KR102135272B1 - Composition for Preventing or Treating Obesity Comprising Exopolysaccharides from Kefir Grains - Google Patents

Abstract

The present invention relates to a functional food and pharmaceutical composition for the improvement, prevention or treatment of obesity containing kefir grain-derived extracellular polysaccharide as an active ingredient, and the composition of the present invention has a shear fluidization property and gel blood performance. It has advantageous properties for application in pharmaceutical compositions. In addition, the composition of the present invention inhibits the differentiation of adipocytes in the cell, exerts an effect of reducing weight gain and reducing fat tissue weight and lowering cholesterol in the blood, especially VLDL, when ingested with a high fat diet, and changes intestinal bacterial flora It has the effect of suppressing obesity.

Description

Composition for Preventing or Treating Obesity Comprising Exopolysaccharides from Kefir Grains

The present invention relates to a functional food and pharmaceutical composition for the improvement, prevention or treatment of obesity containing kefir grain-derived extracellular polysaccharide as an active ingredient.

Obesity, a disorder of lipid metabolism, is often accompanied by metabolic disorders, and its population is increasing worldwide, and the obesity population has recently exceeded the population of low nutritional and infectious diseases. The causes of obesity can range from endocrine, genetic and environmental factors. Recently, some researchers have claimed that the imbalance of the intestinal flora is the cause of obesity. Probiotics are the best known to improve the intestinal flora imbalance.

Probiotics (probiotics) refers to a living microorganism that is beneficial to the health of the intestinal microflora, that is, a living microorganism that provides benefits to the health of the host. In general, probiotics are consumed as part of fermented foods such as yogurt or as dietary supplements. Microorganisms known as probiotics include lactic acid bacteria (LAB), bifidobacteria, and Bacillus. Some of the probiotics are known to have therapeutic effects on obesity, insulin resistance, and non-alcoholic fatty liver.

Kefir is a type of fermented milk derived from the mountainous region of the Caucasus, a popular drink used by Tibetan monks for health. Kefir's constituent nutrients include proteins and polysaccharides, and vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin D, vitamin K2, folic acid, nicotinic acid and calcium, iron, and iodine. This kefir is a yogurt-like beverage obtained by fermenting milk using a bacterial species called kerophile, a complex of lactic acid bacteria and yeast (also called kefir granules), as an initiator.

Kefir grain is a starting material for producing kefir products, and is a gel-like material containing lactic acid bacteria ( Lactobacillus, Leuconostic , Lactococcus , Streptococcus, etc.), acetic acid bacteria ( Acetobacter, etc.) and yeast ( Saccharomyces , Kluyveromyces , Torula, etc.). . By cultivation, Kefir microbes form exo-polysaccharides (EPS) in mucus.

EPS is a component that gives viscosity to dairy products, and has been studied in the dairy industry, mainly focusing on viscosity, stability, texture, gel blood performance, and emulsifying ability. EPS is a polymer polysaccharide produced and released by microorganisms, including lactic acid bacteria, during the growth process.

EPS consists of two types, one of which is a homo-polysaccharide composed of only one type of monosaccharide (e.g., dextran, levan, inulin, etc.), and the other. One is hetero-polysaccharide composed of various kinds of monosaccharides or disaccharides (eg, gellan, xanthan, kefiran, etc.).

Since the industrial use of EPS is determined by the monosaccharide composition, bonding structure, and molecular weight, existing studies have mainly focused on the composition, structure, extraction method, and physical properties of EPS. Recently, studies on the health promoting effects of EPS (antibacterial, anticancer, antioxidant, immune modulating effects, etc.) have been conducted. For example, Republic of Korea Patent No. 10-1751682 describes the cancer cell proliferation inhibitory activity of EPS produced by the Traustokytrid-based mutant strain, and Republic of Korea Patent No. 10-1655882 discloses Seriforia lock It describes a composition for resolving hangover containing an extracellular polysaccharide produced by serata as an active ingredient. However, most studies are still limited to in vitro in vitro experiments.

The present inventors completed the present invention by confirming the results of the study on the health promoting effect of kefir-derived EPS, that EPS separated from kefir grain is effective in preventing or treating obesity in in-vitro and animal experiments. .

An object of the present invention is to provide a functional food for improving obesity containing kefir grain-derived extracellular polysaccharide as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of obesity containing an extracellular polysaccharide derived from kefir grains as an active ingredient.

