CN113797321A

CN113797321A - Composition containing mixed grain fermentation enzyme as effective component for preventing or treating weight loss specific metabolic syndrome

Info

Publication number: CN113797321A
Application number: CN202011153797.0A
Authority: CN
Inventors: 慎慵哲; 朴哲; 申大根; 金爱香; 金垠玲
Original assignee: Amicogen Inc
Current assignee: Amicogen Inc
Priority date: 2020-06-11
Filing date: 2020-10-26
Publication date: 2021-12-17
Also published as: KR102163993B1

Abstract

The present invention relates to a composition for preventing or treating metabolic syndrome, which comprises a mixed grain fermentation enzyme as an active ingredient, and more particularly, to a composition for preventing or treating metabolic syndrome, which comprises a bacillus coagulans fermentation enzyme of a mixed grain consisting of brown rice, barley, soybean, corn, wheat, and pearl barley as an active ingredient. The mixed grain fermentation enzyme of the present invention exhibits a weight-loss, fatty liver-inhibiting effect, a hyperlipemia-ameliorating effect, and a blood glucose-lowering effect, and therefore can be effectively used for the development of a prophylactic or therapeutic agent for metabolic syndrome induced by obesity.

Description

Composition containing mixed grain fermentation enzyme as effective component for preventing or treating weight loss specific metabolic syndrome

Technical Field

The present invention relates to a composition for preventing or treating Weight loss specific metabolic syndrome (Weight-specific metabolic syndrome) comprising a mixed grain fermentation enzyme as an active ingredient, and more particularly, to a composition for preventing or treating Weight loss specific metabolic syndrome comprising a fermentation enzyme obtained by fermenting a mixed grain composed of brown rice, barley, soybean, corn, wheat, and barley using Bacillus coagulans as an active ingredient.

Background

Obesity has emerged as a result of changes in dietary and lifestyle habits with increased levels of industrialization and income, and has become one of the most serious health problems in the world and was prescribed as a disease by the World Health Organization (WHO) in 1996. Also, obesity is known to be directly or indirectly associated with various cancers and adult diseases such as diabetes, hypertension, hyperlipidemia, heart disease, and the like.

The action mechanisms of the obesity therapeutic agents known so far include appetite suppression, promotion of heat metabolism, diuresis, digestion suppression, hormone regulation, and the like. Among them, nominatine (Reducitm, Yapei Co., U.S.A.), which is the most commonly used therapeutic agent for obesity, forms a market for more than 1 billion dollars per year in the U.S.A. However, appetite suppressants for obesity have side effects such as elevation of blood pressure, dependence diarrhea, constipation, insomnia, anxiety, etc., and in order to suppress obesity, attention has been greatly paid to the use of various food materials whose safety and physiological functions have been verified for a long time. Although products containing dietary fibers, crude drugs, and various food materials are being developed as health foods for obese people, it is known that when the amount of a meal is reduced and the health foods are ingested, it is difficult to perform normal life because of a lack of various nutrients, and when the intake is interrupted, the life is rapidly rebounded. Therefore, there is an urgent need to develop an obesity preventive and therapeutic agent that reduces side effects and has excellent safety even when taken for a long period of time.

Metabolic syndrome (metabolic syndrome), also known as insulin resistance syndrome, is also closely associated with diseases such as obesity, type 2 diabetes, hypertension, hypertriglyceridemia, hypercholesterolemia, arteriosclerosis, and the like. Currently, the incidence of metabolic syndrome is increasing worldwide, becoming an important health care medical problem. According to the 2005 korean national health nutrition survey, the prevalence rate of metabolic syndrome (30 years old or older) was 32.3% (male 32.9%, female 31.8%) in general, and thus heart disease and cerebral stroke were ranked second and third among korean death causes, respectively, and it was necessary to improve the morbidity of these diseases as soon as possible by developing metabolic syndrome control techniques. In particular, metabolic syndrome is attracting social attention as a result of early death of patients and a reduction in quality of life of patients due to a variety of vascular diseases caused by arteriosclerosis as complications. Therefore, it is very important to discover and treat the rapidly increasing metabolic syndrome early and further prevent it.

Disclosure of Invention

As a result of intensive studies to develop a composition of natural materials showing a preventive or therapeutic effect on metabolic syndrome, the present inventors have found that a bacillus coagulans fermentation enzyme of mixed grains composed of brown rice, barley, soybean, corn, wheat, and pearl barley has a very excellent effect in improving the overall symptoms of metabolic syndrome, and thus have completed the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating metabolic syndrome, comprising bacillus coagulans fermentation enzyme of mixed grains as an active ingredient, wherein the mixed grains comprise brown rice, barley, soybean, corn, wheat, and pearl barley.

Another object of the present invention is to provide a food composition for preventing or improving metabolic syndrome, comprising bacillus coagulans fermenting enzyme of mixed grains as an active ingredient, wherein the mixed grains comprise brown rice, barley, soybean, corn, wheat and coix seed.

In order to achieve the above objects, the present invention provides a pharmaceutical composition for preventing or treating metabolic syndrome, comprising bacillus coagulans fermenting enzyme of mixed grains as an active ingredient, wherein the mixed grains comprise brown rice, barley, soybean, corn, wheat and pearl barley.

In order to achieve another object of the present invention, the present invention provides a food composition for preventing or improving metabolic syndrome comprising bacillus coagulans fermenting enzyme of mixed grains as an effective ingredient, wherein the mixed grains comprise brown rice, barley, soybean, corn, wheat and pearl barley.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating metabolic syndrome, comprising bacillus coagulans fermenting enzyme of mixed grains as an effective ingredient, wherein the mixed grains comprise brown rice, barley, soybean, corn, wheat and coix seed.

