KR102163993B1 - Weight loss-specific metabolic syndrome prevention or treatment composition using mixed grain fermentation enzyme - Google Patents

Abstract

The present invention relates to a composition for preventing or treating metabolic syndrome comprising a mixed grain fermentation enzymatic product as an active component. More specifically, the present invention relates to a composition for preventing or treating metabolic syndrome containing a Bacillus coagulans fermentation enzymatic product of mixed grains consisting of brown rice, barley, soybean, corn, wheat, and Coix lacryma-jobi as an active component. The mixed grain fermentation enzymatic product according to the present invention exhibits effects of weight loss, inhibiting fatty liver, alleviating hyperlipidemia, and lowering blood sugar, thereby being able to be very usefully used in developing an agent for preventing or treating metabolic syndrome induced by obesity.

Description

Composition for preventing or treating weight loss-specific metabolic syndrome using mixed grain fermentation enzyme as an active ingredient {Weight loss-specific metabolic syndrome prevention or treatment composition using mixed grain fermentation enzyme}

The present invention relates to a composition for preventing or treating metabolic syndrome specialized in weight loss comprising a mixed grain fermentation enzyme as an active ingredient, and more particularly, a mixed grain consisting of brown rice, barley, soybean, corn, wheat, and adlay It relates to a composition for preventing or treating metabolic syndrome specialized in weight loss, comprising as an active ingredient a fermented enzyme obtained by fermentation with Bacillus coagulans .

Obesity has emerged as one of the world's most serious health problems caused by changes in diet and lifestyle due to industrialization and improved income levels. In 1996, it was defined as a disease by the World Health Organization (WHO). In addition, obesity is known to be directly or indirectly related to the induction of various cancers along with adult diseases such as diabetes, hypertension, hyperlipidemia, and heart disease.

Mechanisms of action of obesity treatments known to date include appetite suppression, heat metabolism promotion, diuresis, digestion suppression, and hormone control. Among them, ReductilTM, the most used obesity treatment, is an appetite suppressant, forming a market of over $100 million a year in the United States. However, appetite suppressants for the treatment of obesity have side effects such as increased blood pressure, dependent diarrhea, constipation, insomnia, and anxiety.Therefore, there is a great interest in the use of various food materials whose safety and physiological functions have been verified for many years to suppress obesity. It is rising. Currently, products containing dietary fiber, herbal medicines and various food ingredients are being developed as health foods for obese people.However, if you reduce the amount of food and consume the above health foods, it is difficult to live a normal life due to a lack of various nutrients. It is known to become obese. Therefore, there is an urgent need to develop a prevention and treatment for obesity that reduces side effects and has excellent safety even during long-term use.

Metabolic syndrome, also known as insulin resistance syndrome, is closely related to diseases such as obesity, type 2 diabetes, high blood pressure, hypertriglyceridemia, hypercholesterolemia, and arteriosclerosis. Currently, the incidence of metabolic syndrome is increasing around the world and is emerging as an important health care problem. According to data from the 2005 National Health and Nutrition Survey, the prevalence of metabolic syndrome (over 30 years old) was 32.3% (male 32.9%, female 31.8%), and the resulting heart disease and stroke ranked second and third causes of death in Koreans. It is occupied, and there is a need to promptly improve the incidence of these diseases by developing control technologies for metabolic syndrome. In particular, the reason that metabolic syndrome is of interest to society is that it causes various vascular diseases due to arteriosclerosis as a complication, leading to early death of patients and deterioration of the quality of life of patients. Therefore, it is very important to detect and treat rapidly increasing metabolic syndrome, and further prevent it.

As a result of repeated intensive research to develop a composition exhibiting the effect of preventing or treating metabolic syndrome of natural materials, the present inventors fermented Bacillus coagulans of mixed grains consisting of brown rice, barley, soybeans, corn, wheat and adlay It was found that the enzyme product has a very excellent effect of improving overall symptoms for metabolic syndrome, and the present invention was completed.

Accordingly, an object of the present invention is a pharmaceutical composition for preventing or treating metabolic syndrome comprising a Bacillus coagulans fermented enzyme product of mixed grains including brown rice, barley, soybean, corn, wheat and adlay as an active ingredient Is to provide.

Another object of the present invention is to provide a food composition for preventing or improving metabolic syndrome comprising a Bacillus coagulans fermented enzyme product of mixed grains including brown rice, barley, soybean, corn, wheat and adlay as an active ingredient Is to do.

In order to achieve the above-described object of the present invention, the present invention is to prevent metabolic syndrome comprising a Bacillus coagulans fermented enzyme product of mixed grains including brown rice, barley, soybean, corn, wheat, and adlay as an active ingredient. Or it provides a therapeutic pharmaceutical composition.

In order to achieve another object of the present invention, the present invention is to prevent or improve metabolic syndrome comprising a Bacillus coagulans fermented enzyme product of mixed grains including brown rice, barley, soybean, corn, wheat and adlay as an active ingredient. It provides a food composition for use.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating metabolic syndrome comprising a Bacillus coagulans fermented enzyme product of mixed grains including brown rice, barley, soybeans, corn, wheat, and adlay as an active ingredient.

According to an aspect of the present invention, the mixed grains are mixed in a weight ratio of 1:0.5 to 1.5:0.3 to 1.5:0.05 to 0.5: 0.05 to 0.5: 0.05 to 0.5 of brown rice, barley, soybean, corn, wheat, and adlay There may be.

Preferably, the mixed grain may be mixed in a weight ratio of 1:0.5 to 1.0:0.3 to 1.0:0.05 to 0.4: 0.05 to 0.4: 0.05 to 0.4 of brown rice, barley, soybean, corn, wheat, and adlay.

