KR102296748B1 - Composition for improving, treating or preventing obesity comprising glutinous rice fermented by lactic bacteria as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for improving, treating or preventing obesity containing fermented glutinous rice as an active ingredient, and more particularly, to a composition comprising: yeast fermentation; Alternatively, it is effective for obesity because it inhibits adipocyte differentiation and adipogenesis by including glutinous rice fermented product obtained by fermenting lactic acid bacteria after yeast fermentation of saccharified glutinous rice.

Description

Composition for improving, treating or preventing obesity comprising glutinous rice fermented by lactic bacteria as an active ingredient

The present invention is yeast fermentation of saccharified glutinous rice; Or it relates to a composition for improving, treating, or preventing obesity, comprising as an active ingredient a fermented glutinous rice obtained by fermenting lactic acid bacteria after yeast fermentation of saccharified glutinous rice.

Obesity is a metabolic disease caused by an imbalance in calorie intake and consumption, and refers to a condition in which adipose tissue is abnormally increased due to excessive calorie intake. A man is considered obese when the body fat is 25% of body weight and 30% or more of a woman's body weight. define.

When obesity occurs and the condition continues, it causes various diseases, such as high blood pressure, elevated blood cholesterol, diabetes, kidney disease, stroke, arteriosclerosis, fatty liver, arthritis, cancer, sleep apnea, and diabetes. have.

The causes of obesity include environmental and genetic factors such as a high-fat, high-calorie diet, lack of exercise due to a busy social environment, and endocrine abnormalities. is known, and the remainder is known to be due to genetic factors.

As the world's obese population is increasing, it is recently pointed out as an important factor in causing adult diseases, and the severity of obesity is emerging as an important problem. In order to prevent and improve obesity and maintain a healthy weight, it is necessary to maintain a healthy weight throughout life through a balanced diet and regular exercise. In particular, fat cells can be reduced in size once they are made, but it is impossible to remove them naturally, so it is important to prevent obesity from the beginning because they remain in the body permanently.

For the prevention, improvement or treatment of obesity, methods of reducing intake of calories, increasing activity and exercise, and methods of taking drugs such as reducing fat absorption have been used.

Current treatments for obesity include fenfluramine, which inhibits the serotonin nervous system, ephedrine and caffeine via the noradrenergic nervous system, sibutramine, which acts simultaneously on the serotonin and noradrenergic nervous system, and orli, which reduces fat absorption by inhibiting lipase produced in the pancreas There are drugs such as start. However, among previously used drugs, fenfluramine was banned due to side effects such as primary pulmonary hypertension or heart valve lesions. There is a problem that cannot be done.

Therefore, there is a demand for the development of substances for preventing, improving or treating obesity with fewer side effects using natural substances.

Republic of Korea Patent No. 1802696 Republic of Korea Patent No. 19800816

It is an object of the present invention to provide a pharmaceutical composition capable of treating or preventing obesity by containing fermented glutinous rice as an active ingredient.

In addition, another object of the present invention is to provide a health functional food that can improve or prevent obesity by containing fermented glutinous rice as an active ingredient.

In addition, another object of the present invention is to provide a method for producing fermented glutinous rice.

The pharmaceutical composition for the treatment or prevention of obesity of the present invention for achieving the above object may contain fermented glutinous rice as an active ingredient.

The fermented glutinous rice may be a yeast fermented product obtained by fermenting the saccharified glutinous rice with yeast.

The fermented glutinous rice may be obtained by fermenting the saccharified glutinous rice with yeast and lactic acid bacteria.

The fermented glutinous rice is (a) mixing glutinous rice with water and gelatinizing to obtain a glutinous rice fermented product; (b) treating the gelatinized glutinous rice with a saccharification enzyme and yeast to obtain a saccharified solution of glutinous rice; (c) a primary fermentation step of fermenting the glutinous rice saccharified solution with Saccharomyces sp. yeast; and (d) a secondary fermentation step of fermenting the first fermented glutinous rice fermented product with Weissella sp. lactic acid bacteria.

The gelatinization in step (a) may be performed at a temperature of 90 to 125 °C for 10 to 90 minutes.

In step (b), the gelatinized glutinous rice may be treated with a saccharification enzyme and yeast, and saccharified at 50 to 60° C. for 90 to 150 minutes.

In step (b), the content of the saccharification enzyme may be 0.5 to 5 parts by weight based on 100 parts by weight of the gelatinized glutinous rice.

