Lactobacillus having prophylactic or therapeutic activity for colitis disease and having antibacterial activity against harmful bacteria in the intestine and uses thereof

Abstract

The present invention provides the strain Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), the strain Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or the strain Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP) and the use thereof.

Description

Technology Field

[0001] The present invention relates to a novel Lactobacillus strain and its uses, and specifically, the present invention relates to a novel Lactobacillus strain having antibacterial activity against harmful bacteria in the intestines and an anti-inflammatory effect in the intestines, and its uses. Background Technology

[0002] Probiotics refer to microorganisms possessing antimicrobial and enzymatic activity that help maintain the balance of intestinal microorganisms, as well as the products produced by said microorganisms. Furthermore, probiotics are defined as live bacteria in the form of single or complex strains that are supplied to humans or animals in the form of dried cells or fermentation products to improve the intestinal flora. The characteristics that probiotics must possess include inhabiting the human gut, being non-pathogenic and non-toxic, and surviving during transit to the intestines. Additionally, they must possess resistance to acids, enzymes, and bile in the intestinal environment. Among these probiotics, Lactobacillus species are particularly known as highly useful probiotics because there are strains that produce various antimicrobial substances.

[0003] Meanwhile, 70% of immunity is determined in the gut. Bacteria in the intestines enable the breakdown and digestion of the food we eat and also play a role in training the immune system. Recent studies are increasingly revealing that obesity and various intractable diseases are closely related to these gut bacteria and intestinal immunity. Inflammation is an important immune response that protects against damage or infection by eliminating harmful elements and repairing cells and tissues. However, excessive acute or chronic inflammation can cause serious disorders such as colitis, arthritis, asthma, Parkinson's disease, Alzheimer's disease, and sepsis. Among inflammatory diseases, colitis is a condition in which inflammation occurs in the large intestine, caused by various factors including infection, poor blood supply, and autoimmune reactions. Major symptoms include tenesmus (indigestion), abdominal bloating, lower abdominal pain, and diarrhea, and in some cases, mucus, pus, or blood may be mixed in the stool. Colitis can be broadly classified into infectious and non-infectious colitis depending on the cause, or into acute and chronic colitis depending on the duration of the illness. Acute colitis includes amoebic dysentery, bacterial dysentery, and pseudomembranous enteritis caused by Salmonella or antibiotics, while chronic colitis includes ulcerative colitis, Crohn's disease, tuberculosis, syphilis, and conditions caused by X-rays. Furthermore, colitis encompasses not only inflammatory bowel disease (IBD) but also irritable bowel syndrome (IBS). Ulcerative colitis (UC) and Crohn's disease (CD), which are representative inflammatory bowel diseases, do not yet have clearly identified causes; they can cause severe chronic diarrhea and bloody diarrhea along with abdominal pain, and are characterized by being difficult to cure and repeating cycles of remission and exacerbation.Ulcerative colitis is a disease in which erosions or ulcers form continuously on the mucous membrane of the large intestine, causing bloody stools, bloody stools, diarrhea, and abdominal pain; in severe cases, systemic symptoms such as fever, weight loss, and anemia appear. In addition, pro-inflammatory cytokines such as interleukin and TNF-α are identified in ulcerative colitis. In particular, inflammatory cytokines such as interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α were increased in colon tissues of patients with ulcerative colitis compared to normal controls (Lammers KM, Brigidi P, Vitali B, et al. Immunomodulatory effects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cells. FEMS Immunol Med Microbiol 2003;38:165-172., Pagnini C, Saeed R, Bamias G, Arseneau KO, Pizarro TT, Cominelli F. Probiotics promote gut health through stimulation of epithelial innate immunity. Proc Natl Acad Sci USA 2010; 107:454-459.), and in the case of the anti-inflammatory cytokine IL-10, it was increased in ulcerative colitis (Akagi S, Hiyama E, Imamura Y, Takesue Y, Matsuura Y, Yokoyama T. Interleukin-10 expression in intestine of Crohn disease. Int J Mol Med 2000;5:389-395.).Although significant scientific evidence has been presented regarding not only the pathophysiology but also the diagnosis and treatment of inflammatory bowel disease, it is still insufficient to treat the disease, and a considerable number of patients still do not respond to conventional treatments or experience side effects. Drugs that have been used to treat colitis include steroidal immunosuppressants, 5-aminosalicylic acid (5-ASA) drugs that block the production of prostaglandins (e.g., sulfasalazine), and mesalazine; however, the use of these treatments is restricted because they not only have minimal therapeutic effects on colitis but also cause serious side effects such as abdominal fullness, headache, rash, liver disease, leukopenia, agranulocytosis, and male infertility. Therefore, there is a need for the development of provitotics for the treatment and improvement of colitis that are highly effective, safe, and free from side effects. The problem to be solved

[0005] Therefore, the problem that the present invention aims to solve is to provide a strain having the improvement of intestinal health, the treatment or prevention of intestinal diseases, an antibacterial effect against pathogenic microorganisms, and an anti-inflammatory effect, a probiotic using the same, and a composition for treating or improving symptoms of colitis using the same. means of solving the problem

[0007] To solve the above problem, the present invention provides a strain of Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), a strain of Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or a strain of Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP).

[0008] The present invention provides a food composition containing an effective amount of one or more selected from the group consisting of the strain Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), the strain Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or the strain Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP), a culture of said strain, a lysate of said strain, and an extract of said strain.

[0009] The present invention provides a food composition containing an effective amount of one or more selected from the group consisting of the strain Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), the strain Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or the strain Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP), a culture of said strain, a lysate of said strain, and an extract of said strain.

[0010] The present invention provides a composition for a health functional food for improving intestinal health.

[0011] The present invention provides a pharmaceutical composition for the treatment or prevention of intestinal diseases containing an effective amount of one or more selected from the group consisting of the strain Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), the strain Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or the strain Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP), a culture of said strain, a lysate of said strain, and an extract of said strain.

[0012] The above intestinal diseases may be selected from the group consisting of colitis, infectious diarrhea caused by pathogenic microorganisms, inflammatory bowel disease, nervous enteritis syndrome, irritable bowel syndrome, small intestinal microbial overgrowth, and acute intestinal diarrhea.

[0013] The above colitis may be selected from acute enteritis, bacterial colitis, bacterial dysentery, cholera, typhoid fever, traveler's diarrhea, viral colitis, pseudomembranous colitis, amoebic colitis, inflammatory bowel disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, transformative colitis, Crohn's disease, Behcet's syndrome, drug-induced colitis, microscopic colitis, lymphocytic colitis, radiation colitis, and indeterminate colitis.

