WO2012093755A1 - Composition comprising extracellular vesicles derived from a fermented food and a use therefor - Google Patents

Abstract

The present invention relates to a method or the like for treating or preventing inflammatory diseases and cancers by using extracellular vesicles isolated from a fermented food. More specifically, provided are: a composition for preventing and/or treating inflammatory diseases and cancers, which contains extracellular vesicles derived from a fermented food as an active ingredient; and a vaccine for treating or preventing inflammatory diseases and cancers which contains the extracellular vesicles as an active ingredient. Also, the safety of fermented foods can be diagnosed by using the extracellular vesicles derived from a fermented food of the present invention.

Description

Compositions comprising extracellular vesicles derived from fermented foods and uses thereof

The present invention relates to a composition for the treatment or prevention of inflammatory diseases and cancers containing the extracellular vesicles isolated from fermented foods, and a method for diagnosing safety of foods using the extracellular vesicles.

Inflammation is a local or systemic defense mechanism against damage or infection of cells and tissues. Inflammation is primarily caused by a cascade of biological reactions that occur by the direct response of numerous humoral mediators that make up the immune system or by stimulating local or systemic effector systems. Mediators involved in this inflammatory response include immune cells, such as helper T lymphocytes (Th cells) and cytotoxic T lymphocytes (Tc cells), macrophage cells, neutrophils, eosinophils, and stroma cells. inflammatory cells such as (mast cell) and cytokines secreted from these cells. Inflammatory diseases are particularly characterized by tissue remodeling due to the secretion of inflammatory cytokines, resulting in tissue damage and imbalance of healing. The major inflammatory cytokines known to date include TNF-α (tumor necrosis factor-α), IL-1 (interleukin-1, interleukin-1), IL-6 and IL-8 produced by macrophages and monocytes There is this. In order to treat inflammatory diseases caused by imbalance of inflammatory cytokines, various studies have been made to suppress the inflammatory cytokines.

Major inflammatory diseases include asthma, chronic obstructive pulmonary disease, respiratory diseases such as rhinitis, skin diseases such as atopic dermatitis, gastroenteritis, gastritis, gastric ulcer, inflammatory growthitis, and other infectious rhinitis, allergic rhinitis, chronic rhinitis, Rhinitis and sinusitis, such as acute sinusitis and chronic sinusitis; Otitis media, such as acute purulent otitis media and chronic purulent otitis media; Pneumonia, such as bacterial pneumonia, bronchial pneumonia, lobar pneumonia, regoraella pneumonia and viral pneumonia; Acute or chronic gastritis; Enteritis, such as infectious enterocolitis, Crohn's disease, idiopathic ulcerative colitis, gastric colitis, and the like; Arthritis such as purulent arthritis, tuberculosis arthritis, degenerative arthritis and rheumatoid arthritis; And diabetes mellitus, arteriosclerosis, and the like.

The hypothesis that inflammation is important in the development of cancer has been suggested for centuries, and it has been suggested that cancer occurs due to tissue damage caused by chronic inflammation and a mistake in its healing process. In addition, intestinal bacteria are known to be important for the development of inflammatory bowel disease occurring in the large intestine. Inflammatory growthitis includes Crohn's disease, which is characterized by a Th1 immune response, and ulcerative colitis, which is characterized by a Th17 immune response. Chronic inflammatory growth inflammation is attracting attention as a risk factor for colorectal cancer. In particular, it is known that the incidence of colorectal cancer increases significantly in patients with ulcerative colitis. The role of IL-6 is very important in the development of Th17 immune response, which induces differentiation into Th17 cells that secrete IL-17 to naive T cells via signaling pathways through STAT3.

Recent reports show that Bactroides fragillus, which secretes metalloprotease toxin from intestinal bacteria, induces IL-6 secretion, which induces Th17 immune responses through STAT3 signaling, resulting in colon cancer.

Prokaryotic or eukaryotic cells secrete extracellular vesicles, and recently released extracellular vesicles have several functions. Extracellular vesicles secreted by Gram-negative bacteria contain lipopolysacharide (LPS) and bacteria-derived proteins and are known to induce inflammatory diseases. In addition, it has been reported that extracellular vesicles derived from various cancer cells contain proteins necessary for the growth and metastasis of cancer cells.