In order to achieve the above object, the present invention provides a functional food for obesity improvement comprising an extracellular polysaccharide derived from Kefir grain.

In the present invention, the kefir grain-derived extracellular polysaccharide may include 70 to 90% by weight of carbohydrate and 0.01% by weight or less of protein.

In the present invention, the carbohydrate may include glucose and galactose.

In the present invention, the kefir grain-derived extracellular polysaccharide may have shear fluidization characteristics.

In the present invention, the extracellular polysaccharide can inhibit the differentiation of adipocytes in the cell.

In the present invention, the extracellular polysaccharide may lower blood cholesterol levels.

Functional food of the present invention can increase the bacteria in the intestinal Akkermansia (Akkermansia).

The present invention also provides a pharmaceutical composition for preventing or treating obesity comprising exopolysaccharide derived from Kefir grain.

The composition containing the kefir-derived extracellular polysaccharide according to the present invention as an active ingredient has a shear fluidization property and gel blood performance, and thus has advantageous properties to be applied to food or pharmaceutical compositions.

In addition, the composition of the present invention inhibits the differentiation of adipocytes in the cell, exerts an effect of reducing weight gain and reducing fat tissue weight and lowering cholesterol in the blood, especially VLDL, when ingested with a high fat diet, and changes intestinal bacterial flora It has the effect of suppressing obesity.

1 shows shear stress (a) and apparent viscosity (b) depending on the shear rate of EPS at various concentrations.
Figure 2 shows the apparent viscosity according to the shear rate of 2% EPS and 2% beta-glucan (BG) solution.
Figure 3 (a) shows the change in storage and loss index according to the frequency of the EPS solution,
Figure 3 (b) shows the change of the storage and loss index according to the frequency of the EPS cryogel,
Figure 3 (c) shows the change of the storage index according to the frequency of the EPS solution and EPS cryogel.
Figure 4 shows the cell viability of adipocytes (3T3-L1) according to the concentration of EPS.
Figure 5 (a) is a photograph of the appearance of adipocytes (3T3-L1) according to the concentration of EPS.
Figure 5 (b) shows the relative amount of fat in the cells according to the concentration of EPS.
Figure 6 (a) shows the daily energy intake of the mice according to the type of diet.
Figure 6 (b) shows the increase in the number of insects in the experimental mice according to the type of diet.
7 shows changes in liver tissue weight (a) and adipose tissue weight (b) for each experimental rat group according to dietary types.
Figure 8 shows the blood cholesterol level of each experimental rat group according to the type of diet.
9 shows changes in the gut flora according to the type of diet.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

One embodiment of the present invention relates to a functional food for improving obesity, which contains an extracellular polysaccharide (EPS) derived from kefir grains as an active ingredient.

In the present invention, kefir (kefir) is a type of fermented milk derived from the Caucasus mountainous fat, kefir refers to a yogurt-like beverage obtained by fermenting milk using a lactic acid bacteria and a yeast cell called a kefir grain, which is a yeast complex, as an initiator. .

In the present invention, as the starting material for the production of the cabinet Kane peer grain peer, lactic acid bacteria (Lactobacillus, Leuconostic, Lactococcus, Streptococcus, etc.), gel containing chosangyun (Acetobacter, etc.), and yeast (Saccharomyces, Kluyveromyces, Torula, and so on) It is a form of substance.

In the present invention, extracellular polysaccharide (EPS) is a component that gives viscosity to dairy products, and refers to a polymer polysaccharide produced and discharged by microorganisms including lactic acid bacteria during the growth process.

The kefir-derived extracellular polysaccharide may include 70 to 90% by weight of carbohydrate and 0.01% by weight or less of protein, preferably 79 to 89% by weight of carbohydrate and 0.002% by weight of protein or less. . The carbohydrate is mainly composed of glucose and galactose.

The extracellular polysaccharide dissolving the dried kefir grain in 2 to 20 times boiling water; Centrifuging the kefir grains dissolved in water and adding ethanol to the supernatant to precipitate; And the mixture may be centrifuged again, and may be prepared by dissolving the coagulant in boiling water of about 2 to 20 times, preferably, the method may be separated by repeating 2 to 5 times.

Kefir grain-derived extracellular polysaccharide according to the present invention exhibits a low apparent viscosity as the shear rate increases, and shear stress and apparent viscosity as the concentration of the extracellular polysaccharide increases. Shows an increased shear thinning property. This shear fluidization property facilitates the application of the extracellular polysaccharide of the present invention to foods such as liquid foods.

