WO2023153902A1 - Composition for inhibiting preadipocyte differentiation and lipid accumulation comprising enterococcus faecalis, culture fluid thereof, or killed cells thereof as active ingredient - Google Patents

Abstract

The present invention relates to a composition for inhibiting preadipocyte differentiation and lipid accumulation, comprising Enterococcus faecalis as an active ingredient. Particularly, it was confirmed that killed E. faecalis EF-2001 induces the cell cycle delay of differentiation-induced preadipocytes, induces downregulation of the phosphorylation of downstream signals, such as Akt, Erk, and JNK, in the insulin receptor signaling pathway, has an effect of reducing the protein expression levels of C/EBP-α and PPAR-γ, and has an effect of reducing the amounts of total cholesterol, triglyceride, and non-high density cholesterol in an experimental animal having obesity induced by a high-fat diet. Thus, the E. faecalis, a culture fluid thereof, or killed cells thereof may be used as an active ingredient in the composition for inhibiting preadipocyte differentiation and lipid accumulation.

Description

A composition for differentiation of preadipocytes and inhibition of lipid accumulation comprising Enterococcus faecalis, its culture medium or its dead cells as an active ingredient

The present invention relates to a composition for preadipocyte differentiation and lipid accumulation inhibition comprising Enterococcus faecalis as an active ingredient.

Body fat is an essential nutrient required by all physiological organs and plays an important role in maintaining energy homeostasis. However, Westernized eating habits and lifestyles due to social and economic development lead to an imbalance in energy intake and consumption, resulting in obesity, which is associated with various metabolic diseases such as diabetes, hypertension, hyperlipidemia, arteriosclerosis, heart disease, and stroke. , known as a direct or indirect cause of cardiovascular disease. The accumulation of fat in the body, which is the cause of obesity, occurs through the production and secretion of hormones (adipokine) secreted by fat cells in the body, and differentiation of fat cells occurs, resulting in fat accumulation through hypertrophy and hyperplasia, and lipogenesis. ) is induced by the accumulation of fat globules produced by the action. In addition, adipocyte hyperplasia is induced due to the proliferation and differentiation process of preadipocytes, which are stimulated by DEX (dexamethasone), IBMX (3-isobutyl-1-methylxanthine), insulin, etc. C/EBPβ and C/EBPγ are activated during early differentiation, and they interact to regulate the expression of PPARγ and C/EBPα. In addition, PPARγ and C/EBPα are expressed in the early stage of differentiation and are involved in adipocyte differentiation, and they induce the expression of various adipogenic genes in the late stage of differentiation. Therefore, inhibiting the mechanism is being studied as an important target in anti-obesity research.

Hyperlipidemia, one of the metabolic diseases, is classified into hypercholesterolemia caused by an increase in blood cholesterol concentration, hypertriglyceridemia caused by an increase in blood triglyceride concentration, and high-density lipoprotein hypoplasia caused by a decrease in HLD cholesterol. Hyperlipidemia is known to be a major risk factor for the occurrence of ischemic heart disease caused by atherosclerosis. Serum lipids are closely related to atherosclerosis, coronary artery disease, and cerebrovascular disease, and total cholesterol or triglyceride have a normal relationship with coronary artery disease, and high-density lipoprotein-cholesterol (hereinafter referred to as 'high density lipoprotein-cholesterol'). HDL cholesterol) is inversely correlated. In Korea, interest in the increase in cholesterol due to changes in dietary life is increasing, and research on this is being conducted. Weight gain by high-fat diet, increase in low-density lipoprotein-cholesterol (hereinafter referred to as “LDL cholesterol”) in the blood, and decrease in HDL cholesterol are important factors that cause damage to vascular function.

Until now, attempts have been made to prevent obesity, diabetes, and hyperlipidemia by using the preventive action of natural product-derived ingredients such as fish oil, vitamin C, and vitamin E. Recently, probiotics and post-bio Research on ticks materials is being conducted. (Korean Patent No. 10-2020-0165175) However, these drugs have not yet obtained sufficient results in improving cholesterol and triglyceride levels in vivo.