According to an embodiment of the present invention, in the mixed grain, the ratio of 1: 0.5 to 1.5: 0.3 to 1.5: 0.05 to 0.5: 0.05 to 0.5: 0.05-0.5 weight ratio of brown rice, barley, soybean, corn, wheat and coix seed.

Preferably, in the above mixed grain, the ratio of 1: 0.5 to 1.0: 0.3 to 1.0: 0.05 to 0.4: 0.05 to 0.4: 0.05-0.4 weight ratio of brown rice, barley, soybean, corn, wheat and coix seed.

More preferably, in the above mixed grain, the ratio of 1: 0.5 to 1.0: 0.3 to 0.8: 0.1 to 0.3: 0.1 to 0.3: 0.1-0.3 weight ratio of brown rice, barley, soybean, corn, wheat and coix seed.

According to an embodiment of the present invention, the fermentation enzyme may be prepared by a conventional fermentation method, i.e., inoculating a bacillus coagulans strain culture solution to a mixed grain including brown rice, barley, soybean, corn, wheat, and pearl barley, and then culturing and fermenting the same.

According to a preferred embodiment of the present invention, the present invention is characterized in that the fermentation enzyme is prepared by a method comprising the steps of:

inoculating a bacillus coagulans strain in a strain matrix consisting of rice bran, wheat bran, soybean powder, isolated soybean protein and yeast extract powder to prepare a liquid strain culture solution;

mixing a main culture medium consisting of rice bran, wheat bran, soybean powder, isolated soybean protein and yeast extract powder with the mixed grain, and steaming; and

and (c) adding the liquid seed culture broth prepared in the step (a) to the cooked mixed cereal of the step (b) and fermenting.

The "strain matrix" may comprise soybean powder, rice bran, wheat bran, isolated soybean protein, and yeast extract. Specifically, the seed culture medium may include 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 wheat bran, 0.5 to 5 parts by weight of isolated soybean protein, and 0.1 to 3 parts by weight of yeast extract powder, based on 100 parts by weight of purified water.

Inoculating the bacillus coagulans strain in the strain matrix. Preferably, the above inoculated Bacillus coagulans strain may be the strain deposited under deposit number KCTC13284 BP.

For the inoculation, a preculture solution in which the above-mentioned strain is cultured may be used as it is, or an isolated strain, a strain powdered in a freeze-dried form or the like, or a culture thereof may be used. The amount of the bacillus coagulans strain to be inoculated may be 5 to 20 parts by weight, preferably 5 to 15 parts by weight, and most preferably 7 to 12 parts by weight, to 100 parts by weight of the mixture of the soybean powder, rice bran, wheat bran, isolated soybean protein, and yeast extract.

The "liquid seed culture medium" may be a liquid culture medium. The above-mentioned incubation temperature may be 30 ℃ to 40 ℃ and the incubation time may be 10 hours to 30 hours.

The mixed grain including brown rice, soybean, barley, wheat, corn and pearl barley used in the above step (b) may be obtained by a dipping process in water after suitable screening and washing processes. In this case, the grains may be soaked in the water for 4 to 24 hours depending on the kind of grains, and the soaking time in the water may be adjusted within the above range depending on the kind of grains because the characteristics are different depending on the kind of grains. Specifically, brown rice can be soaked for 14 to 16 hours, soybean can be soaked for 5 to 9 hours, barley can be soaked for 3 to 5 hours, and wheat, corn and coix seed can be soaked for 14 to 16 hours.

The water temperature at the time of impregnation may be specifically 20 ℃ to 35 ℃, and preferably, may be 25 ℃ to 30 ℃. This is because the moisture content varies due to the temperature difference during immersion. The washed and impregnated cereal can be used in a pulverized form, a powdered form or as such without a pulverization process.

The main culture medium used in the step (b) may be composed of soybean powder, rice bran, wheat bran, isolated soybean protein, and yeast extract. Specifically, the main culture medium mixture may include 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 wheat bran, 1 to 5 parts by weight of isolated soybean protein, and 0.5 to 5 parts by weight of yeast extract powder, with respect to 100 parts by weight of the mixed grain including brown rice, soybean, barley, wheat, corn, and pearl barley.

Although the strain can be cultured in the grain without a cooking step, the heat treatment can kill the infectious microbes in the grain and simultaneously destroy the cell walls of the grain, so that gelatinization and protein denaturation are realized, and an environment in which microorganisms can be actively bred is provided, thereby reducing the strain inoculation amount and reducing the cost. The above cooking may be performed using various methods known in the art, for example, using steam (steam) or superheated steam (superheated steam). Specifically, the cooking may be carried out in 2 steps. The first step is a precooking step, which may include a process of precooking at 60 ℃ to 110 ℃ for 10 minutes to 60 minutes. Preferably, it may be carried out at 80 to 120 ℃ for 20 to 40 minutes. The second step is a main cooking step, which may be performed at 90 to 140 ℃ for 10 to 60 minutes, preferably, at 100 to 120 ℃ for 30 to 50 minutes.

In the cooking process, the grains are cooked while adjusting the moisture content while confirming the swollen state of the grains. Preferably, in this step, the grains that have not swelled while maintaining the original form after cooking may account for 50% to 90%, more preferably 60% to 80%, relative to the total weight. This is to maintain the grains in their original shape, thereby forming spaces between the whole grains to ensure air permeability.

The cooking may be followed by a step of cooling the cooked cereal. As for the cooling, natural cooling may be performed after the completion of the cooking, or overheating may be prevented and cooling may be performed uniformly by increasing the cooling rate. Specifically, the cooling temperature may be 35 ℃ to 50 ℃, preferably 40 ℃ to 45 ℃. This is because when cooling below the above range, the material becomes hard and is difficult to mix, and thus cooling to the above temperature range.

