WO2019172598A1 - Nanovesicles derived from lactobacillus sp. bacteria, and use thereof - Google Patents

Abstract

The present invention relates to vesicles derived from Lactobacillus sp. bacteria, and a use thereof. The present inventors experimentally confirmed that: compared to those of a normal person, the vesicles are significantly reduced in samples from patients with stomach cancer, renal failure, dementia and stroke; and the vesicles inhibited the secretion of inflammatory mediators by pathogenic vesicles. Thus, the vesicles derived from Lactobacillus sp. bacteria, according to the present invention, may be usefully employed for the purpose of developing: a method for diagnosing stomach cancer, renal failure, dementia or stroke; and a composition for preventing, improving or treating inflammatory diseases, including said diseases, accompanied by immune dysfunction.

Description

Nanovesicles derived from bacteria of the genus Lactobacillus and uses thereof

The present invention relates to Lactobacillus bacteria-derived nano-vesicles and uses thereof, and more specifically, to a method for diagnosing gastric cancer, kidney failure, dementia, or stroke using nano-vesicles derived from Lactobacillus bacteria and the above-mentioned vesicles. A composition for preventing, ameliorating or treating a disease.

In the 21st century, acute infectious diseases, which were previously recognized as infectious diseases, have become less important, while chronic diseases with immune dysfunctions caused by incompatibility between humans and microbiomes determine the quality of life and human life. As a major disease, the disease pattern has changed. In particular, metabolic diseases such as cancer and diabetes, cardiovascular diseases such as myocardial infarction and stroke, and neuro-psychiatric diseases such as Parkinson's disease, dementia, and depression due to the westernization of dietary habits have become major problems in public health.

The disease is characterized by chronic inflammation, and the occurrence of chronic inflammation is accompanied by abnormalities in immune function against external causative factors. The immune response to the causative agent derived from bacteria is the Th17 immune response that secretes the interleukin (IL) -17 cytokine, and neutrophil inflammation caused by the Th17 immune response occurs when exposed to the bacterial cause. Inflammatory mediators such as tumor necrosis factor-alpha (TNF-α) play an important role in the development of inflammation. In addition, IL-6 secreted by a bacterial causative agent has recently been reported to play an important role in the differentiation into Th17 cells, and to be involved in the pathogenesis of cardiovascular diseases and neuropsychiatric diseases.

The microorganisms symbiotic to the human body reaches 100 trillion times more than human cells, and the number of genes of microorganisms is known to be more than 100 times the number of human genes. Microbiota (microbiota or microbiome) refers to a microbial community including microbes, archaea and eukarya that exist in a given settlement, and the intestinal microbiota is important for human physiology. It plays a role and is known to greatly affect human health and disease through interaction with human cells.

Symbiotic bacteria and archaea, which live in our bodies, secrete nanometer-sized vesicles to exchange information about genes and proteins to other cells. The mucous membrane forms a physical protective film that particles larger than 200 nanometers (nm) in size can't pass through. If the bacteria are symbiotic bacteria, the mucosa cannot pass through the mucous membrane, but the bacteria-derived vesicles are 100 nanometers in size or less. It passes through epithelial cells through the mucous membrane and is absorbed by our body. The pathogenic bacteria-derived vesicles absorbed by our bodies have recently been found to play an important role in the pathogenesis of metabolic diseases such as diabetes and obesity.

Lactobacillus (Lacotobacillus) in bacteria is a Gram-positive bacteria that secrete a legacy, has been used in many bacteria are probiotics products belonging to the genus Lactobacillus. However, there is no report on the diagnosis and treatment technology for diseases such as gastric cancer, renal failure, dementia, and stroke using vesicles derived from Lactobacillus bacteria.

The present inventors earnestly researched to solve the above-mentioned problems. As a result, metagenome analysis significantly reduced the contents of bacteria derived from Lactobacillus bacteria in samples derived from gastric cancer, kidney failure, dementia, and stroke patients compared to normal people. As a result, it was confirmed that the Lactobacillus bacteria-derived vesicles were isolated in vitro to evaluate the therapeutic efficacy, and as a result, the present invention was completed.

Accordingly, an object of the present invention is to provide an information providing method for the diagnosis of gastric cancer, kidney failure, dementia, or stroke.

In addition, another object of the present invention is to provide a composition for preventing, improving or treating inflammatory diseases with an immune dysfunction including Lactobacillus-derived vesicles as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object of the present invention, the present invention provides a method for providing information for the diagnosis of gastric cancer, kidney failure, dementia, or stroke, comprising the following steps:

(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;

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

(c) classifying gastric cancer, renal failure, dementia, or stroke when the content of extracellular vesicles derived from bacteria of the genus Lactobacillus is lower than that of normal people through quantitative analysis of the PCR products.

