Novel Bacillus subtilis strains and methods for the prevention, treatment, or improvement of tuberculosis containing the same.

Abstract

The present invention relates to a novel Bacillus subtilis strain and a method for the prevention, treatment, or improvement of tuberculosis containing the same. More specifically, the present invention relates to Bacillus subtilis strain PMC 205 extracted from kimchi and its uses, for example, an antimicrobial composition having antimicrobial activity against bacteria belonging to the genus Mycobacterium, a pharmaceutical composition or food composition for the improvement, treatment, or prevention of tuberculosis having antimicrobial activity against bacteria belonging to the genus Mycobacterium.

Description

Technical Field

[0001] The present invention relates to a novel Bacillus subtilis strain and a method for preventing, treating or improving tuberculosis comprising the same. More specifically, the present invention relates to the Bacillus subtilis PMC 205 strain extracted from kimchi and its uses, for example, an antibacterial composition having antibacterial activity against bacteria belonging to the genus Mycobacterium, a pharmaceutical composition or a food composition for improving, treating or preventing tuberculosis having antibacterial activity against bacteria belonging to the genus Mycobacterium.

Background Art

[0002] Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Tuberculosis remains a major public health problem worldwide, causing approximately 1.8 million deaths each year. Drug-resistant Mycobacterium tuberculosis, including multidrug-resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant (TDR), continues to pose a threat to public health. Antibiotic-resistant tuberculosis has a low treatment effect when treated with existing antibiotics. In addition, in the case of existing therapeutic drugs, side effects caused by taking the drugs for a long period of time at high doses have caused serious problems.

[0003] Therefore, in order to overcome such problems, the development of a new form of anti-tuberculosis drug different from conventional compound-based antibiotics has become an urgent task.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

[0005] The present invention aims to provide the Bacillus subtilis PMC 205 strain.

[0006] The present invention also aims to provide an antimicrobial composition containing Bacillus subtilis PMC 205 strain.

[0007] The present invention also aims to provide a composition for the prevention, treatment, or improvement of tuberculosis containing the Bacillus subtilis PMC 205 strain. [Means for solving the problem]

[0008] According to the first embodiment, The present invention aims to provide Bacillus subtilis PMC 205 strain or its culture medium, which has antibacterial activity against bacteria belonging to the genus Mycobacterium.

[0009] According to the present invention, the Bacillus subtilis PMC 205 strain may have the deposit number KCTC 15188BP extracted from kimchi.

[0010] According to the present invention, the fungi belonging to the genus Mycobacterium include Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, and Mycobacterium avium complex. Mycobacterium complex (MAC), Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium leprae, Mycobacterium marmoe One or more species may be selected from the group consisting of Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium microti, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium xenopi.

[0011] According to the second embodiment, The present invention aims to provide an antimicrobial composition having antimicrobial activity against bacteria belonging to the genus Mycobacterium, including Bacillus subtilis PMC 205 strain or its culture solution.

[0012] According to the present invention, the Bacillus subtilis PMC 205 strain may have the deposit number KCTC 15188BP extracted from kimchi.

[0013] According to the present invention, the fungi belonging to the genus Mycobacterium include Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, and Mycobacterium avium complex. Mycobacterium complex (MAC), Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium leprae, Mycobacterium marmoe One or more species may be selected from the group consisting of Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium microti, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium xenopi.

[0014] In the present invention, the antibacterial composition can be used in living organisms.

[0015] In the present invention, the antibacterial composition can be used in an in vivo environment.

[0016] According to the third embodiment, The present invention aims to provide a pharmaceutical composition for the treatment or prevention of tuberculosis that has antibacterial activity against bacteria belonging to the genus Mycobacterium, comprising Bacillus subtilis PMC 205 strain or its culture solution as an active ingredient.

[0017] According to the present invention, the Bacillus subtilis PMC 205 strain may have the deposit number KCTC 15188BP extracted from kimchi.

[0018] According to the present invention, the fungi belonging to the genus Mycobacterium include Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, and Mycobacterium avium complex. Mycobacterium complex (MAC), Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium leprae, Mycobacterium marmoe One or more species may be selected from the group consisting of Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium microti, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium xenopi.