In addition, drugs that act on hyperlipidemia include ezetimibe, which lowers blood LDL-cholesterol by inhibiting the reabsorption of cholesterol in the small intestine, and ezetimibe, which lowers LDL-cholesterol by preventing the reabsorption of bile acids in the small intestine. Cholestyramine, which lowers triglyceride and LDL-cholesterol in the blood, but raises HDL-cholesterol, instead of increasing niacin (PPAR alpha), which can cause liver dysfunction and blood sugar control disorder, is effective in lowering triglyceride There are fibrate preparations that cause cholelithiasis, but they generally have side effects, so there is a need for materials that have less side effects and physiological functions.

On the other hand, probiotics were defined by the World Health Organization in 2002 as “living microorganisms that promote health of the host when administered at an effective level.” Lactobacillus represented by Lactobacillus or Bifidobacterium, which includes microorganisms such as Bifidobacterium, Clostridium, Bacillus, and Saccharomyses, accounts for the majority of probiotics and is recognized as a representative health functional food that acts on the intestines in Korea.

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 through screening of the intestinal flora of a 2-year-old girl. Enterococcus faecalis EF-2001 was killed by heat treatment and the cell components were recovered to obtain dead Enterococcus faecalis EF-2001 cells. According to studies reported on the physiological activity of Enterococcus faecalis EF-2001, DSS ( In the case of mice suffering from colitis induced by dextran sulfate sodium), mice fed Enterococcus faecalis EF-2001 showed a DSS alleviation effect, decreased proliferation of transplanted sarcoma cancer cells (Sarcoma-180), and activated NK cells. reported (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 albicans, 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.

Under this background, the present inventors have made efforts to develop a material with low side effects and excellent effects on preadipocyte differentiation and lipid accumulation inhibition, and as a result, Enterococcus faecalis EF-2001 dead cell body differentiation induced fat Inducing cell cycle delay in progenitor cells, inducing down-regulation of phosphorylation of downstream signals such as Akt, Erk and JNK in the insulin receptor signaling pathway, and reducing the protein expression levels of C/EBP-α and PPAR-γ. It was confirmed that there is an effect of reducing the amount of total cholesterol, triglyceride, and non-high-density cholesterol in experimental animals whose obesity was induced by the high-fat diet. Accordingly, the present invention was completed by revealing that the Enterococcus faecalis, its culture medium, or its dead cells can be used as a composition or functional material for preadipocyte differentiation and lipid accumulation inhibition.

An object of the present invention is to provide a composition for differentiating preadipocytes and inhibiting lipid accumulation, comprising Enterococcus faecalis , its culture medium or its dead cells as an active ingredient.

Another object of the present invention is to provide a use of Enterococcus faecalis, its culture medium or dead cells for use as a composition for preadipocyte differentiation and lipid accumulation inhibition.

An object of the present invention is achieved by providing a composition for preadipocyte differentiation and lipid accumulation inhibition comprising at least one selected from the group consisting of Enterococcus faecalis , its culture medium and its dead cells as an active ingredient do.

The object of the present invention is achieved by providing a method for inhibiting differentiation of preadipocytes in vitro by treating preadipocytes with at least one selected from the group consisting of Enterococcus faecalis, culture medium thereof, and dead cells thereof in vitro do.

An object of the present invention is to provide a method for suppressing lipid accumulation in the cells in vitro by treating preadipocytes with at least one selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cells in vitro. is achieved

An object of the present invention is to provide a use of at least one selected from the group consisting of Enterococcus faecalis, a culture medium thereof, and dead cells thereof for use as a composition for preadipocyte differentiation and lipid accumulation inhibition.

In the present invention, enterococcus faecalis EF-2001 ( Enterococcus faecalis EF-2001) killed cells induce cell cycle delay of differentiation-induced preadipocytes, and as shown in FIG. 11, Akt, Erk and JNK in the insulin receptor signaling pathway It induces down-regulation of phosphorylation of downstream signals such as C/EBP-α and PPAR-γ, and has the effect of reducing protein expression levels of C/EBP-α and PPAR-γ. Since it was confirmed that there is an effect of reducing the amount of non-high-density cholesterol, the Enterococcus faecalis, its culture medium, or its dead cells can be used as an active ingredient of a composition for preadipocyte differentiation and lipid accumulation inhibition.

