KR20200024203A - Use of extracellular vesicles from kefir grain - Google Patents

Abstract

The present invention relates to various uses using microvesicles derived from kefir grains. More specifically, the present invention relates to a composition which can effectively prevent, alleviate or treat inflammatory diseases, intestinal diseases and mental disorders by using microvesicles derived from kefir grains.

Description

Use of extracellular vesicles from kefir grain}

The present invention relates to a variety of uses using kefir grain-derived microvesicles (extracellular vesicles), and more specifically to using kefir grain-derived microvesicles effectively prevent inflammatory diseases, intestinal diseases and mental disorders , Compositions that can be improved or treated.

Inflammation is a manifestation of normal and protective in vivo defense mechanisms localized against physical trauma, harmful chemicals, tissue infections caused by microbial infections or irritants in metabolites in vivo. Such inflammation is triggered by various chemical mediators produced from damaged tissues and migrating cells, and these chemical mediators are known to vary according to the type of inflammatory process. In normal cases, the living body restores its normal structure and function by neutralizing or eliminating the pathogenic factors through the inflammatory response and regenerating the upper tissue, but in other cases, the disease progresses to a disease state such as chronic inflammation. In addition, in case of improperly triggered inflammation by harmless substances such as pollen or autoimmune reactions such as asthma and rheumatoid arthritis, the defense reaction itself damages tissues, and thus, an agent for preventing or treating inflammatory diseases is required. In almost all clinical diseases, inflammatory reactions can be observed, and some of these inflammatory diseases are bacterial diseases that can be causatively treated with antibiotics, but most of them are due to tissue damage caused by autoimmune reactions. It is known as an incurable disease.

The most common inflammatory disease preventing or treating agents for treating such inflammatory diseases are largely divided into steroidal and nonsteroidal inflammatory disease preventing or treating agents. Of these, most synthetic inflammatory disease preventing or treating agents have various side effects in addition to the main action. There are many disadvantages that accompany it. In other words, appropriate nonsteroidal inflammatory disease prevention or treatment drugs (NSAIDs) are used to treat inflammatory diseases, and steroid agents are used if severe inflammation or NSAIDs are not effective. I will perform surgery. In this case, NSAIDs are mainly used to prevent or treat inflammatory diseases by inhibiting the production of prostaglandin, which is involved in the inflammatory response by inhibiting cyclooxygenase (COX). It causes side effects such as renal dysfunction and has a problem that is difficult to use for a long time.

Irritable Bowel Syndrome (IBS), on the other hand, is a common bowel dysfunction, usually characterized by persistent or repetitive abdominal pain, abdominal discomfort, and altered bowel habits. Irritable bowel syndrome impairs the patient's social and personal lives, leading to poor quality of life, and the more severe the symptoms, the worse the quality of life.

The cause of the development of irritable bowel syndrome is not yet clear, but stress, sensitive personality, irregular eating habits, chronic fatigue and irritating and fatty foods are known as the main causes. However, the effectiveness of the treatments that have been proposed to date has not been significant, and there are no known basic treatments for the disease, and they rely solely on symptomatic treatments focused on symptom relief.

Recently, the number of patients with irritable bowel syndrome increased to 1.62 million in 2012, increasing by 8.7% (130,000) over the past five years, increasing by 1.7% per year. Most patients (99.4% of all patients) were outpatient. Treating with therapy. Therefore, it is urgent to develop new food bio materials that can activate intestinal function in addition to symptomatic treatment.

An object of the present invention is to provide a composition that can effectively prevent, ameliorate or treat inflammatory diseases using kefir grain-derived extracellular vesicles (extracellular vesicles).

Another object of the present invention is to provide a composition that can effectively prevent, ameliorate or treat intestinal diseases using kefir grain-derived microvesicles.

It is another object of the present invention to provide a composition capable of effectively preventing, ameliorating or treating a mental disorder using a kefir grain-derived microvesicle.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one embodiment of the present invention, it relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising kefir grain-derived microvesicles (extracellular vesicles) as an active ingredient.

In the present invention, the "kefir" (kefir) is a kind of fermented milk derived from the Caucasus mountainous fat, the drink popularized by Tibetan monks for health. Kefir's constituent nutrients include proteins and polysaccharides, and include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin D, vitamin K2, folic acid, nicotinic acid and calcium, iron, and iodine. Such kefir is a yoghurt beverage obtained by fermenting milk with an initiator of a bacterium (also called kefir granules) called lactic acid bacteria and / or yeast complex kefir grains.

In the present invention, the "kefir grain" is a fermentation source used in the manufacturing process of kefir, which essentially includes lactic acid bacteria, and may further include yeast. Specifically, lactic acid bacteria belonging to the kefir grains in the present invention, Lactobacillus (Lactobacillus), Lactococcus (Lactococcus), Leukonostoc (Leuconostoc), Acetobacter (Acetobacter) and Streptococcus (Streptococcus) It may be a lactic acid bacterium belonging to the back, and preferably include at least one selected from the group consisting of Lactobacillus kefiranofaciens, Lactobacillus kefiri and Lactobacillus kefirgranum. It is preferable that the anti-inflammatory effect and the harmful bacterium inhibitory effect is remarkably superior to other strains, and more preferably, may include Lactobacillus kefiranofaciens. In addition, the yeast that may be included in the kefir grains in the present invention is Saccharomyces (Saccharomyces), Lacancea (Lachancea), Hansenia sospora (Hanseniasospora), Zygotorula spora (Zygotorulaspora), It may be yeast belonging to the genus Kluyveromyces, Torula genus and Candida genus, but is not limited thereto.

The microvesicles in the present invention may be a microvesicle derived from the culture of kefir grains.

In the present invention, the culture of kefir grains is obtained by culturing the kefir grains in a medium, and may refer to all materials obtained by culturing medium, kefir grains, and culture. In addition, the culture may be a liquid culture or a solid culture, the shape is not particularly limited. Here, the type of the medium is also not particularly limited, and for example, MRS medium, milk, cheese, and other dairy products may be used.

Specifically, the culture of kefir grains in the present invention, (a) primary culture of kefir grains in solid medium for 12 to 48 hours; And (b) may be obtained by the step of the second culture in suspension for 1 to 48 hours in a liquid medium after the first culture.

In addition, in the present invention, the culture obtained subsequent to step (b) may be subjected to the step of suspension culture in (c) an additional 1-14 days in a liquid medium. Here, it is preferable to suspend culture for 3 to 7 days during the culture (c), more preferably suspension culture for 3 to 4 days during the culture (c).