In the step (c), the Saccharomyces genus (Saccharomyces sp.) yeast is Saccharomyces cerevisiae, Saccharomyces uqvarum, Saccharomyces ellipsoideus ( Saccharomyces ellipsoideus, Saccharomycescarlsbergensis, Saccharomyces sake, Saccharomyces coreanus, Saccharomyces lipolytica, Saccharomyces It may be at least one selected from the group consisting of Myces boulardii (Saccharomyces boulardii) and Saccharomyces pastorianus.

The content of Saccharomyces sp. yeast in step (c) may be 0.5 to 10 parts by weight based on 100 parts by weight of the glutinous rice saccharification solution.

The primary fermentation in step (c) may be performed at 25 to 35 °C for 20 to 36 hours.

In step (d), Weissella sp. lactic acid bacteria are Weissella cibaria, Weissella koreensi, Weissella hanii, Weissella kimchii. ), Weissella soli, and Weissella confusa may be at least one selected from the group consisting of.

In step (d), the content of Weissella sp. lactic acid bacteria may be 0.5 to 10 parts by weight based on 100 parts by weight of the primary fermented product.

Fermentation in step (d) may be performed at 25 to 35 °C for 20 to 36 hours.

filtering and concentrating the secondary fermented glutinous rice obtained in step (d) to obtain a fermented glutinous rice concentrate; mixing the glutinous rice fermentation concentrate with dextrin; and sterilizing the mixture and then spray drying.

In addition, the health functional food for improving or preventing obesity of the present invention for achieving the above other object may contain glutinous rice fermented product as an active ingredient.

In addition, the manufacturing method of the fermented glutinous rice of the present invention for achieving the above another object comprises the steps of (a) mixing glutinous rice with water and gelatinizing to obtain a glutinous rice fermented product; (b) treating the gelatinized glutinous rice with a saccharification enzyme and yeast to obtain a saccharified solution of glutinous rice; (c) a primary fermentation step of yeast-treating the glutinous rice saccharification solution with Saccharomyces sp. yeast; and (d) a secondary fermentation step of fermenting the first fermented glutinous rice fermented product with Weissella sp. lactic acid bacteria.

The composition for improving, treating or preventing obesity of the present invention is a fermented glutinous rice, preferably a saccharified solution of glutinous rice; Alternatively, the fermented glutinous rice obtained by fermentation of lactic acid bacteria after yeast fermentation of glutinous rice saccharification liquid can be used as an active ingredient to inhibit adipocyte differentiation and adipogenesis, so it can be used as a health functional food or pharmaceutical composition.

1 is a graph analyzing the fermented glutinous rice obtained in Example 1 of the present invention.
Figure 2 is a graph showing the viability of cells when the fermented glutinous rice prepared according to Example 1 of the present invention is treated by concentration.
Figure 3 is a Western blot measuring the expression of PPARγ, C/EBPa, AMPK and pAMPK in 3T3-L1 adipocytes when the fermented glutinous rice prepared according to Example 1 of the present invention is treated by concentration.
4 is a graph showing GPDH activity in 3T3-L1 adipocytes when fermented glutinous rice prepared according to Example 1 of the present invention is treated by concentration.
5 is a photograph showing Oil Red O staining of 3T3-L1 adipocytes after processing the fermented glutinous rice prepared according to Example 1 of the present invention by concentration.
6 is a graph showing the measurement of the lipid content of 3T3-L1 adipocytes after treating the fermented glutinous rice prepared according to Example 1 of the present invention by concentration.

The present invention is yeast fermentation of saccharified glutinous rice; Or it relates to a composition for improving, treating, or preventing obesity, comprising as an active ingredient a fermented glutinous rice obtained by fermenting lactic acid bacteria after yeast fermentation of saccharified glutinous rice.

Hereinafter, the present invention will be described in detail.

The fermented glutinous rice of the present invention may be a fermented glutinous rice obtained by fermenting the saccharified glutinous rice with yeast, or a fermented glutinous rice obtained by fermenting the saccharified glutinous rice with yeast after yeast fermentation.

The fermented glutinous rice is (a) mixing glutinous rice with water and gelatinizing to obtain a glutinous rice fermented product; (b) treating the gelatinized glutinous rice with a saccharification enzyme and yeast to obtain a saccharified solution of glutinous rice; (c) a primary fermentation step of fermenting the glutinous rice saccharified solution with Saccharomyces sp. yeast; And (d) the secondary fermentation step of fermenting the first fermented glutinous rice fermented product with Weissella sp. lactic acid bacteria; can be prepared including.

First, in step (a), glutinous rice is mixed with water and gelatinized to obtain a glutinous rice gelatinized product.

