KR20220061555A - Novel Lactobacillus strain and use of extracellular vesicle derived from the strain - Google Patents

Abstract

The present invention relates to a novel strain of the genus Lactobacillus and various uses of extracellular vesicles derived from the strain and, more specifically, to novel Lactobacillus kepirgranum and extracellular vesicles derived therefrom. In addition, it is possible to provide a pharmaceutical composition for preventing, alleviating or treating inflammatory diseases comprising the extracellular vesicles as an active ingredient, and an antiviral composition, and in particular, it is possible to provide the pharmaceutical composition effective for preventing, alleviating or treating inflammatory bowel diseases while having lower side effects than synthetic drugs.

Description

Novel Lactobacillus strain and use of extracellular vesicle derived from the strain

The present invention relates to a Lactobacillus sp. strain and extracellular vesicles derived from the strain, and more particularly, to a novel Lactobacillus sp. strain and various uses of the extracellular vesicles derived from the strain.

Inflammatory bowel disease (inflammatory bowel disease) is a disease that causes chronic, unknown inflammation of the intestine, and can be divided into ulcerative colitis and Crohn's disease, including Behcet's disease. Ulcerative colitis is a disease in which sores (erosion) or ulcers are continuously formed on the mucous membrane of the large intestine, and bloody stools, bloody stools, diarrhea, and abdominal pain occur. In severe cases, systemic symptoms such as fever, weight loss, and anemia appear. In addition, Crohn's disease is a disease in which lesions such as ulcers occur discontinuously in any part of the digestive tract from the mouth to the anus. symptoms appear. Both ulcerative colitis and Crohn's disease are chronic intractable diseases in which symptoms temporarily improve and then recur. Although the cause or pathophysiology of inflammatory bowel disease is not yet clearly known, it is estimated that genetic factors, environmental factors such as intestinal bacteria or food, and immunological factors are involved in the complex development mechanism.

Conventionally, the incidence rate of ulcerative colitis and Crohn's disease was known to be high among Westerners, but the number of patients is rapidly increasing in the East as well as Korea due to changes in lifestyle such as eating habits. Nevertheless, for reasons of unclear causes, etc., a fundamental treatment has not yet been established, so drugs that delay and alleviate the progression of symptoms and maintain this condition for as long as possible are used, rather than aiming for a complete treatment. Aminosalicylic acid preparations, adrenocortical steroids, immunosuppressants, TNF-α monoclonal antibodies, etc. are mainly used as drugs for such popular therapy, but various side effects have been reported. For example, sulfasalazine, which is frequently used as an aminosalicylic acid preparation, has reported side effects such as nausea, vomiting, anorexia, rash, headache, leukopenia, abnormal red blood cells, proteinuria, and diarrhea. Prednisolone, an adrenocortical steroid, is used for oral administration, enema, suppository, and intravenous injection, but it has strong side effects such as gastric ulcer or necrosis of the femoral head due to long-term use.

On the other hand, in recent years, research and development of biological drugs with fewer side effects than synthetic drugs have been actively carried out, and in particular, a number of studies on various therapeutic agents using exosomes are being conducted. However, most of these relate to stem cell-derived exosomes, and studies on lactic acid bacteria-derived exosome therapeutic agents have hardly been made.

Registered Patent Publication No. 10-1862507

In order to solve the above problems, the present invention is to provide a novel Lactobacillus sp. strain and various uses of extracellular vesicles derived from the strain.

In addition, to provide a pharmaceutical composition for preventing, improving or treating an inflammatory disease comprising the extracellular vesicle as an active ingredient, and to provide a method of treating an inflammatory disease using the pharmaceutical composition.

In order to achieve the above object, the present invention provides a novel Lactobacillus sp.

In addition, it provides an extracellular vesicle derived from the Lactobacillus sp. strain and a pharmaceutical composition for preventing, improving or treating an inflammatory disease comprising the same as an active ingredient.

In addition, there is provided a method of treating an inflammatory disease using the pharmaceutical composition.

In addition, the present invention provides an extracellular vesicle derived from the Lactobacillus sp. strain and an antiviral composition comprising the same as an active ingredient.

