KR101564825B1 - Fermented Castanea crenata inner shell extracts for anti-obesity and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a composition for preventing, improving and treating obesity.
The fermentation product of the tuberous enzyme lysate according to the present invention is superior to the 3T3-L1 adipocyte precursor cell line for the cell differentiation inhibitory activity and the high expression of PPAR-γ, CEBP-α and SREBP-1c gene, Inhibition may be useful for prevention, improvement and treatment of obesity.

Description

[0001] The present invention relates to an anti-obesity composition comprising an extract of fermented potato as an active ingredient and a method for producing the same. [0002] Fermented Castanea crenata extracts for anti-

The present invention relates to a method for producing a fermentation product of a tubercleolytic lysate characterized by the expression of PPAR-γ, CEBP-α, and SREBP-1c genes, which are high-level transcription factors involved in cell differentiation inhibitory activity and adipocyte differentiation of 3T3-L1 lipogenic precursor cell line The present invention relates to a composition for preventing, ameliorating or treating fat-accumulating obesity, and more particularly, to a composition for preventing or ameliorating obesity or a composition for preventing or ameliorating obesity.

Obesity is a chronic metabolic disease caused by excessive accumulation of fat in the body due to the accumulation and consumption imbalance of energy, and it is known that diabetes, hypertension, myocardial infarction, cancer, arthritis and stroke are highly likely to cause various complications. According to the National Health and Nutrition Survey, obesity rate increased from 32.7% in 2001 to 34.9% in 2005, and the share of local energy consumption increased from 19.5% in 2001 to 20.3% in 2005. This is due to the increase in the intake of high calorie foods such as meat according to the Westernized diet of modern people, and the obesity population is increasing.

Various methods such as dietary therapy, exercise therapy, behavior modification therapy, drug use, and surgical therapy have been developed and attempted to overcome obesity. Among them, Xenical (Roche Korea) and Reductil (Elysein New Drug) are approved as FDA-approved obesity drugs that are approved by the US FDA. Other non-prescription drugs developed in France include green tea catechol as a main ingredient And exorhizi (Gouju Pharmaceutical). However, in the case of Xenical, gastrointestinal side effects such as lowering of fat, gas production and lowering of fat soluble vitamin are caused, and in the case of reductil, problems such as headache, anorexia, insomnia or constipation are caused by increasing serotonin and noradrenaline concentration in the sympathetic nervous system There is a limit to the treatment of obesity without side effects. In addition, aminophylline, which is known to have a lipid-lowering effect, is known to exhibit a wide range of side effects in the nervous system, circulatory system and digestive system. Penfluramine, dexfenfluramine, topirameye, ephedrine and isoproterenol, And it is currently prohibited from being sold by the Korea Food and Drug Administration.

Due to the above-mentioned side effects due to the pharmacotherapy, there is a growing interest in anti-obesity natural substances using food materials. For example, mulberry leaves that lower the absorption of fats and sugars, fermented red peppers that inhibit pancreatic lipase activity, and foods that enhance fat burning and fat burning are being marketed as dietary health foods. In addition, foods such as brown rice, cereals, herbs and dairy products have been reported to have obesity inhibitory activity (Jin-Nyoung Ho, Mi-Eun Son, Won Chul Lim, Seung Taik Lim, Hong- Antiobesity effects of germinated brown rice extracts on down-regulation of lipogenic genes in high fat diet-induced obese mice, Biosci. Biotechnol. Biochem., 76 (6), 1068-1074, 2012; Kristel Diepvens, Klaas R. Westerterp, and Margriet S. Westerterp-Plantenga, Obesity and thermogenesis related to caffeine, ephedrine, capsaicin, and green tea, Am. J. Physiol. Regul. Integr. Comp. Physiol. 292: 77-85, 2007).

