JP2022512469A - Nanovesicles derived from Corynebacterium and their uses - Google Patents

Abstract

The present invention relates to vesicles derived from a bacterium belonging to the genus Corinebacterium and uses thereof, and the present inventions describe vesicles derived from a bacterium belonging to the genus Corinebacterium to cause liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, and the like. It has been reduced in clinical samples of patients with asthma, dementia and nasal polyps, and it was experimentally confirmed that the secretion of inflammatory mediators by pathogenic vesicles that induce inflammation was effectively suppressed. The vesicles derived from the bacterium of the genus Um can be usefully used as a composition for the prevention or treatment of inflammatory diseases including liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. Be expected. [Selection diagram] FIG. 14

Description

The present invention relates to nanovesicles derived from Corynebacterium bacteria and their uses, and more specifically, liver cirrhosis, stroke, diabetes, asthma, and atopic skin using nanovesicles derived from Corynebacterium bacteria. It relates to a method for diagnosing inflammation, depression, breast cancer, dementia and nasal polyps, and a composition for preventing, ameliorating or treating the disease including the follicles.

This application has priority based on the Republic of Korea Patent Application No. 10-2018-0158622 filed on December 10, 2018 and the Republic of Korea Patent Application No. 10-2019-0132137 filed on October 23, 2019. All content claimed in the application and disclosed in the specification and drawings of the application is incorporated herein by reference.

Since the beginning of the 21st century, the importance of acute infectious diseases, which were previously recognized as infectious diseases, has diminished, while chronic with abnormalities in immune function caused by incongruity between humans and microbiomes. Inflammatory diseases have become the main disease and the disease pattern has changed. In particular, chronic inflammatory diseases such as obesity, diabetes, cardiovascular disease, neuropsychiatric disease and cancer due to westernized eating habits, indoor air pollution due to changes in the structure of the house, and skin and respiratory inflammation diseases due to increased living time indoors. Has become a major problem in national health.

The development of the inflammatory disease is accompanied by abnormalities in immune function against external causative factors. The Th17 immune response, which secretes interleukin (IL) -17 cytokines, is important for the immune response to bacterial causative factors, and when exposed to bacterial causative factors, Th17 immune response neutrophils. Sexual inflammation occurs. Inflammatory mediators such as Tumor Necrosis Factor-alpha (TNF-α), which is secreted by bacterial causative factors during the development of inflammation, play an important role in inflammation and cancer development. Carry. Among the inflammatory mediators, IL-6 secreted by bacterial causative factors plays an important role in the differentiation into Th17 cells, and chronic inflammation due to the Th17 immune response is closely related not only to chronic inflammatory diseases but also to cancer development. It has recently been reported that there is.

It is known that the number of microorganisms coexisting with the human body reaches 100 trillion, which is about 10 times larger than that of human cells, and the number of genes of microorganisms exceeds 100 times the number of human genes. The microbial flora (microbiota or microbiome) refers to a microbial community containing eubacteria, archaea, and eukaryotes present in a given place of residence, and the intestinal microbial flora is important for human physiological phenomena. It is known to play a role and have a great impact on human health and diseases through interaction with human body cells.

Symbiotic eubacteria and archaea in the human body secrete nanometer-sized vesicles to exchange information such as genes and proteins to other cells. The mucosa forms a physical protective membrane that particles larger than 200 nanometers (nm) cannot pass through, and in the case of bacteria that coexist with the mucosa, it cannot pass through the mucosa, but bacterial vesicles are sized. Is less than 100 nanometers in size, so it is relatively free to pass through epithelial cells through the mucosa and be absorbed by the human body. Vesicles derived from pathogenic bacteria absorbed by the human body have recently been shown to play an important role in the etiology of metabolic diseases such as diabetes and obesity.

Corynebacterium genus bacteria are aerobic gram-positive bacteria and are known as bacteria that spread in nature and live in symbiosis with eukaryotes. In particular, Corynebacterium glutamicum is a bacterium that is industrially widely used for the production of amino acids, nucleic acids and the like. However, to date, no therapeutic technique using extracellular vesicles of Corynebacterium spp. Has been reported.

Therefore, in the present invention, by first separating vesicles from Corynebacterium spp. And confirming their characteristics, the vesicles can be used for diagnosis of various inflammatory diseases and for prevention, improvement or therapeutic composition of inflammatory diseases. It was confirmed that it was used as.

As a result of diligent research to solve the above-mentioned conventional problems, the present inventors conducted liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, and cognition as compared with normal humans through metagenome analysis. It was confirmed that the content of vesicles derived from Corinebacterium spp. Was significantly reduced from the samples derived from patients with dementia and nasal polyps. In addition, it was confirmed that vesicles derived from Corynebacterium spp. Efficiently suppress the inflammatory reaction caused by pathogenic vesicles, and the present invention was completed based on this.

Therefore, the present invention provides information for diagnosing one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. The purpose is to provide a method.

Another object of the present invention is to provide a composition for preventing, ameliorating or treating an inflammatory disease containing vesicles derived from a bacterium belonging to the genus Corynebacterium as an active ingredient.