The present invention provides a method for diagnosing gastric cancer, kidney failure, dementia, or stroke, comprising the following steps:

(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;

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

(c) Determining gastric cancer, kidney failure, dementia, or stroke when the content of extracellular vesicles derived from bacteria of Lactobacillus ( Lacotobacillus ) is lower than that of the normal person through quantitative analysis of the PCR product.

In one embodiment of the invention, the sample in step (a) may be blood.

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

In addition, the present invention comprises a bacterium derived from Lactobacillus genus as an active ingredient, accompanied by abnormal immune function It provides a pharmaceutical composition for preventing or treating inflammatory diseases.

In another aspect, the present invention provides a food composition for preventing or improving inflammatory diseases, including immune function abnormalities, including vesicles derived from Lactobacillus bacteria as an active ingredient.

In another aspect, the present invention provides a cosmetic composition for preventing or improving skin inflammatory diseases, comprising a vesicle derived from Lactobacillus bacteria as an active ingredient.

In addition, the present invention is a vesicle derived from the bacteria of the genus Lactobacillus, accompanied by abnormal immune function Provided for the prevention or treatment of inflammatory diseases.

In addition, the present invention comprises the step of administering to a subject a pharmaceutical composition comprising a Lactobacillus bacteria-derived vesicles as an active ingredient, accompanied by immune function Provided are methods for preventing or treating inflammatory diseases.

In one embodiment of the present invention, the vesicles may have an average diameter of 10 to 200 nm.

In another embodiment of the present invention, the vesicles may be secreted naturally or artificially from bacteria of the genus Lactobacillus.

In another embodiment of the present invention, the Lactobacillus ( Lacotobacillus ) bacteria-derived vesicles are derived from Lactobacillus brevis ( Lacotobacillus brevis ) vesicles, Lactobacillus casei ( Lacotobacillus casei ) vesicles, Lactobacillus rhamnosus ( Lacotobacillus rhamnosus ) -derived vesicles And it may be one or more vesicles selected from the group consisting of Lacotobacillus sakei- derived vesicles.

In another embodiment of the present invention, the inflammatory disease may be one or more diseases selected from the group consisting of gastric cancer, kidney failure, dementia, and stroke.

The present inventors confirmed that the intestinal bacteria are not absorbed into the body, but when the bacteria-derived vesicles are absorbed into the body and distributed systemically, they are excreted in vitro through the kidneys, liver, and lungs. Derived vesicles metagenome analysis confirmed that Lactobacillus bacteria-derived vesicles present in gastric cancer, kidney failure, dementia, and stroke patient samples were significantly reduced compared to normal individuals. In addition, when isolated Lactobacillus brevis, Lactobacillus casei, Lactobacillus rhamnosus, and Lactobacillus rhamnosus, and Lactobacillus casei strain belonging to the bacteria of the genus Lactobacillus, cultured in vitro to separate the vesicles and administered to inflammatory cells, IL caused by pathogenic vesicles -6 and TNF-α significantly inhibits the secretion of inflammatory mediators, such that the immune control function, the bacterium derived from Lactobacillus according to the present invention is a method for diagnosing gastric cancer, kidney failure, dementia, or stroke, and It is expected that the present invention may be usefully used in a composition for preventing, ameliorating or treating diseases.

Figure 1a is a picture of the distribution of bacteria and vesicles by time after oral administration of bacteria and bacteria-derived vesicles (EV) to the mouse, Figure 1b is 12 hours after oral administration, blood, kidney Figure shows the distribution of bacteria, vesicles and vesicles in the body, liver and various organs.

Figure 2 is a result of comparing the distribution of bacteria-derived vesicles of the genus Lactobacillus after the analysis of bacteria-derived vesicles metagenome present in gastric cancer patients and normal blood.

3 is a result of comparing the distribution of bacteria-derived vesicles of the genus Lactobacillus after analyzing the bacteria-derived vesicles metagenome present in renal failure patients and normal blood.

4 is a result of comparing the distribution of bacteria-derived vesicles of the genus Lactobacillus after the analysis of bacteria-derived vesicles metagenome present in dementia patients and normal blood.

Figure 5 is a result of comparing the distribution of bacteria-derived vesicles of the genus Lactobacillus after the analysis of bacteria-derived vesicles metagenome present in stroke patients and normal blood.

FIG. 6 shows Lactobacillus plantarum or Lactobacillus brevis (L. plantarum) before treatment of E. coli EV, a pathogenic vesicle, to evaluate the immunomodulatory and anti-inflammatory effects of Lactobacillus brevis-derived vesicles. brevis) -derived vesicles were pretreated to evaluate the effects of E. coli vesicles on secretion of inflammatory mediators IL-6 and TNF-α (PC: positive control; LP: Lactobacillus plantarum EVs; LB: Lactobacillus brevis EVs).