More preferably, the mixed grain may be mixed in a weight ratio of 1:0.5 to 1.0:0.3 to 0.8:0.1 to 0.3: 0.1 to 0.3: 0.1 to 0.3 of brown rice, barley, soybean, corn, wheat, and adlay. .

According to one aspect of the present invention, the fermentation enzyme product is prepared by a general fermentation method in which a Bacillus coagulans strain culture solution is inoculated and fermented in a mixed grain containing brown rice, barley, soybean, corn, wheat, and adlay. It may have been.

According to a preferred aspect of the present invention, the fermentation enzyme product may be characterized in that it is prepared by a method comprising the following steps:

(a) preparing a liquid seed culture solution by inoculating a Bacillus coagulans strain on a seed base consisting of rice bran, bran, soybean powder, isolated soy protein and yeast extract powder;

(b) mixing and increasing a base for main culture consisting of rice bran, bran, soybean powder, isolated soy protein and yeast extract powder with the mixed grain; And

(c) adding and fermenting the liquid seed culture solution prepared in step (a) to the increased mixed grain in step (b).

The'seed base' may include soybean powder, rice bran, bran, soybean protein isolate, and yeast extract. Specifically, the seed base is based on 100 parts by weight of purified water, 1 to 5 parts by weight of soybean powder, 1 to 5 parts by weight of rice bran, 1 to 5 parts by weight of bran, 0.5 to 5 parts by weight of soy protein isolate, 0.1 to 5 parts by weight of yeast extract powder It may be 3 parts by weight.

Bacillus coagulans strain is inoculated on the seed base. The inoculated Bacillus coagulans strain may preferably be a strain deposited with accession number KCTC13284BP.

For the inoculation, the pre-culture solution obtained by culturing the strain may be used as it is, an isolated strain may be used, or a powdered strain or a culture thereof may be used in a form such as lyophilization. The inoculation amount of the Bacillus coagulans strain can be inoculated with 5 to 20 parts by weight based on 100 parts by weight of a mixture of the soybean powder, rice bran, bran, isolated soybean protein and yeast extract, preferably 5 to 15 parts by weight, the most Preferably, 7 to 12 parts by weight may be inoculated.

The'liquid seed culture medium' may be specifically liquid cultured. The culture temperature may be 30 ℃ to 40 ℃, the culture time may be 10 hours to 30 hours.

The mixed grains including brown rice, soybeans, barley, wheat, corn, and adlay used in the step (b) may be subjected to an appropriate selection and washing process, followed by an immersion process called water. At this time, it is called soaking in water for 4 to 24 hours for each type of grain, and since characteristics are different depending on the type of grain, the time to soak in water according to the type can be adjusted within the above range. Specifically, brown rice 14 to 16 hours, soybean 5 to 9 hours, barley 3 to 5 hours, and wheat, corn, and barley may be soaked for 14 to 16 hours.

When immersed, the temperature of the water may be specifically 20°C to 35°C, and preferably 25°C to 30°C. This is because a difference in moisture content occurs due to a temperature difference during immersion. The washed and immersed grain may be used as it is in a pulverized form, a powder form, or without a pulverization process.

The base for main culture used in the step (b) may be made of soybean powder, rice bran, bran, soybean protein isolate, and yeast extract. Specifically, the base mixture for the main culture is based on 100 parts by weight of mixed grains including the brown rice, soybeans, barley, wheat, corn, and adlay, 5 to 20 parts by weight of soybean powder, 1 to 10 parts by weight of rice bran, 1 to 1 bran It may be included in 10 parts by weight, 1 to 5 parts by weight of isolated soy protein, and 0.5 to 5 parts by weight of yeast extract powder.

Although it is possible to cultivate strains in grains without the step of increasing the increase, it is possible to provide an environment in which microorganisms can actively grow by destroying the grain cell walls and denaturing the cell walls of grains and proteins by heat treatment. Cost can be reduced by lowering the amount of strain inoculation. The steaming may be performed using various methods known in the art, for example, using steam or superheated steam. Specifically, capital increase can be carried out in two stages. Step 1 is a preliminary steaming step and may include a process of preliminary steaming at 60°C to 110°C for 10 to 60 minutes. Preferably, it may be carried out at 80°C to 120°C, 20 to 40 minutes. The second step is the main steaming step, which may be increased for 10 to 60 minutes at 90°C to 140°C, and preferably may be performed at 100°C to 120°C for 30 to 50 minutes.

In the process of steaming, the grains are increased by controlling the moisture content while checking the spreading conditions. Preferably, grains that are increased in this step to maintain their shape without spreading may be 50 to 90% of the total weight, and more preferably 60 to 80%. This is to ensure that the grain maintains its shape, so that a space is formed between the whole grains to ensure breathability.

It may further include cooling the additionally steamed grain after the steaming. Cooling can proceed naturally after steaming is over, or by increasing the cooling rate, overheating can be prevented and cooling can be made uniformly. Specifically, the cooling temperature may be 35°C to 50°C, preferably 40°C to 45°C. This is hardened when cooled below the above range, and it is difficult to mix, so that it cools to the above temperature range.

In the step (c), the amount of the liquid seed culture medium added is 100 parts by weight of the mixed grain (including brown rice, barley, soybean, corn, wheat, and barley) containing the base mixture for main culture prepared in step (b). It may be 5 to 20 parts by weight, preferably 5 to 15 parts by weight, and most preferably 7 to 12 parts by weight.