However, the technical issues to be achieved by the present invention are not limited to those mentioned above, and other issues not mentioned above should be clearly understood by those skilled in the art from the description below.

In order to achieve the object of the present invention as described above, the present invention is selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps including the following steps. Providing informational methods for the diagnosis of one or more diseases that have been made:

(A) Step of extracting DNA from vesicles isolated from normal human and subject samples;

(B) After performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA, the steps of obtaining each PCR product; and

(C) Liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and when the content of vesicles derived from Corinebacterium spp. A step of determining one or more diseases selected from the group consisting of nasal polyps.

The present invention also provides a method for diagnosing one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps, which include the following steps. do:

(A) Step of extracting DNA from vesicles isolated from normal human and subject samples;

(B) A step of obtaining each PCR product after performing PCR on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA; and

(C) Liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and when the content of vesicles derived from Corinebacterium spp. A step of determining one or more diseases selected from the group consisting of nasal polyps.

In one embodiment of the present invention, the sample in step (a) may be blood, urine, stool, saliva or nasal mucosa.

In another embodiment of the present invention, the primer pair in step (b) may be a primer pair containing the base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2.

The present invention also provides a composition for preventing, ameliorating or treating an inflammatory disease containing vesicles derived from a bacterium belonging to the genus Corynebacterium as an active ingredient.

The composition can include a pharmaceutical composition, a food composition, a cosmetic composition and an inhalant composition.

The present invention also provides a method for preventing or treating an inflammatory disease, which comprises a step of administering to an individual a composition containing a vesicle derived from a bacterium of the genus Corynebacterium as an active ingredient.

The present invention also provides a prophylactic or therapeutic use for inflammatory diseases of vesicles derived from Corynebacterium spp.

The present invention also provides a prophylactic or therapeutic use for an inflammatory disease of a composition containing a vesicle derived from a bacterium of the genus Corynebacterium as an active ingredient.

The present invention also provides applications for producing agents used for inflammatory diseases of vesicles derived from Corynebacterium spp.

In one embodiment of the present invention, the vesicle may be secreted from Corynebacterium glutamicum.

In another embodiment of the invention, the vesicles may have an average diameter of 10-200 nm.

In yet another embodiment of the invention, the vesicles may be naturally or artificially secreted by a Corynebacterium bacterium.

In still another embodiment of the present invention, the artificial vesicle may be secreted by a bacterium by a method such as heat treatment or pressure treatment.

In yet another embodiment of the invention, the inflammatory disease is one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. There may be.

In the case of bacteria, the present inventors are not absorbed in the body, but in the case of bacterial vesicles, they pass through the protective membrane of the mucosa and are absorbed by the epithelial cells of the mucosa and distributed systemically, and the kidneys, It was confirmed that it was excreted outside the body through the liver and lungs. In addition, corine bacteria present in the blood or nasal mucosa of patients with liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps through metagenome analysis of bacterial vesicles present in the patient's blood. It was confirmed that the number of vesicles derived from the genus Bacteria was significantly reduced compared to normal humans. In addition, when Corynebacterium glutamicum, which is a type of Corynebacterium genus, is cultured in vitro and the vesicles are isolated and administered to inflammatory cells, the pathogenic vesicles secrete inflammatory mediators such as TNF-α. After confirming that it was significantly suppressed, it is expected that the vesicles derived from Corynebacterium genus according to the present invention may be useful for the preventive or therapeutic composition of the inflammatory disease.

FIG. 1a is a photograph of mice administered with bacteria (bacteria) and vesicles derived from the bacteria (EV) orally, and then the distribution aspect of the bacteria and vesicles was photographed by time, and FIG. 1b was administered with the oral cavity. 12 hours later, blood, kidneys, liver and various organs were removed to evaluate the distribution of bacteria and vesicles in the body.

FIG. 2 is a diagram evaluating the presence or absence of bacterial and bacterial vesicle infiltration in mucosal epithelial cells after administration of bacteria (bacteria) and bacterial vesicles (EV) to mice (Lu, lumen; LP, lamina bacterium).

FIG. 3 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of patients with liver cirrhosis and normal humans.

FIG. 4 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of stroke patients and normal humans.

FIG. 5 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenomic analysis of vesicles derived from bacteria present in the blood of diabetic patients and normal humans.

FIG. 6 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of asthmatic patients and normal humans.

FIG. 7 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of patients with atopic dermatitis and normal humans.

FIG. 8 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of depressed patients and normal humans.

FIG. 9 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of breast cancer patients and normal humans.

FIG. 10 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of dementia patients and normal humans.

FIG. 11 is a diagram in which the infiltration aspect of vesicles derived from Corynebacterium spp. In the mucosal tissue of the nasal cavity of patients with allergic and non-allergic nasal polyps and the normal control group was evaluated through metagenome analysis.

FIG. 12 shows the results of evaluating cell killing by treating vesicles derived from Corynebacterium glutamicum with macrophages (Raw264.7 cell) in order to evaluate the cell killing effect of vesicles derived from Corynebacterium glutamicum. (EV, extracellular vesicle; CGT101, Corynebacterium glutamicum EV).