Castanea, which has been used for decanters, crenata ) is a perennial tree, chestnut ( Castanea crenata Lieb. et. Zucc), and the skin that peels off the outer skin and endothelium is mostly starch and contains a small amount of protein and fat (Kim MJ, Lee U, Hwang MS, Kim SC, Lee MH, Blooming, and nut characteristics of chestnut cultivars cultivated in Korea, J. Kor. Food Soc., 2003; 92: 321-32). The endothelium of the night contains a large amount of phenolic acid such as ellagic acid, syringic acid, and protocatechuic acid, and contains mainly coumarin, gallic acid and catechin (J Korean Soc Food Sci Nutr , 2008: 37 (9), 1095-1100 ). It is helpful to wash chestnut leaves with boiled water when itchy with lacquer or skin disease, or when eczema is caused by eczema (Kim CM, Lee JS, An DK, Sim MK, The encyclopedia of Chinese oriental herbal medicine, 1st rev. : Jungdamsa, 2006, 4372-3). Generally, only the pulp is used, but studies for searching for bioactive substances have also been conducted on leaves, flowers, and skin. In addition, studies on the search of physiologically active substances contained in the night showed that the antiallergic effect (Choi OB, Kim KM, Yoo GS, Park KH, Anti-allergic effect of Castanea crenata leaf tea, Kor. J. Food Sci. Technol., 1998; 30: 468-71), antioxidative activity and antioxidant activity of chestnut husk were investigated. Kwon EJ, Kim YC, Chestnut husk, J. Kor. Soc. Food Nutr., 2001; 30: 726-31) Properties of chestnut starch solution, Kor. J, Kim JH, Kim JH, Rheological properties of chestnut starch solution, J Korean Soc Food Nutr 1995; 24: 601-5 (Chung DH, Shim KH, Hur JH, Chung DH, Cho SH, Sung NK, Ki WK, J. Kor. Soc. Food Nutr., 1988; 17: 211-14).

Plant extract fermented products are one of the 32 functional foods, which are listed in the Health Functional Food Program (Korea Food & Drug Administration, 2004). In general, they are added to various vegetable raw materials for natural fermentation Microorganisms such as lactic acid bacteria are added and fermented. Plants contain various enzymes. When the plant extract is fermented, many enzymes are activated and cause various biochemical reactions, so that the nutrients of the plant can be converted into a form that is easily digested and absorbed. (Kim et al., 2003), which is expected to be able to express a new physiological regulatory function by the components produced by enzymatic action. However, the fermented product of the crude enzyme has not yet been used as an active ingredient.

The present invention provides a composition for preventing, ameliorating and treating obesity, which includes a natural plant material exhibiting an obesity pharmacological and physiological activity without adverse effects on the human body.

The present invention provides a pharmaceutical composition for preventing or treating obesity comprising a fermented product of a crude enzyme degraded solution as an active ingredient.

In addition, the present invention provides a food composition for improving obesity comprising a fermented product of a crude oil degrading solution as an active ingredient.

In addition, the present invention provides a method for producing a fermentation product of a crude enzyme degrading solution.

The fermented product of the tuberous enzyme lysate according to the present invention exhibits the cell differentiation inhibitory activity of the 3T3-L1 adipocyte precursor cell line, and the expression of PPAR-γ, CEBP-α and SREBP-1c gene Exhibits high inhibition and exhibits excellent effects in inhibiting fat accumulation, thus being useful as an effective ingredient for prevention, improvement or treatment of obesity.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the lipid accumulation inhibitory activity in the mature adipocyte of the fermented product of the hydrolyzate.
FIG. 2 is a graph showing the activity of inhibiting lipid accumulation in mature adipocytes according to concentration of fermented product of yeast extract using yeast.
FIG. 3 is a photomicrograph showing the fat accumulation inhibitory activity of the fermented product of yeast extract according to yeast concentration. FIG.
Fig. 4 shows the PPAR-y mRNA expression inhibitory activity of the fermentation product of the yeast lipase using yeast.
Fig. 5 shows the inhibitory activity of CEBP-alpha mRNA expression of the fermented product of yeast extract using yeast.
Fig. 6 shows the SREBP-1c mRNA expression inhibitory activity of fermented yeast lyophilized enzyme solution using yeast.