1 is a diagram showing the experimental procedure workflow of an obese laboratory animal induced by a high-fat diet prepared through <Example 2> of the present invention.

Figure 2 is a diagram showing the body weight of the experimental group measured for 6 weeks in <Experimental Example 1> of the present invention.

Figure 3 is a diagram showing the appearance of the experimental animals sacrificed to measure the adipose tissue weight of the experimental group in <Experimental Example 1> of the present invention.

4 is a diagram showing the effect of dead cells of Enterococcus faecalis EF-2001 on the weight of white adipose tissue in the experimental group in <Experimental Example 1> of the present invention. Data are expressed as mean ± SEM (n = 6); *p<0.05, **p<0.01 vs. Control group (no treatment of EF-2001 killed cells in obese experimental animals induced by high fat diet).

Figure 5 shows the total cholesterol (TC), triglyceride (Triglyceride, TG) and non-high-density cholesterol (Non- It is a diagram showing the effect on high density lipoprotein, non-HDL). Data are expressed as mean ± SEM (n = 6); **p<0.01, ***p<0.001 vs. Control group (no treatment of EF-2001 killed cells in obese experimental animals induced by high fat diet).

6 is a diagram showing the effect of dead cells of Enterococcus faecalis EF-2001 on adipogenesis in 3T3-L1 adipocytes in <Experimental Example 3> of the present invention. Data are presented as mean ± SEM (n = 4); **p<0.01, ***p<0.001 vs. Control (no treatment with killed EF-2001 cells in differentiation medium).

7 is a diagram showing the effect of dead cells of Enterococcus faecalis EF-2001 on cell cycle progression in 3T3-L1 adipocytes in <Experimental Example 4> of the present invention. Data are presented as mean ± SEM (n = 4); *p<0.05, **p<0.01, ***p<0.001 vs. Control (no treatment with killed EF-2001 cells in differentiation medium).

8 is a diagram showing the effect of dead cells of Enterococcus faecalis EF-2001 on Akt, Erk, JNK and IR phosphorylation in <Experimental Example 5> of the present invention. Data are expressed as mean ± SEM (n = 3); **p<0.01, ***p<0.001 vs. Control (no treatment with killed EF-2001 cells in differentiation medium).

9 is a diagram showing the effect of dead cells of Enterococcus faecalis EF-2001 on the protein expression levels of C/EBP-α and PPAR-γ in <Experimental Example 6> of the present invention.

10 is a diagram showing the effect of Enterococcus faecalis EF-2001 on the protein expression levels of C/EBP-α and PPAR-γ in experimental animals in <Experimental Example 7> of the present invention. Data are expressed as mean ± SEM (n = 6); *p<0.05 vs. Control group (no treatment of EF-2001 killed cells in obese experimental animals induced by high fat diet).

11 is a diagram schematically illustrating the mechanism of preadipocyte differentiation and lipid accumulation inhibition of dead cells 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.

The present invention provides a composition for differentiation of preadipocytes and inhibition of lipid accumulation, comprising at least one selected from the group consisting of Enterococcus faecalis , a culture medium thereof, and dead cells thereof as an active ingredient.

In addition, the present invention provides a use of at least one selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cells for use as a composition for preadipocyte differentiation and lipid accumulation inhibition.

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 the present invention, the Enterococcus faecalis may be Enterococcus faecalis EF-2001 ( Enterococcus faecalis EF-2001).

In addition, the culture medium refers to a culture medium obtained by culturing Enterococcus faecalis EF-2001 in a culture medium, a concentrated culture medium, a dried culture medium, a dried culture filtrate, a concentrated culture filtrate or a dried culture filtrate. Including, it may be a culture solution from which strains are removed after culturing.

In addition, the dead cells may be prepared by heat-treating corresponding live cells, such as Enterococcus faecalis EF-2001 live cells, or by 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 cells by a conventional method, centrifuging, repeating washing and dehydration as necessary, suspending in distilled water, physiological saline, etc., 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 EF-2001, followed by main culture at pH 4.0 to 9.0 and a temperature of 15 to 45° C., more preferably pH 5.0 to 8.0 at a temperature of 20 to 40° C.;

2) Heat treatment of Enterococcus faecalis EF-2001 viable cells main-cultured in step 1) at a temperature of 60 to 140 ° C for 1 to 40 minutes, more preferably at a temperature of 70 to 130 ° C for 5 to 30 minutes, followed by drying and pulverization step to do.