In the present invention, the culture of kefir grains may include extracellular vesicles, and the microvesicles may be naturally or artificially secreted from lactic acid bacteria belonging to the kefir grains, but are not limited thereto. no.

In the present invention, the "extracellular vesicles" refers to small vesicles of the membrane structure secreted from various cells, also defined as nanovesicles (nanovesicle). The microvesicles have a diameter of approximately 30 to 1,000 nm, preferably an average diameter of 100 to 300 nm, and mean a vesicle which is released into the extracellular environment by fusion of the polycystic body with the plasma membrane.

In the present invention, the amount of the microvesicles included in the pharmaceutical composition is not particularly limited, but may preferably include 1 X 10 ³ to 1 X 10 ⁹ particles. When the content of the microvesicles is less than 1 X 10 ³ particles, the effect may be insignificant, and when the number of microvesicles exceeds 1 X 10 ⁹ particles, it may cause toxicity to the human body.

The microvesicles in the present invention can be obtained by centrifugation of the culture of kefir grains. More specifically, in order to obtain the microvesicles, the step of centrifuging the culture of the kefir grains for 5 minutes to 1 hour at a temperature of more than 0 to 20 ℃ below 100 ~ 1,000g; And the supernatant obtained by recovering the supernatant after the first centrifugation and performing secondary centrifugation for 10 minutes to 1 hour at a temperature of more than 0 to 20 ° C. at 1,000 to 5,000 g.

In addition, in the present invention, in order to obtain the microbubbles, the supernatant obtained after the second centrifugal separation may be used as the supernatant obtained by performing the third centrifugation for 10 minutes to 1 hour at a temperature of more than 0 to 20 ° C at 5,000 to 20,000 g. have.

In the present invention, the micro-vesicles, the supernatant obtained after the third centrifugal centrifugation using an ultracentrifuge, 100,000 ~ 150,000g at a temperature of more than 0 to 20 ℃ or less for 4 hours centrifuged for 1 hour to 3 hours It may be a pellet obtained separately.

In the present invention, the microvesicles having excellent anti-inflammatory effect can be obtained with high purity and content through the four-step centrifugation process as described above.

On the other hand, the microvesicles in the present invention can be obtained by precipitating from the culture of the kefir grains. More specifically, the step of centrifuging the culture of the kefir grains at 3,000 ~ 5,000g for 5 minutes to 1 hour to obtain the microvesicles; To the concentrated culture obtained after the first centrifugation, 0.5 to 2 N sodium acetate was added in a 0.1 to 1.5 volume ratio, preferably 0.1 to 1 volume ratio, more preferably 0.1 to 0.5 volume ratio, and then -10 ° C or more and 0 ° C or less. Precipitation cultures obtained by culturing for 30 to 60 minutes at a temperature can be used.

In addition, in the present invention, the microvesicles may be pellets obtained by secondary centrifugation for 5 minutes to 1 hour at 3,000 to 5,000 g of the precipitate culture obtained after the culture.

In addition, in the present invention, the pellet obtained after the second centrifugal separation is additionally washed with 0.05-0.2N sodium acetate, if necessary, in order to increase the purification efficiency of the microvesicles, and then centrifuged at 3,000-5,000 g for 5 minutes to 1 hour. The pellet obtained by the use can be used.

In the present invention, the microvesicles are preferably obtained by centrifugation of the culture of kefir grains to enhance the therapeutic effect, but those obtained by the precipitation method may be used in consideration of the efficiency of the manufacturing process.

The microvesicles derived from the culture of kefir grains in the present invention have an excellent anti-inflammatory effect, significantly inhibiting the proliferation and killing of harmful bacteria, and effectively prevent, improve or treat inflammatory diseases.

In the present invention, the inflammatory disease is sepsis, alopecia areata, acne, allergies, asthma, asthma, systemic sclerosis, Scleroderma, conjunctivitis, periodontitis, aphthitis, rhinitis, otitis media, sore throat, tonsillitis, chronic thyroiditis (Hashimoto 하시 Thyroiditis Hashimoto's thyroiditis, Pneumonia, Silicosis, Asbestos, Asbestos, Gastric ulcer, Gastritis, Hemorrhoids, Gout, Ankylosing spondylitis, Rheumatoid arthritis, Lupus, Fibromyalgia, Psoriatic arthritis, Osteoarthritis, Rheumatoid Arthritis, periarthritis, tendinitis, hay salt, peritonitis, myositis, hepatitis, cystitis, nephritis, post-streptococcal glomerulonephritis, atherosclerosis, degenerative arthritis, sjogren's syndrome, multiple sclerosis (multiple sclerosis), polymyositis, dermatomyositis, nodular polyarteritis (Polya) rteritis nodosa, ankylosing spondylitis, fibromyalgia syndrome, temporal arteritis, Guilian-Barre syndrome, autoimmune hemolytic anemia, autoimmune encephalomyelitis, It may be selected from the group consisting of Myasthenia gravis, Grave's disease and inflammatory low back pain.

According to another embodiment of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating intestinal diseases comprising kefir grain-derived microvesicles as an active ingredient.

In the present invention, the kefir grains essentially include lactic acid bacteria, and may further include yeast, and the lactic acid bacteria may include Lactobacillus, Lactococcus, Lactococcus, and Leuconostoc. Lactobacillus belonging to the genus, Acetobacter genus and Streptococcus genus, etc., preferably Lactobacillus kefiranofaciens, Lactobacillus kefiri and Lactobacillus kefiri Lactobacillus kefirgranum containing one or more selected from the group consisting of anti-inflammatory effect, improved bowel activity, intestinal immunity and intestinal harmful bacterium is significantly superior to other strains, and more preferably, Lactobacillus kepi It may include Lanobacilli (Lactobacillus kefiranofaciens). In addition, the yeast in the present invention Saccharomyces (Saccharomyces), Lacancea (Lachancea), Hansenia sospora genus, Zygotorulaspora genus, Kluyveromyces (Kluyveromyces) Yeast belonging to the genus, genus Torula and Candida genus, but is not limited thereto.

The microvesicles in the present invention may be a microvesicle derived from the culture of kefir grains.

In the present invention, the culture of kefir grains is obtained by culturing the kefir grains in a medium, and may refer to all materials obtained by culturing medium, kefir grains, and culture. In addition, the culture may be a liquid culture or a solid culture, the shape is not particularly limited. Here, the type of the medium is also not particularly limited, and for example, MRS medium, milk, cheese, and other dairy products may be used.