In FIGS. 13a and 13b, in order to evaluate the pro-inflammatory effect of vesicles derived from Corinebacterium glutamicum (CGT101), vesicles derived from Corinebacterium bacterium are treated with macrophages (Raw264.7 cell) for inflammation. The results of comparing the degree of secretion of the mediator with the pathogenic vesicle E. colli EV, FIG. 13a is a diagram comparing the degree of secretion of IL-6, and FIG. 13b is a diagram comparing the degree of secretion of IL-6. It is a figure comparing the degree of secretion of −α (EV, extracellular inflammation).

FIG. 14 is derived from Corinebacterium vesicles prior to treatment with pathogenic vesicles E. coli EV in order to evaluate the anti-inflammatory effect of vesicles derived from Corinebacterium glutamicum (CGT101). This is the result of pretreating the vesicles of Escherichia coli and evaluating the effect of E. coli vesicles on the secretion of the inflammation mediator TNF-α (EV, extracellular vesicle).

The present invention relates to vesicles derived from Corynebacterium spp. And their uses.

We found that samples from patients with liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps compared to normal humans through metagenome analysis of vesicles derived from the genus Corinebacterium. It was confirmed that the content was significantly reduced. In addition, the present inventors confirmed that treatment of inflammatory cells with vesicles derived from Corynebacterium glutamicum before administration of the pathogenic causative factor efficiently suppresses the inflammatory reaction caused by the pathogenic causative factor. Based on this, the present invention was completed.

Accordingly, the present invention is a method for diagnosing one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps, which include the following steps. Providing informational methods for diagnosis:

(A) Step of extracting DNA from vesicles isolated from normal human and subject samples;
(B) After performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA, the steps of obtaining each PCR product; and (c). ) When the content of vesicles derived from Corinebacterium spp. Is lower than that in normal humans through quantitative analysis of the PCR product, liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. A step of determining one or more diseases selected from the group consisting of.

The term "diagnosis" used in the present invention means to judge the actual condition of a patient's illness in all aspects in a broad sense. The contents of the judgment include the name of the disease, the etiology, the type of the disease, the severity of the disease, the detailed mode of the condition, the presence or absence of complications, and the prognosis. In the present invention, "diagnosis" refers to the presence or absence of onset of one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps, and the degree of the disease. Is to judge.

As used in the present invention, the term "nanovesicle" or "vesicle" means a structure consisting of nano-sized membranes secreted by various bacteria. Gram-positive bacteria-derived vesicles, such as corynebacterium, include peptidoglycan, lipoteichoic acid, and vesicles, which are constituents of the cell wall of bacteria as well as proteins and nucleic acids. It has a variety of low molecular weight compounds within. In the present invention, nanovesicles or vesicles are naturally secreted from bacteria of the genus Corynebacterium or artificially produced by heat treatment, pressure treatment, etc. on the bacteria, and have a diameter of 10 to 200 nm. Has an average diameter.

The term "metagenome" used in the present invention is also referred to as "group gene" and means a synthesis of all genes including all viruses, bacteria, molds, etc. in isolated areas such as soil and animal intestines. It is mainly used as a concept of genetics to explain the identification of many microorganisms at once using a sequencer to analyze uncultured microorganisms. In particular, "metagenomics" does not refer to a kind of genome or genetic body, but to a kind of mixed genetic body as a genetic body of all species in one environmental unit. This arose from the perspective that when defining a species in the process of omics-like development of biology, not only functionally existing species but also diverse species interact with each other to form a complete species. It is a term. Technically, it is the subject of a technique that analyzes all DNA and RNA regardless of species using rapid sequence analysis, identifies all species in one environment, and elucidates interactions and metabolic effects. be.

The vesicles are obtained by centrifuging, ultrafast centrifuging, pressing, ultrasonically decomposing, cell lysing, homogenizing, freezing-thawing, electrophoresing, mechanically decomposing, and treating chemical substances in a culture solution containing bacteria of the genus Corinebacterium. Separation can be performed using one or more methods selected from the group consisting of filtration through a filter, gel filtration chromatography, free-flow electrophoresis and capillary electrophoresis. In addition, additional processes such as washing for removing impurities and concentration of obtained vesicles can be included.

In the present invention, the sample in step (a) may be blood, urine, stool, saliva or nasal mucosa, but is not limited thereto.

In the present invention, the primer pair in the step (b) may be a primer pair containing the base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2, but is not limited thereto.

As another aspect of the present invention, the present invention provides a composition for preventing, ameliorating or treating an inflammatory disease containing a vesicle derived from a bacterium belonging to the genus Corynebacterium as an active ingredient.

The compositions include pharmaceutical compositions, food compositions, cosmetic compositions and inhalant compositions.

As yet another aspect of the present invention, the present invention provides a method for preventing or treating an inflammatory disease, which comprises a step of administering to an individual a composition containing a vesicle derived from a bacterium of the genus Corynebacterium as an active ingredient.