The present invention relates to a pharmaceutical composition for preventing or treating obesity, comprising a fermented product of a crude enzyme degrading solution as an active ingredient.

The present invention also relates to a food composition for improving obesity comprising a fermented product of a crude enzyme decomposition solution as an active ingredient.

The inventors of the present invention found that the fermented product of the crude enzyme hydrolyzate exhibited the proliferation inhibitory activity of lipid precursor cells and the inhibitory activity of lipid accumulation in mature adipocytes in the process of searching for substances exhibiting pharmacological and physiological activities of obesity from natural products Thereby completing the present invention. The fermented product of the lipolytic enzyme solution of the present invention exhibits a similar level to the level of caffeine, which is a positive control, in the proliferation inhibitory ability of lipid precursor cells and the fat accumulation inhibitory effect in mature adipocytes (see Examples to be described later).

Hereinafter, the composition according to the present invention will be described in more detail.

The fermented product of the crude oil lysozyme solution used as the active ingredient in the composition of the present invention is prepared by fermenting the crude oil lysozyme solution. In the present invention, the fermented product of the crude oil degrading solution may be referred to as a fermented milk extract.

The yeast lyophilized lysate may be prepared by adding an enzyme to the yeast lyophilis and enzymatically digesting it. The fermentation may be performed by inoculating bacillus, lactic acid bacteria or yeast into the yeast lyophilized lysate.

At this time, as the enzyme, one or more selected from the group consisting of a sugar chain enzyme, a proteolytic enzyme, a fibrinolytic enzyme, a pectinolytic enzyme, a cellulase and a xylanase may be used.

In addition, during the fermentation, the Bacillus is Bacillus subtilis (Bacillus subtilis) may be used to, lactic acid bacteria are Streptococcus Thermo filler's (Streptococcus thermophilus), Lactobacillus Bulgaria kusu (Lactobacillus bulgaricus , Lactobacillus < RTI ID = 0.0 > acidphilus , Lactobacillus < RTI ID = 0.0 > plantarum , Lactobacillus < RTI ID = 0.0 > casei , Lactobacillus fermentum and Pediococcus spp. pentosaceus ), and the yeast may be selected from the group consisting of Saccharomyces cerevisiae cerevisiae ) can be used.

In one embodiment, the fermentation product of the crude enzyme digest

a) heat-treating the hulls;

b) Enzymatic degradation by adding an enzyme to the cloaca;

c) sterilizing the crude enzyme degrading solution;

d) inoculating bacillus, lactic acid bacteria or yeast into the yeast lyophilized solution and fermenting;

e) sterilizing the fermented fermentation product; And

f) filtration, concentration and drying.

In the present invention, perilla refers to chestnut endothelium surrounding chestnut flesh excluding chestnut husk.

The step (a) is a step of heat-treating the seeds. The seeds may be cooled to 45 to 55 캜 after 10 to 20 times as much water as the seeds is added and heat-treated at 90 캜 or higher for 1 to 3 hours.

Step b) is an enzymatic degradation by adding an enzyme to the yeast, wherein the yeast is heat-treated in step a) and the enzyme is added to the yeast solubles in an amount of 1 to 5% by weight based on the solids content and enzymatically degraded at 50 ° C for 14 to 18 hours. have.

The kind of the enzyme used in the above step is not particularly limited, and the above-mentioned kinds can be used. Specifically, cellulase and amylase can be used.

By the above step, a cytoplasmic enzyme degrading solution is prepared.

Step c) is a step of sterilizing the enzymatic degradation solution, and the enzyme digestion solution prepared in step b) may be sterilized at 100 to 150 ° C, specifically at 120 to 130 ° C for 30 to 60 minutes.

The step (d) is a step of inoculating bacillus, lactic acid bacteria or yeast into the yeast lyophilized lysate, wherein the microorganism (bacillus, lactic acid bacteria, yeast) is inoculated with the bacillus, Lt; / RTI > for 1 to 3 days.