[0019] In the present invention, the tuberculosis may be selected from one or more of the group consisting of ocular tuberculosis, cutaneous tuberculosis, adrenal tuberculosis, renal tuberculosis, epididymal tuberculosis, scrofula, laryngeal tuberculosis, otitis media tuberculosis, intestinal tuberculosis, multi-drug resistant tuberculosis, pulmonary tuberculosis, biliary tuberculosis, bone tuberculosis, pharyngeal tuberculosis, scrofula, pulmonary cachexia, breast tuberculosis, and spinal tuberculosis.

[0020] In the present invention, the tuberculosis may be latent tuberculosis.

[0021] In the present invention, the pharmaceutical composition may further contain at least one selected from the group consisting of rifamficin, rifapentine, isoniazid, pyrazinamide, ethambutol, streptomycin, fluoroquinolone, kanamycin, Cycloserine, Prothionamide, Levofloxacin, Moxifloxacin, Ofloxacin, Rifabutin, Capeomycin, amikacin, ciprofloxacin, protionamide, ethionamide, cycloserine, thioacetazone, clofazimine, amoxicillin / clavulanate, derivative of dianomidiphenylsulphone, clarithromycin, azithromycin, and Linezolid.

[0022] According to the fourth embodiment, The present invention aims to provide a food composition for preventing or improving tuberculosis, which has antibacterial activity against bacteria belonging to the genus Mycobacterium and contains Bacillus subtilis PMC 205 strain or its culture solution as an active ingredient.

[0023] According to the present invention, the Bacillus subtilis PMC 205 strain may have the deposit number of KCTC 15188BP extracted from kimchi.

[0024] According to the present invention, the bacteria belonging to the genus Mycobacterium may be selected from one or more of the group consisting of Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, Mycobacterium avium complex (MAC), Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium leprae, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium microti, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium xenopi.

[0025] In the present invention, the tuberculosis can be selected from one or more of the group consisting of ocular tuberculosis, cutaneous tuberculosis, adrenal tuberculosis, renal tuberculosis, epididymal tuberculosis, lymphadenopathy, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, bile duct tuberculosis, bone tuberculosis, pharyngeal tuberculosis, lymphadenopathy, pulmonary deficiency, breast tuberculosis, and spinal tuberculosis.

[0026] In the present invention, the tuberculosis may be latent tuberculosis. [Effects of the Invention]

[0027] This invention provides a strain of Bacillus subtilis PMC 205, which has antibacterial activity. More specifically, the strain has antibacterial activity against Mycobacterium tuberculosis. Thus, the strain can be used to treat, prevent, or improve tuberculosis.

[0028] In this case, the aforementioned bacterial strain has the advantage of not developing drug resistance or side effects from long-term, high-dose drug therapy compared to compound-based drug therapy. [Brief explanation of the drawing]

[0029] [Figure 1] This invention demonstrates the genetic characteristics and in vitro anti-tuberculosis efficacy of Bacillus subtilis PMC 205, showing (A) the results of high-speed genome sequencing, (B) the results of COG (orthologous gene cluster) classification clusters, and (C) the results of Ortho ANI (average nucleotide identity). [Figure 2] The results of the evaluation of the anti-mycobacterial activity of Bacillus subtilis PMC 205 according to the present invention are shown. [Figure 3] This shows the efficacy evaluation of Bacillus subtilis PMC 205 according to the present invention in a lethal XDR pulmonary tuberculosis mouse model, and presents the results for (A) survival rate, (B) body weight, (C) disease severity score, and (D) CFU / lung. [Figure 4]This invention illustrates the characteristics of the pulmonary microbiome in the treatment of M. tuberculosis with Bacillus subtilis PMC 205, and shows (A) the cytokine content and (B) the results of analyzing the pulmonary microbiome based on the 16S rRNA gene. [Figure 5] This paper shows the efficacy evaluation of Bacillus subtilis PMC 205 according to the present invention on intrapulmonary M. tuberculosis in a mouse model of latent tuberculosis, and presents the results for (A) body weight and (B) CFU / lung. [Modes for carrying out the invention]

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by a person of ordinary skill in the art to which this invention pertains. All publications, patents, and other references referenced herein are included in their entirety for reference only.