As yet another aspect of the invention, the invention provides for the prevention or treatment of inflammatory diseases of vesicles derived from Corynebacterium spp.

As yet another aspect of the present invention, the present invention provides a prophylactic or therapeutic use for an inflammatory disease of a composition containing a vesicle derived from a Corynebacterium genus bacterium as an active ingredient.

As yet another aspect of the invention, the invention provides applications for producing agents used in inflammatory diseases of vesicles derived from Corynebacterium spp.

The term "Inflammatory Disease" as used in the present invention means a disease caused by damage to skin or intestinal epithelial cells and consequent inflammation caused by exposure to a causative factor that induces inflammation. It includes, but is not limited to, metabolic disorders, cardiovascular disorders, neuropsychiatric disorders, and cancers that occur as a result of inflammation.

Examples of the inflammatory disease according to the present invention include, but are not limited to, liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia, and nasal polyps.

As used in the present invention, the term "prevention" means all actions that suppress or delay the onset of inflammatory diseases by administration of the composition according to the present invention.

As used in the present invention, the term "treatment" means any act in which the administration of the composition according to the present invention improves or favorably changes the symptoms of an inflammatory disease.

As used in the present invention, the term "improvement" means any action that at least reduces the degree of symptoms, eg, parameters associated with the condition being treated.

In one embodiment of the invention, bacteria and vesicles derived from the bacteria are orally administered to mice to evaluate the absorption, distribution and excretion aspects of the bacteria and vesicles, and in the case of bacteria, via the intestinal mucosa. It was confirmed that the vesicles were absorbed within 5 minutes of administration, distributed systemically, and excreted through the kidney, liver, etc., as compared with the non-absorbed vesicles (see Example 1).

In another embodiment of the present invention, as a result of directly administering bacteria and vesicles derived from the bacteria to the intestine and evaluating whether or not the vesicles pass through the protective membrane of the intestinal mucosa, in the case of bacteria, they pass through the protective membrane of the intestinal mucosa. However, in the case of vesicles derived from bacteria, it was confirmed that they passed through the protective membrane of the mucosa (see Example 2).

In yet another example of the invention, patients with corynebacterium spp. From clinical samples of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps and normal control groups. As a result of comparing the distributions, it was confirmed that the distribution of vesicles derived from Corynebacterium spp. Was significantly reduced in the clinical sample of the diseased person as compared with the normal control group (see Examples 3 to 12).

In yet another example of the present invention, a strain of Corynebacterium glutamicum belonging to a bacterium belonging to the genus Corynebacterium was cultured, and the pro-inflammatory effect of vesicles secreted from the strain was evaluated. After treating vesicles derived from corinebacterium glutamicum at various concentrations into macrophages, vesicles derived from Escherichia coli, which is a typical pathogenic vesicle, were treated and the degree of secretion of inflammatory mediators was compared. The secretory capacity of the vesicles derived from corynebacterium glutamicum was significantly reduced as compared with the secretion of IL-6 and TNF-α by the vesicles (see Example 14).

In yet another example of the present invention, the anti-inflammatory effect of vesicles derived from the Corynebacterium glutamicum strain was evaluated. Before treating vesicles derived from Escherichia coli, which are pathogenic vesicles, macrophages were treated with vesicles derived from corinebacterium glutamicum at various concentrations, and then the secretion of inflammation mediators was evaluated. It was confirmed that the vesicles derived from corynebacterium glutamicum efficiently suppressed the secretion of TNF-α by the vesicles (see Example 15).

The pharmaceutical composition according to the invention can include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is usually used at the time of preparation, and includes saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes and the like. However, the present invention is not limited to this, and other usual additives such as antioxidants and buffers can be further included, if necessary. In addition, a diluent, a dispersant, a surfactant, a binder, a lubricant, etc. are additionally added to form an injection form such as an aqueous solution, a suspension, an emulsion, a pill, a capsule, a granule, or a tablet. Can be formulated with. Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated with each component using the methods disclosed in the Remington literature. The pharmaceutical composition of the present invention can be formulated into an injection, an inhalant, an external skin preparation, an oral ingestion, or the like, although the dosage form is not particularly limited.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, skin, nasal cavity, airway) according to the method of interest, and the dose is the patient's condition and weight, disease. Depending on the degree of the drug, the form of the drug, the route of administration and the time, it can be appropriately selected by those skilled in the art.

The pharmaceutical composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, a pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level is a patient's disease. It can be determined by type, severity, activity of the drug, sensitivity to the drug, duration of administration, route of administration and rate of excretion, duration of treatment, factors containing the drug used simultaneously and other factors well known in the medical field. The compositions according to the invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, and can be administered single or multiple times. It is important to consider all of the above factors and administer an amount that will give the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, gender, body weight of the patient, and may be increased or decreased depending on the route of administration, severity of obesity, sex, body weight, age and the like.

In the inhaler composition of the present invention, the active ingredient may be added to the inhalant as it is or used in combination with other ingredients, or may be appropriately used by a usual method. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (preventive or therapeutic).