After immersing glutinous rice in water (eg, distilled water), it is gelatinized by applying heat at 90 to 125 ℃, preferably 110 to 125 ℃ for 10 to 90 minutes, preferably 50 to 70 minutes. Through this step, the starch particles of glutinous rice are gelatinized to form a wide space between the micelles of glutinous rice. can

Unlike glutinous rice, glutinous rice is mostly composed of amylopectin, and the content of amylose is about 5% by weight or less. The fermented product obtained by fermenting glutinous rice has superior anti-obesity efficacy compared to the fermented product obtained by fermenting spicy rice, and thus can inhibit adipocyte differentiation and adipogenesis.

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Such glutinous rice can be pulverized and used in powder form to promote gelatinization. In addition, when the glutinous rice is immersed in water for 10 to 60 minutes, since the glutinous rice contains moderate moisture, it is possible to promote the gelatinization action.

The content of water may be 250 to 350 parts by weight based on 100 parts by weight of glutinous rice, but is not limited thereto. The gelatinized glutinous rice obtained through the above-mentioned gelatinization process is a solution containing a translucent colloidal material with increased viscosity and swelling of glutinous rice.

Next, in step (b), the gelatinized glutinous rice is treated with a saccharification enzyme and yeast to obtain a saccharified solution of glutinous rice.

It is a simultaneous saccharification and fermentation (SSF) in which saccharification of glutinous rice starch by saccharification and fermentation by yeast are simultaneously performed by adding saccharification enzyme and yeast to the luxurious glutinous rice. The saccharified solution of glutinous rice of the present invention is a material in which saccharification and fermentation are performed simultaneously, and although it is precisely a saccharified fermented solution, it is described as a saccharified solution to distinguish it from the fermented solution produced later.

According to one example, it may be carried out by adding a saccharification enzyme and yeast to the gelatinized glutinous rice obtained in step (a), and then reacting it at a temperature of 50 to 60° C. for 90 to 150 minutes.

Examples of glycosylation enzymes that can be used in the present invention include alpha-amylase, beta-amylase, glucoamylase, pullulanse, and the like, and yeast that can be used It can be used without restrictions as long as it is a common yeast sold in the market. By using such a saccharification enzyme and yeast together, the saccharification rate is improved compared to the case where only saccharification enzyme treatment is used, and since the resulting saccharification solution is easier for yeast treatment, the starch of glutinous rice is saccharified and fermented at the same time.

The content of the saccharification enzyme and yeast is not particularly limited, and for example, the content of the saccharification enzyme may be 0.5 to 5 parts by weight based on 100 parts by weight of the glutinous rice gelatin, and the content of the yeast is 100 parts by weight of the glutinous rice glutinous rice product. It may be 1 to 20 parts by weight based on the If the content of the saccharification enzyme and the yeast is in the above-mentioned range, it is possible to obtain the optimal reaction conditions to reduce the production cost, and to obtain a high-quality fermented glutinous rice.

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The saccharified glutinous rice solution obtained in this step contains 50 to 55 mg/g of galato oligosaccharides (eg, stachyose, raffinose, etc.).

The saccharified glutinous rice prepared in this way may be sterilized at 110 to 130° C. for 10 to 20 minutes before yeast fermentation, which will be described later.

Next, in the step (c), the glutinous rice saccharification solution is subjected to yeast treatment with Saccharomyces sp. yeast to obtain a first fermented glutinous rice fermented product.

Since the first fermented glutinous rice obtained through this step contains more galactooligosaccharide than the saccharified solution of glutinous rice, it is possible to further inhibit adipocyte differentiation and adipogenesis.

According to one example, the glutinous rice saccharification solution obtained in step (b) is inoculated with yeast of the genus Saccharomyces (Saccharomyces sp.), and then fermented at a temperature of 25 to 35 ° C. for 20 to 36 hours (0 rpm) or Fermentation can be carried out at 50 rpm or less. The temperature, time, and rpm at the time of yeast fermentation are optimized in consideration of the case of being included to a significant degree in improving, preventing and treating anti-obesity after yeast fermentation.

The Saccharomyces genus (Saccharomyces sp.) Yeast includes Saccharomyces cerevisiae, Saccharomyces uqvarum, Saccharomyces ellipsoideus, Saccharomyces carlsbergensis, Saccharomyces sake, Saccharomyces coreanus, Saccharomyces lipolytica, Saccharomyces bouladi (Saccharomyces boulardii) and Saccharomyces pastorianus may be at least one selected from the group consisting of, preferably Saccharomyces cerevisiae (Saccharomyces cerevisiae).