FIG. 7 shows Lactobacillus plantarum or Lactobacillus casei ( L. plantarum ) before treatment with Escherichia coli vesicles ( E. coli EV) to evaluate the immunomodulatory and anti-inflammatory effects of Lactobacillus cassia-derived vesicles . Casei ) derived vesicles were pretreated to evaluate the effects on E. coli vesicles secrete IL-6 and TNF-α secretion (PC: positive control; LP: Lactobacillus plantarum EVs; LC: Lactobacillus casei EVs).

FIG. 8 shows Lactobacillus plantarum or Lactobacillus rhamnosus before treatment of Escherichia coli vesicles (E. coli EV) to evaluate the immunomodulatory and anti-inflammatory effects of Lactobacillus rhamnosus-derived vesicles. L. rhamnosus) vesicles were pretreated to evaluate the effect of E. coli vesicles on the secretion of inflammatory mediators IL-6 and TNF-α (PC: positive control; LP: Lactobacillus plantarum EVs; LR: Lactobacillus rhamnosus EVs). ).

9 is a Lactobacillus bacteria used for evaluating the immunomodulatory and anti-inflammatory effect of the K derived from a parcel, a parcel of pathogenic E. coli vesicles (E. coli EV) Lactobacillus Planta room (L. plantarum) or Lactobacillus four K before the treatment ( L. sakei ) was evaluated by pretreatment of vesicles derived from E. coli vesicles to the secretion of IL-6 and TNF-α, which are inflammation mediators (PC: positive control; LP: Lactobacillus plantarum EVs; LS: Lactobacillus sakei EVs). ).

The present invention relates to vesicles derived from the genus Lactobacillus and uses thereof.

The present inventors have found that the vesicle-derived bacterium from Lactobacillus is significantly reduced in gastric cancer, renal failure, dementia, and stroke patients compared to normal people, thereby confirming that the disease can be diagnosed. In addition, by separating the vesicles from the strains of Lactobacillus brevis, Kasei, Rhamnosus, and Sakei belonging to the genus Lactobacillus and confirmed the characteristics, it was confirmed that the vesicles can be used as a composition for the prevention or treatment of the disease.

Accordingly, the present invention provides an information providing method for diagnosing gastric cancer, kidney failure, dementia, or stroke, comprising the following steps:

(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;

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

(c) classifying gastric cancer, renal failure, dementia, or stroke when the content of extracellular vesicles derived from bacteria of the genus Lactobacillus is lower than that of normal people through quantitative analysis of the PCR products.

As used herein, the term "diagnosis" in the broad sense means to determine the actual condition of the patient's disease in all aspects. The content of the judgment is the name of the disease, the etiology, the type of disease, the seriousness, the detailed mode of the condition, the presence or absence of complications, and the prognosis. Diagnosis in the present invention is to determine the onset of gastric cancer, renal failure, dementia, and stroke and the level of disease.

The term "metagenome" used in the present invention, also referred to as "gunoelectric", refers to the sum total of the genome including all viruses, bacteria, fungi, etc. in an isolated area such as soil, animal intestine, mainly culture It is used as a concept of genome explaining the identification of many microorganisms at once using sequencer to analyze microorganisms that are not. In particular, the metagenome does not refer to one genome or genome, but to a kind of mixed dielectric as the genome of all species of one environmental unit. This is a term from the point of view of defining a species in the course of the evolution of biology in terms of functional species as well as various species that interact with each other to create a complete species. Technically, rapid sequencing is used to analyze all DNA and RNA, regardless of species, to identify all species in one environment, and to identify interactions and metabolism.

In the present invention, the sample may be blood, but is not limited thereto.

As another aspect of the present invention, the present invention provides a composition for the prevention, treatment or improvement of inflammatory diseases, including gastric cancer, kidney failure, dementia, stroke or skin inflammatory diseases, including vesicles derived from Lactobacillus bacteria as an active ingredient. . The composition comprises a food composition, a cosmetic composition and a pharmaceutical composition, in the present invention, the food composition comprises a nutraceutical composition. The composition of the present invention may be a formulation of an oral nebulizer or inhalant.

As used herein, the term "vesicle" refers to a structure of nanoscale membranes secreted by various bacteria. In addition to proteins and nucleic acids, vesicles derived from gram-positive bacteria such as Lactobacillus have peptidoglycan, lipoteichoic acid, and various low molecular weight compounds in the vesicles. have. In the present invention, the nano vesicles or vesicles are naturally secreted or artificially produced by Lactobacillus bacteria, and have an average diameter of 10 to 200 nm.

The vesicles are centrifuged, ultra-fast centrifugation, high pressure treatment, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical degradation, chemical treatment, filtration by a filter containing bacteria of the genus Lactobacillus. It can be separated using one or more methods selected from the group consisting of, gel filtration chromatography, pre-flow electrophoresis, and capillary electrophoresis. In addition, it may further include a process for washing to remove impurities, concentration of the obtained vesicles and the like.