The Lactobacillus sp. strain is not limited, but preferably Lactobacillus kefiranofaciens, Lactobacillus kefiri and any selected from the group consisting of Lactobacillus kefirgranum can be one

The Lactobacillus kefirgranium is preferably a strain having accession number KCTC 14300BP.

The Lactobacillus sp. strain-derived extracellular vesicle has a size of 10 to 1000 nm. The extracellular vesicles include microvesicles, nanovesicles, or exosomes.

The exosome is not limited, but has a size of 20 to 300 nm, preferably 50 to 200 nm, more preferably 70 to 150 nm.

In an embodiment of the present invention, the exosome may have a size of 100 ± 20 nm.

The pharmaceutical composition for preventing, improving, or treating an inflammatory disease according to the present invention comprises an extracellular vesicle derived from a Lactobacillus kefirgranium strain as an active ingredient, and the extracellular vesicle has a particle number of 1.0x10 ⁵ to 1.0x10 ¹⁷ The number of particles of 1.0x10 ⁶ to 1.0x10 ¹⁵ , preferably the number of particles of 1.0x10 ⁷ to 1.0x10 ¹³ , more preferably, the number of particles of 1.0x10 ⁷ to 1.0x10 ¹² , but is not limited thereto.

The inflammatory disease is atopic dermatitis, acne, psoriasis, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory enteritis, irritable bowel syndrome, chronic obstructive pulmonary disease, sepsis, plaque hematologic shock, pulmonary fibrosis, arthritis, inflammatory osteolysis, chronic inflammatory disease caused by viral or bacterial infection, inflammatory bowel disease, arthritis, rheumatoid arthritis, reactive arthritis, osteoarthritis, scleroderma, osteoporosis, atherosclerosis, myocarditis, endocarditis, Pericarditis, Cystic Fibrosis, Hashimoto's Thyroiditis, Graves' Disease, Leprosy, Syphilis, Lyme disease, Borreliosis, Neuro-Borreliosis, Tuberculosis, Sarcoidosis, Lupus, Alumni Lupus, Lupus Tuberculosis , lupus nephritis, systemic lupus erythematosus, macular degeneration, uveitis, irritable bowel syndrome, Crohn's disease, Sjogran's syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immunodeficiency syndrome, myalgic encephalomyelitis, amyotrophic lateral sclerosis, Parkinson's disease, and multiple sclerosis, and the like.

The inflammatory disease may be preferably inflammatory bowel disease, but is not limited thereto.

The inflammatory bowel disease includes Crohn's disease, Behcet's disease, ulcerative colitis, hemorrhagic rectal ulcer, ileal capsulitis, and intestinal lesions accompanying it.

The pharmaceutical composition for preventing, improving, or treating an inflammatory disease comprising the Lactobacillus kefirgranium strain and/or the strain-derived extracellular vesicle as an active ingredient according to the present invention is a pharmaceutically acceptable carrier in addition to the active ingredient; A binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, and the like may be further included, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, Malditol, starch, alginate, calcium phosphate, calcium silicate, maltodextrin, silicon dioxide, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate rate, etc., but is not limited thereto.

In addition, the pharmaceutical composition may be prepared in the form of tablets, capsules, sustained-release preparations, elixirs, suspensions, syrups, wafers, unit dose injection ampoules, suspensions, etc. by methods commonly used for formulation in the art.

The administration method of the pharmaceutical composition is oral administration, intravenous administration, intramuscular administration, intraarterial administration, intramedullary administration, intrathecal administration, intracardiac administration, transdermal administration, subcutaneous administration, intraperitoneal administration, intranasal administration, enteral administration It may be administered by methods such as topical administration, rectal administration, topical administration, dermal administration, mucosal administration, intravertebral administration, etc., including oral and sublingual administration, and preferably oral administration, but is not limited thereto. In addition, at the time of administration, it may be formulated and administered in a formulation appropriate for each administration method, and the formulation may be formulated by a method widely known in the art.

The pharmaceutical composition may be appropriately selected according to several factors including the age, weight, health status, sex, administration time, administration route, excretion rate, drug formulation, and severity of a specific disease to be prevented or treated of the subject to be administered, and the number of administration may be administered once or twice a day, every other day, every other week, every other month, or the like.