In addition, since the extracellular polysaccharide derived from kefir grains has gel-like properties, it can be used as a purpose for giving gelling properties to food. In one embodiment of the present invention, it was confirmed that the storage module (G′) of the kefir grain-derived extracellular polysaccharide was higher than the loss module (G″) in all measurement ranges, and it was confirmed that it exhibits gel-like properties. And when melting to form a gel, it was confirmed that G'and G" increase and show a stronger gel-like property as they tend to be less dependent on frequency.

Since the extracellular polysaccharide derived from kefir grains according to the present invention does not have toxicity, it can be advantageously used in the human body in food or pharmaceutical applications. In one embodiment of the present invention, it was confirmed that the extracellular polysaccharide shows a high level of cell viability regardless of concentration.

In the present invention, it was found that the extracellular polysaccharide isolated from kefir grains can effectively prevent or treat obesity as a result of cell experiments and animal experiments.

Specifically, the kefir grain-derived extracellular polysaccharide of the present invention can prevent or treat obesity by effectively suppressing the differentiation of adipocytes in cells.

In addition, as a result of practical application in animal experiments, the rat group that consumed kefir grain-derived extracellular polysaccharides with a high-fat diet had a reduced weight gain and reduced liver and fat weights compared to the rat group that had only a high-fat diet. It was confirmed that it decreases the cholesterol level in the blood. In one embodiment of the present invention, keratin grain-derived extracellular polysaccharide-derived rats gained less weight, reduced liver and fat weight, and VLDL-C than beta-glucan, which is known to have anti-obesity activity. (very low density lipoprotein cholesterol). It was confirmed that the extracellular polysaccharide of the present invention reduces not only VLDL-C but also LDL-C and TOTAL-C to some extent.

The decrease in VLDL-C concentration is associated with a decrease in triacylglycerol (TG) accumulated in adipocytes. TG is a major component of VLDL produced in the liver and a decrease in fat cells causes the liver to reduce free fatty acids that produce TG and VLDL. In obese people, plasma VLDL-C concentrations are about 40 times higher than in the general population.

In addition, the extracellular polysaccharide derived from kefir grains of the present invention can effectively suppress obesity by generating desirable changes in the intestinal bacterial flora. Specifically, when the extracellular polysaccharide is ingested, bacteria in the intestine increase bacteria of Bacteroidetes , Verrucomicrobia , Proteobacteria phyla, and permicutes ( Firmicutes ) and Actinobacteria ( Bacterial ) bacteria are reduced.

In particular, in the experimental group consuming kefir grain-derived extracellular polysaccharides, the bacteria in Akkermansia are more than doubled compared to beta-glucan. The Akkermansia bacteria are known to be involved in obesity and inflammation as beneficial bacteria in the intestine. For example, Akkermansia muciniphila ) has been found to reduce diet-induced obesity.

The kefir grain-derived extracellular polysaccharide of the present invention has a much lower level of viscosity than beta-glucan, but rather exhibits better anti-obesity activity. This is contrary to that in many conventional studies, a more viscous polysaccharide has a better anti-obesity effect by suppressing appetite, thereby inhibiting energy absorption or absorption of glucose and lipids. This means that the anti-obesity activity of extracellular polysaccharide derived from kefir grain was not exerted from its viscosity.

In the change of the intestinal bacterial flora, the mice ingesting beta-glucan increased the bacteria in the allobaculum in the intestine by about 4 times, but in the case of the EPS ingestion experiment group, the bacteria did not increase at all. Allobacurum bacteria break down tryptophan into an aryl hydrocarbon receptor (AHR) ligand to prevent inflammation in the intestine. That is, it can be seen that the extracellular polysaccharide of the present invention inhibits obesity by a different mechanism from beta-glucan.

The composition containing the kefir grain-derived extracellular polysaccharide according to the present invention as an active ingredient may be applied as a functional food for improving obesity. The composition is useful in food applications because it has shear fluidity and gel-like properties.