In a specific embodiment of the present invention, the inventors induced cell cycle delay of preadipocytes in which dead cells of Enterococcus faecalis EF-2001 were differentiated, and as shown in FIG. 11, Akt, Erk and Akt in the insulin receptor signaling pathway. Downregulation of phosphorylation of downstream signals such as JNK is induced, and the protein expression levels of C/EBP-α and PPAR-γ are reduced. , Since it was confirmed that there is an effect of reducing the amount of non-high-density cholesterol, the Enterococcus faecalis, its culture medium, or its dead cells can be used as an active ingredient of a composition for preadipocyte differentiation and lipid accumulation inhibition. In addition, the composition can be very useful for the prevention, improvement, and treatment of obesity or metabolic syndrome induced by obesity.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating 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. provides

In the present invention, the metabolic syndrome induced from obesity may be hyperlipidemia. Specifically, the Enterococcus faecalis inhibits preadipocyte differentiation and lipid accumulation, and reduces total cholesterol (TC), triglyceride (TG) and non-high density lipoprotein, non-high density cholesterol. By reducing HDL), hyperlipidemia can be prevented or treated.

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 may 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 may 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 is a health functional food composition or food for pre-adipocyte differentiation and lipid accumulation inhibition comprising at least one selected from the group consisting of Enterococcus faecalis , its culture medium and its dead cells as an active ingredient Additives are provided.

In addition, the present invention provides a use of Enterococcus faecalis, its culture medium or its dead cells for use as a health functional food composition or food additive for preadipocyte differentiation and inhibition of lipid accumulation.

In the present invention, the method for obtaining the Enterococcus faecalis, its culture medium, and its dead cells is described in the composition for preadipocyte differentiation and lipid accumulation inhibition comprising the Enterococcus faecalis, its culture medium, or its dead cells as an active ingredient. Since the content is the same, the specific description uses the above content.

In the present invention, the metabolic syndrome induced from obesity may be hyperlipidemia. Specifically, the Enterococcus faecalis inhibits preadipocyte differentiation and lipid accumulation, and reduces total cholesterol (TC), triglyceride (TG) and non-high density lipoprotein, non-high density cholesterol. HDL), it is possible to prevent or improve hyperlipidemia.

There is no particular limitation on the type of food to which Enterococcus faecalis, its culture medium or its dead cells are added according to the present invention. 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, its culture medium, or its dead cells may be suitably determined depending on its purpose of use. In general, the amount of the Enterococcus faecalis, its culture medium or its dead cells in the health food may be added in an amount of 0.001 to 50% by weight of the total weight of the food. 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, its culture medium or its dead cells as essential components in the indicated ratio, and various flavors or natural ingredients like conventional beverages. Carbohydrates and the like may be contained as additional components. 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, its culture medium or its dead cell body 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 ratio of these additives is not so critical, but is generally selected from the range of 0 to about 20 parts by weight per part by weight of Enterococcus faecalis, culture thereof or dead cells thereof according to the present invention.

In addition, the present invention provides a method for inhibiting differentiation of preadipocytes in vitro by treating preadipocytes with at least one selected from the group consisting of Enterococcus faecalis, culture medium thereof, and dead cells thereof in vitro to provide.

In addition, the present invention provides a method of inhibiting lipid accumulation in the cells in vitro by treating preadipocytes with at least one selected from the group consisting of Enterococcus faecalis, culture medium thereof, and dead cells thereof in vitro.

In the present invention, the method for obtaining the Enterococcus faecalis, its culture medium, and its dead cells is described in the composition for preadipocyte differentiation and lipid accumulation inhibition comprising the Enterococcus faecalis, its culture medium, or its dead cells as an active ingredient. Since the content is the same, the specific description uses the above content.