As used herein, the term "prevention" means any action that inhibits or delays the development of an inflammatory disease, including gastric cancer, kidney failure, dementia, stroke, and / or skin inflammatory diseases, by administration of a composition according to the present invention. Means.

As used herein, the term "treatment" means any improvement or beneficial alteration of symptoms for inflammatory diseases, including gastric cancer, kidney failure, dementia, stroke, and / or skin inflammatory diseases, by administration of a composition according to the invention. It means an act.

As used herein, the term “improvement” means any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms.

The term "inflammatory disease" used in the present invention, Inflammatory diseases in the present invention are caused by a series of biological reactions caused by a direct response to the immune media's humoral mediator or by stimulating a local or systemic effector system. It may be a stroke or skin inflammatory disease, and may be accompanied by abnormal immune function, but is not limited thereto.

The pharmaceutical composition according to the invention may comprise a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions and the like. Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, external preparation for skin, oral ingestion, and the like.

The pharmaceutical composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, skin, nasal, airways) according to the desired method, and the dosage is determined by the condition and weight of the patient, disease Depending on the degree, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment, and the effective dose level refers to the type of disease, the severity, the activity of the drug and the drug. Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The composition according to the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a 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, sex and weight of the patient, and generally 0.001 to 150 mg, preferably 0.01 to 100 mg daily or every other day, per kg of body weight Or divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

The food composition of the present invention includes a nutraceutical composition. The food composition according to the present invention may be used as it is, or may be used in combination with other foods or food ingredients, or may be appropriately used according to conventional methods. The mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). Generally, in the preparation of food or beverages the compositions of the invention are added in amounts of up to 15% by weight, preferably up to 10% by weight relative to the raw materials. However, in the case of prolonged intake for health and hygiene purposes or health control purposes, the amount may be below the above range.

The food composition of the present invention, in addition to containing the active ingredient as an essential ingredient in the indicated ratio, there are no particular restrictions on other ingredients, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. . The proportion of the natural carbohydrate can be appropriately determined by the choice of those skilled in the art.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. These 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 include not only vesicles derived from Lactobacillus bacteria, but also components commonly used in cosmetic compositions, for example, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and perfumes. Adjuvants, and carriers.

In addition, the composition of the present invention may be used in addition to the vesicles derived from Lactobacillus bacteria, organic sunscreens that have been conventionally used insofar as it does not impair the skin protection effect by reacting with the vesicles derived from Lactobacillus bacteria. Examples of the organic sunscreen include glyceryl pava, drometrizole trisiloxane, drometrizole, digaloyltrioleate, disodium phenyldibenzimidazole tetrasulfonate, diethylhexyl butamidotriazone, diethylamino Hydroxybenzoylhexylbenzoate, die-methoxycinnamate, a mixture of lowson and dihydroxyacetone, methylenebis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylidene camphor, menthyl anthranilate, benzophenone -3 (oxybenzone), benzophenone-4, benzophenone-8 (dioxyphenbenzone), butylmethoxydibenzoylmethane, bisethylhexyloxyphenol methoxyphenyltriazine, synoxate, ethyldihydroxypropylpava, Octocrylene, ethylhexyldimethylpava, ethylhexylmethoxycinnamate, ethylhexylsalicylate, ethylhexyltrizone, isoamyl-p-methoxycinnamate, polysilicon-15 (dimethicodiethylbenzalmal Ronate), terephthalylidene dicamphor sulfonic acid and salts thereof, thy-salicylate and aminobenzoic acid (Pava) can be used.

Examples of products to which the cosmetic composition of the present invention may be added include, for example, cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, and the like, cleansing agents, soaps, treatments, and essences. Etc. Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, lipsticks, makeup bases, foundations, press powders, loose powders, eye shadows and the like.

In one embodiment of the present invention, the bacteria and bacteria-derived vesicles orally administered to observe the body absorption, distribution, and excretion of the bacteria and vesicles in the body, in the case of bacteria is not absorbed through the intestinal membrane, the vesicles are administered 5 It was confirmed that it was absorbed within minutes and distributed systemically and excreted through the kidney, liver, and the like (see Example 1).

In another embodiment of the present invention, metagenome analysis was performed in gastric cancer, kidney failure, dementia, and stroke patients and normal blood. As a result, it was confirmed that Lactobacillus bacteria-derived vesicles were significantly reduced in the blood of stomach cancer, kidney failure, dementia, and stroke patients compared to normal blood (see Examples 3 to 6).