The functional food is preferably a fermented food, but is not limited thereto. For example, the functional foods may be beverages, gum, tea, vitamin complexes, and dietary supplements, and more specifically, dairy products, confectionery, seasonings, beverages and drinks, snacks, candy, jelly, ice cream, and frozen foods Desserts, breakfast cereals, nutrition bars, snack bars Chocolate products, processed foods, grain products and pasta, soups, sauces and dressings, confectionery products, oil and fat products, dairy drinks and milk drinks, tea, soy milk and soybeans It may be any one selected from the group consisting of soy dairy-like products, frozen foods, cooked and alternative foods, meat products, cheese, yogurt, bread and buns, cakes, cookies and crackers.

In addition, the functional food may be prepared in the form of capsules, tablets, powders, liquid suspensions, pills, granules, etc., containing kefir grain-derived extracellular polysaccharides.

In addition, the functional food may additionally include ingredients that are commonly added when preparing food as an active ingredient in addition to kefir grain-derived extracellular polysaccharide.

Functional foods of the present invention include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof , Organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like, and may contain fruit flesh for fruit juice or vegetable beverage production.

The kefir grain derived extracellular polysaccharide of the present invention can also be applied to a pharmaceutical composition for preventing or treating obesity.

The pharmaceutical composition contains kefir grain-derived extracellular polysaccharide as an active ingredient, and may further include a suitable pharmaceutically acceptable carrier, excipient, or diluent according to a conventional method. The pharmaceutically acceptable carrier is commonly used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like, but is not limited thereto.

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Regarding suitable pharmaceutically acceptable carriers and formulations, it can be preferably formulated for each ingredient using the methods disclosed in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and parenteral administration includes intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.

Formulations for oral administration include, for example, tablets, pills, hard, soft capsules, liquids, suspensions, emulsifiers, syrups, granules, etc.These formulations include diluents (e.g. lactose, dextrose, water) Cros, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg, silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycols). In addition, the tablets may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid Or a disintegrant or boiling mixture such as its sodium salt and/or absorbent, colorant, flavoring and sweetening agent. The formulation can be prepared by conventional mixing, granulating or coating methods.

In addition, typical formulations for parenteral administration are injectable formulations, and water, Ringer's solution, isotonic physiological saline, or suspension are examples of solvents for injectable formulations. The sterile fixed oil of the injectable formulation can be used as a solvent or suspension medium and any non-irritated fixed oil can be used for this purpose, including mono- and di-glycerides. In addition, fatty acids such as oleic acid may be used for the injection preparation.

The composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and an effective dose level is a patient's disease type, severity, and drug activity , The sensitivity to the drug, the time of administration, the route of administration and rate of excretion, the duration of treatment, factors including the drugs used simultaneously, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. In consideration of all the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by a skilled person.

Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, sex, and body weight, and in general, 0.001 to 150 mg per 1 kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day, or 1 to 1 day It can be divided into three doses. However, since the dosage may be increased or decreased depending on the route of administration, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

Example

Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example One: Kefir Grain culture

Kefir grains were purchased from the Food Safety Research Center of the College of Veterinary Medicine, Konkuk University, added to ultra-high temperature sterilized milk (Seoul Milk) equivalent to 10 times the weight, and then cultured in a sealed plastic container at 30°C.

After grinding the kefir grain with fresh sterile milk every day for 6 weeks until 50 g of grain becomes 1000 g, the cultured kefir grain is filtered through a plastic sieve (pore size: 1 mm) and freeze-dried and then -20°C. Were stored at.

Example 2: Kefir From grain Extracellular polysaccharide (EPS) separation

EPS was extracted by slightly modifying known techniques. After measuring the weight of the dried kefir grain, dissolved in 10 times the volume of boiling water and boiled for 1 hour at 500 rpm, the suspension was centrifuged at 20°C for 20 minutes at 10,000×g. The separated supernatant was poured with 2 times the volume of edible ethanol (Daejung Chemical) to precipitate the EPS and maintained at -20 °C overnight. After centrifuging the mixture at 10,000×g for 20 minutes, the coagulum was dissolved by stirring in 10 times the volume of boiling water for 1 hour. This immersion process was repeated three times. The centrifuged coagulum was finally dissolved in 3 times the volume of hot water and lyophilized.

The purity of the EPS is confirmed by the presence of monosaccharides and protein components, reducing sugar test (Miller, GL, chemistry, 31/31(3): p. 426-428.) and protein test (Bradford, MM, Analytical biochemistry, 1976. 72(1-2): p. 248-254.). In addition, the total carbohydrate component content was measured by the phenol sulfate test (Enikeev, R., Food Chem, 2012. 134(4): p. 2437-41).