On the other hand, the present inventors confirmed that dead cells of Enterococcus faecalis EF-2001 induce cell cycle delay in differentiation-induced pre-adipocytes, phosphorylation of IR and downstream signals such as Akt, Erk, and JNK in the insulin receptor signaling pathway. It was confirmed that phosphorylation was down-regulated and the protein expression levels of C/EBP-α and PPAR-γ, which are differentiation control proteins of preadipocytes, were reduced. Accordingly, the Enterococcus faecalis, its culture medium, or its dead cells can be very usefully used in a method of inhibiting preadipocyte differentiation or lipid accumulation of the cells by treating them in vitro.

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) Manufacturing dead cells

Enterococcus faecalis EF-2001 live bacteria (Korea Berm Co., Ltd., Korea) are aerobically or anaerobically cultured in a medium used for general lactobacillus culture, and after pre-culture, pH 5.0 to 8.0, while maintaining 20 to 40 ° C. The main culture was performed to reach a dry weight (DW) of 7.5 × 10 ¹² cfu / g or more by culturing for 3 days. Then, the cells were killed by heat treatment at 70 to 130 ° C. for 5 to 30 minutes, and then the cells were separated and collected by a continuous centrifugal machine, and then lyophilized and powdered.

<Example 2> Production of obese laboratory animals induced by high-fat diet

24 3-week-old male SD (Sprague-Dawley) mice (Orient Bio Co., Ltd., Korea) were purchased, and after giving an adaptation period for 1 week, randomly divided into 4 groups of 6 mice each according to the diet [Table 1] shared with 5L79 (LabDiet, USA) was provided as a standard diet (Standard diet, SD) to the normal group, and D12492 (Research Diets, USA) was provided to the three high fat diet (HFD) groups for 6 weeks, Distilled water and 3 mg/kg or 30 mg/kg of Enterococcus faecalis prepared in <Example 1> were orally administered to each experimental group once a day for 6 weeks (FIG. 1). All experimental procedures were approved by the Yonsei University Animal Laboratory Management Committee and were performed in accordance with the approved guidelines (YWCI-202102-003-01).

experimental group diet sample administered Normal group
(SD) standard diet Oral administration of distilled water once a day for 6 weeks high fat diet
(HFD) high fat diet Oral administration of distilled water once a day for 6 weeks High-fat diet + EF-2001 3 mg/kg administration group
(HFD-EF-2001 (3 mg/kg)) high fat diet Enterococcus faecalis EF-2001 killed cells 3 mg/kg orally once a day for 6 weeks High-fat diet + EF-2001 30 mg/kg administration group
(HFD-EF-2001 (30 mg/kg)) high fat diet Enterococcus faecalis EF-2001 killed cells 30 mg/kg orally once a day for 6 weeks

<Experimental Example 1> Confirmation of the effect of Enterococcus faecalis EF-2001 on reducing body weight and white adipose tissue (WAT)

For each of the experimental groups of <Example 2>, the body weight was measured every day for 6 weeks before and after oral administration of the sample, and then the experimental animals were sacrificed to investigate the weight of white adipose tissue.

As a result, as shown in FIG. 2, the weight of the experimental group consuming the high-fat diet was higher than that of the normal group consuming the standard diet, and 3 mg/kg or 30 mg/kg of Enterococcus faecalis EF-2001 It was confirmed that significant weight loss was observed in all of the experimental groups to which the dead cells were administered.

In addition, as shown in Figures 3 and 4, the weight of white adipose tissue per body weight of the experimental animals was higher in the experimental group consuming a high-fat diet than in the normal group consuming a standard diet, Enterococcus faecalis EF -2001 It was confirmed that the white adipose tissue induced by the high-fat diet was significantly reduced in the experimental group administered with dead cells.

<Experimental Example 2> Confirmation of total cholesterol (TC), triglyceride (TG), non-high density lipoprotein (non-HDL) reduction effect of dead cells of Enterococcus faecalis EF-2001

On the last day of orally administering the sample to each of the experimental groups of <Example 2> for 6 weeks, cardiac blood was collected under ether anesthesia, and serum samples obtained by fractionating blood collected from experimental animals showed total cholesterol, triglyceride, and high density. For the detection of cholesterol (high density lipoprotein, HDL) and non-high density cholesterol, each assay kit (Asan Pharmaceutical, Korea) was used according to the manufacturer's protocol.

