KR20090116188A - A pharmaceutial composition comprising dried power of liquid culture of auricularia auricula (hook.) underw for descending blood sugar hypolycemic agent and health food - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing dried material of Auricularia auricula (Hook.) Underw is provided to use as a therapeutic agent of diabetes. CONSTITUTION: A pharmaceutical composition for reducing blood sugar contains dried material of Auricularia auricula (Hook.) Underw liquid culture as an active ingredient. The Auricularia auricula (Hook.) Underw is cultured in culture medium containing carbon source, organic nitrogen source, and inorganic ion source at 20-30°C for 5-15 days. The carbon source is glucose, D-fructose, D-galactose, D-arabinose, D-xylose, sucrose, mannitol, starch or dextrin. The organic nitrogen source is KNO3, (NH4)2SO4, NH4H2PO4, NH4NO3, NaNO3, NaNO2, yeast extract, casein peptone, isolated soybean protein(ISP) or skim milk.

Description

A pharmaceutial composition comprising dried power of liquid culture of Auricularia auricula (Hook.) Underw for descending blood sugar hypolycemic agent and health food }

The present invention relates to a pharmaceutical composition for lowering blood sugar, a blood sugar lowering agent, and a health food using the dried mushroom culture mycelium liquid culture.

Diabetes, referred to as a representative example of a disease associated with lifestyle, is a disease that causes acute symptoms due to significant hyperglycemia or ketoacidosis or various chronic systemic metabolic abnormalities due to persistent hyperglycemic conditions or decreased glucose tolerance. There are genetic factors and factors such as genetic factors and the environment such as diet and exercise, and because they affect each other, various forms of diabetes mellitus and disease progression. The frequency of occurrence is steadily increasing due to various changes, and the number of people is increasing by 500,000 every year, and the number is increasing every year by 1 person per 10 people.

Diabetes is divided into Insulin Dependent Diabetes Mellitus (hereinafter, IDDM; Type I Diabetes) and Insulin Dependent Diabetes Mellitus (hereinafter, NIDDM; Type II Diabetes). IDDM is caused by genetic factors, viral infections, and autoimmune infections that destroy pancreatic β-cells. It is acute and occurs in young people under 30 years of age. Most are skinny, lose weight, and are absolutely deficient in insulin. NIDDM is caused by obesity, stress, eating faults, or drugs that antagonize insulin. Chronic, common in adults, and the most common form of diabetes, the pancreatic beta cells have no abnormality, and are caused by obesity or lack of intracellular insulin receptors. It is epidemiologically recognized that there is some degree of genetic predisposition to this type, but there are a number of acquired factors, including eating wrong, lack of exercise, stress, heavy metal contamination, pregnancy, obesity, endocrine disorders, and side effects of drugs.

In the case of IDDM, only insulin has been selected as the most ideal and effective drug to date. However, its duration is short, it does not show permanent action, and it has the disadvantage of being used only as an injection. Even if this is normal, blood sugar drops and symptoms of hypoglycemia appear. In severe cases, if not treated, convulsions, unconsciousness, and brain damage may result in death.

In addition, patients with diabetes may have turbid blood and various incompatibilities that cause complications of various circulatory organs such as arteriosclerosis, heart disease, renal failure, and in severe cases, blood supply to the distal parts of the body is not smooth, resulting in necrosis of terminal tissues. May be

In the case of NIDDM, in addition to insulin, a blood glucose lowering agent is used. Representative oral blood glucose lowering agents used to date include sulfonylurea, metformin, butol, such as tolbutamide, chlorpropamide, and glybenclamide. Biguanid-based drugs such as formin. However, sulfonic uric acid-based drugs cause hepatotoxicity and hypoglycemia, and biguanide-based drugs also have side effects that cause lactic acidosis. Particularly, among the above-mentioned diabetic drugs, tolbutamide is used for the purpose of treating diabetes by controlling blood sugar drop. However, when tolbutamide is used as a diabetic drug, pancreatic cancer, pain, blood stains, etc., may be overdosed. If there is a blind spot that can pose a fatal risk to life when the health of the patient is not good, blood glucose lowering effect appears quickly, but the effect is not persistent. Accordingly, studies on herbal compositions excellent in blood sugar lowering effects while having fewer side effects are being actively conducted.