Recently, the present inventors report that Gram-positive bacteria secrete extracellular vesicles, and proteome analysis includes protein causing disease in the endoplasmic reticulum, and reports that atopic dermatitis occurs when administered to the skin.

On the other hand, fermented foods contain ingredients that are completely different from the ingredients before fermentation because the bacteria or yeast metabolize the substrate of the original food. Numerous reports of anti-inflammatory and anti-cancer effects of fermentation products have been reported, but the existence of extracellular vesicles caused by bacteria or yeast causing fermentation in fermented foods, its role and clinical significance are not known.

The present invention is to provide a method for preventing and / or treating inflammatory diseases and cancer using extracellular vesicles derived from fermented foods. Specifically, the present invention provides a composition for preventing and / or treating inflammatory diseases and cancer, a vaccine for treating or preventing inflammatory diseases and cancer, containing the extracellular vesicles as an active ingredient. In addition, the present invention provides a method for diagnosing the stability of fermented food using an extracellular vesicle derived from the fermented food.

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.

The present invention provides a composition for the treatment or prevention of inflammatory diseases and cancer, containing the extracellular vesicles derived from fermented food as an active ingredient. The inflammatory diseases include respiratory diseases, digestive diseases, skin diseases, local inflammatory diseases such as sepsis, systemic inflammatory diseases such as sepsis, arteriosclerosis, arthritis, and brain diseases, and complications thereof.

In an exemplary embodiment of the invention, the extracellular vesicles may be to inhibit the secretion of inflammatory mediators in cells of mammals, but is not limited thereto. The cells include epithelial cells and inflammatory cells. The inflammatory cells include macrophages and eosinophils.

In an exemplary embodiment of the present invention, the inflammatory mediator may be interleukin 6 (IL-6), but is not limited thereto.

In an exemplary embodiment of the present invention, the extracellular vesicles include those derived from fermented foods fermented by bacteria and those derived from fermented foods fermented by mold. The extracellular vesicles may be an extracellular vesicle derived from yeast present in the fermented food, but is not limited thereto. In addition, the extracellular vesicles may be an extracellular vesicle derived from bacteria present in the fermented food, but is not limited thereto.

The extracellular vesicles of the present invention may have an average diameter of 20 nm to 200 nm, but is not limited thereto.

The composition of the present invention may be a pharmaceutical composition, a food composition, a cosmetic composition and the like, but is not limited thereto.

The present invention also provides a vaccine for the treatment or prophylaxis of inflammatory diseases and cancer containing the extracellular vesicles derived from fermented foods as an active ingredient. The vaccine may be used alone or in combination with the extracellular vesicles and the pathogenic vesicles. The pathogenic vesicles include pathogenic vesicles derived from symbiotic bacteria in the body, pathogenic vesicles derived from Gram-positive bacteria, pathogenic vesicles present in the air, and the like.

The present invention also provides a method for preventing or treating a disease, comprising the step of administering an extracellular vesicle derived from fermented food to a mammal at a less than lethal dose. The disease includes inflammatory disease, cancer and the like.

The present invention also provides a method for diagnosing the safety of fermented foods using the fermented food-derived extracellular vesicles.

In an exemplary embodiment of the invention, the method may comprise the steps of: separating the extracellular vesicles from the fermented food; Culturing the isolated extracellular vesicles in cells; And measuring the level of inflammatory mediator in said cell culture.

In an exemplary embodiment of the present invention, the step of separating the extracellular vesicles from the fermented food may include the following: centrifugation of the fermented food to remove impurities, mold and bacteria; And separating the extracellular vesicles by performing ultracentrifugation.

The inventors have found that the extracellular vesicles isolated from fermented foods have anti-inflammatory effects of inhibiting the release of IL-6, an inflammatory mediator associated with inflammatory diseases, and also have an anti-cancer effect of inhibiting cancer in colorectal cancer animal models. It was. Therefore, the composition of the present invention containing the extracellular vesicles derived from fermented foods as an active ingredient can be used as pharmaceutical compositions, food compositions, and cosmetic compositions that can prevent or treat inflammatory diseases and cancer.

In addition, since the extracellular vesicles of the present invention derived from fermented foods can induce secretion of inflammatory mediators of inflammatory cells, the safety of fermented foods can be diagnosed using the extracellular vesicles. In addition, since the extracellular vesicles present in the fermented food can induce inflammation, it is possible to increase the safety of the food by removing the extracellular vesicles in the manufacturing process of the food.