Specifically, the culture of kefir grains in the present invention, (a) primary culture of kefir grains in solid medium for 12 to 48 hours; And (b) may be obtained by the step of the second culture in suspension for 1 to 48 hours in a liquid medium after the first culture.

In addition, in the present invention, the culture obtained subsequent to step (b) may be subjected to the step of suspension culture in (c) an additional 1-14 days in a liquid medium. Here, it is preferable to suspend culture for 3 to 7 days during the culture (c), more preferably suspension culture for 3 to 4 days during the culture (c).

In the present invention, the culture of kefir grains may include extracellular vesicles, and the microvesicles may be naturally or artificially secreted from lactic acid bacteria belonging to the kefir grains, but are not limited thereto. no.

In the present invention, the amount of the microvesicles included in the pharmaceutical composition is not particularly limited, but may preferably include 1 X 10 ³ to 1 X 10 ⁹ particles. When the content of the microvesicles is less than 1 X 10 ³ particles, the effect may be insignificant, and when the number of microvesicles exceeds 1 X 10 ⁹ particles, it may cause toxicity to the human body.

The microvesicles in the present invention can be obtained by centrifugation of the culture of kefir grains. More specifically, in order to obtain the microvesicles, the step of centrifuging the culture of the kefir grains for 5 minutes to 1 hour at a temperature of more than 0 to 20 ℃ below 100 ~ 1,000g; And the supernatant obtained by recovering the supernatant after the first centrifugation and performing secondary centrifugation for 10 minutes to 1 hour at a temperature of more than 0 to 20 ° C. at 1,000 to 5,000 g.

In addition, in the present invention, in order to obtain the microbubbles, the supernatant obtained after the second centrifugal separation may be used as the supernatant obtained by performing the third centrifugation for 10 minutes to 1 hour at a temperature of more than 0 to 20 ° C at 5,000 to 20,000 g. have.

In the present invention, the micro-vesicles, the supernatant obtained after the third centrifugal centrifugation using an ultracentrifuge, 100,000 ~ 150,000g at a temperature of more than 0 to 20 ℃ or less for 4 hours centrifuged for 1 hour to 3 hours It may be a pellet obtained separately.

In the present invention, microvesicles excellent in anti-inflammatory, bowel activity improvement, intestinal immunity, inhibition of proliferation and harmful effects of intestinal harmful bacteria can be obtained with high purity and content through the four-step centrifugation process as described above.

On the other hand, the microvesicles in the present invention can be obtained by precipitating from the culture of the kefir grains. More specifically, the step of centrifuging the culture of the kefir grains at 3,000 ~ 5,000g for 5 minutes to 1 hour to obtain the microvesicles; To the concentrated culture obtained after the first centrifugation, 0.5 to 2 N sodium acetate was added in a 0.1 to 1.5 volume ratio, preferably 0.1 to 1 volume ratio, more preferably 0.1 to 0.5 volume ratio, and then -10 ° C or more and 0 ° C or less. Precipitation cultures obtained by culturing for 30 to 60 minutes at a temperature can be used.

In addition, in the present invention, the microvesicles may be pellets obtained by secondary centrifugation for 5 minutes to 1 hour at 3,000 to 5,000 g of the precipitate culture obtained after the culture.

In addition, in the present invention, the pellet obtained after the second centrifugal separation is additionally washed with 0.05-0.2N sodium acetate, if necessary, to increase the purification efficiency of the microvesicles, and then centrifuged at 3,000-5,000 g for 5 minutes to 1 hour. The pellet obtained by the use can be used.

In the present invention, the microvesicles are preferably obtained by centrifugation of the culture of kefir grains to enhance the therapeutic effect, but those obtained by the precipitation method may be used in consideration of the efficiency of the manufacturing process.

The microvesicles derived from the culture of kefir grains in the present invention have an excellent anti-inflammatory effect, improved bowel activity, enhanced intestinal immunity, inhibition of proliferation and killing of intestinal harmful bacteria, and can effectively prevent, improve or treat intestinal diseases. .

In the present invention, the bowel disease may be irritable bowel syndrome or inflammatory bowel disease.

In addition, in the present invention, the irritable bowel syndrome may be diarrhea predominant irritable bowel syndrome (d-IBS), constipation predominant irritable bowel syndrome, or diarrhea-constipated mixed irritable bowel syndrome.

In the present invention, the inflammatory bowel disease may be selected from the group consisting of Crohn's disease, intestinal lesions associated with Behcet's disease, ulcerative colitis, hemorrhagic rectal ulcer and ileal cystitis.

According to another embodiment of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating mental disorders including kefir grain-derived microvesicles as an active ingredient.

In the present invention, the kefir grains essentially include lactic acid bacteria, and may further include yeast, and the lactic acid bacteria may include Lactobacillus, Lactococcus, Lactococcus, and Leuconostoc. Lactobacillus belonging to the genus, Acetobacter genus and Streptococcus genus, etc., preferably Lactobacillus kefiranofaciens, Lactobacillus kefiri and Lactobacillus kefiri One or more selected from the group consisting of Lactobacillus kefirgranum is significantly superior to other strains, and is significantly superior to the effect of inhibiting the expression of cytokines due to stress, and more preferably, Lactobacillus kefiranofaciens. It may include. In addition, the yeast in the present invention Saccharomyces (Saccharomyces), Lacancea (Lachancea), Hansenia sospora genus, Zygotorulaspora genus, Kluyveromyces (Kluyveromyces) Yeast belonging to the genus, genus Torula and Candida genus, but is not limited thereto.

The microvesicles in the present invention may be a microvesicle derived from the culture of kefir grains.

In the present invention, the culture of kefir grains is obtained by culturing the kefir grains in a medium, and may refer to all materials obtained by culturing medium, kefir grains, and culture. In addition, the culture may be a liquid culture or a solid culture, the shape is not particularly limited. Here, the type of the medium is also not particularly limited, and for example, MRS medium, milk, cheese, and other dairy products may be used.

Specifically, the culture of kefir grains in the present invention, (a) primary culture of kefir grains in solid medium for 12 to 48 hours; And (b) may be obtained by the step of the second culture in suspension for 1 to 48 hours in a liquid medium after the first culture.

In addition, in the present invention, the culture obtained subsequent to step (b) may be subjected to the step of (c) suspension culture for an additional 1 to 14 days in a liquid medium. Here, it is preferable to suspend culture for 3 to 7 days during the culture (c), more preferably suspension culture for 3 to 4 days during the culture (c).

In the present invention, the culture of kefir grains may include extracellular vesicles, and the microvesicles may be naturally or artificially secreted from lactic acid bacteria belonging to the kefir grains, but are not limited thereto. no.