As a result of the experiment, EPS was separated with high purity, and the yield was 569.1±25.3 mg at 100 g of dried kefir grain weight.

The isolated EPS contained 0.0016% protein and no reducing sugar. The total carbohydrate concentration was 84.1 ± 4.6%, and the main carbohydrates were glucose and galactose.

Example 3: Analysis of shear fluidization properties of EPS

3-1: Shear fluidization characteristics according to EPS concentration

To analyze the shear fluidization characteristics of EPS, various concentrations of EPS solutions were prepared. The EPS solution was dissolved in water at 70°C to a desired concentration, and then stored at 25°C until measurement.

RheoStress (thermo Haake) equipped with parallel plates of 1 mm gap and 35 mm diameter for apparent viscosity (η _a ) and flow behavior for EPS at concentrations of 3, 5, 11, 13 and 15%. It was measured by. Shear rate was measured at 25℃ by increasing from 0 to 500S ^-1 , and shear stress was calculated using RheoWin data maganer (RheoWin pro v.2.96, Hakke) by applying experimental data to the power law model. The index (flow index; n) and the consistency index (K) were calculated by applying the equation below.

ⁿ σ = K

ⁿ

는 전단속도(s^-1), 및 n은 유동행동지수(무차원)을 의미한다. σ is the shear stress (Pa), K is the flow viscosity index (Pa·s ⁿ ),

Is the shear velocity (s ^-1 ), and n is the flow behavior index (dimensionless).

The calculated shear stress and apparent viscosity are shown in FIG. 1.

In FIG. 1, the EPS solution showed a low apparent viscosity as the shear rate increased, and both the shear stress and the apparent viscosity increased together as the concentration of the EPS solution increased. Thus, EPS exhibited typical pseudo-plastic or shear thinning properties.

Meanwhile, the physical properties of the EPS solution applied to the power law model are shown in Table 1 below.

In Table 1, the flow behavior index (n) of the EPS was lower than 1, and the apparent viscosity (η, 300S ^-1 ) increased depending on the concentration of the EPS solution.

3-2: Comparison of properties of EPS solution and beta-glucan solution

The viscosity properties of the EPS solution were compared to β-glucan (BG).

2% EPS solution and 2% beta-glucan (Cargil) were added to boiling water, and completely dissolved and cooled for 10 minutes. The flow characteristics of the EPS and BG solutions were measured by increasing the shear rate from 1 to 100s ^-1 at 25°C using a rotary rheometer (AR-2000, TA instruments).

2, as a result of comparing the viscosity of the EPS and BG solutions, it was confirmed that BG (46.74 mPa·s) has a significantly higher viscosity than EPS (7.69 mPa·s).

Example 4: EPS gel Shaping ability Measure

To analyze the gel-forming ability of EPS, an EPS cryogel (gel made by freezing and melting) was prepared using a 2% EPS solution.

The lyophilized EPS was dissolved at 70°C at 2%, cooled at room temperature, poured into a 35 mm diameter cylindrical plastic container, sealed with parafilm, and the EPS solution was left at 4°C for 96 hours, and the EPS cryogel was It was prepared by maintaining at -20°C for the first 48 hours and leaving at 4°C for the next 48 hours.

Small-amplitude oscillatory shear flow characteristics were measured at 1 to 100 Hz and 25°C using a rotary rheometer equipped with a circular cylinder for EPS solutions and a parallel plate for EPS cryogel. The measurement results are shown in FIG. 3.

3(a) shows the change of the storage and loss index according to the frequency of the EPS solution, FIG. 3(b) shows the change of the storage and loss index according to the frequency of the EPS cryogel, and FIG. 3(c) shows the EPS solution And EPS cryogel frequency.

In FIG. 3(a), it was confirmed that the EPS solution showed that the storage module G'was higher than the loss module G'' within the irradiated frequency range, indicating gel-like properties, and was strongly resistant to an increase in frequency. It was confirmed to be dependent.

After freezing and thawing gel formation, EPS cryogel showed stronger gel-like properties as G'and G'' increased and showed less dependence on frequency.

In FIG. 3(c), at the 10 Hz frequency, the G'value of the EPG cryogel is 23.5±0.4 Pa, which is 5 times higher than that of the EPS solution of 5.2±0.5 Pa.

The tanδ (G''/G') values of the EPS solution and cryogel were both less than 1, indicating that the elastic properties were superior to the viscous properties in both samples.

