KR20230122472A - Pharmaceutical composition for the prevention or treatment of obesity or metabolic syndrome induced from obesity containing enteroccocus faecalis as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating obesity or metabolic syndrome induced by obesity, containing Enterococcus faecalis as an active ingredient, and specifically, Enterococcus faecalis EF-2001 is used to treat adipocytes. Since it was confirmed that the effect of inhibiting adipogenesis, the Enterococcus faecalis, its culture medium, or its dead cells prevent, improve, and treat obesity caused by adipogenesis in adipocytes or metabolic syndrome diseases derived from obesity. can be very useful for

Description

Composition for preventing or treating obesity or metabolic syndrome induced by obesity containing Enterococcus faecalis as an active ingredient

The present invention relates to a composition for the prevention or treatment of obesity or metabolic syndrome induced by obesity, containing Enterococcus faecalis as an active ingredient, and more particularly, Enterococcus faecalis, its culture medium or its dead cells It relates to a composition for preventing, improving or treating obesity or metabolic syndrome induced from obesity comprising as an active ingredient.

Metabolic syndrome is a clustering phenomenon of abdominal obesity, impaired glucose tolerance, hypertension, and dyslipidemia. Among them, obesity is regarded as the most basic factor in the onset of the disease, and the obese population is increasing every year. Overeating, stress, and insomnia have been known to be causes of obesity in modernized societies. Gradually interest in the causes of obesity and demands for treatment are also increasing.

From the fact that obesity increases the incidence of various metabolic diseases and that actual weight loss significantly reduces the incidence of these diseases, understanding of the proliferation of fat-containing adipocytes and substances secreted from adipocytes and their in vivo regulation Identification of the mechanism is considered to be the basis for the development of treatments for obesity and various diseases caused by it. Accordingly, studies on the regulation of adipocyte differentiation are being actively conducted.

For example, current research trends are focused on searching for substances capable of inhibiting metabolic processes related to adipocyte differentiation as a method for preventing or treating obesity and lipid-related metabolic diseases. In other words, based on the mechanism of obesity, an attempt is being made to treat obesity through adipocyte regulation, which is to reduce the amount of fat by inhibiting fat synthesis or promoting lipolysis and oxidation, and by suppressing differentiation of adipocytes to reduce fat Substances that mediate or regulate these processes, aimed at reducing cell numbers, are emerging as targets for development.

Meanwhile, microorganisms of the genus Enterococcus exist widely in nature and use carbohydrates aerobically. In general, bacteria such as microorganisms of the genus Enterococcus are known to prevent damage caused by pathogenic microorganisms by in vivo antagonism or secreted antibacterial substances. Among them, Enterococcus faecalis EF-2001 was identified by screening the intestinal flora of a 2-year-old girl. The Enterococcus faecalis EF-2001 was killed by heat treatment and the cell components were recovered.

According to studies reported on the physiological activity of Enterococcus faecalis EF-2001, in the case of mice suffering from colitis induced by DSS (Dextran sulfate sodium), enterococcus faecalis EF-2001 ingested the DSS relief effect in mice. It was reported that the proliferation of transplanted sarcoma cancer cells (Sarcoma-180) was reduced and NK cells were activated (Tadano et al., J. Japan Mibyou System association, 2011). In addition, regarding the suppression of harmful bacteria and the intestinal function, Enterococcus faecalis EF-2001 suppresses the activity of Candida albican , which is the cause of white discharge, and shows symptom improvement and preventive effects (Ishijima et al., Med. Mycol. J, 2014 ), the efficacy of proliferating beneficial bacteria and suppressing harmful bacteria faster than that of the intestinal control in mice administered with antibiotics has been disclosed (Simohashi et al., Medicine and biology, 2002). The various physiological activities of Enterococcus faecalis EF-2001 have the advantage of being unaffected by heat and pH due to the nature of dead cells and can be processed into various types of preparations (Kan, Food industry, 2001). In addition, with the content of 7.5 trillion cells per gram, it is possible to consume a large amount of lactic acid bacteria even with a small amount.