In the present invention, the amount of the microvesicles included in the pharmaceutical composition is not particularly limited, but may preferably include 1 X 10 ³ to 1 X 10 ⁹ particles. When the content of the microvesicles is less than 1 X 10 ³ particles, the effect may be insignificant, and when the number of microvesicles exceeds 1 X 10 ⁹ particles, it may cause toxicity to the human body.

The microvesicles in the present invention can be obtained by centrifugation of the culture of kefir grains. More specifically, in order to obtain the microvesicles, the step of centrifuging the culture of the kefir grains for 5 minutes to 1 hour at a temperature of more than 0 to 20 ℃ below 100 ~ 1,000g; And the supernatant obtained by recovering the supernatant after the first centrifugation and performing secondary centrifugation for 10 minutes to 1 hour at a temperature of more than 0 to 20 ° C. at 1,000 to 5,000 g.

In addition, in the present invention, in order to obtain the microbubbles, the supernatant obtained after the second centrifugal separation may be used as the supernatant obtained by performing the third centrifugation for 10 minutes to 1 hour at a temperature of more than 0 to 20 ° C at 5,000 to 20,000 g. have.

In the present invention, the micro-vesicles, the supernatant obtained after the third centrifugal centrifugation using an ultracentrifuge, 100,000 ~ 150,000g at a temperature of more than 0 to 20 ℃ or less for 4 hours centrifuged for 1 hour to 3 hours It may be a pellet obtained separately.

In the present invention, the microvesicles excellent in suppressing the expression of cytokines due to stress can be obtained with high purity and content through the four-step centrifugation process as described above.

On the other hand, the microvesicles in the present invention can be obtained by precipitating from the culture of the kefir grains. More specifically, the step of centrifuging the culture of the kefir grains at 3,000 ~ 5,000g for 5 minutes to 1 hour to obtain the microvesicles; To the concentrated culture obtained after the first centrifugation, 0.5 to 2 N sodium acetate was added in a 0.1 to 1.5 volume ratio, preferably 0.1 to 1 volume ratio, more preferably 0.1 to 0.5 volume ratio, and then -10 ° C or more and 0 ° C or less. Precipitation cultures obtained by culturing for 30 to 60 minutes at a temperature can be used.

In addition, in the present invention, the microvesicles may be pellets obtained by secondary centrifugation for 5 minutes to 1 hour at 3,000 to 5,000 g of the precipitate culture obtained after the culture.

In addition, in the present invention, the pellet obtained after the second centrifugal separation is additionally washed with 0.05-0.2N sodium acetate, if necessary, in order to increase the purification efficiency of the microvesicles, and then centrifuged at 3,000-5,000 g for 5 minutes to 1 hour. The pellet obtained by the use can be used.

In the present invention, the microvesicles are preferably obtained by centrifugation of the culture of kefir grains to enhance the therapeutic effect, but those obtained by the precipitation method may be used in consideration of the efficiency of the manufacturing process.

Recent studies have shown that physical and psychological stresses increase IL-6 levels, resulting in depressive symptoms (Korean J Psychopharmacol 2000; 11 (4): 304-312). However, in the present invention, the culture-derived microvesicles of kefir grains can reduce the secretion of the stress cytokine IL-6 by oxidative stress induction in the adrenal medulla cells, and thus, anxiety, depression, mood disorders, It can effectively prevent, ameliorate or treat mental disorders such as insomnia, obsessive compulsive disorder or stress.

On the other hand, in the present invention, "prevention" may be used without limitation any action to block, suppress or delay the symptoms of the above-listed diseases using the pharmaceutical composition of the present invention.

In addition, in the present invention, "treatment" may include without limitation any action that improves or advantageously alters the symptoms of the diseases listed above using the pharmaceutical composition of the present invention.

In the present invention, the pharmaceutical composition may be characterized in that the capsule, tablets, granules, injections, ointments, powder or beverage form, the pharmaceutical composition may be characterized in that it is intended for humans.

The pharmaceutical compositions of the present invention may be used in the form of oral dosage forms, such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. have. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, and the like in the case of oral administration, and in the case of injections, buffers, preservatives, analgesic Topical agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, in the case of injections, in unit dosage ampoules or multiple dosage forms. have. And others, solutions, suspensions, tablets, capsules, sustained release preparations and the like.

Examples of suitable carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.

Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal. Oral or parenteral release is preferred.

In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical compositions of the present invention vary depending on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed. The dosage of the pharmaceutical composition may be appropriately selected by those skilled in the art depending on the patient's condition, weight, degree of disease, drug form, route of administration, and duration, and 0.0001 to 50 mg / kg or It may be administered at 0.001 to 50 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

According to another embodiment of the present invention, the present invention relates to a food composition for preventing or ameliorating an inflammatory disease comprising a kefir grain-derived microvesicle as an active ingredient.

According to another embodiment of the present invention, the present invention relates to a food composition for preventing or ameliorating intestinal diseases comprising kefir grain-derived microvesicles as an active ingredient.

 According to another embodiment of the present invention, the present invention relates to a food composition for preventing or improving mental disorders including kefir grain-derived microvesicles as an active ingredient.

Detailed descriptions of the kefir grains, microvesicles, inflammatory diseases, intestinal diseases and psychiatric disorders in the food compositions for each use of the present invention will be omitted from the detailed description of the pharmaceutical composition as described above.

In the present invention, "improvement" means all the actions to improve or benefit the symptoms of the diseases listed above using the food composition of the present invention.

The food composition of the present invention may be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, sweets, rice cakes, breads and the like. Since the food composition of the present invention is composed of plant extracts having almost no toxicity and side effects, it can be used with confidence even for long-term use for prophylactic purposes.

When the microvesicles of the present invention is included in the food composition, the amount may be added at a ratio of 0.1 to 50% of the total weight.

Here, when the food composition is prepared in the form of a drink, there is no particular limitation other than the food composition in the proportion indicated, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like, and common sugars such as polysaccharides, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol. can do. Examples of the flavourant include natural flavourant (tautin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.), synthetic flavors (saccharin, aspartame, etc.).

In addition, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners. , pH adjusters, 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 is not so critical but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

According to another embodiment of the present invention, the present invention relates to a cosmetic composition for preventing or improving an inflammatory disease containing kefir grain-derived microvesicles as an active ingredient.

In addition, in the present invention, the kefir grains, preferably the microvesicles derived from the culture of the kefir grains have an excellent anti-inflammatory effect and can effectively prevent or improve inflammatory diseases such as edema, dermatitis, allergy or atopy.