The above-mentioned bacillus, lactic acid bacteria or yeast may be any of those conventionally used without limitation. Specifically, the above-mentioned species may be used.

A fermentation product of the hydrolyzate is prepared by the above steps.

Step e) is a step of sterilizing the fermentation product, and the fermentation product prepared in step d) may be sterilized at 90 ° C or more and 50 to 100 rpm for 1 to 2 hours.

The step f) is a step of filtration, concentration and drying. The sterilized fermentation broth can be filtered, concentrated and dried by the step e) to prepare a powder of the fermentation product of the hydrolyzate.

In the above step, a filtration aid may be further added to the filtrate. A filter paper can be used for the filtration, and a method such as a reduced pressure filtration or a pressure filtration can be used.

Concentration can be carried out by concentrating the fermentation broth at a reduced pressure of 5 to 10 vol%, and drying can be performed by spray drying or freeze-drying.

An exemplary method of preparing the fermented product powder of the enzymatic degradation solution according to the present invention will be described in detail as follows.

Step a)

To 20 kg of a constant weight of 2 kg of dry noodle (chestnut endothelium), the mixture was mixed in a 30 L fermenter, and heat-treated at 95 ° C and 100 rpm for 1 hour and cooled to 50 ° C.

Step b)

Cellulase and amylase were added to the cooling solution of step a) in an amount of 1% by weight based on the solids content, respectively, and enzymatically degraded at 50 DEG C for 16 hours to obtain a crude enzyme solution.

Step c)

The enzyme-degraded solution obtained in step b) was recovered and sterilized at 121 캜 for 30 minutes.

Step d)

In step c), the sterilized enzyme-degrading solution was inoculated with bacillus, lactic acid bacteria or yeast at a concentration of 2%, and cultured at 37 DEG C for 2 days to obtain a fermented product.

Step e)

The fermentation obtained in step d) was sterilized at 95 DEG C and 100 rpm for 1 hour.

Step f)

The bactericidal fermentation broth of step e) was filtered (≤ 1 mu m). The filtered yeast fermentation broth was concentrated under reduced pressure to 5 to 10 vol% of the volume of the filtrate, and the concentrate was dried to obtain a yeast broth fermentation broth. Drying can be by spray drying or freeze drying.

In one embodiment of the present invention, the composition and food composition for preventing or treating obesity according to the present invention can be used for prevention, improvement and treatment of fat accumulation type obesity.

The term " obesity " in the present invention refers to a state in which fat is generally fat and means a state in which body fat is excessively accumulated.

The type of obesity can be classified according to the cause, the number and size of fat cells, and the area. Depending on the number and size of adipocytes, fat-proliferating obesity and fat-accumulating obesity can be divided into obesity. Fat-growing obesity is an obesity that increases the number of fat cells due to congenital factors. Is a form in which adipose cells are filled with fat due to acquired factors and the size of adipocytes is increased.

On the other hand, adipocytes play a central role in the synthesis, storage and degradation of energy in the form of triglycerides (TG). That is, the amount of calories stored in the form of triglyceride in the case of excessive calorie is decomposed when the calorie consumption is large, and is used as energy. In order to maintain the energy balance of the fat cells, various enzymes and regulatory factors involved in lipogenesis and lipolysis are required. That is, obesity is caused by a failure to maintain balance in the body by the above-mentioned enzymes, which causes excessive accumulation of adipose tissue.

The adipose tissue can be classified into white adipose tissue (WAT) and brown adipose tissue (BAT) according to anatomical location, morphological structure, function and regulation. These two adipose tissues have a common tendency to store energy in the form of triglycerides, but in the case of white fats, this energy is released as needed, and in the case of brown fats, this energy source is converted into heat. The brown fat is distributed more blood vessels than the white fat, and is brown due to the cytochrome pigment, and plays an important role in controlling the body temperature of the hibernating mammal, especially when hibernating. In contrast, white fat plays a pivotal role in maintaining energy homeostasis in the body. Specifically, it stores energy in the form of triglycerides when the amount of energy absorption is greater than consumption, and controls the use of energy sources in case of lack of energy I am responsible. Thus, the distribution and function of the two adipocytes are different, and white adipocytes, commonly referred to as adipocytes, are more commonly distributed subcutaneously and around the organs and are more closely related to obesity as they act as reservoirs of fuel .