As a result, as shown in FIG. 5, the total cholesterol, triglyceride and non-high-density cholesterol levels were significantly higher in the experimental group administered with dead cells of Enterococcus faecalis EF-2001 together with the high-fat diet than in the experimental group consuming only the high-fat diet. There was a decrease, and it was confirmed that no significant difference was observed in the case of high-density cholesterol level.

<Example 3> Culture and differentiation of 3T3-L1 cells

3T3-L1 cells were grown normally in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Bovine serum (BS) (Gibco, USA) and 1% penicillin/streptomycin. was cultured with

Specifically, the differentiation of 3T3-L1 preadipocytes is carried out using a differentiation inducer (MDI) (Isobutylmethylxanthine, IBMX 500 μM, Dexamethasone 10 μM, and Insulin 10 μg/ml). It was induced for 48 hours in a differentiation medium (DM) composed of DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and each well was 2 and 4 days old. was replaced with DM treated with 10 μg/ml of insulin, and the adipogenesis protocol for the 3T3-L1 cells was performed from day 2 to day 6.

<Experimental Example 3> Confirmation of the adipogenesis inhibitory effect of dead cells of Enterococcus faecalis EF-2001 in 3T3-L1 adipocytes

In order to confirm the effect of dead cells of Enterococcus faecalis EF-2001 on adipogenesis of adipocytes, Oil red O (ORO) staining was used.

Specifically, dead cells of Enterococcus faecalis EF-2001 prepared by the method described in <Example 1> were prepared at concentrations of 0, 25, 50, 100, and 250 μg/ml at 100% cell confluence. The 3T3-L1 preadipocytes cultured in Example 3> were treated and cultured in growth medium (GM) or DM for 6 days to perform an adipocyte differentiation induction protocol. Then, 3T3-L1 cells were washed with phosphate-buffered saline (PBS), fixed with 3.7% formaldehyde (Junsei Chemical, Japan), and then stained with 60% ORO diluted in distilled water. Quantification of lipid accumulation was obtained by concentration after treatment with 100% isopropanol in each well treated with dead cells of Enterococcus faecalis EF-2001, and 490 nm microplate measurement (Molecular Devices, USA) collected by performing

As a result, as shown in FIG. 6, it was confirmed that adipogenesis was significantly reduced in a dose-dependent manner in adipocytes treated with dead cells of Enterococcus faecalis EF-2001, and 100 or 250 μg/ml of Enterococcus faecalis EF-2001 It was confirmed that adipogenesis was reduced by 15% and 30% in DM treated with dead cells of 2001 compared to the control group.

<Experimental Example 4> Confirmation of cell cycle progression delay effect of dead cells of Enterococcus faecalis EF-2001 in 3T3-L1 adipocytes

The effect of dead cells of Enterococcus faecalis EF-2001 on cell cycle progression of adipocytes was confirmed.

Specifically, 3T3-L1 cultured in <Example 3> in the presence or absence of 25, 50, 100 or 250 μg/ml of dead cells of Enterococcus faecalis EF-2001 prepared by the method described in <Example 1> Differentiation was induced by treating preadipocytes with dexamethasone and insulin cocktail for 48 hours. Then, the cells were fixed with 70% ethanol at 4°C for more than 2 hours, and a PI solution containing 100 μg/ml of Propidium iodine (PI), 20 μg/ml of RNase A, and 0.1% NP40. After staining with 1 ml, it was washed twice with PBS. The G0/G1, S and G2/M phases of the cell cycle were measured using a Fluorescence-activated cell sorting (FACS) system (BD Bioscience, USA).

As a result, as shown in FIG. 7, as a result of analyzing mitotic clonal expansion (MCE) by flow cytometry, the adipocytes treated with dead cells of Enterococcus faecalis EF-2001 showed S While the ratio of the G2/M phase increased, the ratio of the G2/M phase decreased in a dose-dependent manner, confirming that cell cycle progression was stopped at the G0/G1 phase.

<Experimental Example 5> Confirmation of Insulin Signal Transduction Pathway Inhibitory Effect of Enterococcus faecalis EF-2001 dead cells

Western blotting was performed to examine the effect of dead cells of Enterococcus faecalis EF-2001 on the insulin signal transduction pathway.