The content of such Saccharomyces sp. yeast is not particularly limited, but is preferably 0.5 to 10.0 parts by weight based on 100 parts by weight of saccharified glutinous rice. When the content of yeast is in the above-mentioned range, it is possible to obtain an optimal fermentation rate, and it is possible to reduce the production of by-products, thereby obtaining a high-quality fermented glutinous rice.

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The first fermented glutinous rice obtained in this step contains 55 to 60 mg/g of galato oligosaccharides (eg, stachyose, raffinose, etc.), and 2 to 5 mg/g compared to glutinous rice saccharification solution. g of galato-oligosaccharide content is increased.

The first fermented glutinous rice produced after the yeast fermentation prepared in this way may be sterilized at 110 to 130° C. for 10 to 20 minutes before lactic acid bacteria fermentation, which will be described later.

Next, in the step (d), the second fermented glutinous rice is obtained by secondarily fermenting the first fermented glutinous rice with Weissella sp. lactic acid bacteria.

Since the second fermented glutinous rice obtained through this step contains more galactooligosaccharide than the first fermented glutinous rice, it is possible to further inhibit adipocyte differentiation and adipogenesis.

According to one example, the first fermented glutinous rice obtained in step (c) is inoculated with Weissella sp. lactic acid bacteria, and then fermented for 20 to 36 hours at a temperature of 25 to 35 ° C. (0 rpm) or It can be fermented at 50 rpm or less. The temperature, time and rpm are optimized in consideration of the cases included in the improvement, prevention and treatment of obesity to a significant degree.

The Weissella sp. lactic acid bacteria used in this step are lactic acid bacteria extracted from kimchi, for example, Weissella cibaria, Weissella koreensi, Weissella hanii, Weissella. and at least one selected from the group consisting of kimchi eye (Weissellakimchii), weissellasoli (Weissellasoli), and weissellaconfusa (Weissellaconfusa).

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The content of such Weissella sp. lactic acid bacteria is not particularly limited, but may preferably be 0.5 to 10.0 parts by weight based on 100 parts by weight of the first fermented glutinous rice. When the content of lactic acid bacteria is in the above-mentioned range, it is possible to obtain an optimal fermentation rate, and to reduce the production of by-products, thereby obtaining a high-quality fermented glutinous rice.

The second fermented glutinous rice obtained in this step contains about 73 mg/g or more, specifically, about 73 to 95 mg/g of galato oligosaccharides (eg, stachyose, raffinose, etc.), Compared to the first fermented glutinous rice, the content of galato oligosaccharides of 20 to 35 mg/g is increased.

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In addition, if necessary, the second fermented glutinous rice, which is fermented in step (d), may be filtered and concentrated. At this time, filtration and concentration may be performed according to a conventional method known in the art. According to one example, the second fermented glutinous rice may be filtered and then concentrated at 55 to 65 °C. In addition, the glutinous rice fermentation concentrate may be sterilized and dried. In this case, as the drying method, a method commonly known in the art such as reduced pressure drying, spray drying, freeze drying, etc. may be used. The powdered fermented glutinous rice can be used as a food composition or a pharmaceutical composition.

In addition, the glutinous rice fermentation concentrate may be sterilized and mixed with dextrin before drying. Dextrin has low water absorption and is excellent in dispersibility, solubility and drying properties. Therefore, by mixing the glutinous rice fermentation concentrate with dextrin, it is possible to obtain a powder product of excellent quality by improving the powdering properties of the final product in the drying process. At this time, the use ratio (mixing ratio) of the glutinous rice fermentation concentrate and dextrin is not particularly limited, and may be, for example, a weight ratio of 60:40 to 95:5.

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As used herein, the term 'comprising as an active ingredient' means including an amount sufficient to achieve obesity improvement, treatment or prevention efficacy or activity of the fermented glutinous rice of the present invention.

The health functional food may be a formulation of the fermented glutinous rice in the form of capsules, tablets, powders, granules, liquids, pills, flakes, pastes, syrups, gels, jellies or bars, or beverages, teas It is manufactured in the form of general food by adding it to food materials such as spices, gum, and confectionery, and it means that it has a specific health effect when ingested. It has the advantage that there are no side effects that may occur during the event.

The health functional food is very useful because it can be consumed on a daily basis. The amount of added fermented glutinous rice in such a health functional food cannot be uniformly defined because it varies depending on the type of health functional food, but it can be added within a range that does not impair the original taste of the food. 50% by weight, preferably in the range from 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of capsules, tablets, powders, granules, liquids, pills, flakes, pastes, syrups, gels, jellies or bars, usually 0.1 to 100% by weight, preferably 0.5 to 80% by weight % can be added.