The term "mature adipocytes" in the present invention means a form of complete adipocyte produced in the differentiation process of adipocytes, wherein the mature adipocytes are transformed into adipocytes (pre-adipocytes) And the lipid precursor cells are formed through a process of maturation into complete adipocytes.

The term "lipid precursor cells" in the present invention means cells having the ability to differentiate into adipocytes, which are first produced from stem cells during the differentiation stage of adipocytes. When the fat precursor cells are grown to 100% confluency, the growth is stopped and the differentiation into adipocytes progresses slowly. The process by which lipid precursor cells are differentiated into adipocytes is a phenomenon caused by differentiation inducers in the serum added to the culture medium. Therefore, the addition of differentiation-promoting factors (insulin, dexamethasone, 3-isobutyl-1-methylxanthine) to the culture medium can further promote differentiation of cells. On the other hand, differentiation inhibitors (actinomycin D, tumor necrosis factor α, bromo deoxyuridine, etc.) may be added to inhibit differentiation. Finally, the differentiated lipid precursor cells become mature adipocytes such as accumulation of intracellular fat, and the activation of enzymes inducing adipocyte-specific gene expression and fat accumulation occurs. As a representative example of promoting adipocyte differentiation, insulin-like growth factor-I (IGF-I) and the like have been known so far (Hwang, Hyejung, et al., Effect of alginate on differentiation of 3T3- , Journal of the Korean Fisheries Society 33 (6), 541-545, 2000).

In one embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of obesity comprising a fermented product of a crude enzyme decomposition liquid as an active ingredient.

The fermentation product of the hydrolyzate can be prepared by the method described above and may be in the form of a pharmaceutically acceptable salt.

The salt may be an acid addition salt formed using an organic or inorganic acid. The organic acid may be selected from the group consisting of formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic acid monoamide, glutamic acid, Examples of the inorganic acid include hydrochloric acid, gluconic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxyacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or methanesulfonic acid salt, , Bromic, sulfuric, phosphoric, nitric, carbonic or boric acid based salts. Specifically, it may be in the form of a hydrochloride or an acetate, and more specifically, in the form of a hydrochloride.

Forms which are additionally capable of being additionally salified include, but are not limited to, the salts of gabapentin, gabapentin, pregabalin, nicotinate, adipate, hemimarate, cysteine, acetylcysteine, methionine, arginine, Or aspartate.

In addition, the pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier. The carrier may contain various components such as buffer, injectable sterile water, normal saline or phosphate buffered saline, sucrose, histidine, salt and polysorbate.

The content of the fermentation broth in the composition may be appropriately adjusted according to the symptoms of the disease, the progress of the symptoms, the condition of the patient, the administration method, the form of the preparation, etc. For example, To 10 parts by weight, or 0.001 to 1 part by weight. The content ratio is a value based on the dried amount from which the solvent is removed.

The pharmaceutical composition may be administered orally or parenterally, and may be administered in the form of a general pharmaceutical preparation, for example, in a variety of oral and parenteral formulations upon clinical administration. In the case of formulation, the filler, And may be prepared using diluents or excipients such as binders, wetting agents, disintegrating agents, and surfactants.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like can be prepared.

In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of liquid formulations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are commonly used simple diluents.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

In one embodiment, the present invention provides a food composition for improving obesity comprising a fermented product of a crude enzyme degrading solution as an active ingredient.

The term " food composition " in the present invention means a food prepared by adding a fermented product of the hydrolyzed solution to a food material such as a beverage, a tea, a spice, a gum or a confection or by encapsulating, When taken, it means bringing about a certain effect on health, but unlike general medicine, there is an advantage that there is no side effect that can occur when a medicine is taken for a long time by using food as a raw material.