[0014] The present invention provides an antimicrobial composition containing an effective amount of one or more selected from the group consisting of the strain Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), the strain Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or the strain Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP), a culture of said strain, a lysate of said strain, and an extract of said strain.

[0015] The above composition may be one or more selected from the group consisting of Staphylococcus aureus, Bacillus cereus, Escherichia coli (E. coli), Salmonella enterica, Enterobacter cloacae, and Listeria monocytogenes.

[0016] The above antimicrobial composition may be provided as an antimicrobial composition that is a food, a food additive, or a pharmaceutical composition. Effects of the invention

[0018] The strains Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP) according to the present invention have excellent antimicrobial effects, anti-inflammatory activity, and immunomodulatory functions. In particular, they have an excellent inhibitory effect on pro-inflammatory cytokines. Therefore, they can achieve antibacterial effects, anti-inflammatory effects, immunomodulatory effects, and effects to improve gut health, and can be usefully utilized as probiotic materials. Brief explanation of the drawing

[0020] The following drawings attached to this specification illustrate preferred embodiments of the present invention and serve to further enhance understanding of the technical concept of the present invention together with the aforementioned description; therefore, the present invention should not be interpreted as being limited only to the matters described in such drawings. Figures 1 to 3 are photographs showing the results of hemolytic confirmation tests for Lactobacillus plantarum CLP2001, Lactobacillus plantarum CLP2002, and Lactobacillus paracasei CLB0820. Figures 4 to 6 are photographs showing the Bile salt hydrolase (BSH) activity results of Lactobacillus plantarum CLP2001, Lactobacillus plantarum CLP2002, and Lactobacillus paracasei CLB0820. Figures 7 to 9 show the results of confirming the antibacterial activity of Lactobacillus plantarum CLP2001, Lactobacillus plantarum CLP2002, and Lactobacillus paracasei CLB0820. Figure 10 shows the results of confirming the expression level of tumor necrosis factor-alpha in Raw264.7 (Tumor necrosis factor-alpha in macrophage(Raw264.7)). Figure 11 shows the results of confirming the expression level of Interleukin-1 beta in Raw264.7 (Interleukin-1 beta in macrophage(Raw264.7)). Figure 12 shows the results of confirming the expression level of tumor necrosis factor-alpha in PBMC (Tumor necrosis factor-alpha in PBMC) Figure 13 shows the results of confirming the expression level of Interleukin-1 beta in PBMC (Interleukin-1 beta in PBMC). Figure 14 shows the results of confirming the expression level of interferon-gamma in PBMC (Interferon-gamma in PBMC). Specific details for implementing the invention

[0021] One embodiment of the present invention provides novel strains that were not previously identified, including the Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), the Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), and the Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP).

[0022] In the present invention, the novel strains were identified as Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP) and Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP) strains derived from kimchi, or Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP) strains derived from fruit fermentation liquid, and as a result of analyzing the 16s rDNA of the strains, it was confirmed that the strains are novel strains that have not been previously known.

[0023] Accordingly, in one aspect, the present invention relates to novel probiotic strains, namely Lactobacillus plantarum CLP2001, Lactobacillus plantarum CLP2002, or Lactobacillus paracasei CLB0820, wherein each strain comprises a 16s rDNA sequence represented by SEQ ID NOs 1 to 3.

[0024] The present invention relates to a Lactobacillus paracasei strain, Lactobacillus plantarum, and the uses thereof, and more specifically, to a composition containing an effective amount of one or more selected from the group consisting of a Lactobacillus plantarum CLP2001 strain (accession number KCTC 14848BP), a Lactobacillus plantarum CLP2002 strain (accession number KCTC 14849BP), or a Lactobacillus paracasei CLB0820 strain (accession number KCTC 14850BP), the cell of said strain, a culture of said strain, a lysate of said strain, and an extract of said strain.

[0025] The term "culture" in the present invention refers to the entire medium containing the strain, bacterial extract, metabolites thereof, excess nutrients, etc., obtained by culturing the strains of Lactobacillus plantarum CLP2001, Lactobacillus plantarum CLP2002, or Lactobacillus paracasei CLB0820 for a certain period in a medium capable of supplying nutrients to enable the growth and survival of said strains, but also includes the culture solution from which the strains have been removed after culturing. The culture solution may refer to the liquid of the upper layer obtained by leaving it undisturbed for a certain period to exclude the portion that has settled at the bottom, the bacterial cells removed through filtration, or the liquid of the upper layer obtained by centrifuging the culture solution to remove the sediment at the bottom. The culture solution may also be used as a concentrate by concentrating it using conventional methods. The culture solution or the concentrate of the culture solution may be provided as a dried product by drying it using conventional methods.

[0026] Unless otherwise specified, the term "culture" as used in this specification may include a culture medium of a cell, a concentrate of the culture medium, or a dried product of said culture medium or concentrate. The culture may or may not contain a cell depending on the case, and the inclusion of a cell is not particularly relevant.

[0027] The above "bacterial body" refers to the strain of the present invention itself and includes the strain itself that has been isolated and selected, or the strain isolated from the culture medium by culturing the said strain. The said bacterial body can be obtained by centrifuging the culture medium and taking the portion that settles to the bottom layer, or by letting it sit for a certain period of time and removing the liquid from the top, as it sinks to the bottom layer of the culture medium due to gravity.

[0028] According to one aspect, the culture of the strain of the present invention may use a medium that is easily selected by a person skilled in the art according to the purpose among media used for microbial culture, preferably a medium used for culturing said strain, more preferably a medium used for culturing said strain, an RCM (Reinforced Clostridium Medium) medium, a TSB (Tryptic soy broth) medium, or a BHI (Brain Heart Infusion) medium, but is not limited thereto.

[0029] According to one embodiment of the present invention, a culture of the strain of the present invention can be prepared by inoculating the strain of the present invention into the microbial culture medium and according to a microbial culture method known in the art (e.g., static culture). The culture of the strain may include a culture medium, a concentrate of a culture medium, or a dried product thereof, and the concentrate or dried product may be easily prepared according to a method of concentrating or drying microorganisms or culture medium known in the art.

[0031] In addition, the present invention confirmed that the strains each have antibacterial effects, anti-inflammatory and immunomodulatory effects, and enteritis cytokine inhibitory effects.