The health functional food may include not only fermented glutinous rice as an active ingredient, but also ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatine, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the health functional food of the present invention is manufactured as a drink or beverage, citric acid, high fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included in addition to the fermented rice of the present invention. have.

In addition, in the pharmaceutical composition for the treatment or prevention of obesity comprising fermented glutinous rice as an active ingredient, the fermented glutinous rice is, for example, 0.001 mg/kg or more, preferably 0.1 mg/kg or more, more preferably 10 mg/kg kg or more, even more preferably 100 mg/kg or more, even more preferably 250 mg/kg or more, and most preferably 0.1 g/kg or more. Since the fermented glutinous rice is a natural product and there is no side effect to the human body even when administered in excess, the upper limit of the quantity of the fermented glutinous rice contained in the composition of the present invention may be selected and carried out by those skilled in the art within an appropriate range.

The pharmaceutical composition may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, a lubricant, or A flavoring agent and the like may be used.

The pharmaceutical composition may be formulated to include one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For formulation in the form of, for example, tablets or capsules, the active ingredient may be combined with an orally, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or required, suitable binders, lubricants, disintegrants and color-developers may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol, and these components. One or more of the components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

Furthermore, by using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA by an appropriate method in the art, it can be preferably formulated according to each disease or component.

The pharmaceutical composition may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., preferably oral administration.

A suitable dosage of the pharmaceutical composition varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient, usually skilled A trained physician can readily determine and prescribe an effective dosage for the desired treatment or prevention. According to a preferred embodiment, the daily dose of the pharmaceutical composition is 0.001-10 g/kg.

The pharmaceutical composition may be prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. It may be prepared by introduction into a dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

Hereinafter, preferred examples are presented to aid the understanding of the present invention, but the following examples are merely illustrative of the present invention and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention, It goes without saying that such variations and modifications fall within the scope of the appended claims.

Example 1. Preparation of fermented glutinous rice

After soaking the glutinous rice in 3 times water (about 30 ℃) for 60 minutes and then soaking, 4 kg of the mixture was gelatinized at a temperature of 121 ℃ for 1 hour to obtain a glutinous rice gelatinized product. Thereafter, 20 ml of alpha-amylase, a saccharification enzyme, and 5 g of yeast were added to 4 kg of the gelatinized glutinous rice, and simultaneous saccharification fermentation was performed at a temperature of 55° C. for 2 hours to obtain a saccharified glutinous rice solution. Next, 100 g of yeast, Saccharomyces cerevisiae (ATCC 9804; ATCC, Manassas, VA, USA), was inoculated into 4 kg of the glutinous rice saccharification solution and fermented at 30 ° C. for 24 hours to prepare 1 A fermented glutinous rice was obtained. Then, 200 g of lactic acid bacteria Weissella cibaria (KACC 11845; KACC, Suwon, Korea) was inoculated into 4 kg of the first fermented glutinous rice and fermented at 30 ° C. for 24 hours to ferment the second glutinous rice. got water. Thereafter, the second fermented glutinous rice was filtered, concentrated at a temperature of 60° C., and then dextrin was mixed thereto. Then, the mixture (glutinous rice concentrate: dextrin = 90:10 weight ratio) was sterilized at a temperature of 80 ° C. for 1 hour and spray dried to prepare a fermented glutinous rice (galactooligosaccharide content: 73.02 mg/g).

The results of analysis of the final fermented glutinous rice obtained above are shown in Table 1 and FIG. 1 below.

oligosaccharide Concentration of glutinous rice fermented product (μg/mL) Final volume (mL) Sample collection amount (g) dilution multiple Results (mg/g) Stachyose 419.5390 50 0.8323 One 25.2036 Raffinose 795.9573 50 0.8323 One 7.8167 Total amount of galactooligosaccharides 73.0203

<Test Example>

For induction of adipocyte differentiation, 3T3-L1 adipocytes (purchased from ATCC) were ^{seeded at 1X10 6} cells in a 60 mm plate, and the medium was exchanged 2 days later, and the cells were cultured to 100% confluence on the 4th day. DMEM medium containing 10% fetal bovine serum (FBS) and MDI solution (0.5 mM IBMX, 1 μM dexamethasone, 10 ug/mL insulin) was treated for 2 days. At this time, in order to observe the effect of the fermented glutinous rice on adipocyte differentiation, the fermented glutinous rice was treated with each concentration. After 2 days of differentiation induction, the medium was treated with DMEM containing 10% FBS and 1 ug/mL insulin for each concentration of fermented glutinous rice for 2 days. Cells were differentiated for a total of 6 days by exchanging with DMEM containing penicillin/streptomycin (P/S).