Korean Patent Registration No. 10-1800632 discloses a pharmaceutical composition, food composition and food additive for preventing, improving or treating muscle loss, weakness and muscle atrophy containing Enterococcus faecalis, its culture medium or its dead cells as an active ingredient. Korean Patent Publication No. 10-1996079 discloses a composition for preventing or treating hair loss containing Enterococcus faecalis as an active ingredient. As such, it has not been revealed about its use for the prevention, improvement, or treatment of obesity or obesity-induced metabolic syndrome.

Therefore, the present inventors have made efforts to develop a material with fewer side effects and excellent treatment effects for obesity and metabolic syndrome caused by it. As a result, Enterococcus faecalis EF-2001 inhibits adipogenesis of adipocytes It was confirmed that the present invention exhibits the effect of preventing, improving, and treating Enterococcus faecalis, its culture medium, or its dead cells in obesity or obesity-induced metabolic syndrome caused by adipogenesis in fat cells, or a composition or functional material By revealing that it can be used as, the present invention was completed.

An object of the present invention is to enterococcus faecalis , which has an adipogenesis inhibitory activity and can be very useful for the prevention, improvement, and treatment of obesity caused by adipogenesis in adipocytes or metabolic syndrome diseases derived from obesity. ), to provide a composition for preventing, improving or treating obesity or metabolic syndrome induced by obesity, comprising its culture medium or its dead cells as an active ingredient.

An object of the present invention is to provide a pharmaceutical preparation for the prevention or treatment of obesity or obesity-induced metabolic syndrome, comprising at least one selected from the group consisting of Enterococcus faecalis , its culture medium, and its dead cells as an active ingredient. This is achieved by providing a composition.

In addition, an object of the present invention is to provide a health functional food composition for improving obesity or metabolic syndrome induced from obesity, comprising at least one selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cells as an active ingredient. is achieved by

In addition, the object of the present invention is achieved by providing a food additive for improving obesity or obesity-induced metabolic syndrome, comprising at least one selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cells as an active ingredient. do.

In addition, an object of the present invention is achieved by providing an external preparation for improving obesity or obesity-induced metabolic syndrome, comprising at least one selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cells as an active ingredient. .

In addition, the object of the present invention is achieved by providing a cosmetic for improving obesity or metabolic syndrome induced from obesity, comprising at least one selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cells as an active ingredient. .

In the present invention, since it was confirmed that Enterococcus faecalis EF-2001 exhibits an effect of inhibiting adipogenesis of adipocytes, the Enterococcus faecalis, its culture medium, or its dead cells are It can be very useful for the prevention, improvement, and treatment of obesity that occurs through production or metabolic syndrome diseases induced by obesity.

Figure 1 shows the results of measuring the cytotoxicity of Enterococcus faecalis EF-2001 ( Enterococcus faecalis EF-2001).
Figure 2 shows the adipogenesis inhibitory effect of adipocytes by concentration of Enterococcus faecalis EF-2001.
Figure 3 visually confirms the effect of inhibiting adipogenesis of adipocytes by concentration of Enterococcus faecalis EF-2001.

Hereinafter, a preferred embodiment of the present invention and the physical properties of each component will be described in detail, but this is to be explained in detail so that a person having ordinary knowledge in the art to which the present invention belongs can easily practice the invention, This is not meant to limit the technical spirit and scope of the present invention.

In the present invention, the term "adipocyte" refers to a fat cell differentiated from preadipocytes, which produces fat in vivo. In the present invention, the term "obesity" means to provide a cause of a disease or disease caused by excessive fat production.

In addition, in the present invention, "metabolic syndrome" includes, for example, abdominal fat production, impaired glucose tolerance, hypertension, and comprehensive symptoms of symptoms appearing as a cluster of dyslipidemia.

Preferably, the obesity diseases to be treated, improved or prevented by the present invention are abdominal fat production, impaired glucose tolerance, hypertension, and dyslipidemia belonging to the metabolic syndrome.