There is no particular limitation on the kind of the food. Examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, and includes foods in a conventional sense.

Since the food composition according to the present invention thus obtained can be routinely ingested, a high anti-obesity effect can be expected, which is very useful.

When the fermented product of the crude oil degraded solution is used as a food additive, the fermented product of the crude oil degraded solution can be directly added or used together with other food or other food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed may be within a range that does not impair the inherent taste of the food, and is usually 0.01 to 50 parts by weight or 0.1 to 20 parts by weight based on 100 parts by weight of the total composition. In the case of foods in the form of granules, tablets or capsules, they may be added usually in the range of 0.1 to 100 parts by weight, or 0.5 to 80 parts by weight. However, in the case of long-term consumption intended for health or hygiene purposes or for health control purposes, 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 exceeding the above range.

The food composition may further contain various flavors or natural carbohydrates. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau Martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. In addition to the above, the food composition according to the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the food composition of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings, in which: FIG. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and is defined only by the scope of the claims of the invention.

Example  One: Yeast  origin Julie  Enzyme decomposition solution Fermentation product  Produce

After 20 kg of water was added to 2 kg of UFI, the mixture was mixed in a 30 L fermenter, heat treated at 95 ° C and 100 rpm for 1 hour, and then cooled to 50 ° C.

20 g of each of the cellulase and amylase was dissolved in 200 ml, and the mixture was added to the fermenter. The mixture was reacted at 50 ° C for 16 hours and then sterilized at 121 ° C for 30 minutes.

After cooling to 37 ° C, yeast ( saccharomyces cerevisiae ) was added to the fermenter and cultured at 50 rpm for 36 hours. After completion of the incubation, the cells were sterilized at 95 DEG C for 1 hour.

The mixture was cooled to 50 DEG C, filtered, concentrated, and spray-dried to obtain a fermentation product 620 g was obtained.

Example  2: Julie  Enzyme decomposition solution Fermented  Fat accumulation Low performance

3T3-L1 (mouse embryo fibroblast cell line) was cultured in a 6 well plate at a concentration of 2 × 10 ⁵ cells / well, and the cells were further cultured for 2 days after reaching 100% confluency and then differentiated into mature adipocytes The medium was removed and replaced with 0.5 mM 3-isobutyl-1-methylxanthine, IBMX, 0.1 uM dexamethasone, DEX, 1 ug / ml insulin and 10% fetal bovine serum (0.5 mg / ml) was added to DMEM supplemented with fetal bovine serum (FBS). In the control group, 10 μl of distilled water was added to the DMEM medium, and 0.1 mg / ml of caffeine was added to the positive control group. The cultivation was carried out in a CO ₂ incubator at 37 ° C for 48 hours. After the culture, the original medium was removed again, and the DMEM medium supplemented with 10% fetal bovine serum (FBS) was incubated for 48 hours in a CO ₂ incubator at 37 ° C for 2 times.

After the culture was completed, the culture solution was completely removed, and the cells were washed with phosphate buffer (phosphate buffer, PBS), and the cells were fixed by adding 10% formalin solution (400 μl / well) for 1 hour. After that, a phosphate buffer (phosphate buffer, PBS), and the fat in the adipocyte differentiated for 2 hours was stained with 400 ul / well of Oil red O working solution. After that, the oil red O working solution was removed, and the oil red O working solution on the wall of the well was completely washed using the second distilled water, then dried in the dryer for 5 minutes. After the addition of 500 μl of isopropyl alcohol to the wells, the lipid accumulation was measured by absorbance at 490 nm using a microplate reader (Model 680 microplate reader, Bio-Rad, USA). The fat accumulation capacity was calculated by the following equation (1).