On day 6, differentiated 3T3-L1 pre-adipocytes were applied to Oil Red O solution and used for Western blot analysis and GPDH activity analysis. The fermented glutinous rice prepared according to Example 1 was added to the cell culture medium at concentrations of 0, 100, 200 and 400 μg/ml from day 0 to day 6.

Test Example 1. Measurement of cell viability

3T3-L1 adipocytes were cultured using DMEM medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2. Cell viability was measured using a CCK-8 kit (Tojindo, Kumamoto, Japan) according to the manufacturer's instructions. Briefly, 3T3-L1 adipocytes were plated in 96-well plates at a density of ^{1X10 4 cells/well.} After overnight incubation, cells were tested by treatment by concentration and incubated for 24 hours. After incubation, CCK-8 solution was added to each well and incubated at 37° C. for 3 hours. Absorbance at 450 nm was measured using a microplate reader (Bio-Tek Company, Winooski, VT, USA).

Figure 2 is a graph showing the viability of cells when the fermented glutinous rice prepared according to Example 1 of the present invention is treated by concentration.

As shown in FIG. 2 , it was confirmed that there was no toxicity to the cell growth of 3T3-L1 pre-adipocytes at all concentrations of fermented glutinous rice.

Test Example 2. Measurement of expression of PPARγ, C/EBPa, AMPK and pAMPK

Cytoplasmic RNA was isolated from 3T3-L1 adipocytes using the RNeasy lipid tissue mini kit [Qiagen, Valencia, CA, USA] according to the manufacturer's instructions. 500 ng of cytoplasmic RNA was converted into cDNA using the oligo[dT] primer and reverse transcriptase provided by the Superscript III first-strand synthesis system for RT-PCR (Invitrogen). SYBR Greenbased real-time PCR was performed on an ABI 7500 PCR system [Applied Biosystems, Foster City, CA, USA]. The expression level of the target gene was normalized to the expression level of the housekeeping gene β-actin (Choi et al., 2003).

Figure 3 is a Western blot measuring the expression of PPARγ, C/EBPa, AMPK and pAMPK in 3T3-L1 adipocytes when the fermented glutinous rice prepared according to Example 1 of the present invention is treated by concentration.

In the process of differentiation from preadipocytes to adipocytes through PPARγ and C/EBPa, transcription factors such as PPARγ and C/EBPa are adipocyte-specific proteins such as glucose transporter (GLUT4) or adipocyte-specific fatty acid binding protein 2 (aP2). involved in the continuous expression of The expression of the PPARγ and C/EBPa proteins means that the adipocytes absorb glucose or fatty acids to synthesize or accumulate fat, thereby increasing the size of the adipocytes.

As shown in FIG. 3 , when fermented glutinous rice is treated by concentration, PPARγ and C/EBPa proteins are reduced in a concentration-dependent manner, so it is considered to have the effect of inhibiting adipocyte differentiation.

In addition, AMPK protein responds to changes in the intracellular energy state, as a major regulator of metabolic conversion, and plays an important regulator role in carbohydrate and fat metabolism, and activation of AMPK is a major marker for improving obesity and metabolic diseases. AMPK activation (pAMPK) in adipose tissue inhibits adipogenesis by inactivating actyl-CoA carboxylase (ACC).

As shown in FIG. 3 , when fermented glutinous rice is treated by concentration, activated AMPK (pAMPK) is increased in a concentration-dependent manner, so it is considered to have the effect of inhibiting adipocyte differentiation and adipogenesis.

Test Example 3. GPDH (glycerol-3-phosphate dehydrogenase) activity measurement

4 is a graph showing GPDH activity in 3T3-L1 adipocytes when fermented glutinous rice prepared according to Example 1 of the present invention is treated by concentration.

By confirming the GPDH activity, which increases the activity specifically only in adipocytes, the differentiation of adipocytes and the degree of accumulation of triglycerides according to the treatment of fermented glutinous rice were confirmed. As the differentiation into adipocytes progresses, the content increases, and GPDH activity is measured with glutinous rice fermented water, washed twice with PBS in the differentiated medium for 6 days, and then with a scraper 300 ul of 100 mM triethanolamine/HCl buffer, pH Adipocytes were collected by treatment with 7.5 and 2.5 mM EDTA. Collected adipocytes were sonicated under ice (maximum output power of 250 W) in a DU-250 Bioruptor with 25 ultrasonic bursts of 10 s (Tosho Denki, Co. Ltd., Japan). After centrifugation at 13,000×g for 5 minutes at 4° C., the supernatant was analyzed for GPDH activity according to the method of Wise and Green (Wise & Green, 1979). GPDH activity was measured using a spectrophotometer (Beckman Coulter, DU 530, Indianapolis, IN, U.S.) at 25 °C under zero-order kinetics and optimal substrate and cofactor conditions.