The method of'fermentation' is not limited, but may be cultured using, for example, a liquid culture tank, a rotary drum fermentor, or a tray fermentor. In addition to the fermentor, if it is useful for fermentation of grains or mixtures thereof, it may be used in the method of the present application without limitation in its format, and appropriate equipment may be selected and used according to the production scale. The culture temperature may be 20 ℃ to 50 ℃, 30 ℃ to 45 ℃, during the cultivation humidity may be 40% to 90%, preferably 50% to 85%. The incubation time may be 1-72 hours, 12-72 hours, 36-50 hours or 48 hours.

The method for preparing the fermented enzyme product may further include drying the fermented enzyme product after step (c) and then powdering the fermented enzyme product.

The drying step can be carried out by various methods known in the art, but care should be taken because viable bacteria in the fermented enzyme product of the mixed grain may be killed and the enzyme activity may be reduced when drying at an excessively high temperature. Specifically, it is preferable to dry at a low temperature in which the strain of the present application is not killed and enzyme activity is maintained. The drying temperature may be 30°C to 60°C, and preferably 35°C to 45°C.

The drying time may be 12 to 48 hours, preferably 20 to 28 hours. At this time, the dried fermented enzyme product may be dried to have a moisture content of 3% to 20%, and preferably 3% to 7%.

The pulverization process may be pulverized into various sizes according to the intended use of the mixed grain fermentation enzyme product, crushing, crushing, milling, abrating, striking ( pounding), smashing, grating, or other means used to reduce the food material into powder (flour) or fine particles. Specifically, a hammer mill or the like may be used.

After the powdering process, it may further include the step of optionally formulating a powder. The powder obtained by grinding the dried fermented enzyme product may be formed into any one formulation selected from granules, pills, tablets, and beverages.

According to another aspect of the present invention, the fermentation enzyme product may be characterized in that it is prepared by a method comprising the following steps:

(a1) preparing a liquid seed culture solution by inoculating a Bacillus coagulans strain on a seed base consisting of rice bran, bran, soybean powder, isolated soybean protein and yeast extract powder;

(b1) brown rice; barley; Big head; And mixing and increasing the base for main culture consisting of rice bran, bran, soybean powder, isolated soybean protein and yeast extract powder in each grain mix of corn, wheat and adlay;

(c1) the liquid seed culture solution prepared in the step (a1) and the brown rice increased in the step (b1); barley; Big head; And adding and fermenting to each of the grain mixes of corn, wheat and adlay. And

(d1) each of the fermented brown rice; barley; Big head; And mixing a grain mix of corn, wheat, and adlay.

The execution method of steps (a1) to (c1) is the same as in (a) to (c) described above. However, in the step (b), brown rice in the step (b1), unlike the mixture of grains of brown rice, barley, soybeans, corn, wheat, and adlay to increase the volume; barley; Big head; And it is characterized in that the increase after mixing the base for the main culture in each of the grain mix of corn, wheat and adlay.

In the present invention, the grain mix means that corn, wheat, and adlay are mixed in a ratio of 1:0.5 to 2:0.5 to 2.

Likewise, in step (c1), steamed brown rice prepared in step (b1); barley; Big head; And it characterized in that the fermentation after inoculating the liquid seed culture solution prepared in step (a1) to each of the grain mix of corn, wheat, and barley.

In the step (c1), the amount of the liquid seed culture medium added is each steamed brown rice containing the base mixture for the main culture prepared in step (b1); barley; Big head; And it may be 5 to 20 parts by weight, preferably 5 to 15 parts by weight, and most preferably 7 to 12 parts by weight, based on 100 parts by weight of a grain mix including corn, wheat and adlay.

Brown rice in step (d1) after step (c1); barley; Big head; And it mixes the fermented products of each of the grain mix containing corn, wheat and adlay.

In the step (d1), the brown rice; barley; Big head; And a grain mix including corn, wheat and adlay may be mixed in a weight ratio of 1:0.5 to 1.5:0.3 to 1.5:0.3 to 1.5.

Preferably, in the step (d1), the brown rice; barley; Big head; And a grain mix including corn, wheat, and barley may be mixed in a weight ratio of 1:0.5 to 1.0:0.3 to 1.0:0.3 to 1.0.

More preferably, in the step (d1), the brown rice; barley; Big head; And a grain mix including corn, wheat, and adlay may be mixed in a weight ratio of 1:0.5 to 1.0:0.3 to 0.8:0.3 to 0.8.

In another aspect of the present invention, the fermented enzyme product may be prepared by performing the step of mixing in step (d1) after drying and pulverizing each fermented grain prepared in step (c1).

According to an embodiment of the present invention, the mixed grain fermented enzyme product prepared according to the above method has a weight loss effect, blood lipid improvement effect, fatty liver improvement effect, diabetes improvement effect, etc. in an animal model of metabolic syndrome induced by a high fat diet. It was found to be very effective in improving the overall symptoms of metabolic syndrome.

In the present invention, the'metabolic syndrome' may be selected from the group consisting of obesity, diabetes, arteriosclerosis, hypertension, hyperlipidemia, fatty liver, stroke, myocardial infarction, ischemic disease and cardiovascular disease, preferably obesity, diabetes, fatty liver, It may be selected from the group consisting of hyperlipidemia, myocardial infarction and cardiovascular disease, and most preferably selected from the group consisting of obesity, diabetes, fatty liver and hyperlipidemia.

The pharmaceutical composition according to the present invention may contain the mixed grain fermentation enzyme product alone or may be formulated in a suitable form with a pharmaceutically acceptable carrier, and may further contain an excipient or diluent. The carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery materials used for oral administration. In addition, the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol, and the like, and may further include stabilizers and preservatives. Suitable stabilizers are antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and formulations may be referred to as those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration Can be

The pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration according to the route of administration as described above.