[0031] As used herein, "latent tuberculosis" means inactive tuberculosis, specifically tuberculosis in a dormant state following primary tuberculosis infection, and refers to Mycobacterium tuberculosis infecting a host that does not exhibit symptoms of the disease.

[0032] As used herein, the term "antimicrobial" means killing or inhibiting the growth of microorganisms. For example, it may be used herein to mean killing or inhibiting the growth of specific bacteria. More specifically, it may be used herein to mean killing or inhibiting the growth of bacteria belonging to the genus Mycobacterium. More specifically, it may be used herein to mean Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, and Mycobacterium avium complex. Mycobacterium complex (MAC), Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium leprae, Mycobacterium malmoense, M It may also be used to mean killing or inhibiting the growth of one or more bacteria selected from the group consisting of Icobacterium marinum, Mycobacterium microti, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium xenopi.

[0033] As used herein, the term "prevention" may include, without limitation, any action taken to block, suppress, or delay symptoms caused by tuberculosis using the composition of the present invention, which contains Bacillus subtilis PMC 205 strain as an active ingredient.

[0034] As used herein, the terms “improvement” and “treatment” may include, without limitation, any action that improves or alleviates symptoms caused by tuberculosis using the composition of the present invention, which contains Bacillus subtilis PMC 205 strain as an active ingredient.

[0035] *The specific details of the invention are disclosed below.

[0036] I. Strains Strains obtained from fermented foods The present invention aims to provide the Bacillus subtilis PMC 205 strain, which can be obtained from fermented foods. According to one exemplary embodiment, the Bacillus subtilis PMC 205 strain may have the deposit number KCTC 15188BP, extracted from kimchi.

[0037] Non-cytotoxic strains Antimicrobial compositions used in living organisms and in the extracellular environment. The present invention aims to provide an antimicrobial composition containing the Bacillus subtilis PMC 205 strain. Since this strain is non-cytotoxic, it can be included in compositions used both in vivo and in vitro.

[0038] The aforementioned antimicrobial composition may be an antimicrobial composition administered to a living organism. In this case, the living organism means a living individual, and the individual includes mammals, including humans. That is, as one embodiment, the present invention provides an antimicrobial composition having antimicrobial activity against undesirable microorganisms present in mammals, including humans. As a specific embodiment, the present invention provides an antimicrobial composition having antimicrobial activity against pathogenic bacteria present in mammals, including humans.

[0039] The antimicrobial composition containing the Bacillus subtilis PMC 205 strain may be an antimicrobial composition having antimicrobial activity against undesirable microorganisms present in the extravitae. As a specific example, the antimicrobial composition containing the Bacillus subtilis PMC 205 strain provides an antimicrobial composition having antimicrobial activity against pathogenic bacteria present in the extravitae. Here, "extravitae" may, in one example, mean the surface of a substance that needs to be cleaned. In this case, "cleaning" may mean removing undesirable microorganisms. In this case, "removal" may mean killing some of the bacteria, inhibiting bacterial growth, or reducing the number of bacteria.

[0040] The surface of the aforementioned substance may include, but is not limited to, the surfaces of household goods such as furniture, tableware, and home appliances, and includes all surfaces of substances that require cleaning. In other words, the present invention provides an antimicrobial composition having antimicrobial activity against pathogenic microorganisms present in the extracorporeal environment.

[0041] Antimicrobial methods used in living organisms and the extracellular environment The present invention also aims to provide an antimicrobial method using an antimicrobial composition containing the Bacillus subtilis PMC 205 strain.

[0042] The antimicrobial method may include administering the antimicrobial composition to a living organism. The living organism means a living individual, and the individual includes mammals, including humans. That is, the present invention provides an antimicrobial method that includes administering the composition to a mammal, including humans.