Throat Mushroom ( Auricularia) auricula (Hook.) Underw ) is a fungus of the genus Ophiculus, which grows from old to late autumn and grows in old trees such as mulberry, oak, pine and nursing trees, and is brownish in shape and resembles a human ear. Fruiting bodies are gelatinous and have wrinkling wrinkles.

Thirsty mushrooms are known to have blood purification and anticancer effects, and Korean Patent Registration No. 0698775 prepared fruiting bodies of mushrooms with antidiabetic activity in a form of pickling. However, the conventional technique using the fruiting body contains a thirsty mushroom, there is a problem that there is a limit to increase the anti-diabetic activity using the fruiting body.

Therefore, the present inventors are cultivating through the liquid culture that can cultivate the mycelium mycelium mycelium while mass-producing the mycelium mushroom mycelium, while the mycelium is harmless to the human body and contains β-glucan in the culture medium, thereby excellent blood sugar It confirmed that it exhibits a dropping effect, and completed the present invention.

An object of the present invention is to provide a pharmaceutical composition for lowering blood pressure, which contains a dry mushroom culture mycelium liquid culture dried as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for lowering blood glucose, which contains a liquid cultured polysaccharide of the mycelium mycelium as an active ingredient.

In addition, another object of the present invention is to provide a hypoglycemic agent containing the wood mushroom mycelium culture medium as an active ingredient.

In addition, another object of the present invention is to provide a blood sugar lowering health food containing the wood mushroom mycelium as an active ingredient.

In addition, another object of the present invention provides a method for mass culture of mycelium mushroom mycelium.

In addition, another object of the present invention to provide a method for treating hypoglycemic diseases.

In order to achieve the above object, the present invention provides a pharmaceutical composition for lowering blood sugar, which contains a liquid cultured dry matter of the wood mushroom mycelium as an active ingredient.

Next, the present invention provides a pharmaceutical composition for lowering blood glucose, which contains the liquid culture polysaccharide of the mycelium mushroom mycelium as an active ingredient.

Next, the present invention provides a hypoglycemic agent containing the pharmaceutical composition for hypoglycemic as an active ingredient.

Next, the present invention provides a health food containing the pharmaceutical composition for lowering blood sugar as an active ingredient.

Next, the present invention provides a method for mass-producing mycelium mushroom mycelium.

Finally, the present invention provides a method for treating hypoglycemic diseases.

The pharmaceutical composition for hypoglycemic, blood glucose lowering agent and health food containing the liquid mushroom mycelium mycelium liquid culture liquid or dried product thereof according to the present invention not only cultured the mycelium mushroom mycelium in a large amount, but also cultured mycelium on the human body Although harmless, β-glucan is contained in the culture medium, thus exhibiting an excellent hypoglycemic effect, and thus can be usefully used as a therapeutic agent for hypoglycemic-related diseases, especially diabetes.

Hereinafter, the words used in the present invention are defined.

The mycelium liquid culture dried product of the present invention refers to a powder pulverized by lyophilization or spray drying by inoculating the mycelium in the liquid culture medium and incubating the polysaccharide to produce polysaccharide, followed by vacuum and reduced pressure concentration or thin film concentration (or the culture medium). .

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for lowering blood sugar, which contains Auricularia auricula (Hook.) Underw as an active ingredient.

The mycelium mushroom mycelium can shorten the growth period, can be cultured in large quantities and can be cultured in an economically feasible liquid culture method, the culture conditions are preferably cultured for 5 to 15 days at 20 ~ 30 ℃.

If the culture temperature is out of 20 ~ 30 ℃ there is a problem that mycelium is not cultured, if the incubation time is less than 5 days there is a problem that the amount of mycelium and polysaccharide to be cultured due to insufficient culture of the mycelium does not occur, 15 days If the excess is not cultured anymore, the polysaccharide produced extracellularly consumed has a problem that the yield is lowered.