Example 5: EPS Evaluation of cytotoxicity and adipocyte differentiation inhibitory properties

5-1. Cell culture and differentiation

3T3-L1 adipocytes were purchased from ATCC and used. Dulbecco's modified Eagle's medium containing high glucose (Gibco), 10% bovine serum and 1% penicillin streptomycin (P/S, Gibco) using 6-well plates (Corning Inc.). (Dulbecco's modified Eagle's medium; DMEM) was incubated until confluence.

After 2 days of confluence, 3T3-L1 cells were placed in 10% FBS/DMEM in 1% P/S, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone, and 10 μg/mL insulin solution (Sigma-Aldrich ) And cultured with MDI medium.

At this time, the day of differentiation was set to 0 days, and after 2 days of differentiation, the cells were cultured for 2 days by replacing with DMEM medium containing 10% FBS, 1% P/S and 10 μg/mL insulin solution, and on the 4th day, 10% FBS and 1% Differentiation was completed by culturing in DMEM medium containing P/S for 24 hours.

Cells were cultured in a CO ₂ incubator (Thermo Fisher Scientific Korea) at 37° C., 5% CO ₂ .

5-2. Cell viability analysis

Cells were incubated for 24 hours at a concentration of 1 x 10 ⁴ Cells/well in DMEM medium treated with 10% FBS and 1% P/S in a 96-well plate, and then treated with 20 μL EPS solution or PBS (Lonza) solution as a control. .

After 24 hours, 20 μL of 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich) in PBS was added, and maintained for 4 hours in dark conditions.

Then, the well plate was centrifuged for 5 hours at 1,500 rpm, and the supernatant was removed. Formazan crystals remaining in the well were dissolved in 150 μL of dimethyl sulfoxide (Daejung Chemical) for 15 minutes. Absorbance at 540 nm was measured with a Synergy HT Multi-microplate Reader (BioTek Instruments) to measure formazan content. The results are calculated by cell viability and shown in FIG. 4.

As a result of evaluating the toxicity of EPS to adipocytes, it was confirmed that EPS does not have toxicity by showing a survival rate of 80% or more at all EPS concentrations.

5-3. oil Red O dyed and Intracellular Lipid quantification

Oil red O staining was performed for staining of intracellular lipids.

Cells were cultured in a 6-well plate at a density of 4 x 10 ^-4 cells/well and then differentiated for 6 days in the manner described above.

EPS solutions of 0.01, 0.1 and 1 mg/mL were added to each well except the control group during differentiation. After the differentiation was completed, each well was washed twice with PBS solution and treated with 4% paraformaldehyde (Yakuri Pure Chemicals Co.) in PBS for 1 hour to fix cells on the well surface. After fixation, the cells were rinsed with demineralized water and treated with 50% isopropanol (Daejung Chemical) for 5 minutes. After removing isopropanol, the filtered oil red O solution was added for 10 minutes at room temperature. After washing the wells with water, the pictures were recorded (FIG. 5(a)), and 100% isopropanol was added to each well. The extracted oil red O was centrifuged at 10,000 x g for 2 minutes, and the supernatant was transferred to a 96-well plate.

The absorbance of the oil red O dye was measured at 480 nm using a microplate reader. Relative amount of fat in cells (OD _sample / OD _control ) x 100 is calculated and shown in Figure 5 (b).

In FIG. 5, the relative intracellular fat amount of 80.91%, 59.52% and 42.05% in the EPS solutions of 0.01, 0.1 and 1 mg/mL, respectively, was shown to be concentration-dependent and significantly inhibit fat differentiation.

That is, it was confirmed that EPS derived from kefir grains can prevent or treat obesity by inhibiting the differentiation of fat in cells.

Example 6: Analysis of rat body weight, fat and liver tissue weight, and blood cholesterol change according to EPS intake

6-1: Experimental procedure

After 4 weeks of male C57BL/6J rats were fed an chow diet and water for 1 week to have an adaptation period, the rats were divided into 4 groups of 9 rats each, and the high fat diet containing 5% microcrystalline cellulose (MCC) was high. fat diet, HF), high fat diet with 5% beta-glucan (BG), high fat diet with 5% EPS, and high fat diet with 5% residue (residue after extracting EPS from kefir grains, Res) Was provided for 4 weeks each.

The specific dietary composition is shown in Table 2 below.