Metabolic syndrome includes a comprehensive set of symptoms, such as abdominal fat production, impaired glucose tolerance, high blood pressure, and dyslipidemia. very effective for

In the present invention, the term "prevention" means suppressing the occurrence of a disease or disease in an animal that has never been diagnosed as having the disease or disease, but is prone to such disease or disease. As used herein, the term “treatment” refers to (i) inhibition of a disease or development of a disease; (ii) alleviation of disease or illness; and (iii) the elimination of disease or disease.

The present invention provides a pharmaceutical composition for preventing or treating obesity or obesity-induced metabolic syndrome, comprising, as an active ingredient, at least one selected from the group consisting of Enterococcus faecalis , its culture medium, and its dead cells. to provide.

In the present invention, the Enterococcus faecalis is preferably Enterococcus faecalis EF-2001, but is not limited thereto.

In the present invention, the Enterococcus faecalis, its culture medium or its killed cells may be used either commercially available or prepared by a known method for preparing dead cells, and is non-toxic and harmless to the human body.

In addition, the culture solution refers to a culture solution obtained by culturing Enterococcus faecalis in a culture medium, a concentrated culture solution, a dried culture solution, a culture filtrate, a concentrated culture filtrate, or a dried product of the culture filtrate, including the strain , It may be a culture medium from which strains are removed after culturing.

In addition, the dead cells may be prepared by heat-treating corresponding live cells or treating them together with formalin or other fungicides, and dead cells may be used even if they are substantially dead. In addition, after washing the strain obtained by culturing the dead cell body by a conventional method, centrifuging and repeating washing and dehydration as necessary, it is suspended in distilled water, physiological saline, etc., and the suspension is, for example, Dead cell suspension obtained by heating at 80 to 115 ° C. for 30 minutes to 3 seconds or a dried product thereof, or killed cell suspension obtained by irradiating the dead cell suspension with gamma rays or neutron rays, or a dried product thereof. The method for drying the dead cell suspension is not particularly limited as long as it is a known drying method, but spray drying, freeze drying and the like can be exemplified. In some cases, enzyme treatment, surfactant treatment, grinding/pulverization treatment may be performed before or after the sterilization treatment by heating or the like, or before or after the drying treatment, and those obtained by these treatments are also included in the dead cells of the present invention. do. In addition, the dead cells may be prepared by the following methods, but are not limited thereto:

1) seed culture of Enterococcus faecalis, followed by main culture at pH 4.0 to 9.0 and a temperature of 15 to 45 ° C, more preferably at pH 5.0 to 8.0 and a temperature of 20 to 40 ° C;

2) Heat treatment of Enterococcus faecalis main cultured in step 1) at 60 to 140 ° C for 1 to 40 minutes, more preferably at 70 to 130 ° C for 5 to 30 minutes, followed by drying and pulverization.

In the present invention, the obesity is induced by adipocyte differentiation and proliferation, and metabolic syndrome is induced due to such obesity. More specifically, the metabolic syndrome is in the group consisting of abdominal adipogenesis, impaired glucose tolerance, hypertension and dyslipidemia. more than one selected

In a specific embodiment of the present invention, the present inventors confirmed that Enterococcus faecalis EF-2001 exhibits an effect of inhibiting adipogenesis in adipocytes, and therefore, the Enterococcus faecalis reduces obesity caused by adipogenesis in adipocytes. Or it can be very useful as an active ingredient of a pharmaceutical composition for the prevention, improvement and treatment of metabolic syndrome induced by obesity.

The composition of the present invention may include a strain as an active ingredient in an amount of 10 ⁶ to 10 ¹³ cfu/g based on the total weight of the composition, or may include cultures or dead cells having an equivalent number of viable cells. In addition, by selecting one or two or more pharmaceutically acceptable conventional carriers or one or two or more additives in an effective amount of the main ingredient, Enterococcus faecalis, its culture medium or dead cells of the present invention, composition can be made.

One or two or more carriers may be selected from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and one or two or more additives may be selected from among flavoring agents, vitamins, and antioxidants. and can be used.