The food composition of the present invention includes a health functional food composition. The food composition according to the present invention may be added to the food as it is or used in combination with other foods or food ingredients, or may be appropriately used by a usual method. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement). Generally, during the production of foods or beverages, the compositions of the present invention are added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw materials. However, in the case of long-term ingestion for the purpose of health and hygiene or for the purpose of health regulation, the amount may be less than or equal to the above range.

The food composition of the present invention has no special restrictions on other ingredients other than containing the active ingredient as an essential ingredient in an specified ratio, and has various flavoring agents or natural carbohydrates as in ordinary beverages. Can be contained as an additional ingredient. Examples of natural carbohydrates mentioned above include monosaccharides such as glucose, fructose; disaccharides such as maltose, sucrose; and polysaccharides such as dextrins, cyclodextrins and the like, and xylitol. It is a sugar alcohol such as sorbitol and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrates can be appropriately determined by one of ordinary skill in the art.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), and pectic acids. And its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonizing agents used in carbonated beverages and the like. Such components can be used independently or in combination. The proportion of such additives may also be appropriately selected by those skilled in the art.

The cosmetic composition of the present invention may contain not only vesicles derived from Corynebacterium spp., But also components commonly used in cosmetic compositions, such as antioxidants, stabilizers, and the like. Common auxiliary agents such as solubilizers, vitamins, pigments and fragrances, and carriers can be included.

Further, the composition of the present invention has been conventionally used for organic ultraviolet rays to the extent that it reacts with vesicles derived from Corynebacterium genus bacteria and does not damage the skin protective effect in addition to vesicles derived from Corynebacterium genus bacteria. A blocking agent may be mixed and used. Examples of the organic ultraviolet blocking agent include glyceryl PABA, drometrizoletrisiloxane, drometrizole, digalloyltrioleate, phenyldibenzimidazole tetrasulfonic acid 2Na, iscotridinol, diethylaminohydroxybenzoylhexylbenzoate, and DEA-methoxycinnamate. , Lawson and dihydroxyacetone mixture, Methylenebis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylenekanfa, menthyl anthranilate, benzophenone-3 (oxybenzon), benzophenone-4, benzophenone-8 (dioxybenzone), butylmethoxydi Benzoylmethane, bisethylhexyloxyphenol methoxyphenyltriazine, cinoxate, ethyldihydroxypropyl PABA, octocrylene, ethylhexyldimethyl PABA, ethylhexylmethoxycinnamate, ethylhexyl salicylate, ethylhexyltriazone, isoamyl-p-methoxycinnamate, polysilicon-15 (malon) One or more selected from the group consisting of dimethylodiethylbenzalate), terephthalidenedidicanphasulfonic acid and its salts, TEA-salicylate and aminobenzoic acid (PABA) can be used.

Products to which the cosmetic composition of the present invention can be added include, for example, cosmetics such as astringent lotion, soft lotion, nutritional lotion, various creams, essences, packs, foundations, cleansing, facial cleansers, soaps, etc. There are treatments, beauty essences, etc. Specific dosage forms of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, hand cream, and the like. Essence, nutritional essence, pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, milky lotion, lipstick, makeup base, foundation, press powder, loose powder, eye shadow, etc. include.

Hereinafter, preferred embodiments will be presented to aid in the understanding of the present invention. However, the following examples are provided only for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example 1. Analysis of absorption, distribution and excretion aspects of bacteria and vesicles derived from bacteria]
Experiments were conducted by the following methods to evaluate whether bacteria and vesicles derived from bacteria are systemically absorbed through the mucosa. Bacteria labeled with fluorescence in the gastrointestinal tract of mice and vesicles derived from the bacteria were administered to the gastrointestinal tract at doses of 50 μg each, and fluorescence was measured after 0 minutes, 5 minutes, 3 hours, 6 hours, and 12 hours, respectively. As a result of observing the whole image of the mouse, as shown in FIG. 1a, it was not systemically absorbed in the case of bacteria, but in the case of vesicles derived from bacteria, it was systemically absorbed 5 minutes after administration. 3 hours after administration, a strong fluorescence was observed in the bladder, indicating that vesicles were excreted in the urinary system. It was also found that vesicles were present in the body up to 12 hours after administration (see FIG. 1a).

After systemic absorption of bacteria and bacterial vesicles, 50 μg of fluorescently labeled bacteria and bacterial vesicles were administered as described above to assess the appearance of invasion into many organs. Then, 12 hours after administration, blood, heart, lung, liver, kidney, spleen, fat, and muscle were collected. As a result of observing fluorescence in the collected tissue, as shown in FIG. 1b, bacterial vesicles were distributed in blood, heart, lung, liver, kidney, spleen, fat, and muscle, but the bacteria were not absorbed. Okay (see Figure 1b).