In addition, the thirsty mycelium mycelium according to the present invention is preferably cultured using a carbon source such as glucose, D-fractose, D-galactose, D-arabinose, D-xylose, sucrose, mannitol, starch or dextrin. Do. Among these, the myrtle mycelium is more preferably cultured using glucose as a carbon source.

The concentration of the carbon source can be cultured mycelium mushroom mycelium at 0.1 to 5% by weight / volume%. At this time, if the concentration of the carbon source is out of this range, there is a problem that mycelial growth is poor or polysaccharides are not produced.

Meanwhile, the thirsty mushroom mycelium according to the present invention is potassium nitrate (KNO ₃ ), ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), ammonium phosphate (NH ₄ H ₂ PO ₄ ), ammonium nitrate (NH ₄ NO ₃ ), nitric acid Sodium (NaNO ₃ ), sodium nitrite (NaNO ₂ ), ammonium chloride (NH ₄ Cl), yeast extract, casein peptone, soy flour, soybean meal protein (ISP) or skim milk or mixtures thereof It is preferably cultured using as an organic nitrogen source of the mycelium. Among them, it is more preferable to use yeast extract, casein peptone, soybean meal, soybean protein isolate (ISP), skim milk or a mixture thereof as an organic nitrogen source.

The organic nitrogen source may be cultured mycelium mushroom mycelium at 0.01 to 5% by weight / volume. At this time, when the concentration of the organic nitrogen source is out of this range, there is a problem that the mycelial growth is poor or the polysaccharide is not produced.

Furthermore, the mycelium mushroom mycelium according to the present invention is preferably cultured using potassium phosphate monobasic, potassium diphosphate, magnesium sulfate or a mixture thereof as an inorganic ion source. At this time, the concentration of the inorganic ion source is preferably 0.01 to 1% by weight / volume, if out of this range there is a problem that the mycelial growth is poor or the polysaccharide is not produced.

On the other hand, the thirsty mushroom mycelium according to the present invention preferably contains 0.1 to 2% of a polysaccharide. At this time, if the β-glucan is less than 0.1%, there is a problem in that the blood glucose enhancing effect is weakened. The thirsty mushroom mycelium of the present invention preferably comprises 0.1 to 99.5% of β-glucan, but is not limited thereto.

The present invention provides a pharmaceutical composition for lowering blood sugar, which contains the polysaccharide produced by the liquid culture of mycelium mycelium as an active ingredient.

After cultivating the mycelium mycelium mycelium of the present invention, the crude polysaccharide can be separated by organic solvent deposition on the culture, and the neutral fraction and the acid component fraction can be separated by ion chromatography (see FIG. 10). The separated acidic and neutral fractions may include glucose, mannose, galactose or fucose by performing a high performance liquid chromatograph analysis by hydrolyzing each fraction after gel filtration chromatography. The molecular weight of each polysaccharide can be composed of 10 KDa ~ 1000 KDa.

In another aspect, the present invention provides a blood glucose lowering agent formulated in a pharmaceutically acceptable method using the mycelium mushroom mycelium or a polysaccharide isolated therefrom.

When the pharmaceutical composition containing the dry mushroom mycelium liquid culture dried product as an active ingredient was administered to mice induced with diabetes mellitus with STZ, cholesterol in blood glucose was decreased by 18-22%, and triglyceride was 12-13%. The glucose composition is reduced by 35 to 39%, so that the pharmaceutical composition comprising the mycelium mycelium liquid culture dried product according to the present invention can be usefully used as a hypoglycemic composition, and the mycelium mycelium liquid culture dried product as an active ingredient. Containing pharmaceutical compositions containing insulin-dependent, insulin-independent, and diabetic patients can lower blood sugar levels in a shorter period of time than conventional hypoglycemic agents, and use natural mycelium and its culture components to be safe and useful for diabetics. Can be used.

The method of taking a pharmaceutical composition containing the liquid mushroom culture mycelium mycelia as an active ingredient is taken as a liquid, dried or pulverized in the form of a powder, or mixed with white wheat or syrup to the powder form to prepare a pill You can also take it.