In the present invention, all pharmaceutically acceptable carriers and additives may be used, and specifically, diluents include lactose monohydrate, trehalose, cornstarch, and soybean oil. , Microcrystalline cellulose or mannitol (D-mannitol) is preferable, magnesium stearate or talc is preferable as a lubricant, and polyvinyl pyrrolidone (PVP) or polyvinyl pyrolidone (PVP) or It is preferable to select from hydroxypropylcellulose (HPC). In addition, the disintegrant is preferably selected from carboxymethylcellulose calcium (Ca-CMC), sodium starch glycolate, polacrylin potassium or cross-linked polyvinylpyrrolidone. The sweetener is selected from white sugar, fructose, sorbitol, or aspartame, and the stabilizer is sodium carboxymethylcellulose (Na-CMC: carboxymethylcellulose sodium), β-cyclodextrin, and white lead. It is selected from (white bee's wax) or xanthan gum, and as a preservative, methyl p-hydroxy benzoate (methylparaben), propyl p-hydroxybenzoate (propylparaben), or potassium sorbate ( potassium sorbate), but is not limited thereto.

The pharmaceutical composition of the present invention may be administered to a patient as a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. In the above, 'pharmaceutically effective amount' refers to an amount that exhibits a higher response than that of the negative control group, and preferably refers to an amount sufficient to prevent or treat inflammatory diseases. In addition, the pharmaceutically effective amount may be appropriately changed depending on various factors such as the disease and its severity, the patient's age, weight, health condition, sex, administration route and treatment period.

The composition of the present invention can be formulated in various ways according to the route of administration by a method known in the art together with the pharmaceutically acceptable carrier. In the above, "pharmaceutically acceptable" means a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans and does not usually cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions. says The composition of the present invention can be formulated in various ways according to the route of administration by a method known in the art together with the pharmaceutically acceptable carrier. The route of administration is not limited thereto, but may be administered orally or parenterally.

In addition, the present invention provides a health functional food composition for improving obesity or metabolic syndrome induced from obesity, comprising Enterococcus faecalis, its culture medium or its dead cells as an active ingredient.

In addition, the present invention provides a food additive for improving obesity or metabolic syndrome induced from obesity, comprising Enterococcus faecalis, its culture medium or its dead cells as an active ingredient.

In the present invention, the Enterococcus faecalis is preferably Enterococcus faecalis EF-2001, but is not limited thereto.

In addition, the method for obtaining the Enterococcus faecalis, its culture medium and its dead cells and the obesity or obesity-induced metabolic syndrome types include obesity or obesity comprising the Enterococcus faecalis, its culture medium or its dead cells as an active ingredient. Since it is the same as the content described in the pharmaceutical composition for improvement or treatment of metabolic syndrome derived from, the specific description uses the above content, and hereinafter, only the unique configuration of the health functional food will be described.

On the other hand, since the present inventors confirmed that Enterococcus faecalis EF-2001 exhibits an effect of inhibiting adipogenesis of adipocytes, the Enterococcus faecalis is associated with obesity caused by adipogenesis in adipocytes or metabolic syndrome derived from obesity. It can be very useful as an active ingredient of a health functional food composition or food additive for prevention or improvement.

The type of food to which the Enterococcus faecalis of the present invention is added is not particularly limited. Examples of the food include drinks, meat, sausages, bread, biscuits, rice cakes, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, alcoholic beverages and vitamins There are complex drugs, etc., and includes all health foods in a conventional sense.

The mixing amount of Enterococcus faecalis according to the present invention may be suitably determined depending on its purpose of use (for prevention or improvement). In general, the amount of Enterococcus faecalis in the health food may be 0.001 to 50% by weight of the total food weight. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

The health functional beverage composition of the present invention is not particularly limited in other ingredients except for containing the Enterococcus faecalis as an essential ingredient in the indicated ratio, and contains various flavoring agents or natural carbohydrates as additional ingredients like conventional beverages. can do. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. .

In addition to the above, the food or food additive of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like may be contained. In addition, the Enterococcus faecalis of the present invention may contain fruit flesh for producing natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of Enterococcus faecalis of the present invention.

In addition, the present invention provides an external preparation for improving obesity or metabolic syndrome induced by obesity, comprising Enterococcus faecalis, its culture medium or its dead cells as an active ingredient.