On the last day of the experiment, the mice were fasted for 12 hours, anesthetized with 4% isoflurane, and blood and tissue were collected. Plasma was obtained by centrifuging blood at 2,000 xg for 30 minutes at 4°C. Total cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and ultra low density lipoprotein cholesterol (VLDL-C) were measured by exclusion chromatography.

6-2: Weight change according to diet

Figure 6 (a) shows the daily energy intake of the mice according to the type of diet, Figure 6 (b) shows the weight gain of the mice according to the type of diet.

6(a) and (b), the EPS intake group (7.31±0.46g) was compared to the control group (8.84±1.11g) after 3 weeks of dietary administration, although there was no significant difference in daily energy intake between the experimental groups. It showed a significantly lower weight gain.

After 4 weeks of diet, all groups showed lower weight gain than the control group, and the ingestion of EPS showed the fastest weight loss reduction effect.

6-3: Liver and fat tissue weight change according to diet

7(a) and (b) show changes in liver tissue weight (a) and adipose tissue weight (b) according to dietary types, respectively.

In both the liver and adipose tissues, it can be seen that the weight was the most decreased in the EPS ingestion group. In particular, the fat tissue weight measured through epididymal weight was significantly lower in the EPS intake group.

6-4: Change in blood cholesterol level according to diet

Figure 8 shows the blood cholesterol level of each experimental rat group according to the type of diet.

The blood cholesterol measurement result also showed the lowest cholesterol value in the EPS intake group. In particular, in the case of VLDL-C level, the EPS intake group was 1.28±0.21 mg/dL, while the control group was 1.99±0.22 mg/dL, showing a lower value of about 36%.

In the case of LDL-C and TOTAL-C, the EPS intake group also showed the lowest values of 32.5±4.1mg/dL and 140±6.6mg/dL.

This indicates that EPS has the effect of lowering VLDL-C in the blood and is a material having the potential to lower other blood lipid concentrations.

Example 7: Analysis of changes in the intestinal microflora according to EPS intake

Genomic DNA was extracted from 200 mg of lyophilized fecal sample using QIAamp DNA stool mini kit (QIAGEN).

The V4 and V5 libraries created by generating the V4 and V5 domains of the Bacterial 16S rDNA were isolated and clusters were generated. Operational Taxonomic Units (OUT) tables and alpha diversity were calculated using the QIIME program. The stacked bar and statistical significance were obtained using MEGAN and Explicet program, and the statistical significance of the high-altitude method and other experimental groups was calculated by Wilcoxon method. The results of the analysis of the intestinal microflora calculated from the samples are shown in FIGS. 9 and 4.

In FIG. 9, the EPS intake group had increased bacteria of Bacteroidetes , Verrucomicrobia , and Proteobacteria phyla compared to the control group and the control group, and permicutes ( Firmicutes ) and The bacteria of Actinobacteria were reduced.

Particularly, in Table 3, it was confirmed that the EPS intake group increased the bacteria of the genus Akkermansia more than 2 times as compared to the BG intake group in the Verrucomicrobia statement.

As described above, specific parts of the present invention have been described in detail. For those skilled in the art, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

It is derived from Kefir grain containing lactic acid bacteria, acetic acid bacteria, and yeast, and includes an exopolysaccharide including homo-polysaccharide and hetero-polysaccharide as an active ingredient. Functional food for improving obesity.
According to claim 1,
A functional food for improving obesity, characterized in that the kefir grain-derived extracellular polysaccharide comprises 70 to 90% by weight of carbohydrate and 0.01% by weight or less of protein.
According to claim 2,
Functional food for improving obesity, characterized in that the carbohydrate contains glucose and galactose.
According to claim 1,
Functional food for improving obesity, characterized in that the kefir grain-derived extracellular polysaccharide has shear fluidization properties.
According to claim 1,
A functional food for improving obesity, characterized by suppressing the differentiation of adipocytes in cells.
According to claim 1,
Functional food for improving obesity, characterized by lowering blood cholesterol levels.
According to claim 1,
Functional food for improving obesity, characterized by increasing the bacteria in the intestinal Akkermansia .
It is derived from Kefir grain containing lactic acid bacteria, acetic acid bacteria, and yeast, and includes an exopolysaccharide including homo-polysaccharide and hetero-polysaccharide as an active ingredient. Pharmaceutical composition for preventing or treating obesity.

Images