The pharmaceutical composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodulin solution, glycerol, ethanol, and one or more of these components, as necessary, for example, antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The pharmaceutical compositions of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be weight, age, sex, health of the patient. The range varies depending on the condition, diet, administration time, administration method, excretion rate and severity of the disease. The daily dose of the pharmaceutical extract according to the present invention is 10 to 2,000 mg / kg, preferably 50 to 500 mg / kg.

As a result of oral administration of the pharmaceutical composition of the present invention to mice, 50% lethal dose (LD ₅₀ ) by oral administration toxicity test is judged to be a safe substance of at least 1000 mg / kg or more.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, hormonal therapy, drug treatment and biological response modifiers for the prevention and treatment of diabetes.

In addition, the present invention provides a blood sugar-lowering health food comprising the polysaccharide isolated from the wood mushroom mycelium or liquid culture solution.

The pharmaceutical composition of the present invention may be added to health food for the purpose of improving diabetes disease. When the herbal composition of the present invention is used as a food additive, the pharmaceutical composition may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the preparation of food or beverages, the pharmaceutical composition of the present invention is added in an amount of 0.01 to 10 parts by weight, preferably 0.05 to 1 part by weight based on the raw materials. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

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

The health beverage pharmaceutical composition of the present invention may contain various flavors or natural carbohydrates, and the like as additional ingredients, as in general beverages. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the pharmaceutical composition of the present invention.

In addition to the above, the pharmaceutical composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the pharmaceutical composition of the present invention may contain flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides a method for mass culture of mycelium mushroom mycelium.

The culturing method provides the following steps: i) inoculating the cultivation mycelium mycelium into the culture medium (step 1); ii) liquid culture of the inoculated medium of step 1) (step 2); and iii) drying and pulverizing the culture solution of the incubated mycelium mycelium of step 2).

The culture medium of step 1) according to the present invention is preferably composed of a carbon source, an organic nitrogen source and an inorganic ion source. The conditions of the culture medium have already been described above.

The culture conditions of step 2) according to the present invention, the culture conditions are preferably cultured for 5 to 15 days at 20 ~ 30 ℃. If the culture temperature is out of 20 ~ 30 ℃ mycelia are not cultured problem, if the culture time is less than 5 days there is a problem that the amount of mycelium and polysaccharides cultured due to insufficient culture of mycelium does not occur, 15 days If exceeded, it is no longer cultured and there is a problem that the polysaccharide is consumed and reduced.

The drying method of step 3) according to the present invention is preferably lyophilization or spray drying.

In addition, the present invention provides a method for treating hypoglycemic diseases comprising administering to the patient with hypoglycemic diseases, the pharmaceutical composition for lowering blood sugar as an active ingredient.

The hypoglycemic disease is preferably any one of the group consisting of type 1 diabetes, type 2 diabetes, arteriosclerosis, heart disease and renal failure.

Hereinafter, the present invention will be described in more detail by the following Preparation Examples, Examples, Experimental Examples and Formulation Examples. However, the following Preparation Examples, Examples, Experimental Examples, and Formulation Examples illustrate the present invention, but the contents of the present invention are not limited to the following Preparation Examples, Examples, Experimental Examples, and Formulation Examples.

< Production Example  1> Passage culture and liquid culture of strain Inoculation  Produce

Monarch used for mycelium liquid culture of Auricularia auricula (Hook.) Under the present invention, North Korea strain (Nutra R & BT, South Korea), rapidly frozen and preserved in 20% glycerol (Glycerol), MML-00118 (MML) Biotech strain, Republic of Korea), MML-01055 strain (MT Biotech strain, South Korea) was used. The strains were cultured at 24 ° C. after fragment transplantation into Potato Dextrose Agar (PDA) and passaged at intervals of 2 to 3 weeks. Each strain was passaged is shown in FIG.

As shown in Figure 1, the passage culture of the strain was confirmed that well cultured even in PDA medium medium.

Preparation of the inoculum for liquid culture was aseptically cut 0.1 to 1 cm in length of mycelial sections from the PDA plate cultured the strain (glucose) 3%, yeast extract (Yeast extract) 0.1%, KH ₂ PO ₄ 0.05%, MgSO ₄ 7H ₂ O 0.05% inoculated in 100 ml of the liquid medium and shaken culture at 24 ℃ for 7 days to 10 days, the cultured mycelium homogenized with a homogenizer and then aseptic A certain amount was taken with a pipette and used as an inoculum for liquid culture.