The standard reaction mixture contained 100 mM triethanolamine/HCl buffer, pH 7.5, 2.5 mM EDTA, 0.1 mM/2-mercaptoethanol and 0.12 mM NADH. The reaction was initiated by addition of 0.2 mM dihydroxy acetone phosphate, and the NADH oxidation rate was measured by the change in absorbance at 340 nm for 60 seconds.

Enzyme activity (%) was expressed as a percentage (%) relative to the control.

As a result, as shown in FIG. 4, compared to the control group (C. 0 ug/ml)), the cell group treated with 100, 200 and 40 μg/ml of fermented glutinous rice showed a concentration-dependent decrease in the activity of GPDH. It was confirmed that it has the effect of inhibiting cell differentiation and lipogenesis.

Test Example 4. Oil red O (Oil red O) staining and lipid content confirmation

5 is a photograph showing Oil Red O staining of 3T3-L1 adipocytes after treating the fermented glutinous rice prepared according to Example 1 of the present invention by concentration, and FIG. It is a graph measuring the lipid content of 3T3-L1 adipocytes after fermented glutinous rice was treated by concentration.

The effect of inhibiting lipid synthesis of fermented glutinous rice was confirmed by using Oil Red O staining, a special staining method that can confirm the degree of lipid accumulation by reacting specifically and sensitively to lipid components and showing a red color. On the 6th day of differentiation of 3T3-L1 preadipocytes differentiated by the above method, the medium was removed, the cells were washed with PBS, and then fixed with 60% ethanol at room temperature. After removing the fixative and washing with 60% isopropanol, 1 at room temperature using 0.3% Oil red O (Oil red O) solution that reacts specifically with lipid droplets generated in cells stained for hours. Visualization of lipids was performed using an Olympus IX50 microscope (Olympus, Tokyo, Japan).

As shown in FIG. 5, the result was observed at 200 magnification using an inverted microscope after processing fermented glutinous rice for 6 days in 3T3-L1 preadipocytes induced in differentiation, and as a result, the red color was almost confirmed in a concentration-dependent manner. Therefore, it was confirmed that adipocyte production inhibition and lipid accumulation were inhibited.

In addition, the stained cells were extracted by dissolving Oil Red 0 staining using 100% isopropanol after microscopic observation, and the absorbance was measured at 490 nm. indicated.

As shown in FIG. 6, the lipid content was lower in a concentration-dependent manner in the cell group treated with 100, 200, and 400 μg/ml of fermented glutinous rice compared to the control group (C), which is 3T3-L1 adipocyte differentiated for 6 days. appear. In the cell group treated with 400 μg/ml, the lipid content was low at 73.8%.

From the above results, it was confirmed that the fermented glutinous rice had the effect of inhibiting the production of adipocytes and inhibiting the accumulation of lipids.

Hereinafter, a formulation example of a composition containing the extract of the present invention will be described, but the present invention is not intended to limit the present invention, but to describe it in detail.

Formulation Example 1: Preparation of powder

20 mg of fermented powder of Example 1

Lactose 100 mg

talc 10 mg

The above ingredients are mixed and filled in an airtight bag to prepare a powder.

Formulation Example 2: Preparation of tablets

10 mg of fermented product powder of Example 1

100 mg cornstarch

Lactose 100 mg

2 mg magnesium stearate

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

Formulation Example 3: Preparation of capsules

10 mg of fermented product powder of Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

0.2 mg magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled in a gelatin capsule to prepare a capsule.

Formulation Example 4: Preparation of granules

1,000 mg of fermented product powder of Example 1

appropriate amount of vitamin mixture

70 μg vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

0.2 μg of vitamin B12

Vitamin C 10 mg

Biotin 10 μg

1.7 mg of nicotinic acid amide

50 μg folic acid

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

ferrous sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

potassium phosphate monobasic 15 mg

Dicalcium Phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is mixed with ingredients suitable for health functional food in a preferred embodiment, but the mixing ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health functional food manufacturing method. Next, the granules can be prepared and used in the preparation of a health functional food composition according to a conventional method.