Specifically, 3T3-L1 cells were treated with dead cells of Enterococcus faecalis EF-2001 prepared by the method described in <Example 1>, and lysis buffer (iNtRON Biotechnology) was added at an appropriate stage from day 2 to day 6. , Korea) was added, followed by sonication, and a Bradford assay (Bio-Rad, USA) was used to quantify the protein. The ratio of SDS-polyacrylamide gel is determined according to the kDa of the protein to be identified, and electrophoresis is performed at 100V for about 2 hours, followed by primary antibodies (C/EBP-α, PPAR-γ, p-Erk, Erk, p-JNK, JNK, p-Akt, Akt, p-IR, IR and β-actin) were incubated at a ratio of 1:2500 overnight at 4°C. Then, the cells were washed three times for 10 minutes with Tris-buffered saline (TBS) containing tween 20, and a secondary antibody was added at a ratio of 1:5000 for 2 hours at room temperature. The transcribed protein bands of the PVDF membrane were measured using the LAS 4000 system (GE Healthcare, UK) by inducing an enhanced chemiluminescence response.

As a result, as shown in FIG. 8, it was found that MDI-induced phosphorylation of Erk, JNK, and Akt was significantly inhibited by dead cells of Enterococcus faecalis EF-2001 in a dose-dependent manner, and IR-β at tyrosine residues was stimulated by insulin. Although auto-phosphorylation of the subunit was induced, it was confirmed that the treatment of dead cells of Enterococcus faecalis EF-2001 had the effect of suppressing the phosphorylation level of IR.

<Experimental Example 6> Confirmation of inhibitory effect on C/EBP-α and PPAR-γ protein expression of dead cells of Enterococcus faecalis EF-2001

Western blotting and visualization using confocal microscopy were performed during adipogenesis to investigate the effect of Enterococcus faecalis EF-2001 dead cell treatment on the expression of transcription factors such as C/EBP-α and PPAR-γ.

Specifically, Western blotting was performed by the method described in <Experimental Example 5>. In addition, for visualization using a confocal microscope, 3T3-L1 preadipocytes were cultured and differentiated in a 3 cm cover glass plate (Mattek Corp, USA), and then each dose of Enterococcus faecalis EF-2001 was treated with dead cells. . Then, to facilitate observation of the nuclei, the fluorescent dye DAPI diluted in PBS was fixed with paraformaldehyde at room temperature, incubated for 10 minutes, and then the cells were washed to visualize C/EBP-α and PPAR-γ antibodies. It was incubated with the fluorescent dye GFP diluted in PBS for 30 minutes at room temperature with paraformaldehyde fixation. GFP expression was visualized with an LSM710 confocal microscope (Carl Zeiss, Germany).

As a result, as shown in FIG. 9, the protein expression of C/EBP-α and PPAR-γ was significantly suppressed by dead cells of Enterococcus faecalis EF-2001, and C/EBP-α in the nucleus was observed even under a confocal microscope. And it was confirmed that the protein expression of PPAR-γ was significantly reduced.

<Experimental Example 7> Confirmation of inhibitory effects on C/EBP-α and PPAR-γ protein expression of Enterococcus faecalis EF-2001 killed cells in experimental animals

In order to confirm the effect of Enterococcus faecalis EF-2001 dead cell treatment on the expression levels of C/EBP-α and PPAR-γ proteins in adipose tissue of the normal group consuming a standard diet and the experimental group consuming a high-fat diet, Western blotting was performed on protein samples extracted from white adipose tissue.

Specifically, Western blotting was performed by the method described in <Experimental Example 5>.

As a result, as shown in FIG. 10, oral administration of 30 μg/ml of dead cells of Enterococcus faecalis EF-2001 in the experimental group consuming a high-fat diet significantly increased the protein expression level of Enterococcus faecalis EF-2001. -2001 It was confirmed that the decrease was lower than that of the experimental group not administered, and the protein expression of PPAR-γ in the experimental group consuming a high-fat diet orally administered 3 or 30 μg/ml of dead Enterococcus faecalis EF-2001 It was also confirmed that the level of Enterococcus faecalis EF-2001 decreased to a lower level than that of the experimental group not administered with dead cells.