< Example  1 to Example  4> In badge  Mycelium Liquid Culture According to Glucose Concentration

 The inoculation solution prepared in Preparation Example 1 was inoculated at 2% by weight in 100 ml (using 500 ml flask) of culture medium prepared by varying the glucose concentration, which is the basic carbon source, in the basal medium as shown in Table 1 below. After 10 days of shaking culture, dry mycelial mass and mycelial growth of each medium were observed. At this time, the dry mycelium amount was measured by collecting only the mycelium by filtration and drying at 105 ℃ in a drying oven for at least 5 hours. The flasks in shaken culture were photographed and shown in FIG. 2.

Glucose concentration (w / v%) Default medium Growth form Dry Mycelial Weight (g / l) Example 1 0 YE: 0.1 KH ₂ PO ₄ : 0.05 MgSO ₄ 7H ₂ O: 0.05 Spherical (1 ~ 3cm) or pellet 1.7 Example 2 One 10 Example 3 2 13 Example 4 3 15

< Example  5 to Example  8> In badge  Mycelial Liquid Culture by Yeast Extract Concentration

The inoculation solution prepared in Preparation Example 1 was inoculated at 2% weight ratio in 100 ml (using 500 ml flask) of culture medium prepared by changing the concentration of yeast extract as an organic nitrogen source in the basal medium as shown in Table 2 below. After shaking culture for 10 days, the dry mycelium weight and mycelial growth of each medium were observed.

Yeast Extract (w / v%) Default medium Growth form Dry Mycelial Weight (g / l) Example 5 0 Glucose: 0.1 KH ₂ PO ₄ : 0.05 MgSO ₄ H ₂ O: 0.05 Spherical (1 ~ 3cm) or pellet 6.1 Example 6 0.1 11.1 Example 7 0.2 12.3 Example 8 0.3 13.9

< Example  9 to Example  12> In badge casein  Mycelial Liquid Culture by Peptone Concentration

As shown in Table 3, the inoculum prepared in Preparation Example 1 was changed to 100 ml (using a 500 ml flask) of a culture medium prepared by changing a casein peptone concentration as an organic nitrogen source in a basal medium. Inoculated at a weight ratio of%, and after shaking culture for 10 days, the dry mycelial weight and the mycelial growth of each medium were observed.

Casein peptone (w / v%) Default medium Growth form Dry Mycelial Weight (g / l) Example 9 0 Glucose: 0.1 KH ₂ PO ₄ : 0.05 MgSO ₄ H ₂ O: 0.05 Spherical (1 ~ 3cm) or pellet 6.2 Example 10 0.1 9.1 Example 11 0.2 12.2 Example 12 0.3 11.9

< Example  13 ~ Example  17> In badge  Mycelial Liquid Culture According to Soy Flour Concentration

 Inoculating the inoculum prepared in Preparation Example 1 inoculated at 2% by weight in 100 ml (using 500 ml flask) of culture medium prepared by changing the soy flour concentration as the organic nitrogen source in the basal medium as shown in Table 4 below After shaking culture for 10 days, the dry mycelium weight and mycelial growth of each medium were observed.

Soy flour (w / v%) Default medium Growth form Dry Mycelial Weight (g / l) Example 13 0 Glucose: 0.1 KH ₂ PO ₄ : 0.05 MgSO ₄ H ₂ O: 0.05 Spherical (1 ~ 3cm) or pellet 5.4 Example 14 0.05 9.1 Example 15 0.1 13.9 Example 16 0.2 14.5

< Example  17- Example  20> In badge Soy Protein Isolate  Mycelial liquid culture by concentration

As shown in Table 5, the inoculum prepared in Preparation Example 1 was changed to 100 ml (using a 500 ml flask) of a culture medium prepared by changing the soybean protein (ISP) concentration as an organic nitrogen source in a basal medium. Inoculated at a weight ratio of%, and after shaking culture for 10 days, the dry mycelial weight and the mycelial growth of each medium were observed.