In addition, the present invention provides a cosmetic for improving obesity or metabolic syndrome induced from obesity, comprising Enterococcus faecalis, its culture medium or its dead cells as an active ingredient.

In the present invention, the Enterococcus faecalis is preferably Enterococcus faecalis EF-2001, but is not limited thereto.

In addition, the method for obtaining the Enterococcus faecalis, its culture medium and its dead cells and the obesity or obesity-induced metabolic syndrome types include obesity or obesity comprising the Enterococcus faecalis, its culture medium or its dead cells as an active ingredient. Since it is the same as the content described in the pharmaceutical composition for improvement or treatment of metabolic syndrome derived from, the specific description will use the above content, and hereinafter, only the unique configuration of external preparations and cosmetics will be described.

On the other hand, since the present inventors confirmed that Enterococcus faecalis EF-2001 exhibits an effect of inhibiting adipogenesis of adipocytes, the Enterococcus faecalis is associated with obesity caused by adipogenesis in adipocytes or metabolic syndrome derived from obesity. It can be used very usefully as an external agent for improvement or as an active ingredient in cosmetics.

Ingredients included in the external preparation or cosmetic composition of the present invention may include ingredients commonly used in external preparations or cosmetic compositions in addition to the above active ingredients, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. A phosphorus adjuvant, and a carrier.

In order to formulate the active ingredient of the present invention, it can be easily formulated by conducting it according to a conventional method, and surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspending agents, and other commercially available adjuvants can be appropriately used.

The composition of the present invention can be prepared in any formulation commonly prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, It may be formulated as an oil, powder foundation, emulsion foundation, wax foundation, pack, massage cream and spray, but is not limited thereto. More specifically, it may be prepared in the form of softening lotion, nutrient lotion, nutrient cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as carrier components. can

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, as carrier components, liquid diluents such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, suspending agents such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals Sexual cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. / May contain a propellant such as butane or dimethyl ether.

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

However, the following Examples, Experimental Examples, and Preparation Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Examples, Experimental Examples, and Preparation Examples.

<Example 1> Enterococcus faecalis EF-2001 ( Enterococcus faecalis EF-2001) culture

Enterococcus faecalis EF-2001( Enterococcus faecalis EF-2001) Live bacteria (Nihon BERM Co. Ltd, Japan) are aerobically or anaerobically cultured in a medium used for general lactobacillus culture, and after pre-culture, pH 5.0 ~ 8.0, 20 ~ While maintaining 40 ° C., the culture was performed for 1 to 3 days to reach a dry weight (DW) of 7.5 × 10 ¹² cfu / g or more.

<Example 2> Enterococcus faecalis EF-2001 ( Enterococcus faecalis EF-2001) Manufacturing dead cells

Enterococcus faecalis EF-2001 cultured in <Example 1> was heat-treated at 90° C. for 15 minutes to kill cells, and cells were separated and recovered by a continuous centrifuge, followed by lyophilization and pulverization.

<Experimental Example 1> Cell culture

3T3-L1 (mouse embryonic fibroblast cell line) preadipocytes were purchased from ATCC (American type culture collection), 10% BCS (bovine calf serum, WelGene Biopharmaceuticals, Daegu, Korea) and antibiotics (penicillin/streptomycin 100 units/ml , Bioshop, Burlington, Ontario, Canada) was added to DMEM medium (Dulbecco's modified Eagle's medium, WelGene Biopharmaceuticals, Daegu, Korea), and cultured in a cell culture medium at 37°C and 5% CO ₂ conditions.

<Experimental Example 2> Cytotoxicity evaluation of Enterococcus faecalis EF-2001

The cytotoxicity of Enterococcus faecalis EF-2001 was evaluated using the MTT assay assay.