In the step (c), the liquid seed culture solution may be added in an amount of 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 the mixed grain (including brown rice, barley, soybean, corn, wheat, and pearl barley) including the main culture medium mixture prepared in the step (b).

The method of the above-mentioned "fermentation" is not limited, and for example, the culture may be carried out using a liquid culture tank, a rotary drum fermenter (rotary fermenter), or a tray fermenter (tray fermenter). In addition to the above-described fermentation tank, as long as it is useful for fermentation of grains or a mixture thereof, it can be used in the method of the present application without being limited to its form, and an appropriate apparatus can be selected and used depending on the production scale. The culture temperature may be 20 ℃ to 50 ℃, 30 ℃ to 45 ℃, and the humidity during the culture 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 fermentation enzyme may further comprise a step of drying the fermentation enzyme and powdering the dried fermentation enzyme after the step (c).

The drying step may be performed by various methods known in the art, but may kill viable bacteria in the mixed grain fermentation enzyme and may decrease the activity of the enzyme when dried at an excessively high temperature, and thus it should be noted. Specifically, it is preferable to perform drying at a low temperature that does not kill the strain of the present application and maintains the enzyme activity. The above drying temperature may be 30 to 60 ℃, preferably 35 to 45 ℃.

The drying time may be 12 hours to 48 hours, preferably 20 hours to 28 hours. In this case, the dried fermentation enzyme may be dried to a moisture content of 3% to 20%, preferably 3% to 7%.

In the above-mentioned pulverizing process, it may be pulverized into various sizes according to the intended purpose of the above-mentioned mixed grain fermentation enzyme, including crushing (grinding), grinding (milling), grinding (abrating), beating (smashing), mashing (mashing), grinding (grating) or other different means for reducing the size of the above-mentioned food material into powder (powder) or fine particles. Specifically, a hammer mill (hammer mill) or the like can be used.

After the above-mentioned powdering process, a step of optionally formulating the powder into a dosage form may be further included. The powder obtained by pulverizing the fermented enzyme after drying and aging may be formed into one dosage form selected from the group consisting of granules, pills, tablets and beverages.

According to another embodiment of the present invention, the present invention is characterized in that the above fermentation enzyme can be prepared by a method comprising the steps of:

a step (a1) of inoculating a Bacillus coagulans strain in a strain matrix composed of rice bran, wheat bran, soybean powder, isolated soybean protein and yeast extract powder to prepare a liquid strain culture solution;

a step (b1) of mixing a main culture medium consisting of rice bran, wheat bran, soybean powder, isolated soybean protein and yeast extract powder with each of brown rice, barley, soybean, and a grain mixture containing corn, wheat and pearl barley, and cooking;

a step (c1) of adding the liquid seed culture broth prepared in the step (a1) to each of the cooked brown rice, barley, soybean, and grain mixture including corn, wheat, and pearl barley of the step (b1), and fermenting; and

step (d1), mixing the fermented brown rice, barley, soybean, and grain mixture comprising corn, wheat, and pearl barley.

The steps (a1) to (c1) are performed in the same manner as the steps (a) to (c). However, unlike the process of mixing and cooking the main culture medium in the mixed grains of the brown rice, barley, soybean, corn, wheat and pearl barley in the step (b), the process of mixing and cooking the main culture medium in each of the brown rice, barley, soybean and the grain mixture including the corn, wheat and pearl barley in the step (b1) is characterized in that the cooking is performed.

In the invention, the grain mixture refers to corn, wheat and pearl barley in a ratio of 1: 0.5 to 2: mixed therein in a ratio of 0.5 to 2.

Also, the present invention is characterized in that, in the step (c1), the liquid seed culture solution prepared in the step (a1) is inoculated to each of the cooked brown rice, barley, soybean, and grain mixture including corn, wheat, and pearl barley prepared in the step (b1) and then fermented.

In the step (c1), the liquid seed culture solution may be added in an amount of 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 the grain mixture containing the main culture medium mixture and each of the cooked brown rice, barley, soybean, and corn, wheat, and pearl barley prepared in the step (b 1).

In the above-mentioned step (d1) after the above-mentioned step (c1), the respective fermented products of the brown rice, barley, soybean, and the grain mixture including corn, wheat, and pearl barley are mixed.

In the step (d1), the ratio of 1: 0.5 to 1.5: 0.3 to 1.5: mixing the above brown rice, barley, soybean, and grain mixture comprising corn, wheat and coix seed at a weight ratio of 0.3 to 1.5.

Preferably, in the step (d1) above, the ratio of 1: 0.5 to 1.0: 0.3 to 1.0: mixing the above brown rice, barley, soybean, and grain mixture comprising corn, wheat and coix seed at a weight ratio of 0.3 to 1.0.

More preferably, in the step (d1) above, the ratio of 1: 0.5 to 1.0: 0.3 to 0.8: mixing the above brown rice, barley, soybean, and a grain mixture comprising corn, wheat and pearl barley in a weight ratio of 0.3 to 0.8.

In another embodiment of the present invention, the fermentation enzyme may be prepared by drying and pulverizing each grain fermented product prepared in the step (c1), and then performing a mixing step in the step (d 1).

According to an embodiment of the present invention, it was confirmed that the mixed grain fermentation enzyme prepared according to the above method has a very excellent effect of improving the overall symptoms of metabolic syndrome, and has a weight loss effect, a blood lipid improvement effect, a fatty liver improvement effect, a diabetes improvement effect, and the like, in an animal model of metabolic syndrome induced by high fat diet.

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

The pharmaceutical composition of the present invention may contain the mixed cereal fermentation enzyme alone, or may be formulated in a suitable form together with a pharmaceutically acceptable carrier, and may further contain an excipient or diluent. Such carriers include all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microparticles.