1 is a protocol illustrating a process of extracting extracellular vesicles from doenjang and liver.

Figure 2 shows the result of measuring the density and protein density of each fraction after separation and fractions after density gradient ultrafast centrifugation in the extracellular vesicles extraction process.

3 is a photograph of extracellular vesicles extracted from doenjang (left) and soy sauce (right) observed with a transmission electron microscope (TEM).

Figure 4 shows the effect of extracellular vesicles isolated from Korean traditional doenjang on inflammatory mediator secretion by E. coli-derived extracellular vesicles or LPS To evaluate the inflammatory mediator IL-6 in cell culture after treatment with macrophages (RAW 246.7 cells).

5 shows the effect of extracellular vesicles isolated from traditional Korean liver on secretion of inflammatory mediators by E. coli-derived extracellular vesicles or LPS To evaluate the inflammatory mediator IL-6 in cell culture after treatment with macrophages (RAW 246.7 cells).

Figure 6 shows the effect of extracellular vesicles isolated from Japanese doenjang on the secretion of inflammatory mediators by E. coli-derived extracellular vesicles or LPS To evaluate the inflammatory mediator IL-6 in cell culture after treatment with macrophages (RAW 246.7 cells).

Figure 7 shows the effect of extracellular vesicles isolated from Japanese hepatic on the secretion of inflammatory mediators by E. coli-derived extracellular vesicles or LPS To evaluate the inflammatory mediator IL-6 in cell culture after treatment with macrophages (RAW 246.7 cells).

8 shows the effect of extracellular vesicles isolated from Japanese-style soy sauce on inflammatory mediator secretion by gram-positive S. aureus-derived extracellular vesicles. To evaluate the inflammatory mediator IL-6 in cell culture after treatment with macrophages (RAW 246.7 cells).

9 is an illustration of the size of the endoplasmic reticulum by dynamic light scattering method by separating extracellular vesicles from the Lactobacillus acidophilus lactic acid bacteria beverage.

Figure 10 is a protocol for evaluating the anticancer effect by administering extracellular vesicles contained in Lactobacillus acidophilus lactic acid bacteria beverage to colon cancer animal model by colon cancer cells.

Figure 11 is the result of the extracellular vesicles isolated from Lactobacillus acidophilus lactic acid bacteria beverage showed anticancer effect.

The present inventors found that the extracellular vesicles are present in the fermented food, and the biological activity of the extracellular vesicles present in the fermented food is different according to the method of preparing the fermented food. In this regard, the extracellular vesicles present in the liver prepared by the traditional method of Korea had a significant increase in IL-6 secretion function by the endoplasmic reticulum itself. It has been found that IL-6 secretion by gram negative bacteria-derived extracellular vesicles as well as gram negative bacteria-derived extracellular vesicles is effectively inhibited. In addition, the present inventors confirmed that the extracellular vesicles are present in the lactic acid bacteria beverage, and when it was separated and toured in the colon cancer animal model, the present inventors found that the extracellular vesicles effectively inhibit the development of cancer, thereby completing the present invention. .

Accordingly, the present invention provides a composition for the treatment or prevention of inflammatory diseases and cancer, containing the extracellular vesicles derived from fermented food as an active ingredient.

In the present invention, "fermentation" refers to the conversion of carbohydrates to alcohol and carbon dioxide or organic acids, such as bacteria or yeast in the anaerobic state during the manufacturing process of food, such as beans, cereals, vegetables, fruits, fish, meat, etc. Fermented food can be manufactured using yeast.

In the present invention, "extracellular vesicles derived from fermented food" includes extracellular vesicles present in fermented foods such as beans, grains, vegetables, fruits, fish, meat, characterized in that the size is smaller than the original cells However, the present invention is not limited thereto. The extracellular vesicles of the present invention may be extracellular vesicles secreted by bacteria or fungi present in fermented food. The fungus present in the fermented food includes yeast that induces fermentation.

The present invention also provides a vaccine for the treatment or prophylaxis of inflammatory diseases and cancer containing the extracellular vesicles derived from fermented foods as an active ingredient. The vaccine may be used alone or in combination with the extracellular vesicles and the pathogenic vesicles. The pathogenic vesicles include pathogenic vesicles derived from symbiotic bacteria in the body, pathogenic vesicles derived from Gram-positive bacteria, pathogenic vesicles present in the air, and the like.