Soy flour (w / v%) Default medium Growth form Dry Mycelial Weight (g / l) Example 17 0.0 Glucose: 0.1 KH ₂ PO ₄ : 0.05 MgSO ₄ H ₂ O: 0.05 Spherical (1 ~ 3cm) or pellet 4.9 Example 18 0.05 10.3 Example 19 0.1 13.2 Example 20 0.2 14.7

< Example  21- Example  24> In badge SM Culture of Mycelium Liquids with Different Concentrations

As shown in Table 6, the inoculum cultured in Preparation Example 1 was changed to 100 ml (using a 500 ml flask) of a culture medium prepared by changing skim milk concentration as an organic nitrogen source in a basal medium. Inoculated at a weight ratio of%, and after shaking culture for 10 days, the dry mycelial weight and the mycelial growth of each medium were observed.

SM (w / v%) Default medium Growth form Dry Mycelial Weight (g / l) Example 21 0.0 Glucose: 0.1 KH ₂ PO ₄ : 0.05 MgSO ₄ H ₂ O: 0.05 Spherical (1 ~ 3cm) or pellet 5.7 Example 22 0.05 8.8 Example 23 0.1 12.1 Example 24 0.2 13.0

< Example  25- Example  28> In badge Potassium phosphate In the farm  Mycelium liquid culture

100 ml (using 500 ml flask) of the culture medium prepared by changing the concentration of potassium phosphate (KH ₂ PO ₄ ) into ions in the basal medium as shown in Table 7 below in Preparation Example 1 Was inoculated at a weight ratio of 2%, and dried mycelial weight and mycelial growth of each medium after shaking culture for 10 days.

KH ₂ PO ₄ (w / v%) Default medium Growth form Dry Mycelial Weight (g / l) Example 25 0.0 Glucose: 3 Y.E: 0.1 ISP: 0.05 Spherical (1 ~ 3cm) or pellet 14.5 Example 26 0.04 15.1 Example 27 0.08 15.8 Example 28 0.12 16.3

< Example  29- Example  32> In badge Potassium Diphosphate  Mycelial liquid culture by concentration

100 ml (using 500 ml flask) of the culture medium prepared by changing the concentration of potassium phosphate (K ₂ HPO ₄ ) into ions in the basal medium as shown in Table 8 below in Preparation Example 1 Was inoculated at a weight ratio of 2%, and dried mycelial weight and mycelial growth of each medium after shaking culture for 10 days.

K ₂ HPO ₄ (w / v%) Default badge (%) Growth form Dry mycelial weight (g / l) Example 29 0.0 Glucose: 3 Y.E: 0.1 ISP: 0.05 Spherical (1 ~ 3cm) or pellet 14.9 Example 30 0.04 15.3 Example 31 0.08 16.9 Example 32 0.12 16.7

< Example  33- Example  36> In badge  Culture of Mycelial Liquid by Magnesium Sulfate Concentration

100 ml (using a 500 ml flask) of the culture medium prepared by changing the concentration of magnesium sulfate (MgSO ₄ 7H ₂ O) with ions in a basal medium as shown in Table 9 below. Was inoculated at a weight ratio of 2%, and dried mycelial weight and mycelial growth of each medium after shaking culture for 10 days.

MgSO ₄ H ₂ O (w / v%) Default Medium (%) Growth form Dry mycelial weight (g / l) Example 29 0.0 Glucose: 3 Y.E: 0.1 ISP: 0.05 Spherical (1 ~ 3cm) or pellet 14.9 Example 30 0.04 15.3 Example 31 0.08 16.9 Example 32 0.12 16.7

< Example  37- Example  39> by strain Copolysaccharide  produce

Culture medium consisting of the inoculation solution of the North strain (Example 37), MML-00118 (Example 38) strain, MML-01055 (Example 39) strain prepared by the method of Preparation Example 1 as shown in Table 10 below. 15 L (20 L culture bottle) was inoculated at a 2% weight ratio, respectively, and then incubated at 24 ° C. At this time the initial pH was 6.5 and no further pH adjustment was performed. Photographs of the cultured culture bottles are shown in FIG. 3. In addition, dry mycelium weight, extracellular copolysaccharide and pH change according to aeration culture time were measured and shown in FIGS. 4 to 6. At this time, the extracellular crude polysaccharide was measured by drying at 105 ° C after recovering the polysaccharide deposited by adding 2 times the amount of acetone to the culture filtrate from which the mycelia were removed.