[0032] In another aspect, the present invention relates to a composition for food or food additive for improving intestinal health, preventing, treating, or improving intestinal diseases, containing an effective amount of one or more selected from the group consisting of the strain Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), the strain Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or the strain Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP), the cells of the strains, the cultures of the strains, the lysates of the strains, and the extracts of the strains.

[0033] The above-mentioned composition for food or food additives can be easily utilized as a food effective for improving intestinal health or preventing intestinal diseases, such as a main ingredient, auxiliary ingredient, food additive, health functional food, or functional beverage, but is not limited thereto.

[0034] The term "food" above refers to a natural or processed product containing one or more nutrients, preferably one that has undergone a certain degree of processing to become ready for direct consumption, and in the conventional sense includes all of the following: food, food additives, health functional foods, and functional beverages.

[0035] Foods to which the food composition according to the present invention can be added include, for example, various types of food, beverages, chewing gum, tea, vitamin complexes, functional foods, etc. Additionally, in the present invention, food includes, but is not limited to, special nutritional foods (e.g., infant formula, baby food, etc.), processed meat products, fish products, tofu products, jelly products, noodles (e.g., ramen, noodles, etc.), bread products, health supplements, seasoning foods (e.g., soy sauce, soybean paste, red pepper paste, mixed sauce, etc.), sauces, confectionery products (e.g., snacks), candies, chocolates, chewing gum, ice cream, dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickled vegetables, etc.), beverages (e.g., fruit beverages, vegetable beverages, soy milk, fermented beverages, etc.), and natural seasonings (e.g., ramen soup mix). The above food, beverage, or food additive can be manufactured by conventional manufacturing methods.

[0036] The above-mentioned health functional food refers to a group of foods to which added value has been imparted by utilizing physical, biochemical, or biotechnological methods to enable the function of the food to act or manifest for a specific purpose, or to foods processed and designed to sufficiently express in the body the in vivo regulatory functions regarding the regulation of biological defense rhythms, disease prevention, and recovery possessed by the food composition. The above-mentioned functional food may include food science-acceptable food additives and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods.

[0037] In the present invention, the term "functional beverage" refers to a general term for beverages consumed to quench thirst or enjoy a taste. In addition to including the composition for improving or preventing symptoms of intestinal disease as an essential component in the indicated proportions, there are no special restrictions on other components, and various flavoring agents or natural carbohydrates, etc., may be included as additional components, as in ordinary beverages.

[0038] Furthermore, in addition to what is described above, food containing the food composition for improving or preventing symptoms of intestinal disease according to the present invention may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., and the above components may be used independently or in combination.

[0039] In a food containing the food composition of the present invention, the amount of the composition according to the present invention may be included in an amount of 0.001% to 100% by weight of the total weight of the food, preferably in an amount of 1% to 99% by weight, and in the case of a beverage, may be included in an amount of 0.001g to 10g, preferably 0.01g to 1g based on 100ml, but in the case of long-term consumption for the purpose of health and hygiene or health control, it may be less than the above range, and since the active ingredient has no problem in terms of safety, it may be used in an amount greater than the above range, so it is not limited to the above range.

[0040] The food composition of the present invention may be prepared by adding the strains of Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP) independently or to an acceptable carrier, or in the form of a composition suitable for consumption by humans or animals. That is, it may be used by adding it to foods that do not contain other probiotic bacteria or to foods that already contain several probiotic bacteria. For example, in preparing the food of the present invention, other microorganisms that can be used together with the strains of the present invention are those that are suitable for consumption by humans or animals and possess probiotic activity capable of inhibiting pathogenic harmful bacteria or improving the microbial balance in the intestinal tract of mammals upon consumption, and are not particularly limited. Preferably, the effect can be further enhanced by additionally including a mixed probiotic microbial strain having excellent probiotic activity and an excellent immune-enhancing effect in the food composition of the present invention. Examples of carriers that can be used in the food composition of the present invention may include bulking agents, high-fiber additives, encapsulating agents, lipids, etc., and examples of such carriers are sufficiently known in the art. The Lactobacillus paracasei strain of the present invention may be in a freeze-dried or encapsulated form, or in the form of a culture suspension or a dried powder.

[0041] The composition of the present invention may also be provided in the form of an animal feed additive containing the strain or an animal feed containing the same.

[0042] The animal feed additive of the present invention may be in the form of a dry or liquid formulation and may further include other non-pathogenic microorganisms in addition to the strain.

[0043] The animal feed additive of the present invention may further include one or more enzyme preparations in addition to the strain Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), the strain Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or the strain Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP). The added enzyme preparations may be in either a dry or liquid state, and the enzyme preparations include fat-degrading enzymes such as lipase; phytase, which breaks down phytic acid to produce phosphate and inositol phosphate; amylase, an enzyme that hydrolyzes α-1,4-glycoside bonds contained in starch and glycogen; phosphatase, an enzyme that hydrolyzes organophosphate esters; carboxymethylcellulase, which breaks down cellulose; xylase, which breaks down xylose; maltase, which hydrolyzes maltose into two molecules of glucose; and invertase, which hydrolyzes saccharose to produce a glucose-fructose mixture, etc. The same sugar-producing enzymes can be used.

[0044] When the strains of Lactobacillus plantarum CLP2001 (Deposit No. KCTC 14848BP), Lactobacillus plantarum CLP2002 (Deposit No. KCTC 14849BP), or Lactobacillus paracasei CLB0820 (Deposit No. KCTC 14850BP) of the present invention are used as animal feed additives, the feed raw materials may include various grains and soybean proteins, as well as peanuts, peas, sugar beets, pulp, grain by-products, animal offal powder, and fish meal powder, and these may be used without limitation in either unprocessed or processed form. The processing process is not necessarily limited to this, but, for example, is a process in which the feed raw material is filled and compressed through a specific outlet under pressure; in the case of proteins, it is preferable to use extrusion molding, which denatures the protein and increases its usability. Extrusion offers advantages such as denaturing proteins and destroying antienzymatic factors through a heat treatment process. Furthermore, in the case of soybean protein, extrusion can improve protein digestibility, inactivate anti-nutritional factors—such as trypsin inhibitors, which are one of the proteolytic enzyme inhibitors present in soybeans—and enhance digestibility by increasing the digestibility of proteolytic enzymes, thereby increasing the nutritional value of soybean protein.