The method for separating the extracellular vesicles from fermented foods is not particularly limited as long as the extracellular vesicles can be separated purely, for example, in fermented foods, centrifugation, ultracentrifugation, filtration by filters, gel filtration chromatography, free Extracellular vesicles can be isolated using methods such as flow electrophoresis, capillary electrophoresis, and combinations thereof. In addition, it may further include a process for washing to remove impurities, concentration of the obtained extracellular vesicles and the like.

The extracellular vesicles separated by the above method may have an average diameter of 500 nm or less, and preferably 20 nm to 200 nm.

According to one embodiment of the invention, the composition may be prepared as a pharmaceutical composition. While it may be possible to administer the extracellular vesicles of the present invention for use in treatment and / or prophylaxis, it is preferred that the extracellular vesicles be included as active ingredients of the pharmaceutical composition.

The pharmaceutical composition may contain the isolated extracellular vesicles as an active ingredient, and may include 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 into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Suitable pharmaceutically acceptable carriers and formulations may be preferably formulated according to each component using the methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, or external skin preparation.

The method of administering the pharmaceutical composition of the present invention is not particularly limited, but may be selected by intravenous, subcutaneous, intraperitoneal, inhalation, dermal application, sublingual administration, nasal administration, anal administration, oral administration, etc. .

Dosage varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient. Daily dosage refers to the amount of therapeutic substance of the invention sufficient for treatment for a disease state alleviated by administration to a subject in need thereof. Effective amounts of therapeutic agents depend on the particular compound, disease state and severity thereof, and on the individual in need thereof, and can be routinely determined by one skilled in the art. By way of non-limiting example, the dosage of the composition according to the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient and may be based on an adult patient weighing 70 kg. At this time, it is generally 0.01 to 1000 mg / day, preferably 1 to 500 mg / day, and may be dividedly administered once to several times a day at regular time intervals.

According to another embodiment of the present invention, the composition of the present invention may be prepared as a cosmetic composition. Ingredients included in the cosmetic composition of the present invention, in addition to containing the extracellular vesicles as an active ingredient, include components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. Conventional adjuvants such as, and carriers.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto. More specifically, it may be prepared in the form of a nourishing cream, astringent lotion, a flexible lotion, a lotion, an essence, a nourishing gel or a massage cream.

According to another embodiment of the present invention, the composition of the present invention may be prepared as a food composition. When the composition of the present invention is prepared as a food composition, not only contains the extracellular vesicles as an active ingredient, but also includes components commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients, Flavoring and flavoring agents may be included. For example, when the food composition of the present invention is prepared with a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and the like may be further included in addition to the extracellular vesicles of the present invention.

In the present invention, "treatment or prevention of an inflammatory disease" is meant to include the alleviation, alleviation and improvement of symptoms of an inflammatory disease, and also to reduce the possibility of developing an inflammatory disease.

In the present invention, "inflammatory disease" refers to a disease caused by an inflammatory response in a mammalian body, and representative examples thereof include respiratory diseases such as asthma, chronic obstructive pulmonary disease and rhinitis; Skin diseases such as atopic dermatitis, psoriasis and contact dermatitis; Gastrointestinal diseases such as gastritis, peptic ulcer, and inflammatory bowel disease; Systemic inflammatory diseases such as sepsis, arteriosclerosis, arthritis, brain diseases, and complications thereof. In addition, the inflammatory disease is used in the sense including cancer associated with the inflammatory response, in addition to the general inflammatory disease, and includes, for example, lung cancer, stomach cancer, colon cancer and the like.

The composition of the present invention, by inhibiting the secretion of inflammatory mediators in inflammatory cells of mammals, it is possible to treat or prevent inflammatory diseases. In particular, since the extracellular vesicles isolated from the fermented food of the present invention can inhibit the secretion of IL-6, an important biomarker associated with the development of inflammatory diseases or cancer, the composition of the present invention is mediated by IL-6. It is possible to treat or prevent cancer caused by Th-17 inflammatory disease and IL-6 or Th-17 inflammatory response. In addition, the present inventors confirmed that extracellular vesicles present in fermented foods, such as lactic acid bacteria beverages, effectively inhibit the development of cancer in colorectal cancer animal models. Therefore, by administering a composition comprising extracellular vesicles present in fermented foods such as lactic acid bacteria beverages can be expected to treat and prevent cancer.