Pharmaceutically acceptable carriers may also include, for example, carriers for oral administration or carriers for non-oral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Meanwhile, various drug delivery substances for oral administration may be included. Also, the carrier for non-oral administration may include water, suitable oil, saline, aqueous glucose solution, ethylene glycol, and the like, and may further include a stabilizer and a preservative. Suitable stabilizers are antioxidants, for example sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl or propyl parabens, and chlorobutanol. In addition to the above ingredients, the pharmaceutical composition of the present invention may further comprise lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents and the like. Other pharmaceutically acceptable carriers and preparations can be found in the following references (Remington's Pharmaceutical Sciences,19th ed., Mack Publishing Company, Easton, Pa., 1995).

The compositions of the present invention may be administered to mammals, including humans, by any method. For example, it may be administered orally or non-orally. Non-oral methods of administration may include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or intrarectal administration.

The pharmaceutical composition of the present invention can be formulated into preparations for oral administration or non-oral administration according to the administration route described above.

In the case of preparations for oral administration, the composition of the present invention may be formulated into powders, granules, lozenges, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like using methods well known in the art. For example, with regard to oral formulations, lozenges or dragees can be obtained by compounding the active ingredient with solid excipients, pulverizing them and processing into a mixture of granules after adding suitable auxiliaries. Examples of suitable excipients include: sugars including lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, and the like; starches, including corn starch, wheat starch, rice starch, potato starch, and the like; celluloses including cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose, etc.; fillers such as gelatin, polyvinylpyrrolidone, and the like. Also, as a disintegrating agent, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as the case may be. Further, the pharmaceutical composition of the present invention may further comprise an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

In the case of a preparation for parenteral administration, it can be formulated into injections, creams, emulsions, ointments for external use, oils, moisturizers, gels, aerosols and nasal inhalants by methods well known in the art. These formulations are described in the literature as a general prescription known in all Pharmaceutical chemistry (Remington's Pharmaceutical Sciences,19th ed., Mack Publishing Company, Easton, PA, 1995)).

The total effective amount of the composition of the present invention can be administered to a patient in a single dose (single dose), and can be administered by a fractionated treatment method (fractionated treatment protocol) administered in multiple doses (multiple dose) for a long period of time. The pharmaceutical composition of the present invention may vary the content of the effective ingredient depending on the degree of the disease. Preferably, the preferred total amount of the pharmaceutical composition of the invention may be from about 0.01 μ g to 10000mg, most preferably from 0.1 μ g to 500mg per day per 1kg body weight of the patient. However, the effective dose of the pharmaceutical composition is determined by considering not only the formulation method, the administration route and the number of treatments but also various factors such as age, body weight, health status, sex, severity of disease, diet and excretion rate of the patient, and thus, considering these factors, one of ordinary skill in the art can determine an appropriate effective dose of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, administration route and administration method as long as the pharmaceutical composition exhibits the effects of the present invention.

Further, the pharmaceutical composition of the present invention may be administered concurrently 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 bacillus coagulans fermenting enzyme of mixed grains as an effective ingredient, wherein the mixed grains comprise brown rice, barley, soybean, corn, wheat and pearl barley.

The contents relating to the fermentation enzymes mentioned above can be understood by referring to the above.

The food composition of the present invention includes all forms of functional foods (functional foods), nutritional supplements (nutritional supplements), health foods (health foods) and food additives (food additives). Food compositions of the above type may be prepared in a variety of forms according to conventional methods well known in the art.

For example, the mixed grain fermentation enzyme provided by the present invention may be used as a health food in the form of tea, juice, or beverage, or in the form of granules, capsules, or powder for ingestion. The composition can be prepared by mixing with a known substance or active ingredient having an effect of preventing or ameliorating metabolic syndrome.

The functional food may be prepared by adding the mixed cereal fermentation enzyme of the present invention to a beverage (including alcoholic beverages), a fruit and its processed food (for example, canned fruit, canned food, jam, orange, or lemon paste), a fish, a meat and its processed food (for example, ham, corn beef sausage, etc.), a bread and a noodle (for example, udon noodle, buckwheat noodle, ramen, pasta, macaroni, etc.), a fruit juice, various beverages, a biscuit, a syrup, a dairy product (for example, butter, cheese, etc.), an edible vegetable oil, margarine, a vegetable protein, a distilled food, a frozen food, various seasonings (for example, doenjang, soy sauce, prepared juice, etc.), or the like.

In the food composition of the present invention, the preferable content of the above-mentioned coriomycin C or a pharmaceutically acceptable salt thereof is not limited thereto, and for example, may be 0.001 to 30% by weight in the finally prepared food. Preferably, it may be contained in the finally prepared food in an amount of 0.01 to 20 weight percent.

In addition, the mixed grain fermentation enzyme of the present invention may be used in the form of a powder or a concentrated solution in order to use it as a food additive.

Effects of the invention

The mixed grain fermentation enzyme of the present invention exhibits a weight-loss, fatty liver-inhibiting effect, a hyperlipemia-ameliorating effect, and a blood glucose-lowering effect, and therefore can be effectively used for the development of a prophylactic or therapeutic agent for metabolic syndrome caused by obesity.

Drawings

Fig. 1 is a graph of the administration of each experimental substance to mice induced obesity by High Fat Diet (HFD) and the measurement of the change in animal body weight over time (a, b, c indicate that there is a significant difference in the values with different superscripts between the HFD groups (p <0.05), and the average value of ND is significantly different from the average value of HFD, # p <0.05, # p <0.01, # p < 0.001.).

Fig. 2 is a result of measuring fasting blood glucose after obtaining blood from mice induced obesity by High Fat Diet (HFD) every 3 weeks (a, b, c indicate that there is a significant difference in values with different superscript letters (p <0.05) between HFD groups, and the average value of ND is significantly different from that of HFD,. p < 0.05.).