[0043] The antimicrobial method may also include treating the antimicrobial composition outside of a living organism. "Outside of a living organism" means the surface of a substance that needs to be cleaned, and the present invention provides an antimicrobial method for treating the surface of a substance that needs to be cleaned with the antimicrobial composition.

[0044] Morphology of the bacterial strains contained in the composition The present invention provides Bacillus subtilis PMC 205 strain and antimicrobial compositions containing said strain, pharmaceutical compositions for the treatment, prevention, and improvement of tuberculosis and inflammatory diseases, and food compositions. In one example, the composition may contain Bacillus subtilis PMC 205 strain in the form of live bacteria, dead bacteria, or culture solution thereof. The culture solution may be a stock culture solution containing Bacillus subtilis PMC 205 strain in the present invention, or it may contain its crushed products, centrifugation supernatant, pellets, concentrates, dried products, etc.

[0045] II. Antibacterial composition The present invention provides an antimicrobial composition containing Bacillus subtilis PMC 205 strain. The antimicrobial composition may have antimicrobial activity against bacteria belonging to the genus Mycobacterium. As a specific example, the antimicrobial composition may contain Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, and Mycobacterium avium complex. Mycobacterium complex (MAC), Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium leprae, Mycobacterium malmoens The antibacterial composition may have antibacterial activity against one or more bacteria selected from the group consisting of Mycobacterium ense, Mycobacterium marinum, Mycobacterium microti, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium xenopi. More specifically, the antibacterial composition may have antibacterial activity against Mycobacterium tuberculosis.

[0046] III. Compositions for the improvement, treatment, or prevention of tuberculosis Pharmaceutical compositions for the treatment or prevention of tuberculosis The present invention provides a pharmaceutical composition for the treatment or prevention of tuberculosis, comprising the Bacillus subtilis PMC 205 strain.

[0047] As used herein, the term "tuberculosis" means an infectious disease transmitted by Mycobacterium tuberculosis. For example, tuberculosis may be one or more selected from the group consisting of ocular tuberculosis, cutaneous tuberculosis, adrenal tuberculosis, renal tuberculosis, epididymal tuberculosis, lymphadenopathy, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, bile duct tuberculosis, bone tuberculosis, pharyngeal tuberculosis, lymphadenopathy, pulmonary deficiency, breast tuberculosis, and spinal tuberculosis.

[0048] In the pharmaceutical composition for the treatment or prevention of tuberculosis according to the present invention, the pharmaceutical composition may further include a carrier. For example, the pharmaceutically acceptable carrier may include, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginic acid, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoic acid, propylhydroxybenzoic acid, talc, magnesium stearate, and mineral oil, which are commonly used in formulation. In addition to the above components, the pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0049] In the pharmaceutical composition for the treatment or prevention of tuberculosis according to the present invention, the pharmaceutical composition may be prepared by mixing one or more fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, or other diluents or excipients commonly used in the art. For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and lozenges. Such solid preparations are prepared by mixing the bacterial strain of the present invention or its culture medium with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solution-resistant preparations, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspension solvents, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspension solvents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and scannable esters such as oleic acid. Suppository bases may include witepsol, macrogol, tween 61, cocoa butter, lauric acid butter, glycerol, and gelatin.

[0050] In the pharmaceutical composition for the treatment or prevention of tuberculosis according to the present invention, the pharmaceutical composition may further contain substances that can improve the preventive or therapeutic effect of the disease. For example, the pharmaceutical composition may contain rifampicin, rifapentine, isoniazid, pyrazinamide, ethambutol, streptomycin, fluoroquinolone, kanamycin, cycloserine, prothionamide, levofloxacin, moxifloxacin It may further include at least one selected from the group consisting of, ofloxacin, rifabutin, capreomycin, amikacin, ciprofloxacin, protionamide, ethionamide, cycloserine, thioacetazone, clofazimine, amoxicillin / clavulanate, diaminodiphenylsulfone (derivative of dianomidiphenylsulfone), clarithromycin, azithromycin, and linezolid. According to one exemplary embodiment, the pharmaceutical composition may further comprise rifamficin in the Bacillus subtilis PMC 205 strain.