The present invention also provides a method for diagnosing the safety of foods using extracellular vesicles derived from fermented foods. Extracellular vesicles isolated from fermented foods can induce secretion of inflammatory mediators of inflammatory cells. Thus, the diagnostic method, the step of separating the extracellular vesicles from fermented food; Culturing the isolated extracellular vesicles on inflammatory cells; And measuring the level of an inflammatory mediator in said cell culture. That is, the isolated extracellular vesicles are administered to inflammatory cells in vitro and cultured together, and the type and level of the inflammatory mediator secreted from the inflammatory cells can be measured to diagnose the safety of food.

For example, the isolated extracellular vesicles may be added to the macrophage culture medium and cultured together, and by measuring the level of IL-6 secreted by the macrophages, it is possible to diagnose whether food products develop or worsen inflammatory diseases. In addition, by administering the isolated extracellular vesicles directly to the experimental animals, by measuring the level of inflammatory mediators (eg, IL-6) produced in the body of the experimental animals, It is possible to predict whether it will occur or worsen.

The present invention also provides a method of increasing the safety of food by removing the extracellular vesicles present in the fermented food. Since the extracellular vesicles present in the fermented food can induce inflammation, it is possible to prepare a safer food by removing the extracellular vesicles present in the fermented food in a variety of ways.

Hereinafter, examples are provided to help understand 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 1 Isolation and Characteristics of Extracellular Vesicles Derived from Soy Sauce and Soybean Paste

According to the protocol shown in Fig. 1, 60 ml of soy sauce was placed in a high speed centrifuge tube, followed by two consecutive high speed centrifugations for 15 minutes at 10,000 xg at 4 ° C. It was. The supernatant was placed in a 70 ml ultracentrifuge tube and subjected to ultratrace separation (ultracetrifugation) at 4 ° C., 100,000 × g for 4 hours. The supernatant was discarded and the precipitate present under the tube was dissolved in 50% Optiprep solution. After that, further purification was performed by Optiprep density gradient method. In a 5 ml ultracentrifuge tube, add 50 ml Optiprep solution, 40%, 30%, 20% and 10% Optiprep solution, each containing 1 ml in order, for 4 hours at 4 ° C and 100,000 xg. Ultracentrifugation was performed (see FIG. 1). After ultracentrifugation, the extracellular vesicles were located between 20% Optiprep solution and 30% Optiprep solution layers depending on the density (see Figure 2). The solution was removed from the top of the tube to separate the layer containing extracellular vesicles.

When the extracellular vesicles were separated from doenjang, 10 g of doenjang was dissolved in a beaker containing 100 ml PBS at 4 ° C for 1 hour, and large foreign substances were filtered using gauze. Subsequent procedures were performed in the same way as the separation of extracellular vesicles from liver. As a result of observing the transmission electron microscope (TEM) of the extracellular vesicles extracted by the above method, it was confirmed that the extracellular vesicles consisted of a lipid bilayer, 50-100 nm in size and generally spherical ( 3).

Example 2 Effects on Inflammatory Media Secretion by Extracellular Vesicles Isolated from Korean Doenjang and Soy Sauce (or Chosun Doenjang and Soy Sauce) and on Inflammatory Media Secretion by Extracellular Vesicles Derived from LPS or Escherichia Coli

Mouse macrophages (RAW264.7) were treated with extracellular vesicles (0.1, 1 μg / ml) isolated from Korean doenjang and liver alone or LPS (10 ng / ml) or E. coli-derived extracellular vesicles (100 ng / ml). ) And incubated for 15 hours, the expression level of the inflammatory mediator IL-6 in the cell culture was confirmed, and the results are shown in FIGS. 4 and 5.

4 and 5, it can be seen that the inflammatory mediator IL-6 is increased in the experimental group treated with extracellular vesicles isolated from Korean doenjang and soy sauce compared to the control group. In addition, in the experimental group treated with extracellular vesicles isolated from Korean miso and soy sauce with LPS or E. coli-derived extracellular vesicles, E. coli-derived extracellular vesicles or LPS with the increase in the amount of extracellular vesicles derived from Korean doenjang and soy sauce It was confirmed that IL-6 increases compared to the experimental group treated with only.

The results indicate that Korean doenjang and hepatic-derived extracellular vesicles alone can increase the secretion of inflammatory mediators and can further promote increased expression of inflammatory mediators by E. coli-derived extracellular vesicles and LPS.