[0045] In another aspect, the present invention relates to a pharmaceutical composition for the treatment or prevention of intestinal diseases containing an effective amount of one or more selected from the group consisting of the strain Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), the strain Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or the strain Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP), the cells of the strains, the cultures of the strains, the lysates of the strains, and the extracts of the strains.

[0046] The pharmaceutical composition of the present invention may be provided as a composition that can be combined with a live bacterial cell, a dried bacterial strain, a culture of a bacterial strain, a lysate of a bacterial strain, or a pharmaceutically acceptable carrier or medium thereof. The carrier or medium used may include a solvent, a dispersant, a coating, an absorption promoter, a controlled release agent (i.e., a sustained-release agent), and one or more inert excipients (including starch, polyol, granules, microfine cellulose, microcrystalline cellulose (e.g., Celphere, Celphere beads), a diluent, a lubricant, a binder, a disintegrant, etc.). If necessary, the tablet formulation of the disclosed composition may be coated by a standard aqueous or non-aqueous method. Examples of the pharmaceutically acceptable carrier and the excipient for use as a pharmaceutically acceptable inert carrier, and the additional components mentioned above, include, but are not limited to, binders, fillers, disintegrants, lubricants, antimicrobial agents, and coating agents.

[0047] In the present invention, the intestinal disease may be characterized as being selected from the group consisting of abdominal distension, abdominal discomfort, infectious diarrhea caused by pathogenic microorganisms, gastroenteritis, inflammatory bowel disease, nervous enteritis syndrome, irritable bowel syndrome, small intestinal microbial overgrowth, and acute intestinal diarrhea, and includes diseases caused by damage to the normal intestinal barrier function.

[0048] The effect of improving, treating, or preventing intestinal disease exerted by the strain according to the present invention may be characterized by being induced by one or more of the following mechanisms:

[0049] Inhibiting the expression or secretion of one or more selected from the group consisting of the inflammatory cytokines TNF-α, IL-1β, and IFN-γ.

[0050] In the present invention, the term "effective amount (or effective quantity)" refers to an amount that is sufficient to deliver a desirable effect but small enough to sufficiently prevent serious side effects within the scope of medical judgment. The amount of microorganisms administered into the body by the composition of the present invention can be appropriately adjusted considering the route of administration and the target of administration.

[0051] The composition of the present invention may be administered to a subject at least once daily. A unit dose refers to a physically separated unit suitable for unit administration for human subjects and other mammals, and each unit contains a suitable pharmaceutical carrier and a predetermined amount of the microorganism of the present invention exhibiting a therapeutic effect. For oral administration to adult patients, a unit of the microorganism of the present invention preferably contains at least 0.001 g, and the oral dose of the composition of the present invention is 0.001 to 10 g, preferably 0.01 to 5 g, per dose. The pharmaceutically effective amount of the microorganism of the present invention is 0.01 to 10 g / day. However, the dose is variable depending on the severity of the patient's intestinal disease and the microorganism and auxiliary active ingredients used. Additionally, the total daily dose may be divided into multiple doses and administered continuously as needed. Therefore, the above dose range does not limit the scope of the present invention in any way.

[0053] In addition, the term "pharmaceuticalally acceptable" above refers to a composition that is physiologically acceptable and, when administered to humans, does not typically cause allergic reactions such as gastrointestinal disorders or dizziness, or similar reactions.

[0054] The composition of the present invention may be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to mammals. The formulation may be in the form of powder, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, or sterile powders. Additionally, the composition for the prevention or treatment of intestinal diseases according to the present invention may be administered via various routes including oral, transdermal, subcutaneous, intravenous, or intramuscular, and the dosage of the active ingredient may be appropriately selected according to various factors such as the route of administration, the patient's age, sex, body weight, and the severity of the patient. Furthermore, the composition for the prevention or treatment of gastrointestinal diseases according to the present invention may be administered in combination with known compounds having the effect of preventing, improving, or treating symptoms of intestinal diseases.

[0055] The pharmaceutical composition of the present invention may be provided, in particular, as an enteric-coated formulation as an oral unit formulation. The term “enteric coating” in this specification includes any known type of pharmaceutically acceptable coating that is not degraded by gastric acid and thus maintains the coating, but is sufficiently degraded in the small intestine to allow the active ingredient to be released into the small intestine. The “enteric coating” of the present invention refers to a coating that remains intact for at least 2 hours when contacted with artificial gastric juice, such as an HCl solution of pH 1, at 36°C to 38°C, and preferably degrades within 30 minutes thereafter in artificial intestinal juice, such as a KH2PO4 buffer solution of pH 6.8.

[0056] Specifically, the present invention relates to a composition comprising one or more selected from the group consisting of a strain of Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), a strain of Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or a strain of Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP) for use in the prevention or treatment of intestinal diseases, a culture of said strain, a lysate of said strain, and an extract of said strain.

[0057] The present invention also relates to the use of a composition comprising one or more selected from the group consisting of the strain Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), the strain Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or the strain Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP), a culture of said strain, a lysate of said strain, and an extract of said strain for manufacturing a drug for the prevention or treatment of intestinal diseases.

[0058] The present invention also relates to the use of a composition comprising one or more selected from the group consisting of the strain Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), the strain Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or the strain Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP), a culture of said strain, a lysate of said strain, and an extract of said strain for manufacturing a drug for the prevention or treatment of inflammatory diseases.

[0059] In the present invention, the term 'prevention' relates to preventing, delaying, impeding, or hindering disease.

[0060] In the present invention, the term 'treatment' relates to caring for a subject suffering from a disease in order to improve, heal, or reduce the symptoms of the disease, or to reduce or stop the progression of the disease.

[0061] The present invention relates to a method for treating an intestinal disease comprising the step of administering a therapeutically effective amount of one or more selected from the group consisting of the strain Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), the strain Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or the strain Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP), a culture of said strain, a lysate of said strain, and an extract of said strain to an individual in need thereof.

[0062] The present invention provides an antimicrobial composition containing an effective amount of at least one culture of the strain, at least one lysate of the strain selected from the group consisting of Lactobacillus plantarum CLP2001 (accession number KCTC 14848BP), Lactobacillus plantarum CLP2002 (accession number KCTC 14849BP), or Lactobacillus paracasei CLB0820 (accession number KCTC 14850BP), a culture of the strain, a lysate of the strain, and an extract of the strain. Preferably, the antimicrobial composition may have antimicrobial activity against microorganisms that cause bacterial diarrhea. Preferably, the microorganisms causing bacterial diarrhea may include one or more selected from the group consisting of Staphylococcus aureus, Bacillus cereus, Escherichia coli (E. coli), Salmonella enterica, Enterobacter cloacae, and Listeria monocytogenes. The antimicrobial composition may include an antimicrobial composition that is a food, a food additive, or a pharmaceutical composition.