Specifically, the culture medium was removed from the 100 cm ² culture plate in which the 3T3-L1 preadipocytes were cultured by the method described in <Experimental Example 1>, and the medium was added to CMF-PBS (calcium magnesium free-phosphate buffered saline, pH 7.2). After washing, the cells were detached by treatment with 0.25% trypsin/EDTA, neutralized with cell culture medium, and centrifuged at 1500 rpm for 5 minutes. After adding the culture solution to the remaining cell pellet, aspirate repeatedly with a sterile pipette to make a single cell suspension, and then mix trypan blue with the cell suspension in a weight ratio of 9: 1 to calculate the hemocytometer on an optical microscope. Cells were quantified using a hemocytometer. The quantified 3T3-L1 preadipocytes were dispensed in a 24-well plate to be 5×10 ⁴ cells/well. After culturing it in an incubator at 37°C and 5% CO ₂ conditions, when the cell concentration reached 100% in the plate, differentiation inducers (IBMX, dexamethasone, Insulin) were added to induce differentiation of adipocytes. Enterococcus faecalis EF-2001 cultured in 1> was added at concentrations of 0, 25, 100, and 250 μg/ml, respectively, and cultured for 48 hours to induce differentiation of adipocytes. Thereafter, 50 μl of 5 mg/ml MTT solution was added to measure cell viability, and the cells were incubated at 37° C. for 2 hours. The obtained formazan crystals were dissolved in 150 μl of DMSO, transferred to a 96-well plate, and absorbance was measured at 590 nm using a Victor™ X3 multilabel reader (Perkin Elmer, Waltham, MA, USA).

As a result, as shown in FIG. 1, when Enterococcus faecalis EF-2001 was not added, when the survival rate was 100%, Enterococcus faecalis EF-2001 was added to the group without it. It was confirmed that the survival rate was similar to that of

Through the above results, it was found that Enterococcus faecalis EF-2001 of the present invention was not toxic to adipocytes.

<Experimental Example 3> Confirmation of the adipogenesis inhibitory effect of Enterococcus faecalis EF-2001 in adipocytes

In order to investigate the effect of Enterococcus faecalis EF-2001 on adipogenesis of adipocytes, 3T3-L1 preadipocytes were treated with Enterococcus faecalis EF-2001 and differentiation of adipocytes was induced. It was confirmed using the O fat analysis method.

Specifically, Enterococcus faecalis EF-2001 cultured in <Example 1> in the same manner as described in <Experimental Example 2> was cultured in <Experimental Example 2> at concentrations of 0, 25, 100, and 250 μg/ml. 3T3-L1 preadipocytes cultured in 1> were treated and adipocyte differentiation was induced. Then, the stimulation of adipogenesis was maintained for 48 hours by replacing with a fresh 10% FBS serum medium containing 10 μg/ml of insulin, and the stable period was maintained again for 48 hours with the new medium containing serum. After removing the medium and washing the cells with cold PBS (Gibco, Grand Island, NY, USA), 10% formalin (Formalin, Junsei Chemical, Tokyo, Japan) was applied to each well and fixed for 1 hour at room temperature.

Then, after treatment with 60% isopropanol for 5 minutes, Oil Red O stain solution was applied to each well, stained for 10 minutes, washed with distilled water, and only under the conditions of 0 and 250 μg/ml concentration of Enterococcus faecalis EF-2001, 100 times, 200 Images were taken at 4x and 400x magnification (FIG. 3). Thereafter, each well treated with 0, 25, 100, and 250 μg/ml of Enterococcus faecalis EF-2001 was treated with 100% isopropanol, obtained by concentration, transferred to a 96-well plate, and then measured with a 490 nm microplate reader. was measured.

As a result, as shown in FIG. 2, it was confirmed that adipogenesis of adipocytes significantly increased when only insulin was treated after adipocyte differentiation. On the other hand, when Enterococcus faecalis EF-2001 was treated with insulin, it was confirmed that adipogenesis of adipocytes was significantly reduced. In addition, as the concentration of Enterococcus faecalis EF-2001 increased, it was confirmed that adipogenesis of adipocytes decreased in a concentration-dependent manner.

In addition, as shown in FIG. 3 , when only insulin was treated after adipocyte differentiation, it was confirmed through microscopic observation that the size and number of fat droplets significantly increased. On the other hand, when Enterococcus faecalis EF-2001 was treated together with insulin, it was confirmed that the number of fat droplets was significantly reduced. In addition, it was confirmed that as the concentration of Enterococcus faecalis EF-2001 increased, the number of fat droplets decreased in a concentration-dependent manner.