Fig. 3 is a graph showing the change in respiration rate and energy metabolism rate based on carbon dioxide production and oxygen consumption after 12 weeks of administration of each test substance to mice induced to be obese by High Fat Diet (HFD) (a, b indicate that values with different superscripts are significantly different in the HFD group (p < 0.05); the average value of ND is significantly different from the average value of HFD,. p < 0.05.).

FIGS. 4 to 7 are graphs measuring the expression rate of genes related to lipid metabolism in white adipose tissues around epididymis and small intestine after administering each of the experimental substances to mice induced to obesity by High Fat Diet (HFD) for 12 weeks (a, b indicate that values having different superscript letters are significantly different in the HFD group (p < 0.05); and the average value of ND is significantly different from the average value of HFD,. p < 0.05.).

Detailed Description

The present invention will be described in detail below with reference to the following examples. However, the following examples are merely to illustrate the present invention, and the present invention is not limited thereto.

Experimental methods

1. Preparation of the test substances

(1) Preparation process of liquid strain culture solution

Culturing strains (Seed culture): 1.8mL (0.3%) of a Stock solution (Stock) of a strain (Bacillus coagulans KCTC13284BP) was inoculated into 600mL of LB broth culture medium (LB broth) (Luria Bertani; tryptone (1%), yeast extract (yeast extract) 0.5%, NaCl (1%), and cultured at 37 ℃ and 180rpm for 4-5 hours.

The 600mL of the above-mentioned seed culture was inoculated into a sterilized liquid seed culture medium containing 5.4L of a final volume of a primary fermentation substrate (2 parts by weight of soybean powder, 2 parts by weight of rice bran, 2 parts by weight of wheat bran, 1 part by weight of isolated soybean protein, 0.5 part by weight of yeast extract per 100 parts by weight of purified water) prepared in each of 3 Jar incubators, and then cultured at 35 ℃ and 170rpm for 14 to 15 hours to prepare a seed culture solution suitable for primary fermentation substrate grains.

(2) Grain steeping and cooking process

Brown rice, barley, soybean, grain mixture (corn, wheat and pearl barley) subjected to appropriate screening and washing processes are used and soaked in water. The grains are soaked in the water for 4-15 hours according to the types of the grains, and the soaking time in the water is adjusted within the range according to the types because the grains have different characteristics according to the types. On the other hand, the temperature is maintained at 25 to 30 ℃ because the difference in moisture content occurs due to the temperature difference during immersion.

The brown rice, barley, soybean and grain mixture soaked in the above step were placed in a tray of 4kg, and supplemented with appropriate water to promote the fermentation of grains, and a secondary fermentation substrate (10 parts by weight of soybean powder, 5 parts by weight of rice bran, 5 parts by weight of wheat bran, 2 parts by weight of isolated soybean protein and 1 part by weight of yeast extract) rich in beneficial components such as various minerals was added to each of the grains in 100 parts by weight to mix them into a solid main culture substrate composition.

And steaming the brown rice, barley, soybean and grain mixture added with the secondary fermentation substrate and placed on a tray in a steamer respectively. During the cooking, the grains were cooked while adjusting the moisture content while confirming the swollen state. In this step, it is preferable that the grain which does not swell after cooking and maintains its original form account for 60 to 80% by weight of the total weight. Specifically, the cooking process is carried out by pre-cooking for 15 minutes at 100 ℃, carrying out main cooking for 30 minutes at 121 ℃, and then cooling to 40-45 ℃ in a cooker.

(3) Inoculation step of Strain culture solution

The seed culture solution prepared in the above step (1) was inoculated into each of the cooked grains prepared in the above step (2), and 10 parts by weight of the seed culture solution was inoculated by spraying to 100 parts by weight of each of the cooked grains.

(4) Fermentation step

Fermenting each steamed grain inoculated with the strain culture solution at 35 deg.C and 70% humidity to prepare fermentation enzyme of each grain. It is prepared in sterile ventilation equipment to prevent contamination by other bacteria. In the above step, since the enzyme activity was highest at 48 hours of fermentation completion, 48 hours was defined as the fermentation completion time.

(5) Drying and aging Process

Each grain fermentation enzyme formed through the above-described steps is dried and matured in a drying oven (Dry oven) at 60 ℃ to have an appropriate moisture content. From the viewpoint of enzyme titer and storage stability of the product, it is preferable to maintain 5 to 10% of water.

(6) Grinding and grain mixing process

Each of the dried and matured cereal fermentation enzymes was pulverized (40mesh 60% pass) and powdered by a pin mill. The powdered grain fermentation enzymes (35% of brown rice fermentation enzyme powder, 25% of barley fermentation enzyme powder, 20% of soybean fermentation enzyme powder, and 20% of mixed grains (corn, wheat and coix seed; 1: 1: 1)) are mixed in a mixer according to a predetermined ratio.

2. Animal experiments

(1) Classification of Experimental groups

84C 57BL/6J mice were purchased from Jackson, USA, and acclimatized with a laboratory food (Lab chow) for 1 week, and then grouped as shown in the following table, and each of the experimental substances prepared in the above Experimental example 1 was supplemented to a high fat diet for 12 weeks. Body weight and diet intake were measured once a week during the mice feeding period:

1) ND (Normal diet: general diet) group: low Fat Diet (LFD) containing 10 Kcal% fat

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

3) BC (Bacillus coaguluns: bacillus coagulans) group: HFD + Bacillus coagulans 0.3% (wt/wt)

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

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

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

Group BC was a Bacillus coagulans powder prepared by inoculating 0.1ml (0.1%) of stock solution of the strain in LB broth culture medium (Luria Bertani; tryptone 1%, yeast extract 0.5%, NaCl 1%), mixing maltodextrin with the precipitate (pellet) precipitated by centrifugation, and freeze-drying for 48 hr.

