic effect on respiratory damage defense by administration of a combination drug compared to administration of a single agent, respectively.

Effect of Inflammatory Factor Expression in Bronchoalveolar Lavage (BAL) Experiment Results division Concentration (pg/mL) / Inhibition rate based on trigger group (%) IL-17A TNF-α CXCL-1 normal group 14.9±3.7 55.4±4.9 51.0±4.21 Yu-Gi-Oh 36.3±4.5 131.0±14.0 192.4±16.0 positive control 19.3±5.2 (47%) 60.3±9.7 (54%) 132.0±14 (31%) KC3 lactic acid bacteria sweetener 22.3±2.7 (39%) 70.3±8.4 (46%) 139.2±12 (28%) cold vinegar sweetener 20.3±1.6 (44%) 60.3±7.7 (54%) 88.5±10.7 (54%) KC3+Cold Vinegar Complex 18.3±1.9 (50%) 45.3±5.4 (65%) 71.3±8.9 (63%)

Experimental Example 2. Measurement of biomarkers of chronic obstructive pulmonary disease (COPD) expression in blood

In order to confirm the therapeutic effect of the Example sample on chronic obstructive pulmonary disease (COPD), the method described in the existing literature was applied and tested as follows.

2-1. Experimental method

In order to confirm the effect of the Example sample on the biomarker expression level of Chronic Obstructive Pulmonary Disease (COPD), an experiment was conducted by applying the method described in the literature as follows (Jeremy AS et al., Int. J. Mol. Sci. , 15:6062-6071, (2014)).

An evaluation test was performed using ELISA to measure SDMA (Symmetric Dimethylarginine) in the blood, which is a biomarker of chronic obstructive pulmonary disease (COPD).

Serum was separated from the blood collected from the heart of BALB / c male mouse (7 weeks old, 20-25 g, male, Orient Bio) of Experimental Example 1, and the SDMA antibody (MBS2605912, MyBioSource, SanDiego, Califomia, USA) was added as a buffer solution. After dilution and coating on fine wells (96 wells, SPL 30096, Allforab), they were incubated at 4°C for 16 hours. After washing each well with a buffer solution 3 times, 100 μl of serum diluted 10 times was dispensed.

After leaving it at room temperature for 1 hour, it was washed twice, and 100 μl of Avidin-HRP-conjugated antibody (DY007, R&D Systems, Minneapolis, USA) was treated and left at room temperature for 1 hour, and then washed again. 100 μl of TMB (Tetramethylbenzidine) substrate (DY007, R&D Systems, Minneapolis, USA) was dispensed at a time, left in the dark for 30 minutes, treated with 50 μl of stop solution (DY007, R&D Systems, Minneapolis, USA), and then at 450 nm. Absorbance was measured.

Experiment result

Table 9 shows the results of measuring the content of SDMA (Symmetric Dimethylarginine) in the blood, which is a biomarker of chronic obstructive pulmonary disease (COPD) for the sample.

SDMA, a COPD biomarker increased by air pollutants , was significantly reduced by administration of the combination of 『Lactobacillus Lactobacillus plantarum KC3(KC3)+Cold Herb Extract』, confirming that it had chronic obstructive pulmonary disease inhibitory activity, and each It was found that there was a synergic effect of inhibiting COPD disease by administration of the combination drug compared to administration.

Results of experiments on the effect of chronic obstructive pulmonary disease (COPD) biomarkers in the blood division SDMA level in serum (ug/mL) Inhibition rate (based on trigger group) normal group 8.3 ± 2.4 Yu-Gi-Oh 14.4 ± 3.3 positive control 9.5 ± 2.2 34% KC3 lactic acid bacteria sweetener 11.1 ± 3.4 23% cold vinegar sweetener 10.1 ± 2.2 30% KC3+Cold Vinegar Complex 5.3 ± 2.3 63%

A formulation example of the composition comprising the combination of the present invention will be described, but the present invention is not intended to limit the present invention, but to describe it in detail.

Formulation Example 1. Preparation of powder

VS COMBINATION ------------------------------------------------------- 20 mg

Lactose ----------------------------------------------- 100 mg

talc -------------------------------------------------------------- 10 mg

The above ingredients are mixed and filled in an airtight bag to prepare a powder.

Formulation Example 2. Preparation of tablets

VS Combination --------------------------------------------- 10 mg

Corn Starch --------------------------------------------------- 100 mg

Lactose ----------------------------------------------- 100 mg

Magnesium Stearate ---------------------------------- 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional manufacturing method of tablets.