In the case of a formulation for oral administration, the composition of the present invention may be formulated using a method known in the art as a powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc. I can. For example, in oral preparations, tablets or dragees can be obtained by mixing the active ingredient with a solid excipient, pulverizing it, adding a suitable auxiliary, and processing into a granule mixture. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Fillers such as celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose, gelatin, and polyvinylpyrrolidone may be included. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.

In the case of a formulation for parenteral administration, it can be formulated in the form of injections, creams, lotions, ointments for external use, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995, a formula generally known for all pharmaceutical chemistry.

The total effective amount of the composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol that is administered for a long time in multiple doses. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg, most preferably 0.1 μg to 500 mg per 1 kg of patient body weight per day. However, the dosage of the pharmaceutical composition is determined by considering various factors such as the age, weight, health condition, sex, disease severity, diet and excretion rate of the patient, as well as the formulation method, route of administration and number of treatments. Therefore, considering these points, those of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention.

Furthermore, the pharmaceutical composition of the present invention can be administered in parallel with a known substance having an effect of preventing or treating metabolic syndrome.

The present invention also provides a food composition for preventing or improving metabolic syndrome comprising a Bacillus coagulans fermented enzyme product of mixed grains consisting of brown rice, barley, soybeans, corn, wheat and adlay as an active ingredient.

The contents related to the fermented enzyme product may be understood with reference to the foregoing.

The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the mixed grain fermented enzyme product itself provided by the present invention may be prepared in the form of tea, juice, and drink to be consumed, or granulated, encapsulated, and powdered to be consumed. In addition, it can be prepared in the form of a composition by mixing with known substances or active ingredients known to have an effect of preventing or improving metabolic syndrome.

In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods thereof (eg, canned fruit, canned food, jam, marmalade, etc.), fish, meat and processed foods thereof (eg, ham, sausage corn beef, etc.) ), breads and noodles (e.g. udon, soba, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, Retort foods, frozen foods, various seasonings (eg, soybean paste, soy sauce, sauce, etc.) can be prepared by adding the fermented enzyme product of the mixed grain of the present invention.

The preferred content of the chorismycin C or a pharmaceutically acceptable salt thereof in the food composition of the present invention is not limited thereto, but may be, for example, 0.001 to 30% by weight of the finally prepared food. Preferably, it may be 0.01 to 20% by weight of the finally prepared food.

In addition, in order to use the mixed grain fermentation enzyme product of the present invention in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate.

The mixed grain fermented enzyme product according to the present invention can be very useful for preventing or developing a therapeutic agent for metabolic syndrome induced by obesity because it exhibits weight loss, fatty liver inhibitory effect, hyperlipidemia improving effect, and blood sugar lowering effect.

Figure 1 is a graph measuring the weight change of the animal over time while administering each test substance to a mouse induced obesity with a high fat diet (HFD) ( ^a,b,c Means values with different superscript letter are significantly different among HFD-fed groups(p<0.05); Mean values are significantly different for ND from those of HFD, *p<0.05, **p<0.01, ***p<0.001.).
Figure 2 is a result of measuring fasting blood glucose after obtaining blood every 3 weeks from a mouse induced obesity by a high fat diet (HFD) ( ^a,b,c Means values with different superscript letter are significantly different among HFD-fed groups(p<0.05); Mean values are significantly different for ND from those of HFD, *p<0.05.).
3 is ^a graph showing changes in energy metabolism according to respiratory rate, carbon dioxide production, and oxygen consumption after administering each test substance to mice inducing obesity with a high fat diet (HFD) for 12 weeks ( ^a,b Means values with different superscript letter are significantly different among HFD-fed groups(p<0.05); Mean values are significantly different for ND from those of HFD, *p<0.05).
Figures 4 to 7 are graphs measuring the level of expression of lipid metabolism-related genes in white adipose tissue around the epididymis and small intestine after 12 weeks of administration of each test substance to a mouse induced obesity with a high fat diet (HFD) ( ^a,b Means values with different superscript letter are significantly different among HFD-fed groups(p<0.05); Mean values are significantly different for ND from those of HFD, *p<0.05).

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are for illustrative purposes only, and the present invention is not limited thereto.

Experiment method

1. Preparation of test material

(1) Manufacturing process of liquid seed culture solution

Seed culture culture: 1.8 ml (0.3%) seed culture stock ( Bacillus coagulans KCTC13284BP) inoculated in 600 mL LB broth (Luria Bertani; tryptone 1%, yeast extract 0.5%, NaCl 1%), and cultured at 37° C. 180 rpm for 4-5 h.

Final volume 5.4L prepared in each of the three incubators in each of the three jars (based on 100 parts by weight of purified water, 2 parts by weight of soybean powder, 2 parts by weight of rice bran, 2 parts by weight of bran, 1 part by weight of isolated soy protein, 0.5 parts by weight of yeast extract) Part) was inoculated into the sterilized liquid seed culture medium containing the above 600 mL of seed culture and cultured at 170 rpm for 14 to 15 hours at 35°C to prepare a seed culture solution adapted to the primary fermentation base grain.

(2) Grain immersion and increase process

A mix of brown rice, barley, soybeans, and grains (corn, wheat, and barley) that had been properly screened and washed was soaked in water. Each type of grain is soaked in water for 4 to 15 hours, and since the characteristics are different depending on the type of grain, the time for soaking in water according to the type was adjusted within the above range. On the other hand, the temperature was maintained at 25 to 30°C because a difference in moisture content occurred due to the temperature difference during immersion.