[0064] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the embodiments according to the present invention may be modified in various different forms, and the scope of the present invention should not be interpreted as being limited to the following embodiments. The embodiments of the present invention are provided to more completely explain the present invention to those with average knowledge in the field to which the present invention pertains. Unless otherwise specified, % in this specification may be understood to mean weight %.

[0066] 1. 균주 기탁번호

[0067] - CLP2001 KCTC 14848BP

[0068] - CLP2002 KCTC 14849BP

[0069] - CLB0820 KCTC 14850BP

[0071] 2. Sequencing (16S sequencing)

[0072] 1) CLP2001 (1439bp)

[0073] ⇒ Identification Result : Lactobacillus plantarum

[0075] 2) CLP2002 (1432bp)

[0076] ⇒ Identification Result : Lactobacillus plantareum

[0078] 3) CLB0820 (1440bp)

[0079] ⇒ Identification Result : Lactobacillus paracasei

[0081] 3. 유산균의 Thanksgiving Application Api kit installation(API kit installation)

[0082] - Verify sugar usability using the API 50 CHL kit.

[0083] < Test Method >

[0084] a. Inoculate each strain into 5 ml of MRS broth and incubate at 37°C for 24 hours.

[0085] B. Suspend the culture medium in 10 ml of API suspension medium. (Adjust to the concentration of McFarland Standard 2.)

[0086] c. Dispense approximately 120 µl of the strain suspension into each well of the API 50CHL kit, then add mineral oil on top of the suspension.

[0087] D. After incubating at 37℃ for 48 hours, observe whether there is a change in color to observe whether each carbon source is utilized.

[0088] E. Verify the identification results by entering the above result values ​​into the identification program, API web.

[0090] 1) CLP2001

[0091] ⇒ Identification Result : Lactobacillus plantarum

[0092] Substrate Result Substrate Result Substrate Result Control(Negative) - Inositol - Melezitose + Glycerol - Mannitol + Raffinose - Erythritol - Sorbitol + Starch - D-arabinose - α-methyl-D-mannoside + Glycogen - L-arabinose - α-methyl-D glucoside - Xylitol - D-ribose + N-acethyl-glucosamine + Gentiobiose + D-xylose - Amygdalin + D-turanose + L-xylose - Arbutin + D-lyxose - D-adonitol - Esculin + D-tagatose - Methyl-β D-xylopyranoside - Salicin + D-fructose - D-galactose + Cellobiose + L-fructose - D-glucose + Maltose + D-arabitol - D-fructose + Lactose + L-arabitol - D-Mannose + Melibiose + Gluconate - L-sorbose - Sucrose + 2-keto-gluconate - Rhamnose - Trehalose + 5-keto-gluconate - Dulcitol - Inulin -

[0094] 2) CLP2002

[0095] ⇒ Identification Result : Lactobacillus plantarum

[0096] Substrate Result Substrate Result Substrate Result Control(Negative) - Inositol - Melezitose + Glycerol - Mannitol + Raffinose - Erythritol - Sorbitol + Starch - D-arabinose - α-methyl-D-mannoside + Glycogen - L-arabinose - α-methyl-D glucoside - Xylitol - D-ribose + N-acethyl-glucosamine + Gentiobiose + D-xylose - Amygdalin + D-turanose + L-xylose - Arbutin + D-lyxose - D-adonitol - Esculin + D-tagatose - Methyl-β D-xylopyranoside - Salicin + D-fructose - D-galactose + Cellobiose + L-fructose - D-glucose + Maltose + D-arabitol - D-fructose + Lactose + L-arabitol - D-Mannose + Melibiose + Gluconate - L-sorbose - Sucrose + 2-keto-gluconate - Rhamnose - Trehalose + 5-keto-gluconate - Dulcitol - Inulin -

[0098] 3) CLB0820

[0099] ⇒ Identification Result : Lactobacillus paracasei

[0100] Substrate Result Substrate Result Substrate Result Control(Negative) - Inositol - Melezitose + Glycerol - Mannitol + Raffinose - Erythritol - Sorbitol + Starch - D-arabinose - α-methyl-D-mannoside - Glycogen - L-arabinose - α-methyl-D glucoside - Xylitol - D-ribose + N-acethyl-glucosamine + Gentiobiose + D-xylose - Amygdalin + D-turanose + L-xylose - Arbutin + D-lyxose - D-adonitol - Esculin + D-tagatose + Methyl-β D-xylopyranoside - Salicin + D-fructose - D-galactose + Cellobiose + L-fructose - D-glucose + Maltose + D-arabitol - D-fructose + Lactose - L-arabitol + D-Mannose + Melibiose - Gluconate - L-sorbose + Sucrose + 2-keto-gluconate - Rhamnose - Trehalose + 5-keto-gluconate - Dulcitol - Inulin -

[0101] As a result of identification, CLP2001 and CLP2002 were identified as Lactobacillus plantarum species, and CLB0820 as Lactobacillus paracasei species, and it was confirmed that they are new strains that had not been previously identified.

[0103] 4. Application Application(API Zym kit 실험)

[0104] < Test Method >

[0105] a. Inoculate each strain into 5 ml of MRS broth and incubate at 37°C for 24 hours.

[0106] B. Suspend the culture medium in 2 ml of physiological saline (0.85% NaCl). (Adjust to a concentration of McFarland standard 5–6.)

[0107] c. Dispense 65 µl of the strain suspension into each well of the API Zym kit, then add mineral oil on top of the suspension.

[0108] D. Drop Zym A and Zym B reagents into each well and observe the results after 5 minutes.