Example 3 Influence on Inflammatory Media Secretion by Extracellular Vesicles Isolated from Japanese Soybean Paste (or Korean Doenjang) and on Inflammatory Media Secretion by LPS or Escherichia Coli-derived Extracellular Vesicles

Mouse macrophages (RAW264.7) were treated with extracellular vesicles (0.1, 1, 10 μg / ml) isolated from Japanese miso alone, or LPS (10 ng / ml), E. coli-derived extracellular vesicles (10 ng / ml) and incubated for 15 hours, the expression level of the inflammatory mediator IL-6 in the cell culture was confirmed, and the results are shown in FIG. 6.

As shown in FIG. 6, IL-6 secretion did not increase when only extracellular vesicles isolated from Japanese doenjang were administered, but the concentration of 10 μg / ml of Japanese doenjang-derived extracellular vesicles together with LPS or E. coli-derived extracellular vesicles. It can be seen that IL-6 is increased in the experimental group treated with the control group compared to the control group (LPS or experimental group treated with E. coli-derived extracellular vesicles only).

The results indicate that Japanese miso-derived extracellular vesicles can increase the expression of inflammatory mediators by LPS and E. coli-derived extracellular vesicles that induce an inflammatory response.

Example 4 Influence on Inflammatory Media Secretion by Extracellular Vesicles Isolated from Japanese Soy Sauce (or Waco Hepatic) and on Inflammatory Media Secretion by LPS, Escherichia Coli-derived Extracellular Vesicles, or Staphylococcus aureus-Derived Extracellular Vesicles

Mouse macrophages (RAW264.7) were treated with extracellular vesicles (0.1, 1, 10 μg / ml) isolated from Japanese liver alone or LPS (10 ng / ml), E. coli-derived extracellular vesicles (10 ng / ml), or treated with Staphylococcus aureus-derived extracellular vesicles (1 μg / ml) and incubated for 15 hours to determine the expression level of inflammatory mediator IL-6 in cell culture. As a result, IL-6 secretion did not increase when only the extracellular vesicles isolated from Japanese liver were administered, but 10 μg of the Japanese hepatic-derived extracellular vesicles with LPS or E. coli-derived extracellular vesicles or Staphylococcus aureus-derived extracellular vesicles. In the experimental group treated with / ml concentration it was confirmed that IL-6 was significantly reduced compared to the control group (LPS, experimental group treated with E. coli-derived extracellular vesicles or staphylococci-derived extracellular vesicles only) (see Figs. 7 and 8).

The above results indicate that Japanese hepatic-derived extracellular vesicles can antagonize the expression of inflammatory mediators by LPS, E. coli-derived extracellular vesicles, or Staphylococcus aureus-derived extracellular vesicles that induce an inflammatory response.

Example 5. Isolation and Characterization of Extracellular Vesicles from Lactobacillus acidophilus Lactic Acid Bacteria Beverage

In order to separate the extracellular vesicles from the lactic acid bacteria beverage by Lactobacillus acidophilus, 1 L of the lactic acid bacteria beverage was filtered with gauze. The lactic acid bacteria fermented by gauze was centrifuged for 30 minutes at a rate of 12,000 x g to obtain a supernatant excluding lactic acid bacteria and other suspended solids. The supernatant obtained after centrifugation was removed 0.22㎛ excess material using a 0.22㎛ filter. Ultra-high centrifugation was performed for 2 hours at a rate of 150,000 × g to obtain extracellular vesicles from the obtained solution of less than 0.22 μm. After ultra-fast centrifugation, the extracellular vesicles that had sunk were extracted with 200 μl of physiological saline and quantified by Bradford assay. Dynamic light scattering was used to measure the size of the extracellular vesicles. As a result, the obtained extracellular vesicles were confirmed to have an average size of 29.34nm (see Fig. 9).

Example 6 Anticancer Effect of Extracellular Vesicles Isolated from Lactobacillus acidophilus Lactic Acid Bacteria Drink

In order to determine whether the Lactobacillus acidophilus lactic acid bacteria beverage-derived extracellular vesicles isolated by the method of Example 5 had an anticancer effect, the activity was confirmed in an in vivo tumor model using colon 26 tumor cells.