Specific details of the kefir grains and microvesicles in the cosmetic composition of the present invention is overlapped with those described in the pharmaceutical composition, and the detailed description thereof will be omitted below.

In the present invention, the cosmetic composition is a lotion, nutrition lotion, nutrition essence, massage cream, beauty bath additives, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen Creams, suntan creams, skin lotions, skin creams, sunscreen cosmetics, cleansing milk, depilatory {cosmetic}, face and body lotions, face and body creams, skin whitening creams, hand lotions, hair lotions, cosmetic creams, jasmine Oil, Bath Soap, Water Soap, Beauty Soap, Shampoo, Hand Cleanser (Hand Cleaner), Medicated Soap {Non-Medical}, Cream Soap, Facial Wash, Systemic Cleanser, Scalp Cleaner, Hairrin, Cosmetic Soap, Tooth Whitening Gel, Toothpaste Or the like. To this end, the composition of the present invention may further include a solvent or a suitable carrier, excipient or diluent commonly used in the preparation of the cosmetic composition.

The kind of solvent that can be further added in the cosmetic composition of the present invention is not particularly limited, but, for example, water, saline, DMSO, or a combination thereof may be used, and as the carrier, excipient or diluent, purified water, oil, wax , Fatty acids, fatty alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, and the like. In addition, a whitening agent, a moisturizing agent, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, antifungal agents may be included as necessary.

The oil may be hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, avocado oil, wax, wax, carnauba, candelilla, montan, ceresin, liquid paraffin, lanolin Can be used.

Stearic acid, linoleic acid, linolenic acid, oleic acid may be used as the fatty acid, cetyl alcohol, octyl dodecanol, oleyl alcohol, pantenol, lanolin alcohol, stearyl alcohol, hexadecanol may be used as fatty acid alcohol. Isopropyl myristate, isopropyl palmitate, butyl stearate may be used as the fatty acid ester. As surfactants, cationic surfactants, anionic surfactants and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred as much as possible.

In addition, it may include a hygroscopic agent, a thickener, an antioxidant, and the like, which are widely known in the cosmetic field, and their types and amounts are known in the art.

The composition provided by the present invention has an excellent anti-inflammatory effect and can effectively prevent, ameliorate or treat various inflammatory diseases.

In addition, the composition provided by the present invention has an excellent effect of inhibiting the expression of inflammatory factors in colon cells, improve bowel activity, enhance intestinal immunity and inhibit the proliferation of intestinal harmful bacteria and induce death, effectively preventing and improving intestinal diseases Or can be treated.

In addition, the composition provided in the present invention can significantly reduce the expression level of IL-6 due to physical or mental stress, thereby effectively preventing, ameliorating or treating mental disorders caused by stress.

Figure 1 (a) shows the results of measuring the particle size and the average size of the microvesicles contained in the concentrated culture obtained by centrifuging the Lactobacillus kepyri culture in Experimental Example 1 by NTA.
Figure 1 (b) shows the results of measuring the particle size and the average size of the microvesicles contained in the concentrated culture obtained by centrifuging the Lactobacillus kepyranofaxens culture in Experimental Example 1 by NTA.
Figure 1 (c) shows the results of measuring the number of particles and the average size of the microvesicles contained in the concentrated culture obtained by centrifuging the Lactobacillus kefirranum culture in Experimental Example 1 by NTA.
Figure 2 (a) shows the content of the protein contained in the culture of Lactobacillus kepyri, Lactobacillus kepyranofaxiens and Lactobacillus kepyranum by incubation time in Experimental Example 2.
Figure 2 (b) shows the results of measuring the number of particles of microvesicles contained in the culture of Lactobacillus kepyri, Lactobacillus kepyranofaxiens and Lactobacillus kepyranum by incubation time in Experimental Example 2 by NTA It is shown.
Figure 3 shows the results of measuring the number of particles of the microvesicles according to the separation method from the culture of Lactobacillus kepyri, Lactobacillus kepyranofaxiens and Lactobacillus kepirranum in Experimental Example 3 by NTA.
Figure 4 shows the results of measuring the mRNA expression level of IL-8 and TNF-α after the microvesicle treatment according to the present invention to the inflammation-induced colon cell line CaCo-2 in Experimental Example 5.
Figure 5 shows the results of measuring the expression level of IL-6 after stimulation with LPS after the microvesicle according to the present invention to macrophages (Raw246.7 cells) in Experimental Example 6.
Figure 6 shows the results of measuring the expression level of TNF-α after stimulation with LPS after the microvesicles according to the present invention to macrophages (Raw246.7 cells) in Experimental Example 6.
Figure 7 shows the results of measuring the expression level of IL-8 after treating the microvesicles according to the present invention after treatment with CaN-2 cells TNF-α in Experimental Example 7.
Figure 8 shows the results of measuring the expression level of TNF-α after treatment with CaN-2 cells TNF-α in Experimental Example 7 and the microvesicles according to the present invention.
Figure 9 shows the results of measuring the secretion rate of IL-8 compared to the negative control after treating the microvesicles according to the present invention after treatment with CaN-2 cells TNF-α in Experimental Example 7.
FIG. 10 shows colon tissue photographs after microvesicle treatment according to the present invention in a TNBS-induced colitis mouse model in Experimental Example 8. FIG.
Figure 11 shows the results of histological evaluation of colon tissue after the microvesicle treatment according to the present invention in the TNBS-induced colitis mouse model in Experimental Example 8.
Figure 12 shows the results of evaluating the stool homeostasis after the treatment of the mixed strain culture-derived microvesicle (123_v) according to the present invention in the TNBS-induced colitis mouse model in Experimental Example 8.
Figure 13 shows the results of evaluating the degree of colon bleeding after treatment of the mixed strain culture-derived microvesicles (123_v) according to the present invention in the TNBS-induced colitis mouse model in Experimental Example 8.
Figure 14 shows the results of evaluating the stool homeostasis after treatment of the culture-derived microvesicles (1_v) of the Lactobacillus kefir granum according to the present invention in the TNBS-induced colitis mouse model in Experimental Example 8.
Figure 15 shows the results of evaluating the degree of colon bleeding after treatment of the culture-derived microvesicles (1_v) of the Lactobacillus kefirranum according to the present invention in the TNBS induced colitis mouse model in Experimental Example 8.
Figure 16 shows the results of measuring the change in MPO activity after microvesicle treatment according to the present invention in a mouse model of TNBS induced colitis in Experimental Example 8.
Figure 17 shows the results of evaluating the degree of colon bleeding after treating the microvesicles according to the present invention to the acetic acid-induced colitis mouse model in Experimental Example 9.
Figure 18 shows the results of measuring the number of viable bacteria after treatment with microvesicles according to the present invention in Staphylococcus epidermidis in Experimental Example 10.
19 shows the results of measuring the expression level of IL-6 after treatment with hydrogen peroxide (H ₂ O ₂ ) in adrenal medulla-derived PC-12 cells in Experimental Example 11 and after treatment with microvesicles according to the present invention.
Figure 20 (a) shows the results of measuring the cell viability after the microvesicle treatment according to the present invention to 3T3-L1 cells in Experimental Example 12.
20 (b) shows the results of measuring the cell viability after the micro-vesicle treatment according to the present invention to CaCo-2 cells in Experimental Example 12.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited by the following examples.