In another embodiment of the present invention, the immunomodulatory and anti-inflammatory effects of Lactobacillus brevis strain-derived vesicles were evaluated, and Lactobacillus plantarum or Lactobacillus brevis-derived vesicles of various concentrations were treated before treating the pathogenic vesicle-derived E. coli-derived vesicles. After treatment with macrophages, the secretion of inflammatory mediators was evaluated. As a result, it was confirmed that the lactobacillus brevis-derived vesicles by E. coli-derived vesicles suppressed the vesicles derived from Lactobacillus plantarum more efficiently. See examples 7 and 8).

In another embodiment of the present invention, the immunomodulatory and anti-inflammatory effects of the vesicles derived from Lactobacillus casei strains were evaluated, and treated with Escherichia coli-derived vesicles, which are pathogenic vesicles, before the Lactobacillus plantarum or various concentrations of Lactobacillus casei-derived vesicles. After treatment with macrophages, the secretion of inflammatory mediators was evaluated, and it was confirmed that the TNF-α secretion by E. coli-derived vesicles was suppressed more efficiently than the Lactobacillus plantarum-derived vesicles (Lactobacillus plantarum-derived vesicles). See examples 7 and 9).

In another embodiment of the present invention, the immunomodulatory and anti-inflammatory effects of Lactobacillus rhamnosus strain-derived vesicles were evaluated, and Lactobacillus plantarum or various concentrations of Lactobacillus rhamnosus before treatment with Escherichia coli-derived vesicles, which are pathogenic vesicles. After treating the derived vesicles with macrophages and evaluating the secretion of inflammatory mediators, it was confirmed that the TNF-α secretion by E. coli-derived vesicles suppressed Lactobacillus rhamnosus-derived vesicles more efficiently than Lactobacillus plantarum-derived vesicles. (See Examples 7 and 10).

In another embodiment of the present invention, the immunomodulatory and anti-inflammatory effects of the vesicles derived from Lactobacillus Sakei strains were evaluated, and Lactobacillus plantarum or various concentrations of Lactobacillus sakei before treatment of Escherichia coli-derived vesicles, which are pathogenic vesicles After treating the derived vesicles with macrophages and evaluating the secretion of inflammatory mediators, it was confirmed that the LN-α secretion by E. coli-derived vesicles effectively inhibited Lactobacillus plant-derived vesicles compared with Lactobacillus plantarum-derived vesicles. (See Examples 7 and 11).

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example  1. Analysis of absorption, distribution, and excretion of intestinal bacteria and bacterial-derived vesicles

In order to evaluate whether the intestinal bacteria and bacteria-derived vesicles are absorbed systemically through the gastrointestinal tract, experiments were performed as follows. Fluorescently labeled enterobacteriaceae and enteric bacteria-derived vesicles were administered to the gastrointestinal tract at doses of 50 μg, respectively, and the fluorescence was measured after 0, 5, 3, 6 and 12 hours. As a result of observing the whole image of the mouse, as shown in FIG. 1A, the bacteria were not absorbed systemically, but in the case of bacterial-derived vesicles, they were absorbed systemically 5 minutes after administration, and the bladder fluoresced 3 hours after administration. This was observed strongly, indicating that the vesicles were excreted by the urinary system. In addition, the vesicles were found to exist in the body until 12 hours of administration.

In addition, after the intestinal bacteria and enteric bacteria-derived vesicles are absorbed systemically, 50 μg of fluorescently labeled bacteria and bacteria-derived vesicles are administered in the same manner as described above in order to evaluate the infiltration into various organs. After 12 hours, blood, heart, lungs, liver, kidneys, spleen, fat and muscle were collected. As a result of fluorescence observed in the collected tissue, as shown in Figure 1b, the bacteria-derived vesicles were distributed in the blood, heart, lung, liver, spleen, fat, muscle, kidney, it can be seen that the bacteria are not absorbed.

Example 2 Bacterial-derived Vesicular Metagenome Analysis in Clinical Samples

Blood was first placed in a 10 ml tube and the suspension was allowed to settle by centrifugation (3,500 × g, 10 min, 4 ° C.) and only the supernatant was transferred to a new 10 ml tube. After removing the bacteria and foreign substances using a 0.22㎛ filter, and transferred to centrifugal filters (centrifugal filters 50 kD) and centrifuged at 1500 x g, 4 ℃ for 15 minutes to discard the material smaller than 50 kD concentrated to 10 ml. Once again, the bacteria and foreign substances were removed using a 0.22㎛ filter, and the supernatant was discarded using ultrafast centrifugation for 3 hours at 150,000 xg and 4 ℃ with a Type 90ti rotor, and the pulverized pellet was physiologically It was dissolved in saline (PBS).