[0051] Treatment methods for tuberculosis The present invention provides a method for treating tuberculosis, comprising administering a composition containing the Bacillus subtilis PMC 205 strain to an individual. In this case, the individual may mean a mammal, including a human.

[0052] In the method for treating tuberculosis according to the present invention, the composition can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) depending on the method to be used, and the dosage will vary depending on the patient's condition and weight, the severity of the disease, the form of the drug, the route of administration and the time, but can be appropriately selected by those skilled in the art.

[0053] In the method for treating tuberculosis according to the present invention, the composition is administered in a pharmaceutically effective amount. The “pharmaceutically effective amount” means an amount sufficient to treat the disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined by factors including the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as a standalone therapeutic agent, in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. It is important to administer an amount that takes all of the above factors into consideration and provides the greatest effect with the minimum amount without side effects, which can be easily determined by those skilled in the art. In this case, the composition of the present invention and rifampicin may be administered together. When rifampicin is administered together, the preventive and therapeutic effect of the composition of the present invention may be enhanced. Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, sex, and weight. Generally, 0.1 to 100 mg per kg of body weight, preferably 0.5 to 10 mg, may be administered daily or every other day, or divided into 1 to 3 doses per day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., so the aforementioned dosage does not limit the scope of the present invention by any means.

[0054] Food composition The present invention provides a food composition for the prevention or improvement of tuberculosis containing the Bacillus subtilis PMC 205 strain. In this case, the food composition includes a functional health food composition, and the functional health food is not particularly limited as long as it contains the Bacillus subtilis PMC 205 strain, but preferably it may contain 0.01 to 20% by weight of the strain in the total weight of the functional health food composition.

[0055] In the food composition for the prevention or improvement of tuberculosis according to the present invention, the type of food in the food composition is not particularly limited as long as it is a food that is normally manufactured and / or sold. For example, it includes meats, sausages, bread, chocolates, candies, snacks, confectionery, pizzas, ramen and other noodles, gums, dairy products including ice cream, various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, and can be used in the form of pills, powders, granules, infusions, tablets, capsules or beverages, and includes all health functional foods in the usual sense.

[0056] In the food composition for the prevention or improvement of tuberculosis according to the present invention, the food composition is not particularly limited in its liquid component, other than containing the aforementioned bacterial strain, and may include various flavorings or natural carbohydrates as additional components, as in ordinary beverages. Examples of natural carbohydrates include ordinary sugars such as monosaccharides, e.g., glucose, fructose; disaccharides, e.g., maltose, sucrose; and polysaccharides, e.g., dextrin, cyclodextrin; as well as sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to the aforementioned flavorings, natural flavorings (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.) can be advantageously used.

[0057] In the food composition for the prevention or improvement of tuberculosis according to the present invention, the food composition may contain, in addition to the above-mentioned additional components, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and enhancers (such as cheese and chocolate), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages.

[0058] The present invention will now be described in detail through the following examples. It will be obvious to those with ordinary skill in the art to which the present invention pertains that these examples are merely for the purpose of illustrating the present invention more concretely, and that the scope of the present invention is not limited by these examples.

[0059] <Experimental Method> 1. Isolation, identification, and elucidation of the genetic characteristics of probiotic strains from kimchi. The probiotic strain was isolated from napa cabbage kimchi. The liquid portion of the napa cabbage kimchi was streaked onto MRS agar plates (de Man, Rogosa and Sharp, 288210, BD difco, USA) using a loop. The plates were cultured in an aerobic incubator (general incubator, N-Biotek, Korea) at 37°C. After culturing, the colonies were cultured in MRS broth (288130, BDdifco, USA) and stored at -80°C in a 15% glycerol stock. The strain was identified by 16s rRNA gene sequencing analysis.