<Formula 1>

Lipid accumulation (%) = [(Sample OD - Blank OD) / (Control OD - Blank OD)] × 100

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the activity of inhibiting lipid accumulation in mature adipocytes of a fermented product of a lipolytic enzyme solution. FIG. In FIG. 1, Control is a control group in which distilled water is added in place of the uri extract, Caffeine is a positive control group in which caffeine is added instead of a tubular extract, A is a group in which a tubular hot water extract is added and B is a yeast ( saccharomyces cerevisiae ), and C was a fermented product of Lactobacillus yulpi of enzyme solution using acidophilus fermented addition group, D is a group of the added fermented yulpi enzyme solution using the fermentation, the addition of the group E yulpi enzyme solution with L. plantarum L. casei is, F is L , fermented with fermentum , and G was added with Bacillus The results of this study are as follows.

As shown in FIG. 1, the fat accumulation capacity of caffeine (0.1 mg / ml) as a positive control was 68%, that of the control group (control) was 100% (A) was 91%, yeast, Lactobacillus acidophilus , L. plantarum , L. casei , L. fermentum , Bacillus The fermented products (B to G) of the hydrolyzate of subtilis were 58%, 71%, 67%, 70%, 70% and 66%, respectively.

In other words, the fermentation products of the lipolytic enzyme showed a similar inhibitory effect on lipid accumulation in the mature adipocytes and the caffeine positive control group. In particular, fermentation of the lipolytic enzyme solution with yeast showed the highest inhibitory effect on the lipid accumulation of mature adipocytes Respectively.

Example  3: Yeast Using Julie  Enzyme decomposition solution Fermented  Fat accumulation by concentration Low performance

Yeast ( saccharomyces In order to investigate the lipid accumulation inhibitory activity in the mature adipocytes of the fermented product of the periplasma lysate with the use of cerevisiae , the extracts were treated with 1.0, 0.5, 0.1 and 0.01 mg / ml, Similarly, adipocytes were differentiated.

2 is a graph showing the activity of inhibiting lipid accumulation in mature adipocytes according to concentration of fermented product of yeast extract using yeast.

As shown in FIG. 2, the fat accumulation capacity of the positive control group (added caffeine instead of the fermented crude fat hydrolyzate) was 76%, the fat accumulation capacity of the fermented hydrolyzate by yeast was 38%, 62 %, 82% and 88%, respectively. The fermented product of the hydrolyzate showed a concentration - dependent inhibition of lipid accumulation and a similar fat - accumulation inhibitory effect as the positive control caffeine.

Example  4: Yeast Using Julie  Enzyme decomposition solution Fermented  Fat accumulation by concentration Inferior  Microscopic observation

Yeast ( saccharomyces In order to observe the lipid accumulation inhibitory effect of the fermented product of Saccharomyces cerevisiae by concentration, the adipocytes were differentiated according to the method of Example 3. Cells cultured for 8 days were stained with Oil red O staining method and adipocytes were observed.

3 is a photomicrograph showing the lipid accumulation inhibitory activity of the fermented product of the yeast extract according to the concentration by yeast. In this experiment, A was the control group, B was the Caffeine 0.1 mg / ml in the positive control group, C was the fermented product of the yeast extract solution 1.0 mg / ml, D was the fermented product of the yeast- ml, E is the fermented product of the yeast extract and 0.1 mg / ml of the yeast extract, and F is the fermented yeast extract solution of 0.01 mg / ml.

As shown in FIG. 3, it can be seen that the area of the red-stained fat is decreased in a concentration-dependent manner depending on the concentration of the fermented product of the yeast-enzyme-decomposing solution.

Example  5: Yeast Using Julie  Enzyme decomposition solution Fermented  Inhibition of transcription factor related to adipocyte differentiation

As in Example 2, yeast ( saccharomyces and CEBP-α and SREBP-1c, which are the major transcription factors involved in adipocyte differentiation, were extracted from the cells obtained by treating and culturing the fermented product of the tuberous lysozyme hydrolyzate with the S. cerevisiae Reverse transcriptase-PCR (RT-PCR) was performed as follows and the degree of expression was compared by agarose electrophoresis.