Example

Experimental Example 1 Confirmation of the Size and Particle Size of Microvesicles from Cultures of Kefir Grain

Lactobacillus kefiranofaciens, Lactobacillus kefiri, and Lactobacillus kefirgranum, which are included in kefir grains, were each streaked onto solid MRS medium at 37 ° C. After 24 hours of incubation, the suspension was incubated for 24 hours on a small scale in a liquid MRS medium. Thereafter, OD values were adjusted and cultured in a large scale in liquid MRS medium. On day 4 of the culture, the cultures were collected and the cultures were centrifuged at 300 g for 10 minutes at 4 ° C., the supernatant was taken, centrifuged at 1,200 g for 20 minutes at 4 ° C., and the supernatant was taken at 40,000 g. Centrifuged at 30 ℃ for 30 minutes, the supernatant was taken and centrifuged for 1 hour and 10 minutes at 4 ℃ at 110,000g using an ultracentrifuge and the supernatant was removed to suspend the pellet (pellet) with PBS. The number and average size of the microvesicles contained in 1 ml of the concentrated culture of lactic acid bacteria thus obtained were measured using NTA (Nanosight NS300), and the results are shown in FIGS. 1A to 1C and Table 1 below. .

Strain Total number
(particles / ml) Lactobacillus kefiranofaxiens 6.0 X 10 ¹¹ Lactobacillus kepiri 3.0 X 10 ¹¹ Lactobacillus kefir granum 3.0 X 10 ¹¹

As shown in (a) to (c) of FIG. 1 and Table 1, it was confirmed that the microvesicles were contained in an average number of particles 4.5 × 10 ¹¹ particles for 1ml of lactic acid bacteria culture, the average size is 120 to 190nm It could be confirmed as.

Experimental Example 2 Yield Change of Microvesicles by Culture Period of Lactic Acid Bacteria

Each of Lactobacillus kepyranofaxens, Lactobacillus kepyri and Lactobacillus kepirranum strains contained in kefir grains was streaked on solid MRS medium and incubated at 37 ° C. for 24 hours, followed by liquid MRS medium. Suspension incubation for 24 hours on a small scale. Thereafter, OD values were adjusted and cultured in a large scale in liquid MRS medium. Cultures were harvested on Day 1, Day 2, Day 3, Day 4, and Day 7, respectively, and centrifuged with an ultracentrifuge to discard the supernatant and recover the concentrated culture of lactic acid bacteria. NTA (Nanosight NS300) was used to measure the particle number and protein content of the microvesicles contained in 1ml of the culture and the results are shown in (a) and (b) of FIG.

As shown in (a) and (b) of FIG. 2, as a result of confirming the microvesicles by the number of cultures and the number of particles for each culture day, it was confirmed that the number of particles has a constant pattern for each culture day compared to the protein. It was confirmed that the microvesicles contained in the culture at the 4th day of culture in all three lactic acid bacteria on the basis of the number of particles.

Experimental Example 3 Change in Yield of Microvesicles by Separation Methods

Microvesicles were separated from the 4th day culture obtained in Experimental Example 1 using the following three methods.

1. Separation of microvesicles using ultracentrifuge (ultracentrifuge method)

The culture was centrifuged at 300 g for 10 minutes at 4 ° C., the supernatant was taken and centrifuged at 1,200 g at 4 ° C. for 20 minutes, then the supernatant was taken and centrifuged at 10,000 g at 4 ° C. for 30 minutes, The supernatant was taken and centrifuged at 110,000 g for 1 hour and 10 minutes at 4 ° C. using an ultracentrifuge. The supernatant was removed, and the pellet was suspended in PBS.

2. ExoQuick ^TM-TC separation of the fine ER with a little precipitate a solution of exo (Exoquick method)

The culture was centrifuged at 3,000 g for 15 minutes, and the ExoQuick-TC ^TM exosome precipitation solution was added at a rate of 2 ml per 10 ml of culture and then incubated overnight at 4 ° C. After incubation, the supernatant was removed after centrifugation at 1,500g for 30 minutes, and the supernatant was removed after centrifugation at 1,500g for 30 minutes, and the pellets were suspended in PBS.

3. Separation of Microvesicles by Precipitation Method

The culture was centrifuged at 5,000 g for 10 minutes, 1N sodium acetate was added at a rate of 1 ml per 10 ml of culture and incubated for 45 minutes on ice. After incubation, the mixture was centrifuged at 5,000 g for 10 minutes, the supernatant was removed, and the precipitate was washed with 0.1 N sodium acetate. After centrifugation at 5,000 g for 10 minutes, the supernatant was removed and the pellet was suspended in PBS.

NTA (Nanosight NS300) was used to measure the particle number of the microbubbles contained in the precipitate obtained by the above three methods and the results are shown in FIG.

As shown in FIG. 3, it can be seen that the microvesicles can be obtained by the method using the ultracentrifuge at the highest yield from the lactic acid bacteria culture.

Experimental Example 4 Acquisition of Kefir Grain-Derived Microvesicles

Lactobacillus kefiranofaciens (Lactobacillus kefiranofaciens), Lactobacillus kefiri (Lactobacillus kefiri) and Lactobacillus kefirgranum (Lactobacillus kefirgranum) mixed cultures of the same strain as in Experimental Example 1 to obtain a culture , Microvesicles were separated by the method using the ultracentrifuge of Experimental Example 3.

Experimental Example 5 Confirmation of Anti-inflammatory Effect of Kefir Grain-derived Microvesicles (1)

Inflammation-induced colon cell line CaCo-2 with 20ng TNF-α was plated at 1 X 10 ⁶ cells per well in a 12-well plate, and the microvesicles isolated from the ultracentrifuge in Experimental Example 3 were each 1 X. Treatment was carried out for 24 hours in the amount of 10 ³ , 1 X 10 ⁶ , 1 X 10 ⁹ particles. After 48 hours of incubation, RNA was extracted from the cells of each group, followed by synthesis of complementary DNA (cDNA) using intron premix, and real-time PCR (RT-PCR) using the same. Was performed. Expression of IL-8 and TNF-α as inflammatory factors in the cells of each treatment group was measured and the results are shown in FIG. 4.