[0060] 16s rRNA gene sequencing was performed at Biofact (Korea), and compared with the GenBank database using BLAST (basic local alignment search tool) at NCBI (National Center for Biotechnology Information). For whole-genome sequencing of the strain, gDNA was extracted from the strain, sequenced using PacBio RSII (PacBio, USA) at CJ Bioscience (Korea), and then analyzed on the Ezbiocloud server. The genomic information of the strain Bacillus subtilis PMC 205 was deposited on the NCBI website under accession number PRJNA865470. The probiotic strains were cultured in food-grade medium (20 g / l glucose, 25 g / l yeast peptone F, 1 g / l Twin 80, 0.2 g / l magnesium sulfate, pH 6.3).

[0061] 2. In vitro antifungal activity test against macrophages Macrophages from Murine Raw 264.7 that had reached confluence were infected with Mycobacterium tuberculosis (M. tuberculosis), treated with the probiotic or drug according to the present invention, cultured for 3 days, and then subjected to CFU analysis. The Mycobacterium tuberculosis strains H37Rv and XDR used in this study were purchased from ATCC (American Type Culture Collection, USA) and KMRC (Korean Mycobacterium Resource Center, Korea), respectively.

[0062] 3. Evaluation of the in vivo anti-tuberculosis efficacy of probiotics. The in vivo anti-tuberculosis effect of the probiotics according to the present invention was evaluated in an XDR pulmonary tuberculosis mouse model and a latent tuberculosis mouse model. First, 6-week-old Balb / c mice were infected with a high dose of the XDR strain via lung inhalation using a sprayer, and then the probiotic strain was administered by lung inhalation three times a day starting one week after infection. In the latent pulmonary tuberculosis mouse model, mice were infected with a low dose of the XDR strain, and one month later, the probiotic strain was administered by lung inhalation once a day, twice a week for eight months. Rifampicin was used as a control drug and administered orally at a dose of 30 mg / kg. In this animal experiment, saline was used as the vehicle. For additional analysis, lungs were extracted and M. tuberculosis was quantitatively analyzed by CFU and real-time PCR. Primers used were KY18 (forward, 5'-CACATGCAAGTCGAACGGAAAGG-3'; reverse, 5'-CCTTTCCGTTCGACTTGCATGTG-3') and KY75 (forward, 5'-GCCCGTATCGCCCGCACGCTCACA-3'; reverse, 5'-TGTGAGCGTGCGGGCGATACGGGC-3'). Perfused lungs were used to investigate the pulmonary microenvironment in an XDR pulmonary tuberculosis mouse model. Microbiome and metabolite analysis was performed using previously reported methods. Lung cytokines were analyzed using the Mouse ELISA Kit (Thermo Fisher Scientific, USA), and H&E (Hematoxylin and eosin) staining analysis was performed at KPNT (Korea Pathology Technical Center, Korea).

[0063] <Result> 1. Analysis of the genetic characteristics of probiotic strains After identifying a probiotic strain isolated from kimchi, a traditional fermented food, based on its 16s rRNA gene sequence, its identity was confirmed by whole-genome analysis. The results showed that the isolated probiotic strain's genome consisted of a single circular chromosome of 4,096,449 bp with 4,036 coding sequences (CDS) (Figure 1A), and a total of 3,691 putative proteins were grouped into clusters according to the Orthologous Groups (COG) functional classification (Figure 1B). Similarity analysis using the Ortho ANI (Orthologous Average Nucleotide Identity) method revealed a high similarity to Bacillus subtilis (B. subtilis), while showing low similarity to other Bacillus species, ultimately confirming that the probiotic strain isolated from kimchi was indeed a Bacillus subtilis strain (Figure 1C). Furthermore, the isolated Bacillus subtilis variety is a new variety that is genetically distinct from previously reported varieties, and has been named PMC 205.

[0064] 2. Evaluation of the anti-mycobacterial activity of Bacillus subtilis PMC 205 The antimycobacterial activity of Bacillus subtilis PMC 205 according to the present invention was confirmed using an in vitro macrophage infection model. Raw264.7 cells were infected with the H37Rv strain, treated with PMC 205 extract for 3 days, and then quantified by CFU (colonyforming unit) analysis. The result was 2.3 × 10⁶ 6 When extracts of PMC 205 containing CFU / ml or higher were treated, the growth of M. tuberculosis was significantly suppressed compared to a control group that received no treatment. This effect was similar to that of treatment with 1 μg / ml of rifampicin (RIF) (Figure 2).