100 μl of the vesicles separated by the above method was boiled at 100 ° C. to let the internal DNA come out of the lipid and then cooled on ice for 5 minutes. And centrifuged at 10,000 x g, 4 ℃ 30 minutes to remove the remaining suspended solids and collected only the supernatant. The amount of DNA was quantified using Nanodrop. Thereafter, PCR was performed with the 16s rDNA primer (primer) shown in Table 1 to confirm whether the bacteria-derived DNA exists in the extracted DNA, and it was confirmed that the bacteria-derived gene is present in the extracted gene.

primer		order	SEQ ID NO:
16S rDNA	16S_V3_F	5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3 '	One
	16S_V4_R	5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3	2

DNA extracted by the above method was amplified using the above 16S rDNA primers, followed by sequencing (Illumina MiSeq sequencer), and the results were outputted in a Standard Flowgram Format (SFF) file, using GS FLX software (v2.9). After converting the SFF file into a sequence file (.fasta) and a nucleotide quality score file, the credit rating of the lead is checked, and the window (20 bps) has an average base call accuracy of less than 99% (Phred score <20). It was. For operational taxonomy unit (OTU) analysis, clustering is performed according to sequence similarity using UCLUST and USEARCH, genus 94%, family 90%, order 85%, class 80%, phylum 75% sequence similarity Clustering is based on the phylum, class, order, family, and genus levels of each OTU, and BLASTN and GreenGenes' 16S RNA sequence database (108,453 sequences) is used to identify bacteria with greater than 97% sequence similarity at the genus level. Was profiled (QIIME).

Example  3. Vesicles derived from gastric cancer patients and normal blood bacteria Metagenome  analysis

In the method of Example 2, the blood of 66 gastric cancer patients and 198 normal blood patients whose age and sex were matched were extracted from the vesicles in the blood and subjected to metagenomic analysis, and then derived from the bacteria of the genus Lactobacillus. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Lactobacillus significantly decreased in the blood of gastric cancer patients compared to the normal blood (see Table 2 and Figure 2).

blood	Control		Stomach cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Lactobacillus	0.0325	0.0246	0.0242	0.0150	0.0015	0.75

Example  4. Renal switch and normal blood-derived vesicles Metagenome  analysis

In the method of Example 2, the blood of 21 renal convertors and 19 normal blood patients whose age and sex were matched were extracted from the vesicles present in the blood and subjected to metagenomic analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that Lactobacillus bacteria-derived vesicles were significantly reduced in the blood of the renal converter compared to normal blood (see Table 3 and FIG. 3).

blood	Control		Kidney failure		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Lactobacillus	0.0552	0.0268	0.0034	0.0133	<0.0001	0.06

Example  5. Vesicles derived from dementia patients and normal blood bacteria Metagenome  analysis

In the blood of 45 dementia patients and 49 blood of normal people whose age and sex were matched by the method of Example 2, genes were extracted from vesicles in the blood and subjected to metagenomic analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that Lactobacillus bacteria-derived vesicles were significantly reduced in the blood of dementia patients compared to normal blood (see Table 4 and FIG. 4).

blood	Control		dementia		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Lactobacillus	0.0557	0.0117	0.0313	0.0170	0.0051	0.56

Example  6. Vesicles derived from stroke patients and normal blood bacteria Metagenome  analysis

In the method of Example 2, the blood of 115 stroke patients and 109 normal blood patients whose age and sex were matched were extracted from the vesicles present in the blood and subjected to metagenomic analysis, followed by derivation of bacteria of the genus Lactobacillus. The distribution of vesicles was evaluated. As a result, it was confirmed that the bacteria-derived Lactobacillus-derived vesicles significantly reduced in the blood of stroke patients compared to normal blood (see Table 5 and FIG. 5).

blood	Control		stroke		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Lactobacillus	0.0361	0.0354	0.0046	0.0225	<0.0001	0.13

Example  7. Lactobacillus  Isolation of Vesicles from Genus Bacterial Cultures

Lactobacillus brevis , Lactobacillus casei , Lactobacilus Lactobacilus rhamnosus ), and Lactobacillus sakei strains were isolated from the environmental samples, cultured, and then their vesicles were isolated and characterized. Each Lactobacillus strain was cultured in de Man-Rogosa and Sharpe (MRS) medium until absorbance (OD ₆₀₀ ) was 1.0 to 1.5 at 37 ° C aerobic conditions, and then sub-cultured to Luria-Bertani (LB) medium. It was. Then, the medium containing the strain was recovered, centrifuged at 10,000 g, 4 ° C. for 20 minutes to remove the strain, and filtered through a 0.22 μm filter. The filtered supernatant was concentrated to 50 ml volume by microfiltration using a MasterFlex pump system (Cole-Parmer, US) with a 100 kDa Pellicon 2 Cassette filter membrane (Merck Millipore, US). The concentrated supernatant was once again filtered through a 0.22 μm filter. Thereafter, the protein was quantified using the BCA assay, and the following experiment was performed on the obtained vesicles.