As shown in Figure 4, when the microvesicles derived from the lactic acid bacteria culture according to the present invention can be seen that the expression level of IL-8 and TNF-α is significantly reduced, in particular, the anti-inflammatory effect of Lactobacillus kepyranofaxiens derived microvesicles You can see the excellent.

Experimental Example 6 Confirmation of Anti-inflammatory Effect of Kefir Grain-derived Microvesicles (2)

Raw 246.7 cells, which are macrophages of mice, were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin for 48 hours, and then 200 μg / ml (1 × 10 ⁹ nanomicrovesicles obtained in Experiment 4) were obtained. Particles / ml) and incubated for 24 hours, treated with LPS 100ng / ml for 6 hours, and then measured the expression levels of IL-6 and TNF-α using RT-PCR and ELISA. 5 and 6 are shown. However, as a positive control curcumin (curcumin) was treated in an amount of 10μM.

As shown in Figures 5 and 6, when treated with the microvesicles according to the present invention to the macrophages and induction of inflammation using LPS, the IL-6 and TNF-α of the case than the case of no treatment or curcumin treatment It was confirmed that the expression level was significantly reduced by more than half.

Experimental Example 7 Confirmation of Anti-inflammatory Effect of Kefir Grain Derived Microvesicles (3)

5% CO ₂ -95% air in a 37 ° C. incubator with 10% FBS, 100 units / mL penicillin, 100 μg / mL streptomycin, 0.1 mmol / l non-essential amino acid, 1 mmol / l sodium pyruvate in MEM medium Caco-2 cells were cultured as a composition. When cultured cells grew 80% confluent, cells were detached using 0.25% trypsin, and 24 wells were inoculated with 2 × 10 ⁴ cells per well. Thereafter, each well was treated with TNF-α in an amount of 20 ng / ml for 6 hours, and then the microvesicle obtained in Experimental Example 4 was treated in an amount of 200 µg / ml (1 X 10 ⁹ nanoparticles / ml) to 24 Incubated for hours. The expression level of IL-8 and TNF-α and the rate of secretion of IL-8 in the microvesicle treated group compared to the negative control group were measured using RT-PCR and ELISA (100 µg / ml). 9 is shown.

As shown in Figures 7 to 9, treatment of the microvesicles according to the present invention after inducing inflammation by treating TNF-α in intestinal epithelial cells significantly reduced the mRNA expression levels of inflammatory factors IL-8 and TNF-α IL-8 secretion was also markedly reduced.

Experimental Example 8 Confirmation of Anti-inflammatory Effects of Kefir Grain-derived Microvesicles (4)

BALB / c mice induced colitis with 2,4,6-trinitrobenzenesulfonic acid (TNBS). Specifically, the mouse was anesthetized with ether, and 2.5 g of TNBS (2,4,6-Trinitrobenzene sulfonic acid) solution was mixed with 50% ethanol. Inflammation was induced by administering ㎖ and vertically held for 30 seconds. In contrast, the normal group was orally administered with 0.1 ml of saline solution. Then, orally administer the microvesicles obtained in Experiment 4 at a dose of 10 μg / horse or 1 mg / horse once daily for three days from the next day, and the day after the end of the sample administration, suffocate the mice with carbon dioxide and euthanize them. The large intestine from the middle caecum to the area immediately before the anus was extracted. However, as a positive control, prednisolone, a therapeutic agent for colitis, was administered orally in an amount of 2 mg / kg.

(1) organization appearance evaluation

Cross-section photographs of the extracted colon tissues after H & E staining are shown in FIG. 10, and the scores were scored according to the criteria of Table 2 (Hollenbach et al., 2005 colitis degree) by observing the length and appearance of the colon. The results are shown in FIG.

Macroscopic Score standard 0 No ulcers and inflammations found One Congestion without bleeding is found 2 Ulcers with congestion found 3 Sheep and inflammation found only in one place 4 Ulcers and inflammations found in more than one place 5 Ulcers that extend beyond 2 cm

As shown in FIG. 10, in the large intestine of the colitis mouse model, severe epithelial necrosis, bleeding, infiltration of inflammatory cells, edema and ulcers, etc. were observed, but the symptoms of the above-described colitis were dose-dependently when the microvesicles were administered according to the present invention. It was confirmed that the relief was significantly.

In addition, as shown in Figure 11, the administration of the microvesicles according to the present invention in the colitis mouse model was reduced intestinal bleeding in a concentration-dependent manner, this therapeutic effect is administered in a large amount of prednisolone 2mg / kg as a positive control It was confirmed to be superior to one case.

(2) DAI evaluation

The activity activity index (DAI) is a method of scoring the symptoms caused by TNBS that induces acute colitis, starting from the time of providing the microvesicles according to the present invention and the concentration and color of the stool at the same time every day. It was measured by checking. Fecal homeostasis and bleeding were measured and the results are shown in FIGS. 12 to 15. However, the evaluation criteria of the DAI is shown in Table 3 below. In the evaluation of DAI, in addition to the microvesicles (123_v) derived from the culture of the mixed strain obtained in Experimental Example 4, the microvesicles (1_v) derived from the Lactobacillus kefirgranum culture obtained using an ultracentrifuge in Experimental Example 3 were administered. Further experiments.

Stool consistency Stool bleeding 0 Formed 0 Normal color 2 Loose stool 2 fecal occult blood test positive 4 Diarrhea 4 Gross bleeding

As shown in FIGS. 12 to 15, when the culture microvesicles (123_v) or lactobacillus-derived kefir granum culture-derived microvesicles (1_v) of the mixed strain according to the present invention were administered to the colitis mouse model, as a positive control. It was confirmed that the bowel activity was improved by lowering the DAI value to a level equivalent to or higher than that of prednisolone.