Through the above results, it can be seen that Enterococcus faecalis EF-2001 of the present invention exhibits an effect of inhibiting adipogenesis of adipocytes in a concentration-dependent manner.

Therefore, since it was confirmed through the above results that Enterococcus faecalis EF-2001 according to the present invention exhibits an effect of inhibiting adipogenesis of adipocytes, the Enterococcus faecalis, its culture medium, or its dead cells are By reducing the possibility of obesity occurring through production, it can be very useful for the early prevention, improvement, and treatment of obesity and metabolic syndrome diseases derived from obesity.

Preparation examples for the composition of the present invention are exemplified below.

<Preparation Example 1> Enterococcus faecalis EF-2001 of the present invention Preparation of pharmaceutical preparations containing active ingredients

<1-1> Preparation of powder

Enterococcus faecalis of the present invention EF-2001 10 mg

1 g lactose

A powder was prepared by mixing the above components and filling in airtight bags.

<1-2> Manufacture of tablets

Enterococcus faecalis of the present invention EF-2001 0.1 mg

Corn Starch 100 mg

100 mg milk sugar

Magnesium stearate 2 mg

After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet manufacturing method.

<1-3> Preparation of capsules

Enterococcus faecalis of the present invention EF-2001 0.1 mg

Corn Starch 100 mg

100 mg milk sugar

Magnesium stearate 2 mg

After mixing the above ingredients, capsules were prepared by filling gelatin capsules according to a conventional capsule preparation method.

<1-4> Preparation of ring

Enterococcus faecalis of the present invention EF-2001 1 mg

lactose 1.5 g

1 g of glycerin

0.5 g xylitol

After mixing the above components, it was prepared to be 4 g per ring according to a conventional method.

<1-5> Preparation of granules

Enterococcus faecalis of the present invention EF-2001 0.15 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60° C. to form granules, which were then filled into bags.

<Preparation Example 2> Enterococcus faecalis EF-2001 of the present invention Manufacture of health food containing active ingredients

Enterococcus faecalis of the present invention EF-2001 1 mg

Appropriate amount of vitamin mixture

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

10 μg of biotin

Nicotinamide 1.7 mg

50 mg folic acid

Calcium Pantothenate 0.5 mg

Appropriate amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium Carbonate 25.3 mg

Potassium Phosphate Monobasic 15 mg

Dibasic Calcium Phosphate 55 mg

Potassium citrate 90 mg

Calcium Carbonate 100 mg

Magnesium Chloride 24.8 mg

Although the composition ratio of the above vitamin and mineral mixture was prepared by mixing ingredients suitable for relatively healthy food in a preferred embodiment, the mixing ratio may be arbitrarily modified, and the above ingredients are mixed according to a normal health food manufacturing method, and then , Granules can be prepared and used in the preparation of health food compositions according to conventional methods.

<Preparation Example 3> Enterococcus faecalis EF-2001 of the present invention Manufacture of health drinks containing active ingredients

Enterococcus faecalis of the present invention EF-2001 1 mg

Citric Acid 1000 mg

100 g of oligosaccharides

2 g plum concentrate

1 g of taurine

Total 900 ml with purified water added

After mixing the above components according to the usual health drink manufacturing method, stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered and collected in a sterilized container, sealed and sterilized, and then refrigerated. Health drink composition used for manufacturing.

Although the composition ratio is a mixture of ingredients suitable for a relatively favorite beverage in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the class of demand, the country of demand, and the purpose of use.