Formulation Example 3. Preparation of capsules

VS Combination ---------------------------------------------- 10 mg

crystalline cellulose ------------------------------------- 3 mg

Lactose ----------------------------------------------------- 14.8 mg

Magnesium Stearate ------------------------------- 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled in a gelatin capsule to prepare a capsule.

Formulation Example 4. Preparation of injection

VS Combination -------------------------------------------- 10 mg

mannitol -------------------------------------------- 180 mg

Sterile distilled water for injection ------------------------------ 2974 mg

Na ₂ HPO ₄ 12H ₂ O -------------------------------------- 26 mg

According to a conventional method for preparing injections, it is prepared with the content of (2) above ingredients per 1 ampoule.

Formulation Example 5. Preparation of liquid formulation

CB5 compound --------------------------------------------------- 10 mg

Isomerized sugar --------------------------------------------- 10 g

Mannitol -------------------------------------------------------------- 5 g

Purified water ----------------------------------------------- Appropriate amount

According to a conventional method for preparing a liquid, add each component to purified water to dissolve it, add an appropriate amount of lemon flavor, mix the above components, add purified water to adjust the total volume to 100 ml, fill a brown bottle, and sterilize the solution manufacture

Formulation Example 6. Preparation of health food

VS Combination ------------------------------------------- 1000 mg

Vitamin mixture ----------------------------------- Appropriate amount

Vitamin A Acetate -------------------------------- 70 ug

Vitamin E ---------------------------------------------------- 1.0 mg

Vitamin B1 -------------------------------------------------- 0.13 mg

Vitamin B2 ---------------------------------------- 0.15 mg

Vitamin B6 ----------------------------------------- 0.5 mg

Vitamin B12 ---------------------------------------- 0.2 ug

Vitamin C ------------------------------------------------------ 10 mg

Biotin --------------------------------------------- 10 ug

Nicotinamide ------------------------------------ 1.7 mg

folic acid ----------------------------------------------- 50 ug

Calcium Pantothenate ------------------------------------- 0.5 mg

Mineral mixture ----------------------------------- Appropriate amount

ferrous sulfate ---------------------------------------- 1.75 mg

Zinc Oxide --------------------------------------------------- 0.82 mg

Magnesium carbonate ------------------------------------- 25.3 mg

Potassium Phosphate ---------------------------------------- 15 mg

Dicalcium Phosphate ------------------------------------ 55 mg

Potassium Citrate ----------------------------------------- 90 mg

Calcium carbonate ---------------------------------------------------- 100 mg

Magnesium Chloride ----------------------------------------------- 24.8 mg

The composition ratio of the vitamin and mineral mixture is a composition that is relatively suitable for health food in a preferred embodiment, but the mixing ratio may be arbitrarily modified, and the ingredients are mixed according to a conventional health food manufacturing method. , to prepare granules, and can be used for preparing health food compositions according to a conventional method.

Formulation Example 7. Preparation of a health drink

VS Combination ----------------------------------------- 1000 mg

citric acid ---------------------------------------------------- 1000 mg

Oligosaccharide ------------------------------------------ 100 g

Plum Concentrate ------------------------------------------ 2 g

Taurine ---------------------------------------------- 1 g

Add purified water --------------- Total 900 ml

After mixing the above ingredients according to a conventional health drink manufacturing method, after stirring and heating at 85° C. for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2ℓ container, sealed and sterilized, and then refrigerated. used in the manufacture of health beverage compositions of