[Example 2. Evaluation of the presence or absence of permeation of the mucosal protective membrane of bacteria and vesicles derived from bacteria]
In order to evaluate whether bacteria and vesicles derived from bacteria pass through the mucosal defense membrane and invade epithelial tissue, vesicles derived from bacteria and bacteria are directly administered to the intestine and then immunohistochemistry is used. Invasion into epithelial tissue through the mucosal defense membrane was evaluated. In order to evaluate the presence of bacteria and vesicles in mucosal tissue, antibodies against the bacteria and vesicles were prepared and used with GFP (Green Fluorescent protein) and stained with DAPI (4,6-diamidino 2-phenylindole). Later, it was observed under a microscope.

As a result, it was confirmed that in the case of bacteria, the vesicles derived from the bacteria did not pass through the mucosal protective membrane, while the vesicles derived from the bacteria passed through the mucosa and infiltrated the epithelial tissue (see FIG. 2).

[Example 3. Metagenomic analysis of bacterial-derived vesicles present in clinical samples]
Blood or nasal mucosa was first placed in a 10 ml tube, the suspended matter was submerged in a centrifuge (3,500 × g, 10 min, 4 ° C.), and then only the supernatant was transferred to a new 10 ml tube. After removing bacteria and foreign matter using a 0.22 μm filter, transfer to a centriplegic filter (50 kD), centrifuge at 1500 × g at 4 ° C for 15 minutes, discard substances smaller than 50 kD, and concentrate to 10 ml. rice field. Once again, after removing bacteria and foreign matter using a 0.22 μm filter, ultrafast centrifugation was performed at 150,000 × g with a Type 90ti rotor at 4 ° C. for 3 hours to remove the supernatant, and then solidified. The pellet was dissolved in Phosphate buffered saline (PBS).

100 μl of the vesicles separated by the above method were boiled at 100 ° C. to allow the internal DNA to come out of the lipid, and then cooled with ice for 5 minutes. Then, in order to remove the remaining suspended matter, the mixture was centrifuged at 10,000 × g at 4 ° C. for 30 minutes, only the supernatant was collected, and then the amount of DNA was quantified using Nanodrop. Then, in order to confirm whether or not bacterial-derived DNA is present in the extracted DNA, PCR is performed with the 16s rDNA primers shown in Table 1 below, and the bacterial-derived gene is present in the extracted gene. It was confirmed.

The DNA extracted by the above method is amplified using the above 16S rDNA primer and then sequenced (Illumina MiSeq sequencer), the result is output as an SFF (Standard Flowgram Form) file, and GS FLX software (v2.9) is used. After converting the SFF file into a sequence file (.fasta) and a nucleice quality score file using, the credit rating of the lead was confirmed, and the window (20 bps) average base call currency was less than 99% (Phred score <20). The part was removed. For OUT (Operational Taxonomy Unit) analysis, clustering was performed by sequence similarity using UCLUST and USEARCH, 94% for genus, 90% for family, 85% for order, and net. (Class) is 80%, phylum (phylum) is clustered based on the sequence similarity of 75%, and each OTU phylum (phylum), net (class), order (order), family (family), genus (genus). ) Level classification was performed and bacteria with a sequence similarity of 97% or higher at the genus level were profiled using the 16S RNA sequence database (108,453 sequences) of BLASTN and GreenGenes (QIIME).

[Example 4. Metagenomic analysis of bacterial vesicles in blood of patients with cirrhosis]
In the blood of 97 patients with liver cirrhosis and 171 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles existing in the blood and metagenome analysis was performed, and then corynebacterium was performed. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of patients with liver cirrhosis and liver cancer as compared with the blood of normal humans (see FIG. 3).

[Example 5. Metagenomic analysis of bacterial vesicles in the blood of stroke patients]
In the blood of 79 stroke patients and 158 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles existing in the blood and metagenome analysis was performed, and then corynebacterium was performed. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of stroke patients as compared with the blood of normal humans (see FIG. 4).

[Example 6. Metagenomic analysis of bacterial vesicles in the blood of diabetic patients]
In the blood of 81 diabetic patients and 126 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles present in the blood and metagenome analysis was performed, and then corynebacterium was performed. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of diabetic patients as compared with the blood of normal humans (see FIG. 5).

[Example 7. Metagenomic analysis of bacterial vesicles in the blood of asthmatic patients]
In the blood of 182 asthma patients and 180 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles existing in the blood and metagenome analysis was performed, and then corynebacterium was performed. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of asthma patients as compared with the blood of normal humans (see FIG. 6).

[Example 8. Metagenome analysis of bacterial vesicles in the blood of patients with atopic dermatitis]
After extracting genes from vesicles present in the blood and performing metagenome analysis on the blood of 42 patients with atopic dermatitis and 40 normal humans whose age and gender were matched by the method of Example 3 above. , The distribution of vesicles derived from Corynebacterium spp. Was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of patients with atopic dermatitis as compared with the blood of normal humans (see FIG. 7).

[Example 9. Metagenomic analysis of bacterial vesicles in the blood of depressed patients]
In the blood of 72 depressed patients and 80 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles existing in the blood and metagenome analysis was performed, and then corynebacterium was performed. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of depressed patients as compared with the blood of normal humans (see FIG. 8).