The RMG group is a raw mixed grain prepared by washing 6 kinds of grains (brown rice, barley, soybean, mixed grains (corn, wheat and pearl barley)) and then hot air-drying at 60 ℃ for 24 hr.

(2) Collection of samples and measurement of tissue weights

After fasting the mice for 12 hours, the mice were first anesthetized by isoflurane inhalation (isoflurane, Baxter, USA) and fasting blood was collected from the inferior vena cava (affeior vena cava). The collected blood was immediately collected in heparin-treated tubes and centrifuged at 1000 KHz g at 4 ℃ for 15 minutes, and then plasma was collected and stored at-70 ℃ until sample analysis.

Also, the liver and muscle of each experimental animal were washed several times in a Phosphate Buffered Saline (PBS) solution, and then the water was removed and the weight was measured. Perirenal white adipose tissue (perirenal white adipose tissue), mesenteric white adipose tissue (mesenteric white adipose tissue), subcutaneous white adipose tissue (subcontaneous white adipose tissue) were also excised to be washed in PBS solution and removed of water, and then weighed separately.

(3) Blood glucose measurement and measurement of glucose tolerance

Blood was collected through the tail vein after fasting for 16 hours at 2-week intervals at 12 weeks as the entire experimental period, and then blood glucose was measured by the glucose oxidase (glucose oxidase) method.

To measure glucose tolerance, each kg of body weight of 0.5g of glucose solution was administered to the abdominal cavity after 12 hours of fasting, and then blood was collected through the tail vein after 0, 30, 90, and 120 minutes, respectively. The collected blood was subjected to glucose concentration measurement by the glucose oxidase method and used for comparison of glucose tolerance.

(4) Plasma lipid analysis

1) Quantification of Total-cholesterol (TC)

For the quantification of total plasma cholesterol, a test solution for measurement (asanpharm kit (kit)) using the enzyme method of Allain et al (1974) was used.

2) Quantification of HDL-cholesterol (cholestrol)

Plasma HDL-cholesterol (HDL-C) was measured using a HDL-C measurement test solution (asanpharm kit). If 100. mu.L of plasma is taken and treated with 500. mu.g of sodium phosphotungstate and 1mg of magnesium chloride, LDL and VLDL containing apolipoprotein B (apo B) are precipitated in lipoproteins by the action of phosphotungstic acid and magnesium cations (Warnick, 1982). After centrifugation, the same method as that for total cholesterol remaining in HDL of the supernatant was used for color development, and absorbance was measured at 500nm and compared with a cholesterol standard solution (50mg/dL) for quantification.

3) Calculation of non HDL-C and AI (arteriosclerosis index)

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

AI＝([Total-C]-[HDL-C])/[HDL-C]

(5) Measurement and calculation of energy metabolism rate (EE)

Animal metabolism measuring devices (Oxylet; Panlab, Cornella, Spain) are used for measuring the energy metabolism rate. To measure the metabolic rate, the flow rate in the cage was adjusted to 3L/min after calibration with oxygen and carbon (calibretion), the mice were placed in separate metabolic cages, and the energy consumption (oxygen uptake) was measured for 24 hours to show the metabolic rate. The energy metabolism rate was calculated by the following formula.

EE (kcal/day/body weight 0.75) ═ VO2 Piperdill 1.44 Piper KHz [3.815+ (1.232 Piper KHz VO2/VCO2) ]

(6) Analysis of Gene expression Rate associated with lipid metabolism

In order to analyze the gene expression rate related to lipid metabolism in the white adipose tissues around epididymis and small intestine, real-time PCR was performed after isolating RNA from the tissues and synthesizing cDNA.

1) RNA isolation of tissue

After adding 1mL of TRIzol per 0.1g of tissue and disrupting the cells using a glass homogenizer (glass homogenizer), chloroform (chloroform) was added and centrifuged at 12000 KHz g at 4 ℃ for 20 minutes. The supernatant was added with isopropanol (isoproapanol), centrifuged again at 4 ℃ under 12000 KHg for 15 minutes to obtain RNA precipitate, which was washed 2 times with isopropanol and centrifuged at 10000 KHg to remove the supernatant. The RNA precipitate is diluted to a concentration of 0.5-10. mu.g/. mu.L and stored at-70 ℃.

2) cDNA Synthesis

mu.L of previously isolated total RNA was taken and first strand cDNA was synthesized using the QuantiTect reverse Transcription kit (Qiagen, Germany).

3)Real-Time PCR

The previously obtained template cDNA was diluted in RNAse free water using 1. mu.g/reaction, and for analysis of gene expression, the expression of the target gene was observed by a real-time quantitative PCR method using QuantiTect SYBR Green PCR kit (QIAGEN, Germany). Primers (primers) capable of analyzing the expression of each gene were synthesized by Macrogen (Korea). As for the composition of the reaction solution, 10. mu.L of SYBR Green (double-stranded chimeric fluorescent Fuel), 2. mu.L of template and 200. mu.M of primer were added, respectively, and RNAse free water was added so that the final volume reached 20. mu.L, and then 40 times of reactions were performed in one cycle of 15 seconds at 94 ℃, 30 seconds at 58 ℃, 30 seconds at 72 ℃ and 15 seconds at 65 ℃. In this case, the fluorescence signal was monitored for each cycle, the threshold cycle (Ct) that occurred was analyzed, and the mRNA expression between experimental groups was quantitatively analyzed using a CFX96 real-time system (Bio-rad, USA). As an Internal transcription marker (Internal transcription marker), GAPDH was used.