[0109] When Enzyme CLP2001 CLP2002 CLB0820 1 Control 2 Alkaline phosphatase - - - 3 Esterase(C4) - - + 4 Esterase Lipase(C8) - - + 5 Lipase(C14) - - - 6 Leucine arylamidase - - + 7 Valine arylamidase - - v 8 Crystine arylamidase - - - 9 Trypsin - - - 10 α-chymotrypsin - - - 11 Acid phosphatase + + + 12 Naphtol-AS-BI-phosphohydrolase + + + 13 α-galactosidase - - - 14 β-glucuronidase + + - 15 β-glucosidase - - - 16 α-glucosidase + + + 17 β-glucosidase + + - 18 N-acetyl-β-glucosaminidase + + - 19 α-mannosidase - - - 20 α-fucosidase - - -

[0111] 5. Hemolytic Confirmation Test

[0112] Test Method

[0113] (1) Prepare a blood agar medium by adding 5% defibrinated blood to Tryptic soy agar.

[0114] (2) Spread the target strain on blood agar medium and incubate at 37°C for 48 hours.

[0115] (3) After culture, observe whether a clear ring forms around the colony.

[0117] <Result>

[0118] (1) L. plantarum CLP2001

[0119] In cases where β-hemolysis related to pathogenicity occurs, red blood cells are completely destroyed on blood agar medium, and a clear halo forms around the colony, as shown in the right part of the medium in Figure 1 (positive control, B. cereus smear). Since no clear area appeared on the left side of the medium in which L. plantarum CLP2001 was cultured, it was confirmed that there was no β-hemolysis.

[0120] (2) L. plantarum CLP2002

[0121] In cases where β-hemolysis related to pathogenicity occurs, red blood cells are completely destroyed on blood agar medium, and a clear halo forms around the colony, as shown in the right part of the medium in Figure 2 (positive control, B. cereus smear). Since no clear area appeared on the left side of the medium in which L. plantarum CLP2002 was cultured, it was confirmed that there was no β-hemolysis.

[0122] (3) L.paracasei CLB0820

[0123] In cases where β-hemolysis related to pathogenicity occurs, red blood cells are completely destroyed on blood agar medium, forming a clear halo around the colony as shown in the right part of the medium in Figure 3 (positive control, B. cereus smear). Since no clear area appeared on the left side of the medium in which L. paracasei CLB0820 was cultured, it was confirmed that there was no β-hemolysis.

[0125] 6. acid resistance and Internal bile acidity test

[0126] Test Method

[0127] 1) Acid resistance

[0128] (1) Each strain is inoculated into 10 ml of MRS broth and cultured for 24 hours to be used as a test solution.

[0129] (2) Inoculate 100 µl of the test solution into 10 ml of artificial gastric fluid (pH 2) and incubate at 37°C.

[0130] (2) Sampling is performed using a rotator at 0 hours, 30 minutes, 1 hour, and 2 hours, serially diluted, and plated on MRS agar.

[0131] (3) Check the number of viable cells after incubation at 37℃ for 48 hours.

[0133] 2) Bile acidity

[0134] (1) Prepare MRS agar and MRS agar with 0.3% oxgall added, respectively.

[0135] (2) Dilute the culture solution of each strain cultured overnight by an appropriate amount and spread it onto the two media prepared above. After incubating at 37°C, compare the colony formation on both media.

[0137] <Result>

[0138] (1) L. plantarum CLP2001

[0139] Since probiotics are exposed to an acidic environment caused by gastric acid in the stomach and bile salts in the small intestine after ingestion, acid resistance and bile acid resistance are important characteristics. The survival rate of L. plantarum CLP2001 at pH 2.5 was 89% at 2 hours, and the survival rate on 0.3% oxgall MRS agar was 13.1% compared to MRS agar.

[0141] 1) Acid resistance

[0142] hour CLP2001 CFU / ml Survival rate (%) 0hr 2.34E+07 100% 1hr 2.37E+07 101% 2hr 2.08E+07 89%

[0143] 2) Bile acidity

[0144] CFU / ml Survival rate (%) MRS agar 1.80×10 9 　- MRS agar with 0.3% oxgall added 1.58×10 9 88%

[0145] (2) L. plantarum CLP2002

[0146] Since probiotics are exposed to the acidic environment caused by gastric acid in the stomach and bile salts in the small intestine after ingestion, acid resistance and bile acid resistance are important characteristics. L. planrarum The survival rate of CLP2002 at pH 2.5 was 87% at 2 hours, and the survival rate on 1% oxgall MRS agar was 97% compared to MRS agar.

[0148] 1) Acid resistance

[0149] hour CLP2002 CFU / ml Survival rate (%) 0hr 1.74E+07 100% 1hr 1.86E+07 107% 2hr 1.52E+07 87%

[0150] 2) Bile acidity

[0151] CFU / ml Survival rate (%) MRS agar 2.05×10 9 　- MRS agar with 1% oxgall added 1.98×10 9 97%

[0153] (3) L.paracasei CLB0820

[0154] Since probiotics are exposed to the acidic environment caused by gastric acid in the stomach and bile salts in the small intestine after ingestion, acid resistance and bile acid resistance are important characteristics. L. paracasei The survival rate of CLB0820 at pH 2.5 was 89% after 2 hours, and the survival rate on 1% oxgall MRS agar was 68% compared to MRS agar.

[0156] 1) Acid resistance

[0157] hour CLB0820 CFU / ml Survival rate (%) 0hr 5.05E+07 100% 1hr 5.45E+07 108% 2hr 4.50E+07 89%

[0158] 2) Bile acidity

[0159] CFU / ml Survival rate (%) MRS agar 6.65×10 9 　- MRS agar with 1% oxgall added 4.55×10 9 68%

[0161] 7. Bile salt deconjugation

[0162] Test Method

[0163] (1) Prepare a medium containing 0.5% (w / v) of taurodeoxycholic acid and store it under anaerobic conditions for at least 48 hours before use.

[0164] (2) Spread 10 µl of overnight culture onto a line using a loop.

[0165] (3) After incubating at 37℃ for more than 72 hours, check if precipitation has occurred around the colony.

[0167] <Result>

[0168] (1) L. plantarum CLP2001

[0169] Microorganisms with bile salt hydrolase (BSH) activity cleave the amino acid bonds of conjugated bile acid, converting it into unconjugated bile acid, which results in a precipitate appearing around the colony. In the case of L. plantarum CLP2001, no precipitate was observed around the colony, confirming the absence of BSH activity.

[0170] (2) L. plantarum CLP2002

[0171] Microorganisms with bile salt hydrolase (BSH) activity cleave the amino acid bonds of conjugated bile acid, converting it into unconjugated bile acid, which results in a precipitate appearing around the colony. In the case of L. plantarum CLP2002, no precipitate was observed around the colony, confirming the absence of BSH activity.