Example 8. Immunomodulation and Anti-inflammatory Effects of Lactobacillus Brevis-derived Vesicles

In order to evaluate the immunomodulatory and anti-inflammatory effects of Lactobacillus brevis-derived vesicles, lactobacillus brevis-derived vesicles of various concentrations (0.1, 1, 10 ㎍ / ml) were pretreated in macrophage lines for 12 hours, followed by Escherichia coli, a pathogenic vesicle. After 12 hours of treatment with vesicles, the secretion of inflammatory cytokines was measured by ELISA. To perform ELISA, the capture antibody was diluted in phosphate buffered saline (PBS), 50 μl aliquots were applied to 96 well polystyrene plates at a working concentration, and then reacted at 4 ° C. overnight. .

After washing three times with 100 μl of PBST (PBS containing 0.05% tween-20), 100 μl of RD (PBS containing 1% BSA) solution was dispensed and blocked for 1 hour at room temperature. And 50 μl of the standard was adjusted according to the concentration, and reacted at room temperature for 2 hours. After washing three times with 100 μl of PBST, the detection antibody was diluted in RD, 50 μl was dispensed to the working concentration for 2 hours at room temperature, washed three times with 100 μl of PBST, and then Strpetavidin-HRP. (R & D system, USA) was diluted 1/40 in RD, 50 μl was dispensed and reacted at room temperature for 20 minutes. Finally, after washing three times with 100 μl of PBST, 50 μl of TMB substrate (SurModics, USA) is dispensed, and when the color development progresses after 5 to 20 minutes, 50 μl of 1 M sulfuric acid solution is stopped to stop the reaction. Absorbance was measured at 450 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA).

As a result, as shown in Figure 6, when the Lactobacillus brevis-derived vesicles pretreated, it was confirmed that the secretion of IL-6 and TNF-α by E. coli-derived vesicles is significantly suppressed. In particular, when the Lactobacillus brevis-derived vesicles pretreated, it was confirmed that the effect of inhibiting the secretion of TNF-α is significantly more effective than Lactobacillus plantarum , a useful microorganism control.

Example 9. Immunomodulation and Anti-inflammatory Effects of Lactobacillus Casei-derived Vesicles

In order to evaluate the immunomodulatory and anti-inflammatory effects of Lactobacillus casein-derived vesicles, Lactobacillus casei-derived vesicles of various concentrations (0.1, 1, 10 ㎍ / ml) were pretreated in macrophage lines for 12 hours, followed by Escherichia coli, a pathogenic vesicle. After 12 hours of treatment with vesicles, the secretion of inflammatory cytokines was measured by ELISA. As a result, when the Lactobacillus casei-derived vesicles were pretreated, it was confirmed that the secretion of IL-6 and TNF-α by E. coli-derived vesicles was significantly inhibited (see FIG. 7). In particular, when the Lactobacillus casei-derived vesicles pretreated, it was confirmed that the effect of inhibiting TNF-α secretion is significantly more effective than Lactobacillus plantarum ( Lactobacillus plantarum ), which is a useful microorganism control (see Fig. 7b).

Example 10. Immunomodulation and Anti-inflammatory Effects of Lactobacillus rhamnosus-derived Vesicles

In order to evaluate the immunomodulatory and anti-inflammatory effects of Lactobacillus rhamnosus-derived vesicles, Lactobacillus rhamnosus-derived vesicles of various concentrations (0.1, 1, 10 ㎍ / ml) were pretreated in macrophage lines for 12 hours, followed by pathogenic vesicles. The secretion of inflammatory cytokines was measured by ELISA 12 hours after treatment with 1 μg / ml E. coli-derived vesicles. As a result, as shown in Figure 8, when the Lactobacillus rhamnosus-derived vesicles pretreated, it was confirmed that the secretion of IL-6 and TNF-α by E. coli-derived vesicles is significantly suppressed. In particular, when the Lactobacillus rhamnosus-derived vesicles were pretreated, it was confirmed that the effect of inhibiting TNF-α secretion was significantly more effective than the Lactobacillus plantarum , a useful microorganism control group.

Example 11. Immunomodulation and Anti-inflammatory Effects of Lactobacillus Sakei-derived Vesicles

In order to evaluate the immunomodulatory and anti-inflammatory effects of Lactobacillus sakei-derived vesicles, Lactobacillus sakei-derived vesicles of various concentrations (0.1, 1, 10 μg / ml) were pretreated in macrophage lines for 12 hours and then pathogenic vesicles The secretion of inflammatory cytokines was measured by ELISA 12 hours after treatment with 1 μg / ml E. coli-derived vesicles. As a result, when pre-treatment of the Lactobacillus Sakei-derived vesicles as shown in Figure 9, it was confirmed that the secretion of IL-6 and TNF-α by E. coli-derived vesicles is significantly suppressed. In particular, when the Lactobacillus Sakei-derived vesicles were pretreated, it was confirmed that the effect of inhibiting the secretion of TNF-α is significantly more effective than the Lactobacillus plantarum , a useful microorganism control group.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Lactobacillus bacteria-derived vesicles according to the present invention can be used as a diagnostic method for gastric cancer, renal failure, dementia or stroke, as well as a composition for the prevention, improvement or treatment of the above diseases, so as to be useful in related medical, cosmetic and food industries It is expected to be available.