[0065] 3. Efficacy evaluation of Bacillus subtilis PMC 205 in a fatal XDR pulmonary tuberculosis mouse model. The in vivo efficacy of Bacillus subtilis PMC 205 according to the present invention was evaluated using an XDR pulmonary tuberculosis mouse model. Six-week-old Balb / c mice were infected with a high dose of the XDR strain via lung inhalation using a sprayer. One week after infection, Bacillus subtilis PMC 205 was administered once daily, three times a week, using the same method, and the mortality rate was observed. As a result, infected control mice began to die from week 5, and all died by week 12. The survival rate when administered RIF 30 mg / kg was 40%, while all mice administered Bacillus subtilis PMC 205 according to the present invention were confirmed to be alive at the end of the experiment. Interestingly, no anti-tuberculosis effect was observed when Lactobacillus, Leuconostoc, and Pediococcus were treated under the same conditions (Figure 3A). Furthermore, while weight loss was observed in mice treated with Lactobacillus, Leuconostoc, and Pediococcus strains or RIF, no significant weight loss was observed in mice administered Bacillus subtilis PMC 205 according to the present invention (Figure 3B). In addition, in terms of disease severity scores, unlike in mice treated with Lactobacillus, Leuconostoc, and Pediococcus strains or RIF, mice administered Bacillus subtilis PMC 205 according to the present invention showed results similar to those of uninfected mice (Figure 3C). Further analysis of extracted lung tissue revealed that M. tuberculinosis was significantly reduced in the lungs of mice administered Bacillus subtilis PMC 205 according to the present invention, with Log 3.6 and Log 2.6 at 6 and 10 weeks, respectively, compared to mice administered Lactobacillus, Leuconostoc, and Pediococcus strains or RIF (Figure 3D).

[0066] 4. Characteristics of the pulmonary microbiome in the treatment of M. tuberculosis with Bacillus subtilis PMC 205 To investigate the characteristics of the pulmonary microbiome in the treatment of M. tuberculosis with Bacillus subtilis PMC 205 according to the present invention, cytokines in the lungs extracted from a lethal XDR pulmonary tuberculosis animal model were quantified. As a result, all infectious cytokines (IFN-γ, TNF-α, IL-6, and IL-10) tested after M. tuberculosis infection increased, but in mice administered with Bacillus subtilis PMC 205 according to the present invention, these cytokines were significantly reduced (Figure 4A). Analysis of the pulmonary microbiome based on the 16S rRNA gene revealed that in mice infected with M. tuberculosis, the number of Mycobacterium species increased and species diversity decreased in the lungs, whereas in mice administered with Bacillus subtilis PMC 205 according to the present invention, mycobacteria decreased, species diversity increased again, and the bacterial flora recovered to a normal state (Figure 4B).

[0067] 5. Evaluation of the efficacy of Bacillus subtilis PMC 205 on M. tuberculosis of the lung in a mouse model of latent tuberculosis. The anti-tuberculosis effect of Bacillus subtilis PMC 205 according to the present invention was also confirmed in a mouse model of latent pulmonary tuberculosis. M. tuberculosis was inhaled into the lungs of mice at a low dose, and after one month, Bacillus subtilis PMC 205 was administered by lung inhalation once daily, twice weekly for eight months. As a result, mice administered with Bacillus subtilis PMC 205 according to the present invention did not show weight loss (Figure 5A), and analysis of the excised lungs revealed that M. tuberculosis decreased by approximately log 1.3 in mice administered with Bacillus subtilis PMC 205 according to the present invention, which was similar to the effect observed in mice administered 30 mg / kg / day RIF (Figure 5B).

[0068] Although specific parts of the present invention have been described in detail above, it will be clear to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and therefore do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0069] Depository name: Korea Institute of Biotechnology, Bioresource Center (KCTC) Deposit number: KCTC15188BP Deposit date: November 9, 2022 [Industrial applicability]

[0070] This invention provides the Bacillus subtilis PMC 205 strain, which has antibacterial activity. More specifically, the strain has antibacterial activity against Mycobacterium tuberculosis. It is therefore expected that the strain may be used to treat, prevent, or improve tuberculosis.