Formulation Example 5: Preparation of beverage formulations

1,000 mg of fermented product powder of Example 1

1,000 mg citric acid

100 g of oligosaccharides

2 g of plum concentrate

1 g taurine

Add purified water to total 900 mL

After mixing the above ingredients according to a conventional beverage manufacturing method, stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and then refrigerated. used in the manufacture of functional beverage compositions.

Claims (16)

It contains fermented glutinous rice as an active ingredient,
The rice fermented product is a pharmaceutical for the treatment or prevention of overweight characterized by a. Containing galacto-oligosaccharides going fermented in which rice Hydrolyzate fermenting rice with saccharification enzymes and yeast as yeast and lactic acid bacteria in order of 73 to 95 mg / g composition. delete delete The method according to claim 1, wherein the fermented glutinous rice product comprises: (a) mixing glutinous rice with water and gelatinizing to obtain a glutinous rice fermented product;
(b) treating the gelatinized glutinous rice with a saccharification enzyme and yeast to obtain a saccharified solution of glutinous rice;
(c) a primary fermentation step of yeast-treating the glutinous rice saccharification solution with Saccharomyces sp. yeast; and
(D) a second fermentation step of fermenting the first fermented glutinous rice fermented product with Weissella sp. lactic acid bacteria; The pharmaceutical composition for the treatment or prevention of obesity according to claim 4, wherein the gelatinization in step (a) is performed at a temperature of 90 to 125 °C for 10 to 90 minutes. [Claim 5] The pharmaceutical composition for the treatment or prevention of obesity according to claim 4, wherein in step (b), the gelatinized glutinous rice is treated with a saccharification enzyme and yeast and saccharified at 50 to 60 ° C. for 90 to 150 minutes. The pharmaceutical composition for the treatment or prevention of obesity according to claim 4, wherein the content of the saccharification enzyme in step (b) is 0.5 to 5 parts by weight based on 100 parts by weight of the glutinous rice. According to claim 4, wherein in step (c) Saccharomyces genus (Saccharomyces sp.) yeast is Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces uqvarum (Saccharomyces uqvarum), Saccharo Saccharomyces ellipsoideus, Saccharomycescarlsbergensis, Saccharomyces sake, Saccharomyces coreanus, Saccharomyces lipolytica (Saccharomyces lipolytica) Saccharomyces boulardii (Saccharomyces boulardii) and Saccharomyces pastorianus (Saccharomyces pastorianus) A pharmaceutical composition for the treatment or prevention of obesity, characterized in that at least one selected from the group consisting of. [Claim 5] The treatment or prevention of obesity according to claim 4, wherein the content of Saccharomyces sp. yeast in step (c) is 0.5 to 10 parts by weight based on 100 parts by weight of the glutinous rice saccharification solution. pharmaceutical composition. The pharmaceutical composition for the treatment or prevention of obesity according to claim 4, wherein the primary fermentation in step (c) is carried out at 25 to 35° C. for 20 to 36 hours. According to claim 4, wherein in step (d) Weissella genus (Weissella sp.) lactic acid bacteria Weissella cibaria (Weissella cibaria), Weissella koreensi (Weissella koreensi), Weissella hanii (Weissella hanii), wey Cella kimchii (Weissella kimchii), Weissella soli (Weissella soli), and Weissella confusa (Weissella confusa) A pharmaceutical composition for the treatment or prevention of obesity, characterized in that at least one selected from the group consisting of. [Claim 5] The pharmaceutical for the treatment or prevention of obesity according to claim 4, wherein the content of Weissella sp. lactic acid bacteria in step (d) is 0.5 to 10 parts by weight based on 100 parts by weight of the primary fermented product. composition. The pharmaceutical composition for the treatment or prevention of obesity according to claim 4, wherein the fermentation in step (d) is carried out at 25 to 35° C. for 20 to 36 hours. 5. The method of claim 4, further comprising: filtering and concentrating the secondary fermented glutinous rice obtained in step (d) to obtain a fermented glutinous rice concentrate; mixing the glutinous rice fermentation concentrate with dextrin; And sterilizing the mixture and then spray-drying; A pharmaceutical composition for the treatment or prevention of obesity, characterized in that it further comprises. It contains fermented glutinous rice as an active ingredient,
The fermented glutinous rice is a fermented glutinous rice saccharified solution obtained by fermenting glutinous rice with a saccharification enzyme and yeast in the order of yeast and lactic acid bacteria to contain galactooligosaccharide in an amount of 73 to 95 mg/g. Health function for improvement or prevention of obesity food. delete

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