In addition, the antiviral composition according to the present invention includes the Lactobacillus kefirgranium strain and/or extracellular vesicles derived from the strain as an active ingredient.

The antiviral composition contains an extracellular vesicle derived from Lactobacillus kefirgranium strain (Accession No. KCTC 14300BP) as an active ingredient, and the extracellular vesicle is 1.0x10 ⁵ to 1.0x10 ¹⁷ of particles 1.0x10 ⁶ to The number of particles of 1.0x10 ¹⁵ , preferably the number of particles of 1.0x10 ⁷ to 1.0x10 ¹³ , and more preferably the number of particles of 1.0x10 ⁷ to 1.0x10 ¹² may be included, but is not limited thereto.

The virus is foot-and-mouth disease virus (FMDV), Leishmania, human immunodeficiency virus (Human Immunodeficiency virus, HIV), hepatitis C virus (Hepatitis C virus, HCV), hepatitis E virus (Hepatitis E virus, HEV) , hepatitis A virus (HAV), hepatitis B virus (HBV), tuberculosis (tuberculosis), herpes simplex virus (HSV), malaria causing parasites , Human papilloma virus (HPV), influenza virus (influenza virus), measles virus (measles virus), mumps virus (mumps virus), Ebola virus (Ebola virus), respiratory syncytial virus (RSV) , West Nile virus (WNV), coronavirus, and the like.

The corona virus includes alpha corona virus, beta corona virus, gamma corona virus, delta corona virus, and the like.

Examples of alphacoronaviruses are alphacoronavirus 1, bat coronavirus CDPHE15, bat coronavirus HKU10, human coronavirus 229E, human coronavirus NL63, Miniopterus bat coronavirus 1, bat coronavirus HKU8, mink coronavirus 1, porcine epidemic diarrhea virus, tuberculous bat coronavirus HKU2, and Scotophilus bat coronavirus 512, and the like.

Examples of betacoronaviruses include murine coronavirus, betacoronavirus 1, hedgehog coronavirus 1, human coronavirus HKU1, Middle East respiratory syndrome-associated coronavirus, Pipistrellus bat coronavirus HKU5, Rousettus ) bat coronavirus HKU9, severe acute respiratory syndrome-associated coronavirus, and Tylonycteris bat coronavirus HKU4.

Examples of gamma coronaviruses include avian coronavirus and Beluga whale coronavirus SW1.

Examples of deltacoronaviruses are Firebug coronavirus HKU11, Common moorhen coronavirus HKU21, coronavirus HKU15, Munia coronavirus HKU13, Heron coronavirus HKU19, thrush coronavirus HKU12, guinea pig coronavirus HKU16. , and Wigeon coronavirus HKU20.

The antiviral composition includes prevention, treatment or alleviation of symptoms caused by viral infection, inhibition of virus proliferation in the human body, inhibition of infection, and the like.

The antiviral composition may be any one of a pharmaceutical composition, a cosmetic composition, and a food composition, but preferably a pharmaceutical composition.

When the pharmaceutical composition is prepared as an oral dosage form, powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc. It can be prepared in a dosage form. Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, Cellulose such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil etc. are mentioned. In the case of formulation activity, if necessary, the formulation may include a diluent and/or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant.

When the pharmaceutical composition is prepared for parenteral use, it may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories together with suitable carriers according to methods known in the art. In the case of formulation for injection, a suitable carrier may be sterile water, ethanol, polyol such as glycerol or propylene glycol, or a mixture thereof, but is not limited thereto.

When formulated for transdermal administration, it can be formulated in the form of an ointment, a cream, a lotion, a gel, an external solution, a pasta agent, a liniment agent, an air roll, and the like. In the case of a nasal inhalant, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, and the like. witepsol), tween 80, tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters, and the like can be used.

The present invention can provide novel Lactobacillus kefirgranium and extracellular vesicles derived therefrom. In addition, it is possible to provide a pharmaceutical composition for the prevention, improvement or treatment of inflammatory diseases comprising the extracellular vesicles as an active ingredient, in particular, a pharmaceutical composition effective for the prevention, improvement or treatment of inflammatory bowel disease with lower side effects than synthetic drugs. Or an antiviral composition may be provided.