Each of the brown rice, barley, soybean and grain mix immersed in the above process is placed in a 4 kg tray to supplement proper moisture to promote fermentation of grains, and a secondary fermentation base rich in beneficial ingredients such as various minerals (100 weights of each grain above) Based on parts, 10 parts by weight of soybean powder, 5 parts by weight of rice bran, 5 parts by weight of bran, 2 parts by weight of isolated soy protein, and 1 part by weight of yeast extract) were added to the solid main medium composition and mixed.

Each of the brown rice, barley, soybean and grain mix contained in a tray to which the secondary fermentation base was added was put in a steamer and steamed. In the process of steaming, the grains were increased by adjusting the moisture content while checking the spreading conditions. In this step, it is desirable to increase the amount of grains that do not spread and maintain their shape to be 60 to 80% of the total weight. Specifically, the steaming process was preliminary steaming at 100℃ for 15 minutes, main steaming at 121℃ for 30 minutes, and then cooled to 40~45℃ in the steaming machine.

(3) Inoculation process of seed culture solution

The seed culture solution prepared in step (1) was inoculated into each of the boosted grains prepared in step (2), and 10 parts by weight of the seed culture solution compared to 100 parts by weight of each increased grain were sprayed and inoculated.

(4) Fermentation process

Each steamed grain inoculated with the seed culture solution was fermented under a humidity of 35°C and 70% to prepare a fermented enzyme product for each grain. It was manufactured in a sterile ventilation facility to prevent contamination by other bacteria. In the above process, it was confirmed that the fermentation was completed for 48 hours had the highest enzyme activity, and the fermentation was completed.

(5) Drying and aging process

Each grain fermentation enzyme product formed through the above process was dried and aged in a dry oven at 60° C. to have an appropriate moisture content. Maintaining 5-10% moisture is desirable for the enzyme titer and preservation properties of the product.

(6) Grinding and grain mixing process

Each of the dried and aged grain fermentation enzymes was pulverized with a pin mill grinder (40 mesh 60% passed) and powdered. Each powdered grain fermentation enzyme has a certain ratio (35% of brown rice fermentase powder, 25% of barley fermentase powder, 20% of soybean fermentase powder, and 20% of mixed grains (corn, wheat and barley; 1:1:1) ) Into the mixer and mixed.

2. Animal testing

(1) Classification of experimental group

84 C57BL/6J mice were purchased from Jackson, USA and acclimated to Lab chow for 1 week, and then each of the experimental materials prepared in Experimental Example 1 was added to the high fat diet by dividing the groups as shown in the following table. It was administered weekly. Body weight and dietary intake were measured once a week during the breeding period of mice:

1) ND (Normal diet) group: Low-fat diet (LFD) containing 10 Kcal% fat

2) HFD (High fat diet) group: High-Fat Diet (HFD) containing 40 Kcal% fat

3) BC (Bacillus Coagulans) group: HFD + Bacillus Coagulans 0.3% (wt/wt)

4) RMG (Raw mixed grain) group: HFD + 0.3% (wt/wt) of raw mixed grain

5) L-FMG (Low-dose fermented mixed grain) group: HFD + 0.3% (wt/wt) of fermented enzyme product of mixed grain

6) H-FMG (High-dose fermented mixed grain) group: HFD + 0.9% (wt/wt) of fermented enzyme product of mixed grain

The BC group was Bacillus coagulans powder, inoculated with 0.1 ml (0.1%) seed stock in LB broth (Luria Bertani; tryptone 1%, yeast extract 0.5%, NaCl 1%), cultured, and centrifuged. It was prepared by mixing maltodextrin in the settled pellet and freeze-drying for 48 hours.

The RMG group was prepared by washing 6 kinds of grains (brown rice, barley, soybeans, mixed grains (corn, wheat, and barley)) as a raw material of mixed grains and then hot air drying at 60℃ for 24 hours.

(2) Sample collection and tissue weight measurement

After fasting for 12 hours, the mice were subjected to primary anesthesia through inhalation of isoflurane (Baxter, USA), and fasting blood was collected from the inferior vena cava. The collected blood was collected directly in a test tube treated with heparin, centrifuged at 1,000 Хg, 4° C. for 15 minutes, and then plasma was collected and stored at -70° C. until sample analysis.

In addition, the liver and muscles of each experimental animal were rinsed several times in a phosphate buffered saline (PBS) solution and then dried to measure the weight. Perirenal white adipose tissue, mesentric white adipose tissue, and subcutaneous white adipose tissue were also removed, rinsed in PBS solution, and then dried and weighed.

(3) Blood glucose measurement and glucose tolerance measurement

After fasting for 16 hours at 2-week intervals for 12 weeks, blood was collected through the tail vein, and blood glucose was measured by the glucose oxidase method.

To measure glucose tolerance, 0.5 g of glucose solution per kg body weight was administered intraperitoneally after fasting for 12 hours, and blood was collected through the tail vein after 0, 30, 90, and 120 minutes, respectively. The collected blood was used to compare glucose tolerance by measuring the sugar concentration by the glucose oxidase method.

(4) plasma lipid analysis

1) Total-cholesterol (TC) quantification

For the determination of plasma total cholesterol, a test solution (Asan Pharmaceutical's kit) applied to the enzyme method of Allain et al. (1974) was used.