[Example 10. Metagenomic analysis of bacterial vesicles in the blood of breast cancer patients]
In the blood of 102 breast cancer patients and 100 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles existing in the blood and metagenome analysis was performed, and then corynebacterium was performed. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of breast cancer patients as compared with the blood of normal humans (see FIG. 9).

[Example 11. Metagenomic analysis of bacterial vesicles in the blood of dementia patients]
In the blood of 73 dementia patients and 70 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles existing in the blood and metagenome analysis was performed, and then corynea. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of dementia patients as compared with the blood of normal humans (see FIG. 10).

[Example 12. Infiltration aspect of vesicles derived from Corynebacterium spp. Through metagenome analysis of nasal mucosa in patients with nasal polyps and normal controls]
Metagenome analysis was performed by the method of Example 3 to evaluate the infiltration aspect of bacterial vesicles in the nasal mucosa of nasal polyp patients (43 non-allergic patients and 45 allergic patients) and 39 normal control groups. went. As a result, in the nasal tissues of patients with allergic nasal polyps and patients with non-allergic nasal polyps, vesicles derived from Corinebacterium spp. There was no difference between the presence or absence and the degree of infiltration of vesicles derived from Polynebacterium spp. (See FIG. 11).

[Example 13. Vesicle separation from Corynebacterium glutamicum culture medium]
After culturing a strain of Corynebacterium glutamicum, the vesicles were isolated and analyzed for their characteristics. First, the Corynebacterium glutamicum strain was cultured in an MRS (de Man-Logosa and Sharpe) medium in an incubator at 37 ° C. until the absorbance (OD 600) reached 1.0 to 1.5, and then LB (Luria-). Subculture (sub-culture) was carried out in Bertani) medium. Then, the culture broth containing the strain was collected, centrifuged at 10,000 × g at 4 ° C. for 20 minutes to remove the cells, and filtered through a 0.22 μm filter.

The filtered supernatant is concentrated by microfiltration in a 100 kDa Cellicon 2 Cassette filter membrane (Merck Millipore, US) using a MasterFlex pump system (Cole-Parmer, US) to a volume of 50 ml or less. The supernatant was once again filtered through a 0.22 μm filter. Then, the protein was quantified using BCA (Bicinchoninic acid) assay, and the following experiment was carried out on the obtained vesicles.

[Example 14. Anti-inflammatory effect of vesicles derived from Corynebacterium glutamicum]
In order to confirm the effect of Corynebacterium glutamicum-derived vesicles (Corynebacterium glutamicum EV) on the secretion of inflammatory mediators (IL-6, TNF-α) in inflammatory cells, a mouse macrophage strain, Raw 264.7 cells, was used. After treatment of vesicles derived from Corynebacterium glutamicum at various concentrations (0.1, 1, 10 μg / ml), cell killing and ELISA proceeded.

More specifically, various concentrations of corynebacterium diluted in DMEM (Dulvecco's Minimum Essential Medium) serum-free medium in Raw264.7 cells dispensed in 5 × 10 ⁴ cells in a 48-well cell culture plate. Vesicles derived from Glutamicum were treated and cultured for 12 hours. Then, the cell culture solution was collected in a 1.5 ml tube and centrifuged at 3000 × g for 5 minutes, and the supernatant was collected and stored at −80 ° C., and then ELISA was proceeded.

To perform ELISA, the capture antibody was diluted with PBS and dispensed into a 96-well polystyrene plate in 50 μl increments according to the concentration of action, followed by overnight reaction at 4 ° C. Then, wash 3 times with 100 μl of PBST (PBS containing 0.05% Tween-20) solution, and then dispense 100 μl of RD (PBS containing 1% BSA) solution for 1 hour at room temperature. Blocked for a while. After dispensing 50 μl of the sample and standard according to the concentration and reacting at room temperature for 2 hours, washing with 100 μl of PBST three times, diluting the detected antibody with RD and dividing by 50 μl according to the action concentration. It was poured and reacted at room temperature for 2 hours. After washing 3 times with 100 μl of PBST, Streptavidin-HRP (R & D system, USA) was diluted 1/40 with RD, dispensed in 50 μl portions, and reacted at room temperature for 20 minutes. Finally, after washing 3 times with 100 μl of PBST, 50 μl of TMB (3,3', 5,5'-Tetramethylbenzidine) substrate (SurModics, USA) was dispensed and color development progressed 5 to 20 minutes later. The reaction was stopped by dispensing 50 μl of a 1 M sulfuric acid solution at a time, and the absorbance was measured at 450 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA).

As a result, as shown in FIG. 12, no cell death was observed by the treatment of the vesicle (CGT101) derived from Corynebacterium glutamicum (see FIG. 12). In addition, as shown in FIGS. 13a and 13b, as a result of evaluating the secretory aspect of the inflammatory mediator in the inflammatory cells, compared with the treatment of vesicles derived from Escherichia coli (E. coli EV 1 μg / ml), which is a positive control group. It was confirmed that the secretion of IL-6 (FIG. 13a) and TNF-α (FIG. 13b) was further reduced during the treatment of vesicles (CGT101) derived from Escherichia coli glutamicum.