1 shows the IL-6 inhibitory effect of Lactobacillus kefirgranium-derived extracellular vesicles according to the present invention.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

<Example 1> Lactobacillus kefirgranum strain identification according to the present invention

The kefir grains (Tibetan mushroom) used for isolating the strain were cultured at home in Korea and used for free distribution through the Internet. Each of the kefir grains was suspended and diluted in sterile physiological saline, 0.1 ml each was taken and spread on Difco Lactobacilli MRS agar medium. Thereafter, the plate was incubated for 2 days in an incubator at 35°C. Each produced colony was streaked on an MRS agar plate, and colonies showing white colonies were obtained by culturing at 35° C. for 2 days. Lactobacillus kefir granium PRCC-1301 was isolated from kefir grains in the same manner as above. The isolated strain was deposited at the Microbial Resource Center of the Korea Research Institute of Bioscience and Biotechnology and was given an accession number KCTC 14300BP.

<Example 2> Lactobacillus kefirgranium-derived extracellular vesicle isolation according to the present invention

In this example, using a Lactobacillus sp. strain obtained from kefir grains as lactic acid bacteria, extracellular vesicles were isolated therefrom. More specifically, Lactobacillus keifrgranum PRCC-1301 (Accession No.: KCTC 14300BP) strain was used to prepare lactic acid bacteria-derived extracellular vesicles.

(Step 1) The culture solution of the cultured Lactobacillus sp. strain was centrifuged at 3,000 × g at 4° C. for 20 minutes to separate and remove the Lactobacillus sp. strain from the culture solution. That is, the cells and the supernatant were separated through a low-speed centrifugation process.

(Step 2) The culture solution centrifuged in step 1 was centrifuged at 4° C. at 10,000 × g for 20 minutes using a high-speed centrifuge to additionally remove cells or cell-related debris and residues.

(Step 3) The purity of the culture solution was increased by filtering the culture solution centrifuged in step 2 through a 0.45 μm bottle top filter.

(Step 4) Extracellular vesicles were separated by ultracentrifugation of the culture solution filtered in step 3 at 110,000 × g at 4° C. for 1 hour and 10 minutes.

(Step 5) After ultra-high speed centrifugation in step 4, the supernatant was removed and the remaining extracellular vesicle pellet was resuspended in a buffer solution (HEPES buffer).

(Step 6) The extracellular vesicles (EV) resuspended in the buffer solution in step 5 were stored at -70° C. and used for the following test.

<Experimental Example 1> Evaluation of the anti-inflammatory effect of the Lactobacillus kefirgranium strain according to the present invention and the extracellular vesicles isolated from the strain

The Lactobacillus kefirgranium strain of Example 1 and the extracellular vesicles isolated in Example 2 were prepared. IL-6 inhibitory ability was tested to evaluate the anti-inflammatory effect of the strain and extracellular vesicles, and the specific method is as follows.

After culturing Raw246.7 cells, which are macrophages of mice, in DMEM medium containing 10% FBS and 1% penicillin-streptomycin for 24 hours, the obtained exosomes were freeze-dried samples were prepared. 1 mg/ml of the sample was simultaneously treated with LPS (100 ng/ml) and cultured for 24 hours, and the expression level of IL-6 was measured using ELISA, and the results are shown in FIG. 1 . However, as a positive control, curcumin was treated with 10 μM, and as a comparative example, a normal strain of Lactobacillus kefirgranium was treated.

As a result of the experiment, the IL-6 inhibitory ability of the Lactobacillus kefirgranium-derived extracellular vesicles according to the present invention was more than 2.5 times superior to that of curcumin, and the IL-6 inhibitory ability was higher than that of the general Lactobacillus kefirgranium-derived extracellular vesicles. It was found to be more than twice as good

<Experimental Example 2> Evaluation of the antiviral effect of the Lactobacillus kefirgranium strain according to the present invention and extracellular vesicles isolated from the strain

The Lactobacillus kefirgranium strain of Example 1 and the extracellular vesicles isolated in Example 2 were prepared.

The coronavirus was isolated from a domestic patient in Korea, and was purchased from the Korea Centers for Disease Control and Prevention and used under strict management at the Pasteur Korea Institute for Biosafety Level 3 (BSL-3) research facility. Viruses were titered using plaque assay. The compound used in the experiment was purchased in powder form from Sigma-Aldrich or Tokyo Chemical Industry, and was used by dissolving it in DMSO at a concentration of 10 mM.