Claims (17)

1. A method of providing information for diagnosing gastric cancer, kidney failure, dementia, or stroke, comprising the following steps:

   (a) extracting DNA from extracellular vesicles isolated from normal and subject samples;

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

   (c) classifying gastric cancer, renal failure, dementia, or stroke when the content of extracellular vesicles derived from bacteria of the genus Lactobacillus is lower than that of normal people through quantitative analysis of the PCR products.
2. The method of claim 1,

   The sample in step (a) is characterized in that the blood, information providing method.
3. Lactobacillus ( Lacotobacillus ) comprising a bacterium derived from the bacteria as an active ingredient, a pharmaceutical composition for preventing or treating inflammatory diseases with an immune function abnormality.
4. The method of claim 3,

   The vesicles are characterized in that the average diameter of 10 to 200 nm, pharmaceutical composition.
5. The method of claim 3,

   The vesicles are characterized in that the natural or artificial secretion from Lactobacillus ( Lacotobacillus ) bacteria, pharmaceutical composition.
6. The method of claim 3,

   The Lactobacillus (Lacotobacillus) in bacterial-derived vesicles is Lactobacillus brevis (Lacotobacillus brevis) derived vesicles, Lactobacillus Kasei (Lacotobacillus casei) derived vesicles, Lactobacillus ramno Versus (Lacotobacillus rhamnosus) derived vesicles and Lactobacillus four K (Lacotobacillus sakei) A pharmaceutical composition, characterized in that it is one or more vesicles selected from the group consisting of derived vesicles.
7. The method of claim 3,

   Inflammatory disease accompanied by immune dysfunction is characterized in that at least one disease selected from the group consisting of gastric cancer, kidney failure, dementia, and stroke, pharmaceutical composition.
8. Lactobacillus (Lacotobacillus) comprising a bacterial-derived vesicles as an active ingredient, food composition for preventing or improving inflammatory diseases accompanied with immune function abnormalities.
9. The method of claim 8,

   The Lactobacillus (Lacotobacillus) in bacterial-derived vesicles is Lactobacillus brevis (Lacotobacillus brevis) derived vesicles, Lactobacillus Kasei (Lacotobacillus casei) derived vesicles, Lactobacillus ramno Versus (Lacotobacillus rhamnosus) derived vesicles and Lactobacillus four K (Lacotobacillus sakei) Food composition, characterized in that one or more vesicles selected from the group consisting of the derived vesicles.
10. The method of claim 8,

    Inflammatory disease with an immune dysfunction is characterized in that at least one disease selected from the group consisting of gastric cancer, kidney failure, dementia, and stroke, food composition.
11. Lactobacillus ( Lacotobacillus ) comprising a bacterial-derived vesicles as an active ingredient, cosmetic composition for preventing or improving skin inflammatory diseases.
12. The method of claim 11,

    The Lactobacillus (Lacotobacillus) in bacterial-derived vesicles is Lactobacillus brevis (Lacotobacillus brevis) derived vesicles, Lactobacillus Kasei (Lacotobacillus casei) derived vesicles, Lactobacillus ramno Versus (Lacotobacillus rhamnosus) derived vesicles and Lactobacillus four K (Lacotobacillus sakei) Cosmetic composition, characterized in that one or more vesicles selected from the group consisting of the derived vesicles.
13. A method of diagnosing gastric cancer, kidney failure, dementia, or stroke, comprising the following steps:

    (a) extracting DNA from extracellular vesicles isolated from normal and subject samples;

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

    (c) Determining gastric cancer, kidney failure, dementia, or stroke when the content of extracellular vesicles derived from bacteria of Lactobacillus ( Lacotobacillus ) is lower than that of the normal person through quantitative analysis of the PCR product.
14. Lactobacillus (Lacotobacillus) bacteria belonging to the genus pharmaceutical composition for preventing or treating an inflammatory disorder accompanied by a method, the immune dysfunction comprising administering to the object that contains the packet originated, as an active ingredient.
15. The method of claim 14,

    The inflammatory disease is gastric cancer, renal failure, dementia, and stroke, characterized in that at least one disease selected from the group consisting of, inflammatory disease prevention or treatment method.
16. Lactobacillus (Lacotobacillus) in the bacteria-derived vesicles, an inflammatory disease preventing or treating purpose accompanied by immune dysfunction.
17. The method of claim 16,

    The inflammatory disease is gastric cancer, renal failure, dementia, and stroke, characterized in that at least one disease selected from the group consisting of, inflammatory disease prevention or treatment use.

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