[Example 15. Anti-inflammatory effect of vesicles derived from Corynebacterium glutamicum]
Based on the results of Example 14, various concentrations (0.1, 1, 10 μg / ml) derived from Corynebacterium glutamicum were used to evaluate the anti-inflammatory effect of the vesicles derived from Corynebacterium glutamicum. (CGT101) was pretreated with a mouse macrophage strain for 12 hours, followed by treatment with 1 μg / ml of vesicles derived from Escherichia coli, which is a pathogenic causative factor, and 12 hours later, the secretion of inflammatory cytokines was measured by ELISA.

As a result, it was confirmed that when pretreatment of vesicles derived from Corynebacterium glutamicum, the amount of TNF-α secreted into inflammatory cells stimulated by vesicles derived from Escherichia coli was significantly suppressed (FIG. 14). reference). This means that vesicles derived from Corynebacterium glutamicum can efficiently suppress inflammatory reactions induced by pro-inflammatory factors such as vesicles derived from Escherichia coli, which are pathogenic vesicles.

The above description of the present invention is for illustration purposes only, and a person who has ordinary knowledge in the technical field to which the present invention belongs may use other specific materials without changing the technical idea or essential features of the present invention. It can be understood that it can be easily transformed into a typical form. Therefore, it should be understood that the examples described above are exemplary in all respects and are not limiting.

It was confirmed that the vesicles derived from the genus Corinebacterium according to the present invention pass through the protective membrane of the mucosa, are absorbed by the epithelial cells of the mucosa, are distributed systemically, and are excreted from the body through the kidneys, liver, and lungs. It was confirmed that there was a significant decrease in the blood or nasal mucosa of patients with liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps, and TNF-α due to pathogenic vesicles. Since it can significantly suppress the secretion of inflammatory mediators such as inflammatory mediators, it is expected to have great industrial utility value in that it can be usefully used in compositions for the prevention or treatment of inflammatory diseases.

Claims (17)

Information method for diagnosing one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps, including the following steps:
(A) Step of extracting DNA from vesicles isolated from normal human and subject samples;
(B) After performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA, the steps of obtaining each PCR product; and (c). ) When the content of vesicles derived from Corynebacterium spp. Is lower than that of normal humans through quantitative analysis of the PCR product, liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia. And a step of determining one or more diseases selected from the group consisting of nasal polyps. The information providing method according to claim 1, wherein the sample in step (a) is blood, urine, stool, saliva, or nasal mucosa. A pharmaceutical composition for the prevention or treatment of inflammatory diseases, which comprises a vesicle derived from a bacterium belonging to the genus Corynebacterium as an active ingredient. The pharmaceutical composition for preventing or treating an inflammatory disease according to claim 3, wherein the vesicles derived from the genus Corynebacterium are secreted from Corynebacterium glutamicum. The pharmaceutical composition for preventing or treating an inflammatory disease according to claim 3, wherein the vesicles have an average diameter of 10 to 200 nm. The pharmaceutical composition for preventing or treating an inflammatory disease according to claim 3, wherein the vesicles are naturally or artificially secreted from a bacterium belonging to the genus Corynebacterium. 3. The inflammatory disease is one or more selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. A pharmaceutical composition for the prevention or treatment of inflammatory diseases according to. A food composition for preventing or ameliorating an inflammatory disease, which comprises a vesicle derived from a bacterium belonging to the genus Corynebacterium as an active ingredient. The food composition for preventing or ameliorating an inflammatory disease according to claim 8, wherein the vesicles derived from the genus Corynebacterium are secreted from Corynebacterium glutamicum. The food composition for preventing or ameliorating an inflammatory disease according to claim 8, wherein the vesicles have an average diameter of 10 to 200 nm. The food composition for preventing or ameliorating an inflammatory disease according to claim 8, wherein the vesicles are naturally or artificially secreted from a bacterium belonging to the genus Corynebacterium. 8. Claim 8 characterized in that the inflammatory disease is one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. A food composition for preventing or ameliorating an inflammatory disease according to. A cosmetic composition for preventing or ameliorating an inflammatory disease, which comprises a vesicle derived from a bacterium belonging to the genus Corynebacterium as an active ingredient. An inhaler composition for preventing or treating an inflammatory disease, which comprises a vesicle derived from a bacterium belonging to the genus Corynebacterium as an active ingredient. Diagnostic method of one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps including the following steps:
(A) Step of extracting DNA from vesicles isolated from normal human and subject samples;
(B) After performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA, the steps of obtaining each PCR product; and (c). ) When the content of vesicles derived from Corynebacterium spp. Is lower than that of normal humans through quantitative analysis of the PCR product, liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia. And a step of determining one or more diseases selected from the group consisting of nasal polyps. A method for preventing or treating an inflammatory disease, which comprises a step of administering to an individual a composition containing a vesicle derived from a bacterium belonging to the genus Corynebacterium as an active ingredient. Uses for producing drugs used for inflammatory diseases of vesicles derived from Corynebacterium spp.

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