2) HDL-cholesterol quantification

Plasma HDL-cholesterol (HDL-C) was measured using a test solution for HDL-C measurement (Asan Pharmaceutical's kit). When 100 μL of plasma was taken and treated with 500 μg of sodium phosphotungsten and 1 mg of magnesium chloride, LDL and VLDL containing apo B in lipoprotein were precipitated by the action of phosphotungstic acid and magnesium cation (Warnick, 1982). After centrifugation, the color development reaction was carried out in the same manner as the total cholesterol in HDL remaining in the supernatant, and the absorbance was measured at 500 nm, and quantified by comparison with a standard cholesterol solution (50 mg/dL).

3) NonHDL-C and AI (arteriosclerosis index) calculation

Plasma nonHDL cholesterol concentration was calculated by subtracting HDL-cholesterol concentration from total cholesterol concentration, and arteriosclerosis index and HTR were calculated by the following formula (Yamajaki, 1990).

AI = ([Total-C]-[HDL-C]) / [HDL-C]

(5) Energy expenditure (EE) measurement and calculation

An animal metabolism measuring device (Oxylet; Panlab, Cornella, Spain) was used to measure the energy metabolism rate. After performing calibration with oxygen and carbon to measure the metabolic rate, the flow rate in the cage was adjusted to 3 L/min, and the mouse was placed in a separate metabolic cage and the energy consumption (oxygen intake) for 24 hours was measured to indicate the metabolic rate. Done. The energy metabolism rate was calculated by the following formula.

EE(kcal/day/bodyweight0.75) = VO2Х1.44Х[3.815+(1.232ХVO2/VCO2)]

(6) Analysis of gene expression related to lipid metabolism

In order to analyze the expression level of genes related to lipid metabolism in white adipose tissue around the epididymis and small intestine, real-time PCR was performed after the RNA was isolated and cDNA was synthesized.

1) tissue RNA isolation

After adding 1 mL of TRIzol per 0.1 g of tissue and destroying the cells using a glass homogenizer, chloroform was added and centrifuged at 4° C. and 12,000 Хg for 20 minutes. After taking the supernatant, adding isopropanol, and then centrifuging for 15 minutes at 4°C and 12,000 Хg, the resulting RNA pellet was washed twice with isopropanol, and then centrifuged at 10,000 Хg to remove the supernatant. The RNA pellet was diluted to a concentration of 0.5 to 10 μg/μL and stored at -70°C.

2) cDNA synthesis

First-strand cDNA was synthesized by taking 1 μL of the total RNA isolated previously and using the QuantiTect Resverse Transcription kit (Qiagen, Germany).

3) Real-Time PCR

The template cDNA obtained previously was diluted in RNAse free water to use 1 μg/reaction, and for gene expression analysis, the expression of the target gene was investigated by real-time quantitative PCR using the QuantiTect SYBR Green PCR kit (QIAGEN, Germany). I did. A primer capable of analyzing the expression of each gene was synthesized by Macrogen (Seoul, Korea). For the composition of the reaction solution, 10 μL of SYBR Green, 2 μL of template, and 200 μM of primer were added, and RNAse free water was added so that the final volume was 20 μL, followed by 15 seconds at 94℃, 30 seconds at 58℃, and 30 at 72℃. The reaction was carried out 40 times in 1 cycle of seconds and 15 seconds at 65°C. At this time, the threshold cycle (Ct) displayed by monitoring the fluorescence signal in each cycle was analyzed, and the mRNA expression between each experimental group was quantitatively analyzed with the CFX96 Real time system (Bio-rad, USA). GAPDH was used as an internal transcription marker.

3. Statistical analysis

All experimental results of this study were calculated using the SPSS package program (Statistical Package for the Social Sciences, SPSS Inc., Chicago), one of the computer statistics programs. One-way analysis of variance (ANOVA) was performed for the significance test for the mean difference between each group, and for the difference between multiple groups, a post-test was performed at a level of p<0.05 or higher by Duncan's multiple range test. Student's t-test was performed to test the significance between HFD groups. All results are mean±S.D. It was expressed as (standard deviation).

Experiment result

1. Body weight and tissue weight of high fat diet (HFD)-induced obese mice

Fig. 1 shows the weight change of obese mice with dietary induction during 12 weeks of animal breeding.

Referring to FIG. 1, the weight of the high fat diet group (HFD) was significantly increased compared to the weight of the normal diet group (ND) from week 1 of the test diet to the end of the test, whereby the obesity induction due to the high fat diet was well achieved. I could confirm that I lost.

In the comparison between the high fat diet groups (HFD, BC, RMG, L-FMG, H-FMG), the high-dose functional grain fermented food group (H-FMG) had a significant weight at week 12 compared to the HFD group and the grain raw material group (RMG). It was found that the weight loss effect of the functional fermented grain food group was dose dependent.

The weight of the organs (liver and muscle) harvested after the animal was sacrificed after the 12-week breeding period was completed is converted into a unit weight (100 g body weight), and is shown in Table 1 below.

[Table 1]

^a,b Means values with different superscript letter are significantly different among HFD-fed groups (p<0.05); Mean values are significantly different for ND from those of HFD, ***p<0.001; Mean values are significantly different for FMGs from those of HFD, ?p<0.05.

Referring to Table 1, as a result of comparison between the ND group and the HFD group, it was found that intake of a high fat diet significantly reduced the weight of muscles per unit body weight.

In the comparison between the high fat diet groups, it was found that the liver weight of the H-FMG group was significantly lower than that of the HFD group and the RMG group, and in the comparison of the HFD group and the L-FMG or H-FMG group, compared to the HFD group It was found that the liver and muscle weight of the L-FMG and H-FMG groups were significantly decreased and increased in the L-FMG and H-FMG groups.