<Preparation Example 4> Enterococcus faecalis EF-2001 of the present invention Preparation of external preparations containing active ingredients

<4-1> Preparation of ointment

An ointment containing Enterococcus faecalis EF-2001 of the present invention was prepared by a conventional method according to the composition shown in Table 1 below.

ingredient content(%) Enterococcus faecalis EF-2001 of the present invention 1.0 glycerin 8.0 Butylene Glycol 4.0 liquid paraffin 15.0 Ketaglucan 7.0 carbomer 0.1 Caprylic/Capric Triglyceride 3.0 squalane 1.0 Cetearyl Glucoside 1.5 Sorbitan Stearate 0.4 cetearyl alcohol 1.0 beeswax 4.0 antiseptic a very small amount Spices a very small amount Purified water balance Sum 100

<Preparation Example 5> Enterococcus faecalis EF-2001 of the present invention Preparation of cosmetic composition containing as an active ingredient

<5-1> Lotion

A nutrient lotion containing Enterococcus faecalis EF-2001 of the present invention was prepared in a conventional manner with the component ratios shown in [Table 2] below.

ingredient Content (% by weight) Enterococcus faecalis EF-2001 of the present invention 2.0 Glyceryl Stearate SE 1.5 Cetearyl Alcohol 1.5 lanolin 1.5 Polysorbate 60 1.3 Sorbitastearate 0.5 hydrogenated vegetable oil 4.0 mineral oil 5.0 trioctanoin 2.0 dimethicone 0.8 Tocopherol Acetate 0.5 carboxyvinyl polymer 0.12 glycerin 5.0 1,3-butylene glycol 3.0 sodium hyaluronate 5.0 triethanolamine 0.12 Uniside-U 13 0.02 incense a very small amount Distilled water balance Sum 100

<5-2> Nutritional cream

A nutritional cream containing Enterococcus faecalis EF-2001 of the present invention was prepared in a conventional manner with the ingredient ratios shown in [Table 3].

ingredient Content (% by weight) Enterococcus faecalis EF-2001 of the present invention 2.0 Pro-type glycerin monostearate 1.5 Cetearyl Alcohol 1.5 stearic acid 1.0 Polysorbate 60 1.5 Sorbitastearate 0.6 isostearyl isosterate 5.0 squalane 5.0 mineral oil 35.0 dimethicone 0.5 Hydroxyethyl Cellulose 0.12 glycerin 6.0 triethanolamine 0.7 Uniside-U 13 0.02 incense a very small amount Distilled water balance Sum 100

Claims (9)

Enterococcus faecalis ( Enterococcus faecalis ) EF-2001, a culture medium thereof, and a pharmaceutical composition for preventing or treating obesity or metabolic syndrome induced by obesity, comprising at least one selected from the group consisting of dead cells thereof as an active ingredient.
The method of claim 1,
The dead cell body is cultured as a spawn of Enterococcus faecalis EF-2001, then cultured at pH 4.0 to 9.0 and a temperature range of 15 to 45 ° C, followed by heat treatment to kill cells, characterized in that, obesity or obesity A pharmaceutical composition for the prevention or treatment of metabolic syndrome derived from.
The method of claim 2,
The heat treatment is a pharmaceutical composition for the prevention or treatment of obesity or obesity-induced metabolic syndrome, characterized in that made at a temperature of 60 ~ 140 ℃ for 1 to 40 minutes.
The method of claim 1,
The obesity is characterized by being induced by adipocyte differentiation and proliferation, a pharmaceutical composition for improvement or treatment of obesity or metabolic syndrome induced from obesity.
The method of claim 1,
The metabolic syndrome induced from obesity is a pharmaceutical composition for improvement or treatment of obesity or metabolic syndrome induced from obesity, characterized in that at least one selected from the group consisting of abdominal adipogenesis, impaired glucose tolerance, hypertension and dyslipidemia.
Enterococcus faecalis EF-2001, a culture thereof, and a health functional food composition for improving obesity or metabolic syndrome induced from obesity, comprising at least one selected from the group consisting of its dead cells as an active ingredient.
A food additive for improving obesity or obesity-induced metabolic syndrome, comprising at least one selected from the group consisting of Enterococcus faecalis EF-2001, its culture medium, and its dead cells as an active ingredient.
Enterococcus faecalis EF-2001, a culture medium thereof, and an external preparation for improving obesity or metabolic syndrome induced by obesity, comprising at least one selected from the group consisting of dead cells thereof as an active ingredient.
A cosmetic for improving obesity or metabolic syndrome induced from obesity, comprising at least one selected from the group consisting of Enterococcus faecalis EF-2001, its culture medium, and its dead cells as an active ingredient.