(3) MPO activity evaluation

In addition, using the MPO ELISA kit (Hycult Biotech, HK105) to measure the activity of Myeloperoxidase (MPO) as an indicator of inflammation in the colon tissues are shown in Figure 16 the results. However, the MPO activity is 100 μl of the supernatant obtained by centrifugation of homogenously grounded intestinal tissue with a homogenizer for 15 minutes and a standard solution prepared in advance to the well to which the antibody is attached. , The plate was sealed with a cover to prevent air from entering and then reacted at room temperature for 1 hour. After 1 hour after the reaction, the wash buffer was washed four times using a wash buffer to be completely removed. Thereafter, 100 μl of tracer solution was added to each well, and a cover was attached and reacted at room temperature for 1 hour, and washed 4 times as well. Then, diluted streptavidin-peroxidase solution was added to all wells by 100 μl, and the cover was attached and reacted at room temperature for 1 hour. After completion of the reaction, each well was washed four times, and 100 μl of TMB substrate was added to all wells, followed by reaction for 30 minutes under shading, and then 100 μl of the same amount of stop solution was added. Since the absorbance at 450 nm was measured to obtain the MPO activity value of the sample using a standard curve (standard curve) prepared as a standard solution.

As shown in FIG. 16, when the microvesicles according to the present invention were administered to the colitis mouse model, the MPO activity was markedly reduced to a level equivalent to or higher than that of the administration of prednisolone as a positive control.

Experiment 9 Confirmation of anti-inflammatory effect of kefir grain-derived microvesicles (5)

BALB / c mice induced irritable bowel syndrome with acetic acid. Specifically, mice were anesthetized with ether, and then 5% acetic acid was administered 0.1 ml into the large intestine through the anus using a 1 ml syringe with a rounded tip and held vertically for 30 seconds to cause irritable bowel syndrome. In contrast, the normal group was orally administered with 0.1 ml of saline solution. Thereafter, the microvesicles obtained in Experiment 4 were administered orally at a dose of 10 μg / dose for 3 days once daily from the next day. Starting from the time of providing the microvesicles according to the present invention, the color of the stool is checked at the same time every day while providing and the bleeding degree of the large intestine is measured based on the criteria shown in Table 3, and the results are shown in FIG. It was. However, as a positive control, mixed strains of prednisolone (2 mg / kg), Lactobacillus kefiranofaciens, Lactobacillus kefiri, and Lactobacillus kefirgranum (5X10) ⁷ CFU), commercially available lactic acid bacteria products on the market (5 X 10 ⁷ CFU) and commercially available lactic acid bacteria products on the market (5 X 10 ⁷ CFU) and microvesicles of the present invention (10 ㎍ / horse) was orally administered.

As shown in Figure 17, when treated with the microvesicles according to the present invention to acetic acid-induced irritable bowel syndrome mouse model, the microvesicle-derived mixed strain itself, or significantly when compared to the conventional commercially administered lactic acid bacteria It was confirmed to reduce intestinal bleeding. In addition, when the micro-vesicles according to the present invention is treated with lactic acid bacteria previously sold commercially, it was confirmed that the degree of reduction of colon bleeding is further increased.

Experimental Example 10 Confirmation of Antimicrobial Effects of Kefir Grain Derived Microvesicles

In order to confirm the antimicrobial effect against enteric harmful bacteria of kefir grain-derived microvesicles according to the present invention, the experimental example after incubating the Staphylococcus epidermidis belonging to the enteric harmful bacteria in a liquid nutrition medium for 24 hours The microvesicles obtained in 4 were treated with an amount of 500 µg / ml, and further cultured at 37 ° C. for 3 hours, and the number of viable bacteria was measured. The results are shown in FIG. 18. However, the negative control was treated with distilled water, and the positive control was treated with penicillin (Penicillin) in an amount of 10 ㎍ / ml.

As shown in Figure 18, when treated with microvesicles according to the present invention can be confirmed to inhibit the proliferation of intestinal harmful bacteria Staphylococcus epidermidis and induces death to the same level as when treated with a positive control penicillin there was.

Experimental Example 11 Stress Relieving Effect of Kefir Grain Derived Microvesicles

In order to evaluate the stress relaxation effect of the kefir grain-derived microvesicles according to the present invention, each well of a 24-well plate contains 10% serum (FBS: Fetal Bovine Serum) and 1% penicillin / streptomycin antibiotic. MEM (Minimal Essential Medium) was dispensed, inoculated with 5 × 10 ⁴ cells of adrenal medulla-derived PC-12 cells per well and treated with hydrogen peroxide (H ₂ O ₂ ) for 1 hour to induce the expression of IL-6. . Thereafter, the microvesicles obtained in Experimental Example 4 were treated in amounts of 0.1 µg / ml, 1 µg / ml, 10 µg / ml and 100 µg / ml, respectively, and the expression level of IL-6 was measured by ELISA. The results are shown in FIG. 19.

As shown in Figure 19, when treated with microvesicles according to the present invention it was confirmed that the expression level of IL-6 was significantly reduced compared to the untreated group, the decrease was increased in proportion to the concentration.

By using the kefir grain-derived microvesicles according to the present invention it can be clearly predicted that can also prevent or treat mental disorders such as depression by significantly reducing the expression level of IL-6 by stress.

Experimental Example 12 Confirmation of Safety of Kefir Grain-derived Microvesicles

In order to confirm the safety of the kefir grain-derived microvesicles according to the present invention, the microvesicles obtained by using the ultracentrifuge in Experiment 3 in 3T3-L1 and CaCo-2 normal cells 1 X 10 ³ to 1 X 10 ⁹ After treatment with the amount of particles, dilute the MTS reagent (Promega, USA) and the cell basal medium (FBS free, 1% p / s) at a ratio of 1: 5, and dispense 500 µl of each well for 1 hour 30 minutes. After the reaction, the absorbance was measured at a wavelength of 490 nm to evaluate the change in cell viability, and the results are shown graphically in FIGS. 20A and 20B. However, no treatment was performed as a control.

As shown in (a) and (b) of FIG. 20, even when the microvesicles according to the present invention were treated to an amount of 1 X 10 ⁹ particles, there was no cytotoxicity.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (4)

A pharmaceutical composition for preventing or treating mental disorders comprising kefir grain or extracellular vesicles derived from kefir grain culture as an active ingredient,
The kefir grains include one or more selected from the group consisting of Lactobacillus kefiranofaciens, Lactobacillus kefiri, and Lactobacillus kefirgranum. The method of claim 1,
The mental disorder is anxiety, depression, mood disorders, insomnia, obsessive compulsive disorder or stress. Food composition for the prevention or improvement of mental disorders comprising kefir grain or extracellular vesicles derived from kefir grain culture as an active ingredient,
The kefir grains include one or more selected from the group consisting of Lactobacillus kefiranofaciens, Lactobacillus kefiri, and Lactobacillus kefirgranum. The method of claim 3,
The mental disorder is anxiety, depression, mood disorders, insomnia, obsessive compulsive disorder or stress.

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