[Table 2]

^a,b Means values with different superscript letter are significantly different among HFD-fed groups (p<0.05); Mean values are significantly different for ND from those of HFD, ***p<0.001.

Referring to Table 2, as a result of comparison between the ND group and the HFD group, it was found that the intake of a high fat diet significantly reduced the weight of white fat around the kidney, white mesenteric fat, and white subcutaneous fat per unit body weight.

In the comparison between the high fat diet groups, it was found that the weight of peri-renal fat in the L-FMG and H-FMG groups was significantly lower than in the HFD group, and the weight of mesenteric fat was significantly reduced in the H-FMG group. It was confirmed to be. In addition, it was found that the weight of subcutaneous fat in the H-FMG group was lower than that of the HFD group.

As described above, the L-FMG group and the H-FMG group exhibited anti-obesity activity through weight loss effect, fat liver suppression effect and muscle mass increase effect, weight reduction effect of white fat around kidney, mesenteric white fat, and subcutaneous white fat. It could be confirmed that it was shown.

2. Plasma lipid concentration

The plasma lipid concentration measured through the plasma obtained after sacrifice is shown in Table 3 below.

[Table 3]

^a,b,c Means values with different superscript letter are significantly different among HFD-fed groups (p<0.05); Mean values are significantly different for ND from those of HFD, *p<0.05, **p<0.01.

Referring to Table 3, intake of a high-fat diet includes total cholesterol (TC), high-density cholesterol (HDL-C), non-high-density cholesterol (nonHDL-C), apoprotein A-1 (apo A-1), and low-density cholesterol. The concentration of (LDL-C) was significantly increased.

In comparison between the high fat diet groups, the concentrations of TC, nonHDL, apo A-1 and LDL-C were significantly decreased in the L-FMG and H-FMG groups, and these effects were compared with the BC and RMG groups. It was found to be remarkable.

3. Measurement of blood sugar index

Fasting blood glucose was measured with blood obtained through tail blood sampling every 3 weeks during the breeding period of experimental animals, and the results are shown in FIG. 2.

Referring to FIG. 2, it was found that the fasting blood glucose of the HFD group was significantly higher than that of the ND group from the 3rd week of the test diet to the 12th week of the end of the test. It was confirmed that it rises.

In the comparison between the high-fat diet groups, the fasting blood glucose of the L-FMG group and the H-FMG group was lower than those of the other high-fat diet groups from the 3rd week of receiving the test diet, and in particular, the HFD group, BC It was found that the fasting blood glucose of the L-FMG group and the H-FMG group was significantly lower than that of the group and RMG group.

The concentrations of plasma glucose, insulin, and glucagon were measured through plasma obtained after sacrifice to calculate HOMA-IR, which is a representative type 2 diabetes indicator, and is shown in Table 4 below.

[Table 4]

Mean values are significantly different for ND from those of HFD, *p<0.05.

Referring to Table 4 above, it was confirmed that plasma insulin concentration and HOMA-IR were significantly increased by ingestion of a high fat diet, through which type 2 diabetes, which is affected from obesity, was caused by a long-term experimental diet. I could confirm.

In the comparison between the high fat diet groups, the L-FMG group and the H-FMG group showed significantly lower values in all measurement items compared to other groups.

4. Energy metabolic rate measurement

Fig. 3 shows the change in energy metabolism according to the respiration rate and the amount of carbon dioxide production and oxygen consumption for 24 hours according to the 12-week diet intake.

Referring to FIG. 3, it was confirmed that the amount of carbon dioxide generation and oxygen consumption during the day in the ND group was significantly higher than that of the HFD group.

In the comparison between the high fat diet groups, it was confirmed that the energy consumption of the RMG and H-FMG groups increased significantly compared to the HFD group, and the carbon dioxide production amount of the H-FMG group increased significantly compared to the HFD group.

5. Analysis of gene expression related to lipid metabolism

The expression of genes related to lipid metabolism in white adipose tissue around the epididymis and small intestine was analyzed using Real-Time PCR. As a result of the white adipose tissue around the epididymis, FIG. 4 shows a fatty acid oxidation-related gene, FIG. 5 shows a fat synthesis related gene, and FIG. The expression of genes related to heat generation was shown, and the expression of genes related to lipid absorption and excretion was shown in FIG.

Referring to FIG. 4, in comparison between the high-fat diet groups, PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) and PPARα (Peroxisome proliferator-activated receptor alpha) gene expression levels are increased in the H-FMG group compared to the HFD group. It was confirmed that the expression of SIRT1 (Sirtuin 1) gene was significantly increased. SIRT1 is activated by deacetylating PGC-1α, which increases the production of mitochondria and fatty acid oxidation, which plays a role in energy production, and PGC-1α becomes a co-activator and is known to improve PPARα activity. It was confirmed that grain fermentation enzyme intake contributes to fatty acid oxidation improvement due to the complementary action of these genes.

Referring to FIG. 5, in the comparison between the high fat diet groups, the expression level of the FAS (Fatty acid synthase) gene synthesizing fatty acids in all of the experimental substance groups compared to the HFD group was significantly reduced, and compared to the HFD group, the L-FMG group and H -In the FMG group, the expression of HMGCR (3-Hydroxy-3-Methylglutaryl-CoA Reductase) and SREBP2 (Sterol regulatory element binding protein) genes, which contribute to the increase in cholesterol synthesis, tended to decrease. It was confirmed that it contributed to.

Referring to Figure 6, it was confirmed that the expression of the CIDEA (Cell Death Inducing DFFA Like Effector A) gene, which induces UCP1 (Uncoupling Protein 1) expression and regulates AMPK activity in the HFD group, significantly decreased compared to the ND group.