[0172] (3) L.paracasei CLB0820

[0173] Microorganisms with bile salt hydrolase (BSH) activity cleave the amino acid bonds of conjugated bile acid, converting it into unconjugated bile acid, which results in a precipitate appearing around the colony. In the case of L. paracasei CLB0820, no precipitate was observed around the colony, confirming the absence of BSH activity.

[0175] 8. D- / L- lactic acid generation ability

[0176] Test Method

[0177] (1) Inoculate the strain into MRS broth and culture at 37°C for 24 hours.

[0178] (2) Filter the culture solution to 0.22 µm and dilute it to an appropriate concentration to use as a test solution.

[0179] (3) Use uninoculated MRS broth as a blank.

[0180] (4) Measure the absorbance at 340 nm after testing according to the Megazyme kit (K-DLATE) test method.

[0181] (5) Calculate the amount of lactic acid based on the measured absorbance.

[0183] <Result>

[0184] (1) L. plantarum CLP2001

[0185] L. salivarius When CLS0420 was cultured in MRS broth for 24 hours, the amount of D-lactic acid was 9.74 g / L and the amount of L-lactiacid was detected as 7.08 g / L.

[0186] lactate concentration ratio (g / L) D-lactic acid 9.74 57.9% L-lactic acid 7.08 42.1%

[0187] (2) L. plantarum CLP2002

[0188] L. plantarum When CLP2002 was cultured in MRS broth for 24 hours, the amount of D-lactic acid was 9.64 g / L and the amount of L-lactiacid was detected as 7.01 g / L.

[0189] lactate concentration ratio (g / L) D-lactic acid 9.64 57.9% L-lactic acid 7.01 42.1%

[0190] (3) L.paracasei CLB0820

[0191] L. paracasei When CLB0820 was cultured in MRS broth for 24 hours, the amount of D-lactic acid was 1.12 g / L and the amount of L-lactiacid was detected as 18.36 g / L.

[0192] lactate concentration ratio (g / L) D-lactic acid 1.12 5.7% L-lactic acid 18.36 94.3%

[0194] 9. Antibiotic resistance

[0195] Test Method

[0196] (1) After culturing the strain on MRS agar, inoculate the colony into 10 ml of Mueller Hinton broth and culture for 4 to 6 hours.

[0197] (2) The prepared culture medium was prepared using sterile physiological saline McFarland 0.5 (OD 0.08~0.1, ~1x10 8 Adjust to approximately CFU.ml, then dilute 10 times (~1x10 7 CFU / ml)

[0198] (3) Add antibiotic stock to Mueller Hinton broth to achieve the highest antibiotic concentration to be tested, and add 200 µl to the 12th column of a sterile 96-well plate.

[0199] (4) Dilute the antibiotics to the concentrations shown in the table using 1 / 2 serial dilution. Finally, add 100 µl of the antibiotic dilution solution to each well.

[0200] (5) Inoculate 10 µl of the bacterial dilution prepared above into each well. (~5 x 10 4 CFU / 110ul)

[0202] <Result>

[0203] (1) L. plantarum CLP2001

[0204] <L. plantarum CLP2001의 MIC 평가 결과>

[0205] Antibiotics MIC Cut-off value (mg / L) by EFSA guide MIC Result(mg / L) Susceptible / Resistant Ampicillin 2 0.5 S Vancomycin not required Gentamycin 16 1 S Kanamycin 64 4 S Streptomycin not required Erythromycin 1 0.032 S Clindamycin 2 P S Tetracycline 32 32 S Chlormaphenicol 8 4 S

[0206] Judgment criteria

[0207] MIC>cut-off value : Evaluated as Resistant(R);

[0208] MIC cut-off value: Evaluated as Susceptible(S)

[0210] When evaluated according to the EFSA guide, regarding the 7 antibiotics tested as above (ampicillin, gentamicin, kanamycin, erythrolmycin, clindamycin, tetracycline, chloramphenicol) L. plantarum CLP2001 did not show antibiotic resistance. .

[0212] (2) L. plantarum CLP2002

[0213] < L. plantarum CLP2002 MIC Evaluation Results>

[0214] Antibiotics MIC Cut-off value (mg / L) by EFSA guide MIC Result(mg / L) Susceptible / Resistant Ampicillin 2 0.5 S Vancomycin not required Gentamycin 16 1 S Kanamycin 64 4 S Streptomycin not required Erythromycin 1 0.032 S Clindamycin 2 P S Tetracycline 32 32 S Chlormaphenicol 8 4 S

[0215] Judgment criteria

[0216] MIC>cut-off value : Evaluated as Resistant(R);

[0217] MIC cut-off value: Evaluated as Susceptible(S)

[0219] When evaluated according to the EFSA guide, regarding the 7 antibiotics tested as above (ampicillin, gentamicin, kanamycin, erythrolmycin, clindamycin, tetracycline, chloramphenicol) L. plantarum CLP2002 did not show antibiotic resistance. .

[0221] (3) L.paracasei CLB0820

[0222] < L. paracasei CLB0820 MIC Evaluation Results>

[0223] Antibiotics MIC Cut-off value(mg / L) by EFSA guide MIC Result(mg / L) Susceptible / Resistant Ampicillin 4 1 S Vancomycin not required Gentamycin 32 2 S Kanamycin 64 8 S Streptomycin 16 16 S Erythromycin 1 P S Clindamycin 1 P S Tetracycline 4 2 S Chlormaphenicol 4 4 S

[0224] Judgment criteria

[0225] MIC>cut-off value : Evaluated as Resistant(R);

[0226] MIC cut-off value: Evaluated as Susceptible(S)

[0228] When evaluated according to the EFSA guide, regarding the 8 antibiotics tested as above (ampicillin, gentamicin, kanamycin, streptomycin, erythrolmycin, clindamycin, tetracycline, chloramphenicol) L. paracasei CLB0820 did not show antibiotic resistance. .

[0230] 10. 항균력

[0231] Test Method

[0232] A. Preparation of lactic acid bacteria

[0233] (1) Inoculate each lactic acid bacteria strain into 30 ml of MRS broth and culture overnight at 37°C.

[0234] (2) Centrifuge the cultured lactic acid bacteria to obtain the pellet.

[0235] (3) Wash with 20 ml of sterile physiological saline (0.85% NaCl) and centrifuge, then discard the supernatant.