According to the protocol shown in FIG. 10, 1 × 10 ⁶ colon 26 cells were injected subcutaneously into 5 week old Balb / c (male) mice to create an in vivo tumor model using Colon 26 tumor cells. In order to confirm the anticancer effect of the lactic acid bacteria beverage-derived extracellular vesicles, 10 ㎍ of lactic acid bacteria beverage-derived extracellular vesicles were injected through the tail vein at intervals of 3 days from 7 days after tumor injection. Three days after the last injection, mice were dissected to excise the tumor and the tumor volume was measured to evaluate the anticancer effect of the lactic acid bacteria-derived extracellular vesicles.

As a result, it was confirmed that the tumor size was significantly reduced in the group injected with lactic acid bacteria beverage-derived extracellular vesicles compared to the group not injected (see FIG. 11).

Through the above examples, the present inventors first identified that extracellular vesicles isolated from Japanese soy sauce, which is a representative fermented food, inhibit IL-6 secretion, which is an important biomarker for causing inflammatory diseases and cancers. In addition, Lactobacillus acidophilus It was the first time that extracellular vesicles isolated from lactic acid bacteria drink effectively inhibited the development of colorectal cancer.

In addition, the present inventors secrete extracellular vesicles in the process of fermentation of bacteria such as soy sauce in fermented foods such as soy sauce using a substrate such as soybean, which causes the development of inflammatory diseases and cancers mediated by IL-6 and the like. And it was confirmed that the progression can be prevented.

The extracellular vesicles of the invention have been isolated from, but not limited to, miso and soy. In addition, the extracellular vesicles isolated from Korean liver (or Chosun soy sauce) induce inflammatory mediator secretion from inflammatory cells is similar to that caused by LPS or E. coli-derived extracellular vesicles. It means that it induces an inflammatory disease, which means that the safety of fermented food can be diagnosed by measuring the secretion of IL-6 and the like from inflammatory cells caused by extracellular vesicles in fermented food. This focuses on fermented foods such as soy sauce, but is not limited thereto.

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.

The present invention is to provide a method for preventing and / or treating inflammatory diseases and cancer using extracellular vesicles derived from fermented foods. In addition, the composition of the present invention containing the extracellular vesicles derived from fermented foods as an active ingredient can be used as pharmaceutical compositions, food compositions, and cosmetic compositions that can prevent or treat inflammatory diseases and cancer. In addition, since the extracellular vesicles present in the fermented food can induce inflammation, it is possible to increase the safety of the food by removing the extracellular vesicles in the manufacturing process of the food.

Claims (47)

1. A composition for the treatment or prevention of inflammatory diseases, comprising as an active ingredient extracellular vesicles derived from fermented foods.
2. A composition for the treatment or prevention of cancer, comprising as an active ingredient extracellular vesicles derived from fermented foods.
3. The method of claim 1,

   The inflammatory disease is selected from the group consisting of respiratory diseases, digestive diseases, skin diseases, sepsis, arteriosclerosis, arthritis, brain diseases and complications thereof.
4. The method of claim 3,

   Wherein the respiratory disease comprises rhinitis, asthma, and chronic obstructive pulmonary disease.
5. The method of claim 3,

   Wherein the digestive disease comprises gastritis, peptic ulcer, and inflammatory colitis.
6. The method of claim 3,

   Wherein said skin disease comprises atopic dermatitis, psoriasis, and contact dermatitis.
7. The method of claim 2,

   Wherein the cancer comprises lung cancer, gastric cancer, and colorectal cancer.
8. The method according to claim 1 or 2,

   The extracellular vesicles are fermented food-derived extracellular vesicles fermented by bacteria, compositions.
9. The method according to claim 1 or 2,

   The extracellular vesicles are fermented food-derived extracellular vesicles fermented by the fungus.
10. The method according to claim 1 or 2,

    Wherein said extracellular vesicles inhibit the secretion of inflammatory mediators in mammalian cells.
11. The method of claim 10,

    The inflammatory mediator is interleukin 6 (IL-6).
12. The method of claim 10,

    The cell is an epithelial cell or an inflammatory cell.
13. The method of claim 12,

    Wherein said inflammatory cell is a macrophage or eosinophil.
14. The method according to claim 1 or 2,

    Wherein said extracellular vesicles have an average diameter of 20 nm to 200 nm.
15. The method according to claim 1 or 2,