24 hours before the experiment, 1.2 × 10 ⁴ Vero cells were put into each well of a 384-well black μClear plate. Test compounds were added to each well immediately before coronavirus infection. The sample compound was treated at a concentration of 10 μM, and the DMSO concentration was maintained below 0.5%. After transferring the plate containing the cells and test compound to a biosafety level 3 test area, the virus was infected with a multiplicity of infection (MOI) of 0.0625, and the cells were fixed with 4% paraformaldehyde 24 hours later, followed by immunofluorescence staining. carried out. Virus infection was observed using a primary antibody against spike isolated from rabbit and AlexFluor ^TM 488 fluorescent secondary antibody (Invitrogen, #A-11008), and cell viability was performed through Hoechst® 33342 (Invitrogen, #H3570) staining. was evaluated.

[Accession number]

Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14300BP

Deposit date: 20200907

Claims (12)

Lactobacillus kefirgranium (KCTC 14300BP) strain. Extracellular vesicles derived from Lactobacillus kepiranofaciens (KCTC 14300BP) strain. The extracellular vesicle according to claim 2, wherein the extracellular vesicle is any one of microvesicles, nanovesicles, and exosomes. The extracellular vesicle according to claim 2, wherein the extracellular vesicle has a size of 30 to 300 nm. The extracellular vesicle according to claim 2, wherein the extracellular vesicle has a size of 100±20 nm. A pharmaceutical composition for preventing, improving or treating an inflammatory disease comprising the Lactobacillus kefirgranium strain of claim 1 and/or the extracellular vesicles according to claim 2 to 5 as an active ingredient. The pharmaceutical composition for preventing, improving or treating an inflammatory disease according to claim 6, wherein the extracellular vesicles contain a particle number of 1.0x10 ⁵ to 1.0x10 ¹⁷ . According to claim 6, wherein the inflammatory disease is atopic dermatitis, acne, psoriasis, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory enteritis, irritable bowel syndrome, chronic obstructive Pulmonary disease, sepsis, septic shock, pulmonary fibrosis, arthritis, inflammatory osteolysis, chronic inflammatory disease caused by viral or bacterial infection, inflammatory bowel disease, arthritis, rheumatoid arthritis, reactive arthritis, osteoarthritis, scleroderma, osteoporosis, atherosclerosis Sclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, Borreliosis, neuro-borreliosis, tuberculosis, Sarcoidosis, Lupus, Lupus alumni, lupus tuberculosis, lupus nephritis, systemic lupus erythematosus, macular degeneration, uveitis, irritable bowel syndrome, Sjogren's syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immunodeficiency syndrome, myalgic encephalomyelitis, amyotrophic lateral sclerosis, parky A pharmaceutical composition for the prevention, improvement or treatment of inflammatory diseases, characterized in that at least one selected from Seon's disease and multiple sclerosis. According to claim 8, wherein the inflammatory bowel disease is Crohn's disease, Behcet's disease, or a pharmaceutical composition for the prevention, improvement or treatment of an inflammatory disease, characterized in that the ulcerative colitis. An antiviral composition comprising the Lactobacillus kefirgranium strain of claim 1 and/or the extracellular vesicles according to claim 2 to 5 as an active ingredient. The antiviral composition according to claim 10, wherein the extracellular vesicles contain a particle number of 1.0x10 ⁵ to 1.0x10 ¹⁷ . 11. The method of claim 10, wherein the antivirus is foot-and-mouth disease virus (FMDV), Leishmania (Leishmania), human immunodeficiency virus (Human Immunodeficiency virus, HIV), hepatitis C virus (Hepatitis C virus, HCV), hepatitis E virus (Hepatitis E virus, HEV), hepatitis A virus (HAV), hepatitis B virus (HBV), tuberculosis, Herpes Simplex virus (HSV), parasites Malaria causing parasites, Human Papilloma virus (HPV), influenza virus, measles virus, mumps virus, Ebola virus, respiratory syncytial virus (Respiratory Syntial virus, RSV), West Nile virus (WNV), antiviral composition, characterized in that at least one selected from the group consisting of coronavirus.

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