In comparison between the high-fat diet groups, the expression of the UCP1 (Uncoupling Protein 1) gene and the PRDM16 (PR-domain containing 16) gene that activates UCP1 expression in the L-FMG group and the H-FMG group, compared to the HFD group, generates heat in the mitochondria. Was shown to increase, and the CIDEA gene expression was found to increase in the H-FMG group, so it was confirmed that the intake of mixed grain fermentation enzymes contributed to the promotion of heat generation. In addition, PRDM16 is known to be induced by PPARα, and it was confirmed that the increase of PRDM16 was suggested to be caused by PPARα, which showed an increasing tendency in FIG.

Referring to FIG. 7, it was confirmed that the expression of ApoB48 (Apolipoprotein B-48) gene, which is an apoprotein present in ultra-low density and low density lipoproteins, was significantly increased in the HFD group compared to the ND group. Since ApoB48 is known to play a role in transporting fat molecules to tissues, it suggests that the absorption of fat from the small intestine to tissues of the HFD group was increased compared to that of the ND group.

In comparison between the high fat diet groups, the expression levels of PPARα and FATP4 (Long-chain fatty acid transport protein 4) genes tended to decrease in the L-FMG and H-FMG groups compared to the HFD group, and the ApoB48 gene expression level was significantly decreased. Was confirmed. PPARα is known as a high-level factor regulating ApoB48 and FATP4 (long-chain fatty acid transport protein 4), which are responsible for transporting fat molecules to tissues. In addition, it was confirmed that the expression level of the ABCG8 (ATP Binding Cassette Subfamily G Member 8) gene that transports and excretes cholesterol in the H-FMG group compared to the HFD group increased. Therefore, it was confirmed that the intake of fermented enzyme products of mixed grains contributed to the reduction and increase of lipid absorption and excretion in the body.

The fermented enzyme product of mixed grain according to the present invention exhibits weight loss, fatty liver inhibitory effect, hyperlipidemia improvement, and blood sugar lowering effect, so it can be very usefully used in the prevention or development of a therapeutic agent for metabolic syndrome induced by obesity, and thus industrial applicability Is high.

Claims (12)

A group consisting of obesity, diabetes, arteriosclerosis, hypertension, hyperlipidemia, and fatty liver, containing as an active ingredient a Bacillus coagulans fermented enzyme product of mixed grains including brown rice, barley, soybeans, corn, wheat and adlay A pharmaceutical composition for preventing or treating metabolic syndrome selected from.
The method of claim 1, wherein the mixed grain is mixed in a weight ratio of 1:0.5 to 1.5:0.3 to 1.5:0.05 to 0.5:0.05 to 0.5:0.05 to 0.5 of brown rice, barley, soybean, corn, wheat, and adlay. Pharmaceutical composition characterized by.
The pharmaceutical composition according to claim 1, wherein the fermentation enzyme product is prepared by a method comprising the following steps:
(a) preparing a liquid seed culture solution by inoculating a Bacillus coagulans strain on a seed base consisting of rice bran, bran, soybean powder, isolated soy protein and yeast extract powder;
(b) mixing and increasing a base for main culture consisting of rice bran, bran, soybean powder, isolated soy protein and yeast extract powder with the mixed grain; And
(c) adding and fermenting the liquid seed culture solution prepared in step (a) to the increased mixed grain in step (b).
The pharmaceutical composition according to claim 3, wherein the amount of the liquid seed culture medium added in step (c) is 5 to 20 parts by weight based on 100 parts by weight of the increased mixed grain.
The pharmaceutical composition according to claim 3, further comprising the step of (d) drying and powdering the fermented enzyme fermented in step (c) after step (c).
The method of claim 3, wherein the seeding base of step (a) is based on 100 parts by weight of purified water, 1 to 5 parts by weight of soybean powder, 1 to 5 parts by weight of rice bran, 1 to 5 parts by weight of bran, 0.5 to 5 parts by weight of isolated soy protein. 5 parts by weight, a pharmaceutical composition, characterized in that the yeast extract powder 0.1 to 3 parts by weight.
The method of claim 3, wherein the base for main culture in step (b) is based on 100 parts by weight of the mixed grain, 5 to 20 parts by weight of soybean powder, 1 to 10 parts by weight of rice bran, 1 to 10 parts by weight of bran, and separated soybeans Pharmaceutical composition, characterized in that 1 to 5 parts by weight of protein, 0.5 to 5 parts by weight of yeast extract powder.
The pharmaceutical composition according to claim 1, wherein the Bacillus coagulans strain is deposited under the accession number KCTC13284BP.
delete A group consisting of obesity, diabetes, arteriosclerosis, hypertension, hyperlipidemia, and fatty liver, containing as an active ingredient a Bacillus coagulans fermented enzyme product of mixed grains including brown rice, barley, soybeans, corn, wheat and adlay Food composition for preventing or improving metabolic syndrome selected from.
The food composition according to claim 10, wherein the fermentation enzyme product is prepared by a method comprising the following steps:
(a) preparing a liquid seed culture solution by inoculating a Bacillus coagulans strain on a seed base consisting of rice bran, bran, soybean powder, isolated soy protein and yeast extract powder;
(b) mixing and increasing a base for main culture consisting of rice bran, bran, soybean powder, isolated soy protein and yeast extract powder with the mixed grain; And
(c) adding and fermenting the liquid seed culture solution prepared in step (a) to the increased mixed grain in step (b).
The food composition according to claim 11, further comprising the step of (d) drying and powdering the fermented enzyme fermented in step (c) after step (c).

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