3. Statistical analysis

All experimental results in this study were calculated using a SPSS Package program (Statistical Package for the Social science Sciences), SPSS inc. One-way analysis of variance (ANOVA) was performed as to the significance test of the average difference between each group, post-tests were performed at a level of p <0.05 or more by Duncan's multiple range test as to the difference between groups, and student's t-test was performed to test the significance between ND group and HFD group. All results are expressed as mean ± s.d. (standard deviation).

Results of the experiment

1. High Fat Diet (HFD) -induced body and tissue weight in obese mice

Figure 1 shows the diet-induced body weight change in obese mice over a 12-week animal feeding period.

Referring to fig. 1, it was confirmed that the body weight of the high fat diet group (HFD) was significantly increased from 1 week of the test diet supply to the end of the test, compared to the body weight of the normal diet group (ND), and thus the effect of inducing obesity was good.

Through comparison among a plurality of high fat diet groups (HFD, BC, RMG, L-FMG, H-FMG), it was confirmed that the body weight of the functional cereal fermented food group (H-FMG) at the 12 th week was significantly lower than that of the HFD group and the original cereal group (RMG) at the 12 th week, and the weight loss efficacy of the functional cereal fermented food group was dependent on the usage amount.

After the 12-week rearing period was completed, the weight of organs (liver and muscle) excised after sacrifice is shown in table 1 in terms of unit weight (100g of body weight).

TABLE 1

a, b indicate that the values with different superscript letters differ significantly (p <0.05) between the HFD groups; the average value of ND is significantly different from the average value of HFD, { p } 0.001; the mean values of FMGs are significantly different from the mean values of HFD, # p < 0.05.

Referring to table 1 above, the results of comparison between the ND group and the HFD group showed that the intake of high fat diet significantly reduced the muscle weight per body weight.

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

TABLE 2

a, b indicate that the values with different superscript letters differ significantly (p <0.05) between the HFD groups; the average values of ND were significantly different from the average values of HFD,. p < 0.001.

Referring to table 2 above, the results of comparison between the ND group and the HFD group showed that intake of high fat diet significantly reduced the weight of perirenal white fat, mesenteric white fat, subcutaneous white fat per unit body weight.

In comparison between the high fat diet groups, it was confirmed that the weight of perirenal fat was significantly lower in the L-FMG group and the H-FMG group than in the HFD group, and the weight of mesenteric fat was significantly reduced in the H-FMG group. Also, a tendency that the weight of subcutaneous fat of the H-FMG group was lower than that of the HFD group was exhibited.

As described above, it was confirmed that the L-FMG group and the H-FMG group exhibited anti-obesity activity by weight loss, fatty liver suppression and muscle mass increase, and weight loss effects of perirenal white fat, mesenteric white fat and subcutaneous white fat.

2. Plasma lipid concentration

Plasma lipid concentrations measured using plasma obtained after sacrifice are shown in table 3 below.

TABLE 3

a, b, c indicate that the values with different superscript letters differ significantly between the HFD groups (p < 0.05); the average values of ND were significantly different from those of HFD, # p <0.05, # p < 0.01.

Referring to Table 3 above, the intake of a high fat diet significantly increased the concentrations of Total Cholesterol (TC), high density cholesterol (HDL-C), non-high density cholesterol (nonHDL-C), apolipoprotein A-1(apoA-1), and low density cholesterol (LDL-C).

In comparison between the high-fat diet groups, it was confirmed that the concentrations of TC, nonHDL, apoA-1 and LDL-C were significantly reduced in the L-FMG-administered group and the H-FMG-administered group, and the effect was more significant than that in the BC group and the RMG group.

3. Measurement of blood glucose indicators

During the raising of the experimental animals, fasting blood glucose was measured using blood obtained by tail blood sampling every 3 weeks, and the results thereof are shown in fig. 2.

Referring to fig. 2, from 3 weeks to 12 weeks after the end of the test, the HFD group showed significantly higher fasting plasma glucose than the ND group, and it was confirmed that fasting plasma glucose gradually increased with the increase of the test diet period.

In comparison between the high-fat diet groups, it was confirmed that fasting blood glucose was significantly lower in the L-FMG group and the H-FMG group than in the other high-fat diet groups, particularly at week 12 as the test end time point, from week 3 of the test diet supply, than in the HFD group, BC group, and RMG group.

HOMA-IR as an index of representative type 2 diabetes was calculated after measuring the concentrations of plasma glucose, insulin and glucagon using plasma obtained after sacrifice and is shown in table 4.

TABLE 4

The average value of ND was significantly different from the average value of HFD, # p < 0.05.

Referring to table 4 above, it can be confirmed that plasma insulin concentration and HOMA-IR are significantly increased by high fat diet intake, and thus it can be confirmed that type 2 diabetes induced by obesity is caused by long-term experimental diet supply.

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 than the other groups.

4. Measurement of energy metabolism rate

Fig. 3 shows the respiration rate and the change in energy metabolism rate based on carbon dioxide production and oxygen consumption at 24 hours after 12 weeks dietary intake.

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

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

5. Analysis of expression level of lipid metabolism-related Gene

The expression levels of genes related to lipid metabolism of white adipose tissues around the epididymis and small intestine were analyzed using real-time PCR, and as a result of white adipose tissues around the epididymis, fig. 4 shows the expression levels of genes related to fatty acid oxidation, fig. 5 shows the expression levels of genes related to fat synthesis, fig. 6 shows the expression levels of genes related to caloric production, and as a result of white adipose tissues around the small intestine and fig. 7 shows the expression levels of genes related to lipid absorption and excretion.