<Example 4> Statistical analysis

All values are expressed as mean ± SEM. Data were analyzed by One-way ANOVA (Student's t-test) using PRISM version 5.0. Differences between groups were assessed using Dunnett's test. Statistical significance was indicated by p values *p<0.05, **p<0.01, ***p<0.001.

Through the above results, the enterococcus faecalis EF-2001 killed cells according to the present invention induced cell cycle delay of differentiation-induced preadipocytes, and as shown in FIG. 11, in the insulin receptor signal pathway, such as Akt, Erk and JNK It induces down-regulation of downstream signal phosphorylation, has the effect of reducing the protein expression levels of C/EBP-α and PPAR-γ, and has the effect of reducing total cholesterol, triglyceride, and high-density Since it was confirmed that there is an effect of reducing the amount of cholesterol, the Enterococcus faecalis, its culture medium or its dead cell body can be used as an active ingredient of a composition for preadipocyte differentiation and lipid accumulation inhibition, and the composition is obese or obese. It can be used very usefully for the prevention, improvement and treatment of metabolic syndrome derived from.

The present invention relates to a composition for differentiation of enterococcus faecalis and inhibition of lipid accumulation containing enterococcus faecalis as an active ingredient. It induces cell cycle delay, induces down-regulation of phosphorylation of downstream signals such as Akt, Erk and JNK in the insulin receptor signaling pathway, and has the effect of reducing the protein expression levels of C/EBP-α and PPAR-γ. Since it is excellent in reducing the amount of total cholesterol, triglyceride, and non-high-density cholesterol in laboratory animals whose obesity was induced by this method, it can be usefully used as an active ingredient in a composition for preadipocyte differentiation and lipid accumulation inhibition.

Claims (12)

Enterococcus faecalis ( Enterococcus faecalis ), a culture medium thereof, and a composition for inhibiting lipid accumulation and differentiation of preadipocytes, comprising at least one selected from the group consisting of dead cells thereof as an active ingredient. The composition for preadipocyte differentiation and lipid accumulation inhibition according to claim 1, wherein the Enterococcus faecalis is Enterococcus faecalis EF-2001. The method of claim 1, wherein the killed cells are cultured at pH 4.0 to 9.0 and a temperature range of 15 to 45 ° C. after culturing Enterococcus faecalis as a seed, and then killed by heat treatment. Characterized in that, A composition for preadipocyte differentiation and inhibition of lipid accumulation. The composition for differentiation of preadipocytes and inhibition of lipid accumulation according to claim 3, wherein the heat treatment is performed at a temperature range of 60 to 140° C. for 1 to 40 minutes. The method of claim 1, wherein the Enterococcus faecalis is characterized by reducing total cholesterol (TC), triglyceride (TG) and non-high density lipoprotein (non-HDL) To, a composition for preadipocyte differentiation and inhibition of lipid accumulation. The composition for preadipocyte differentiation and lipid accumulation inhibition according to claim 1, wherein the Enterococcus faecalis induces cell cycle delay. The composition for inhibiting preadipocyte differentiation and lipid accumulation according to claim 1, wherein the Enterococcus faecalis inhibits phosphorylation of Erk, JNK, Akt and IR. The composition for inhibiting preadipocyte differentiation and lipid accumulation according to claim 1, wherein the Enterococcus faecalis inhibits the expression of a differentiation regulatory protein of preadipocytes. The composition for preadipocyte differentiation and lipid accumulation inhibition according to claim 8, wherein the differentiation regulatory protein of preadipocytes is C/EBP-α or PPAR-γ. A method of suppressing differentiation of preadipocytes in vitro by treating preadipocytes with at least one selected from the group consisting of Enterococcus faecalis, culture medium thereof, and dead cells thereof in vitro. A method of suppressing lipid accumulation of the cells in vitro by treating pre-adipocytes with at least one selected from the group consisting of Enterococcus faecalis, a culture thereof, and dead cells thereof in vitro. Any one or more uses selected from the group consisting of Enterococcus faecalis, its culture medium, and its dead cell body for use as a composition for differentiation of preadipocytes and inhibition of lipid accumulation.

Images