[0071] Sequence List agtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactcta aggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaagggcagcgaaa ccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaa tacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtgaggtaaccttttaggagccagccgccgaaggtgggacagatgattg

Claims

1. A strain of Bacillus subtilis PMC 205 or its culture medium having antibacterial activity against bacteria belonging to the genus Mycobacterium, The aforementioned strain is characterized by having the deposit number KCTC 15188BP extracted from kimchi, and is either a strain of Bacillus subtilis PMC 205 or its culture medium.

2. The fungi belonging to the genus Mycobacterium mentioned above include Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium asiaticum, and Mycobacterium avium complex. (MAC), Mycobacterium bovis (M. bovis), Mycobacterium chelonae (M. chelonae), Mycobacterium fortuitum (M. fortuitum), Mycobacterium gordonae (M. gordonae), Mycobacterium haemophyllum (M. haemophyllum), Mycobacterium intracellulare (M. intracellulare), Mycobacterium kansasi (M. kansasii), Mycobacterium lentiflavum (M. lentiflavum), Mycobacterium leprae (M. leprae), Mycobacterium malmoeens (M. malmoeens), My Bacillus subtilis PMC 205 strain or culture solution thereof according to claim 1, characterized in that one or more are selected from the group consisting of Cobacterium marinum, Mycobacterium microti, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium xenopi.

3. An antimicrobial composition having antimicrobial activity against bacteria belonging to the genus Mycobacterium, including the Bacillus subtilis PMC 205 strain described in claim 1 or 2 or its culture solution.

4. The antibacterial composition according to claim 3, characterized in that the antibacterial composition is used in a living organism.

5. A pharmaceutical composition for the treatment or prevention of tuberculosis having antibacterial activity against bacteria belonging to the genus Mycobacterium, comprising the Bacillus subtilis PMC 205 strain or its culture solution as an active ingredient, as described in claim 1 or 2.

6. The pharmaceutically active composition according to claim 5, characterized in that the tuberculosis is selected from one or more of the group consisting of ocular tuberculosis, cutaneous tuberculosis, adrenal tuberculosis, renal tuberculosis, epididymal tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, bile duct tuberculosis, bone tuberculosis, pharyngeal tuberculosis, lymphatic tuberculosis, pulmonary deficiency, breast tuberculosis, and spinal tuberculosis.

7. The pharmaceutical composition according to claim 5, characterized in that the tuberculosis is latent tuberculosis.

8. The aforementioned pharmaceutical compositions include rifampicin, rifapentine, isoniazid, pyrazinamide, ethambutol, streptomycin, fluoroquinolone, kanamycin, cycloserine, prothionamide, levofloxacin, moxifloxacin, and off. Ofloxacin, Rifabutin, Capreomycin, Amikacin, Ciprofloxacin, Prothionamide, Ethionamide, Cycloserine, Thioacetazone, Clofazimine, Amoxicillin / Clavulanate, Diaminodiphenylsulfone derivatives The pharmaceutical composition according to claim 5, further comprising at least one selected from the group consisting of dianomidiphenylsulphone, clarithromycin, azithromycin, and linezolid.

9. A food composition for the prevention or improvement of tuberculosis having antibacterial activity against bacteria belonging to the genus Mycobacterium, comprising the Bacillus subtilis PMC 205 strain or its culture solution as an active ingredient, as described in claim 1 or 2.

10. The food composition according to claim 9, characterized in that the tuberculosis is selected from one or more of the group consisting of ocular tuberculosis, cutaneous tuberculosis, adrenal tuberculosis, renal tuberculosis, epididymal tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, bile duct tuberculosis, bone tuberculosis, pharyngeal tuberculosis, lymphatic tuberculosis, pulmonary deficiency syndrome, breast tuberculosis, and spinal tuberculosis.

11. The food composition according to claim 9, characterized in that the tuberculosis is latent tuberculosis.

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