Specifically, in order to extract the RNA of the differentiated cells, the cultured cells were washed twice with PBS, mixed with Easyblue and chloroform, and centrifuged. RNA was separated from the uppermost layer, precipitated with isopropyl alcohol and centrifuged. The precipitate was dissolved in 75% DEPC-ethanol. After centrifugation again, the precipitated RNA was dissolved in 0.1% DEPC distilled water to extract RNA.

For cDNA synthesis, mix the same amount of Oligo (dT) ₁₅ primer with random hexamers, extracted RNA and RNase-free distilled water, heat-cool, add 5X first-strand buffer, dNTP mix, DTT and reverse transcriptase 42 ° C for 90 minutes, and 70 ° C for 15 minutes. The synthesized first-strand cDNA (Taq DNA polymerase), dNTP, 10X PCR buffer, forward primer and reverse primer were added to PCR tubes. After pre-denaturation at 95 ° C for 5 minutes, denaturation at 95 ° C for 30 seconds and annealing at 50-65 ° C for 30 seconds were performed for 45 cycles, and finally, extension was performed at 72 ° C for 30 minutes. The PCR product was electrophoresed in 2.5% agarose gel containing 0.002% ethidium bromide at 100 V for a period of time, and the degree of expression was confirmed by ultraviolet light. The intensity of the band was analyzed and analyzed by Gelpro software. At this time, β-actin was used as an internal standard.

4 to 6 are graphs showing the inhibitory activity of PPAR-γ mRNA expression (FIG. 4), CEBP-α mRNA expression inhibitory activity (FIG. 5) and SREBP-1c mRNA expression inhibitory activity of fermented yeast extract 6).

In the case of PPAR-γ (FIG. 4), 84% of the caffeine and 1.0%, 0.5, 0.1 and 0.01 mg / ml, respectively, the expression rates were 32%, 36%, 73% and 84%, respectively.

In the case of CEBP-α shown in FIG. 5, caffeine was 76%, yeast fermented product was 53%, 70%, and 84% when treated with 1.0, 0.5, 0.1 and 0.01 mg / And 99%, respectively. In the case of SREBP-1c of FIG. 6, 68% of caffeine and 0.5%, 0.1, and 0.01 mg / ml of fermented yeast- Respectively, and 22%, 55%, 67%, and 81%, respectively.

From these results, yeast-derived fermentation broth of fermented lysozyme degrades transcription factors involved in adipocyte differentiation in a concentration-dependent manner and at the same time inhibits similar expression of caffeine as a positive control (yeast) The mechanism of adipocyte differentiation and lipid accumulation inhibitory activity of the fermented product of the hydrolyzate of the used uricase can be identified at the gene expression level.

Claims (9)

A pharmaceutical composition for preventing or treating obesity comprising primary fermentation with tuberous cellulase and amylase and secondary fermentation using yeast (saccharomyces cerevisiae) as an active ingredient. The pharmaceutical composition for preventing or treating obesity according to claim 1, wherein the obesity is fat-accumulating obesity. The method according to claim 1, wherein the fermented product is first fermented with a cellulase and an amylase and then fermented with yeast (saccharomyces cerevisiae) in an amount of 0.0001 to 10 parts by weight relative to 100 parts by weight of the pharmaceutical composition. Lt; / RTI &gt; A food composition for improving obesity comprising a fermented product of yeast (saccharomyces cerevisiae) firstly fermented with cellulase and amylase, and fermented by fermentation with yeast (saccharomyces cerevisiae) as an active ingredient. [Claim 5] The composition according to claim 4, wherein the fermented product is first fermented with a cellulase and an amylase, and then fermented with yeast (saccharomyces cerevisiae) in an amount of 0.01 to 50 parts by weight relative to 100 parts by weight of the food composition . A method for producing a fermentation product of a crude enzyme according to claim 1, wherein the fermentation product is a fermentation product comprising a primary fermentation with a cellulase and an amylase, and a secondary fermentation with yeast (saccharomyces cerevisiae).
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