Referring to FIG. 4, in comparison between the high fat diet groups, a tendency was shown that the expression of PGC-1 a (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha: Peroxisome proliferator-activated receptor gamma-co-activator 1-alpha) and PPAR a (Peroxisome proliferator-activated receptor alpha: Peroxisome proliferator-activated receptor alpha) genes was increased in the H-FMG group compared to the HFD group, and it was confirmed that the expression of SIRT1(Sirtuin 1) gene was significantly increased. SIRT1 is activated by deacetylation of PGC-1 α, which increases production of mitochondria and oxidation of fatty acids, where mitochondria function to produce energy. Since PGC-1 α is known to increase PPAR α activity by acting as a co-activator, it was confirmed that the intake of a high content of mixed grain fermentation enzyme contributes to the enhancement of fatty acid oxidation due to the complementary action of these genes.

Referring to fig. 5, in comparison between the high fat diet groups, the expression level of FAS (Fatty acid synthase) gene, which synthesizes Fatty acids, was significantly reduced in all the experimental material groups compared to the HFD group, while the expression levels of HMGCR (3-Hydroxy-3-Methylglutaryl-CoA Reductase: 3-Hydroxy-3-Methylglutaryl coenzyme a Reductase) and SREBP2(Sterol regulation element binding protein) genes, which contribute to the increase of cholesterol synthesis, showed a decreasing trend in the L-FMG group and the H-FMG group compared to the HFD group, so that it could be confirmed that the intake of fermentation enzymes of mixed grains contributes to the reduction of the amount of fat in vivo.

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

In comparison between the high fat diet groups, the expression levels of UCP1 gene that generates calories in mitochondria and PRDM16(PR-domain stabilizing 16: PR domain protein 16) gene that activates UCP1 expression showed a tendency to increase in the L-FMG group and the H-FMG group compared to the HFD group, and a tendency to increase the expression level of CIDEA gene was confirmed in the H-FMG group, and thus it was confirmed that the intake of mixed grain fermentation enzymes contributes to the promotion of the production of calories. Also, PRDM16 is known to be induced by PPAR α, suggesting that the increase in PRDM16 is caused by the aforementioned PPAR α showing an increasing trend in fig. 4.

Referring to fig. 7, in the HFD group, it was confirmed that the expression level of apolipoprotein ApoB48(ApolipoproteinB-48) gene present in the ultra-low-density and low-density lipid proteins was significantly increased compared to the ND group. Since ApoB48 is known to function to transport fat molecules to tissues, this suggests an increased absorption of fat from the small intestine to tissues in the HFD group compared to the ND group.

In comparison between the high fat diet groups, it was confirmed that the expression levels of PPAR α and FATP4(Long-chain fatty acid transport protein 4) genes showed a decrease in the L-FMG and H-FMG groups, while the expression level of ApoB48 gene was significantly decreased in the HFD group. PPAR α is known to be a high-level factor that regulates ApoB48 and FATP4 that transport fat molecules to tissues. Also, a tendency was confirmed that the expression level of ABCG8(ATP Binding Cassette surface GMember 8: ATP Binding Cassette Subfamily G member 8) gene, which transports and excretes cholesterol, was increased in the H-FMG group compared to the HFD group. Thus, it was confirmed that the intake of the mixed grain fermentation enzyme contributes to the reduction and increase of the absorption and discharge of the body fat.

The mixed grain fermentation enzyme of the present invention exhibits a weight-loss, fatty liver-inhibiting effect, hyperlipidemia-ameliorating effect, and blood glucose-lowering effect, and therefore, is very useful for the development of a preventive or therapeutic agent for metabolic syndrome induced by obesity, and thus has very high industrial applicability.

Claims

1. A pharmaceutical composition for preventing or treating metabolic syndrome,

comprises Bacillus coagulans fermenting enzyme as effective component of mixed grains, wherein the mixed grains comprise brown rice, barley, soybean, corn, wheat and Coicis semen.

2. The pharmaceutical composition of claim 1,

in the mixed grain, the ratio of 1: 0.5 to 1.5: 0.3 to 1.5: 0.05 to 0.5: 0.05 to 0.5: 0.05-0.5 weight ratio of brown rice, barley, soybean, corn, wheat and coix seed.

3. The pharmaceutical composition of claim 1,

the fermentation enzyme is prepared by a method comprising the following steps:

4. The pharmaceutical composition of claim 3,

in the step (c), the liquid seed culture solution is added in an amount of 5 to 20 parts by weight based on 100 parts by weight of the cooked mixed cereal.

5. The pharmaceutical composition of claim 3,

the method further comprises a step (d) of drying and powdering the fermentation enzyme fermented in the step (c) after the step (c).

6. The pharmaceutical composition of claim 3,

in the culture medium of the step (a), the soybean powder is 1 to 5 parts by weight, the rice bran is 1 to 5 parts by weight, the wheat bran is 1 to 5 parts by weight, the isolated soybean protein is 0.5 to 5 parts by weight, and the yeast extract powder is 0.1 to 3 parts by weight, based on 100 parts by weight of purified water.

7. The pharmaceutical composition of claim 3,

in the main culture medium of the step (b), the soybean powder is 5 to 20 parts by weight, the rice bran is 1 to 10 parts by weight, the wheat bran is 1 to 10 parts by weight, the isolated soybean protein is 1 to 5 parts by weight, and the yeast extract powder is 0.5 to 5 parts by weight, based on 100 parts by weight of the mixed grain.

8. The pharmaceutical composition of claim 1,

the above-mentioned Bacillus coagulans strain was deposited under accession number KCTC13284 BP.

9. The pharmaceutical composition of claim 1,

the metabolic syndrome is selected from the group consisting of obesity, diabetes, arteriosclerosis, hypertension, hyperlipidemia, fatty liver, cerebral apoplexy, myocardial infarction, ischemic diseases and cardiovascular diseases.

10. A food composition for preventing or improving metabolic syndrome,

11. The food composition of claim 10,

the fermentation enzyme is prepared by a method comprising the following steps:

12. The food composition of claim 11,