    The composition is a pharmaceutical composition, a food composition, or a cosmetic composition.
16. A vaccine for the treatment or prevention of inflammatory diseases, which contains an extracellular vesicle derived from fermented food as an active ingredient.
17. A vaccine for the treatment or prevention of cancer, comprising an extracellular vesicle derived from fermented food as an active ingredient.
18. The method of claim 16,

    The inflammatory disease is selected from the group consisting of respiratory diseases, digestive diseases, skin diseases, sepsis, arteriosclerosis, arthritis, brain diseases and complications thereof.
19. The method of claim 18,

    Wherein the respiratory disease comprises rhinitis, asthma, and chronic obstructive pulmonary disease.
20. The method of claim 18,

    The gastrointestinal disease includes gastritis, peptic ulcer, and inflammatory colitis.
21. The method of claim 18,

    Wherein said skin disease comprises atopic dermatitis, psoriasis, and contact dermatitis.
22. The method of claim 17,

    Wherein the cancer comprises lung cancer, gastric cancer, and colon cancer.
23. The method according to claim 16 or 17,

    The vaccine is to use extracellular vesicles alone.
24. The method according to claim 16 or 17,

    The vaccine is to use a mixture of the extracellular vesicles and pathogenic vesicles.
25. The method of claim 24,

    The pathogenic vesicles are pathogenic vesicles derived from symbiotic bacteria in the body, vaccine.
26. The method of claim 24,

    Wherein said pathogenic vesicles are pathogenic vesicles derived from Gram-positive bacteria.
27. The method of claim 24,

    Wherein said pathogenic vesicles are pathogenic vesicles present in air.
28. The method according to claim 16 or 17,

    The extracellular vesicles have a mean diameter of 20 nm to 200 nm.
29. A method for preventing or treating a disease comprising administering an extracellular vesicle derived from fermented food to a mammal at a less than lethal dose.
30. The method of claim 29,

    The disease is selected from the group consisting of respiratory diseases, digestive diseases, skin diseases, sepsis, arteriosclerosis, arthritis, brain diseases and complications thereof, and cancer.
31. The method of claim 30,

    Wherein the respiratory disease comprises rhinitis, asthma, and chronic obstructive pulmonary disease.
32. The method of claim 30,

    Wherein said digestive disease comprises gastritis, peptic ulcer, and inflammatory colitis.
33. The method of claim 30,

    Wherein said skin disease comprises atopic dermatitis, psoriasis, and contact dermatitis.
34. The method of claim 30,

    Wherein the cancer comprises lung cancer, gastric cancer, and colorectal cancer.
35. The method of claim 29,

    The method is to use extracellular vesicles alone.
36. The method of claim 29,

    The method is to use a mixture of the extracellular vesicles and pathogenic vesicles.
37. The method of claim 36,

    The pathogenic endoplasmic reticulum is a pathogenic endoplasmic reticulum derived from symbiotic bacteria in the body.
38. The method of claim 36,

    Said pathogenic endoplasmic reticulum is a pathogenic endoplasmic reticulum derived from gram positive bacteria.
39. The method of claim 36,

    Wherein said pathogenic vesicles are pathogenic vesicles present in air.
40. The method of claim 29,

    Wherein said administration is selected from the group consisting of subcutaneous injection, intravenous injection, nasal administration, sublingual administration, inhalation administration, oral administration, anal administration, and skin administration.
41. A method for diagnosing the safety of fermented foods using the fermented food-derived extracellular vesicles, the method comprising the following steps:

    (a) separating extracellular vesicles from fermented foods;

    (b) treating the isolated extracellular vesicles with cells and culturing the cells; And

    (c) measuring the level of inflammatory mediator in said cell culture.
42. The method of claim 41, wherein

    The step (a) is the step of centrifuging the fermented food to remove impurities, mold and bacteria; And separating the extracellular vesicles by performing ultracentrifugation after the removal.
43. The method of claim 41, wherein

    The cell of step (b) is epithelial cells or inflammatory cells.
44. The method of claim 41, wherein

    The inflammatory mediator of step (c) is interleukin 6 (IL-6).
45. The method of claim 41, wherein

    The extracellular vesicles are extracellular vesicles derived from fermented foods fermented by bacteria.
46. The method of claim 41, wherein

    The extracellular vesicles are extracellular vesicles derived from fermented foods fermented by the fungus.
47. The method of claim 41, wherein

    Wherein said extracellular vesicles have an average diameter of 20 nm to 200 nm.

Images