[0236] (4) Suspend in 10 ml of phosphate buffer solution (pH 7.3).

[0238] B. Preparation of pathogenic strains

[0239] (1) Inoculate each strain into a medium suitable for culture and then culture overnight before use.

[0241] C. Antimicrobial activity test

[0242] Experiments were conducted to confirm the antimicrobial activity against pathogenic microorganisms capable of causing inflammation in the large intestine. The test method is as follows.

[0244] Each lactic acid bacterium is inoculated into MRS broth and cultured overnight at 37°C. After culture is complete, the lactic acid bacteria are centrifuged, the cells are washed twice with PBS (pH 7.0), and then resuspended in 10 ml of PBS to prepare them.

[0245] Pathogenic microorganisms are prepared by inoculating each into a suitable medium and culturing overnight.

[0246] The antibacterial activity experiment was conducted in the following manner.

[0247] The lactic acid bacteria prepared above are mixed with MRS agar (agar 0.8%) in a 1:1 ratio, 20 µl is dropped onto an MRS agar plate, and the mixture is left to stand at room temperature until it solidifies, followed by overnight incubation under anaerobic conditions at 37°C. Select a medium appropriate for each pathogenic microorganism, prepare Top agar media (agar concentration: 0.8%), and add 100 µl of the pathogenic microorganism culture solution prepared by overnight incubation into 4 ml of Top agar media. Then, dispense the mixture onto an MRS agar plate containing cultured lactic acid bacteria and shake to ensure even distribution. Leave the mixture to stand at room temperature until the Top agar solidifies, followed by overnight incubation at the growth temperature of the pathogen.

[0248] Antibacterial activity was confirmed by examining the clearing zone on the plate after culture was completed.

[0250] <Result>

[0251] As can be seen in Figures 7 to 9, it was confirmed that the strains L. plantarum CLP2001, L. plantarum CLP2002, and L. paracasei CLB0820 have antibacterial activity against Staphylococcus aureus, Bacillus cereus, E. coli, Salmonella enterica, Enterobacter cloacae, and Listeria monocytogenes.

[0253] 11. 유산균의 과민면역반응 완화시험

[0254] Experiments to mitigate hypersensitivity immune responses were conducted using peripheral blood mononuclear cells (PBMC), splenocytes, and the macrophage cell line Raw264.7, which are immune cells isolated from Balb / c mice.

[0255] The test group consisted of a normal control group that was not treated with a stimulant, a negative control group that was treated with a stimulant, a positive control substance 1 (L. rhamnosus GG) treatment group, a positive control substance 2 (Dexamethasone) treatment group, and the test substance on PBMC, splenocyte, and Raw264.7.

[0256] Stimulant and test substance were administered to all test groups except the normal control group in 5x10 doses 7 CFU / ml or 10x10 7 Each was treated at CFU / ml and cultured for 24 to 96 hours. Then, the amount of cytokines in the culture medium was measured using an ELISA kit to evaluate the test substance that alleviates the hypersensitivity immune response induced by the stimulant.

[0257] 1) The hypersensitivity immune response of lactic acid bacteria was evaluated using peripheral blood mononuclear cells (PBMC), splenocytes, and the macrophage cell line Raw264.7, which are immune cells isolated from mice.

[0258] 2) The test groups consisted of PBMCs and splenocytes isolated from mice, as well as the mouse macrophage cell line Raw264.7, comprising a normal control group without stimulant treatment, a negative control group treated with stimulant, a group treated with a positive control (Lactobacillus rhamnosus GG), a group treated with a positive control (Dexamethasone), and test substances (Lactobacillus plantarum CLP2001, Lactobacillus plantarum CLP2002, Lactobacillus paracasei CLB0820). For all test groups except the normal control, the stimulant and test substance were administered at a dose of 5 x 10⁶ 7 CFU or 10x10 7 Treated with CFU and cultured for 24 to 96 hours.

[0260] <Test Results>

[0261] 1) In the case of Raw264.7, it was confirmed that a hypersensitive immune response was induced, as TNF-α and IL-1β significantly increased in the negative control group treated with the stimulant LPS compared to the normal control group. As a result of treatment with the test substance under these conditions, TNF-α and IL-1β showed a significant decrease in all test groups, demonstrating an effect equivalent to or greater than that of the positive control substance.

[0262] 2) In the case of PBMC, it was confirmed that a hypersensitive immune response was induced, as TNF-α, IL-1β, and IFN-γ significantly increased in the negative control group treated with the stimulant LPS compared to the normal control group. When the test substance was applied under these conditions and the changes in each cytokine were measured, TNF-α, IL-1β, and IFN-γ significantly decreased in all test substance treatment groups compared to the negative control group.

[0264] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC14848BP Date of Deposit: 2022-01-13 Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC14849BP Date of Deposit: 2022-01-13 Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC14850BP Date of Deposit: 2022-01-13

Claims

Claim 1 Lactobacillus plantarum CLP2001 strain deposited under accession number KCTC 14848BP, having antimicrobial activity against one or more species selected from the group consisting of Bacillus cereus, Enterobacter cloacae, and Listeria monocytogenes. Claim 2 delete Claim 3 delete Claim 4 A food composition for preventing colitis caused by one or more selected from the group consisting of Bacillus cereus, Enterobacter cloacae, and Listeria monocytogenes, containing an effective amount of one or more selected from the group consisting of the strain of claim 1, a culture of the strain, a lysate of the strain, and an extract of the strain. Claim 5 delete Claim 6 A composition according to claim 4, characterized in that the above composition is a composition for health functional foods. Claim 7 A pharmaceutical composition for the treatment or prevention of colitis caused by one or more selected from the group consisting of Bacillus cereus, Enterobacter cloacae, and Listeria monocytogenes, containing an effective amount of one or more selected from the group consisting of the strain of claim 1, a culture of the strain, a lysate of the strain, and an extract of the strain. Claim 8 delete Claim 9 delete Claim 10 An antimicrobial composition containing an effective amount of one or more selected from the group consisting of the strain of claim 1, a culture of the strain, a lysate of the strain, and an extract of the strain, wherein the composition has antimicrobial activity against one or more selected from the group consisting of Bacillus cereus, Enterobacter cloacae, and Listeria monocytogenes. Claim 11 delete Claim 12 In claim 10, the above antimicrobial composition is an antimicrobial composition that is a food, a food additive, or a pharmaceutical composition.

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