<110> KT & G CORPORATION CKD Bio Corp. <120> a composition comprising a combination consisting of an extract of Veronicastrum sibiricum L. Pennell and Lactobacillus plantarum KC3 as an active ingredient for preventing or treating immune disorders, respiratory organ disease, allergy or asthma <130> DIF/2020-06-002/EK <160> 1 <170> KoPatentIn <210> 1 <211> 1509 <212> RNA <213> Artificial Sequence <220> <223> 16s RNA of L. plantarum KC3 (KCTC13375BP) <400> 1 TATGGCTCAG GACGAACGCT GGCGGCGTGC CTAATACATG CAAGTCGAAC GAACTCTGGT 60 ATTGATTGGT GCTTGCATCA TGATTTACAT TTGAGTGAGT GGCGAACTGG TGAGTAACAC 120 GTGGGAAACC TGCCCAGAAG CGGGGGATAA CACCTGGAAA CAGATGCTAA TACCGCATAA 180 CAACTTGGAC CGCATGGTCC GAGTTTGAAA GATGGCTTCG GCTATCACTT TTGGATGGTC 240 CCGCGGCGTA TTAGCTAGAT GGTGGGGTAA CGGCTCACCA TGGCAATGAT ACGTAGCCGA 300 CCTGAGAGGG TAATCGGCCA CATTGGGACT GAGACACGGC CCAAACTCCT ACGGGAGGCA 360 GCAGTAGGGA ATCTTCCACA ATGGACGAAA GTCTGATGGA GCAACGCCGC GTGAGTGAAG 420 AAGGTTTCG GCTCGTAAAA CTCTGTTGTT AAAGAAGAAC ATATCTGAGA GTAACTGTTC 480 AGGTATTGAC GGTATTTAAC CAGAAAGCCA CGGCTAACTA CGTGCCAGCA GCCGCGGTAA 540 TACGTAGGTG GCAAGCGTTG TCCGGATTTA TTGGGCGTAA AGCGAGCGCA GGCGGTTTTT 600 TAAGTCTGAT GTGAAAGCCT TCGGCTCAAC CGAAGAAGTG CATCGGAAAC TGGGAAACTT 660 GAGTGCAGAA GAGGACAGTG GAACTCCATG TGTAGCGGTG AAATGCGTAG ATATATGGAA 720 GAACACCAGT GGCGAAGGCG GCTGTCTGGT CTGTAACTGA CGCTGAGGCT CGAAAGTATG 780 GGTAGCAAAC AGGATTAGAT ACCCTGGTAG TCCATACCGT AAACGATGAA TGCTAAGTGT 840 TGGAGGGTTT CCGCCCTTCA GTGCTGCAGC TAACGCATTA AGCATTCCGC CTGGGGAGTA 900 CGGCCGCAAG GCTGAAACTC AAAGGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT 960 GGTTTAATTC GAAGCTACGC GAAGAACCTT ACCAGGTCTT GACATACTAT GCAAATCTAA 1020 GAGATTAGAC GTTCCCTTCG GGGACATGGA TACAGGTGGT GCATGGTTGT CGTCAGCTCG 1080 TGTCGTGAGA TGTTGGGTTA AGTCCCGCAA CGAGCGCAAC CCTTATTATC AGTTGCCAGC 1140 ATTAAGTTGG GCACTCTGGT GAGACTGCCG GTGACAAACC GGAGGAAGGT GGGGATGACG 1200 TCAAATCATC ATGCCCCTTA TGACCTGGGC TACACACGTG CTACAATGGA TGGTACAACG 1260 AGTTGCGAAC TCGCGAGAGT AAGCTAATCT CTTAAAGCCA TTCTCAGTTC GGATTGTAGG 1320 CTGCAACTCG CCTACATGAA GTCGGAATCG CTAGTAATCG CGGATCAGCA TGCCGCGGTG 1380 AATACGTTCC CGGGCCTTGT ACACACCGCC CGTCACACCA TGAGAGTTTG TAACACCCAA 1440 AGTCGGTGGG GTAACCTTTT AGGAACCAGC CGCCTAAGGT GGGACAGATG ATTAGGGTGA 1500 AGTCGTACA 1509 ------------------------------------------------------------ ---------------------------

Claims (10)

Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number: KCTC13375BP), a culture thereof, a dry weight relative of at least one selected from the group consisting of a concentrate or a dry matter of the culture and a combination consisting of a cold herb extract Any one respiratory tract selected from the group consisting of rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, asthma and chronic obstructive pulmonary disease, comprising a combination consisting of a combination by weight of 1: 0.01 to 100 (w/w) as an active ingredient. A pharmaceutical composition for the prevention or treatment of inflammatory diseases. delete delete delete delete Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number: KCTC13375BP), a culture thereof, a dry weight relative of at least one selected from the group consisting of a concentrate or a dry matter of the culture and a combination consisting of a cold herb extract Any one respiratory tract selected from the group consisting of rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, asthma and chronic obstructive pulmonary disease, comprising a combination consisting of a combination by weight of 1: 0.01 to 100 (w/w) as an active ingredient. Health functional food for the prevention or improvement of inflammatory diseases. 7. The method of claim 6,
The health functional food is a health functional food in the form of powder, granule, tablet, capsule, pill, suspension, emulsion, syrup, tea bag, leached tea, or health drink. Lactobacillus plantarum KC3 strain (also referred to as “CKDB-KC3”, accession number: KCTC13375BP), a culture thereof, a dry weight relative of at least one selected from the group consisting of a concentrate or a dry matter of the culture and a combination consisting of a cold herb extract Any one respiratory tract selected from the group consisting of rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, asthma and chronic obstructive pulmonary disease, comprising a combination consisting of a combination by weight of 1: 0.01 to 100 (w/w) as an active ingredient. A health supplement for the prevention or improvement of inflammatory diseases. delete delete

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