Glucose (%) Y.E (%) ISP (%) Potassium phosphate (%) Potassium Diphosphate (%) Magnesium sulfate (%) Example 37 3 0.1 0.1 0.1 0.1 0.05 Example 38 3 0.1 0.1 0.1 0.1 0.05 Example 39 3 0.1 0.1 0.1 0.1 0.05

As shown in FIGS. 4 to 6, the strain cultured MML-00118 (see FIG. 5) and strain MML-01055 (see FIG. 6) were maintained in dry mycelial mass and extracellular copolysaccharide with increasing incubation time. When increased to, and cultured for about 12 days, 15 ~ 17 g / L of dry mycelium was produced. At this time, the extracellular crude polysaccharide was produced at a level of 1.6 g / L. On the other hand, as the incubation time of the North Korean strain (see FIG. 4), the dry mycelium mass increased similarly to other strains, and when cultured for 12 days, about 18 g / L of dry mycelium was produced, but the extracellular microsaccharide was Was about 0.6 g / L, which showed lower polysaccharide production capacity than other strains.

In addition, the initial pH of all the culture medium was about 6.5, but decreased to about 5 after incubation, showing continuous acid production characteristics. In the case of artificially adjusting the initial pH or pH during incubation using acid or alkaline buffer solution (pH 4 ~ 7 range), the production of dry mycelium and extracellular polysaccharides tends to decrease, resulting in polysaccharide production according to culture pH. No optimal pH could be found in relation to this.

< Example  40- Example  42> Soy Protein Isolate  Except In badge  According to mycelial type Copolysaccharide  produce

The inoculum of the North Korean strain (Example 40), MML-00118 strain (Example 41), and MML-01055 strain (Example 42) prepared in Preparation Example 1 includes the isolated soy protein as shown in Table 11 below. Inoculated at a weight ratio of 2% to 15 liters (20 L culture bottle) of the culture medium was not incubated at 24 ℃ aeration. At this time, the initial pH is 6.5, and the dry mycelium mass, extracellular copolysaccharide and pH change according to the aeration culture time were measured and shown in FIGS. 7 to 9.

Glucose (%) Y.E (%) Potassium phosphate (%) Potassium Diphosphate (%) Magnesium sulfate (%) Example 40 5 0.1 0.1 0.1 0.05 Example 41 5 0.1 0.1 0.1 0.05 Example 42 5 0.1 0.1 0.1 0.05

As shown in FIGS. 7 to 9, all of the extracellular microsaccharides were increased in small amounts, but the dry mycelial weight of all strains cultured in the culture medium containing the isolated soybean protein was compared to the dry mycelium cultured in the culture medium containing the isolated soy protein. Compared to 1/2 level.

Thus, the composite nitrogen source medium is advantageous for the production of mycelia and extracellular polysaccharides, it was confirmed that the medium composed only of the hydrolysis nitrogen source is only advantageous for the production of extracellular microsaccharides.

In addition, the initial pH of all cultures was 6.5 but decreased to about 5 after incubation.

According to the above results, the amount of dried mycelium and the amount of copolysaccharide of the mycelium increased continuously as the incubation time increased in Example 38, and when cultured for about 12 days, the most mycelium was incubated in the following experiment. Used.

< Example  43> Polysaccharide Separation and Purification

Example 38 was purified separately in the order shown in Figure 10, the molecular weight and properties of the fractions were analyzed according to the fraction number.

<43-1> Ion Exchange Chromatography

In separating and purifying the present invention, ion-exchange chromatography was used to separate neutral polysaccharides from acidic polysaccharides. The ion exchange chromatography used was filled with DEAE (diethylaminoethyl) cellulose in a glass column with an inner diameter of 2.8 cm and a length of 80 cm, and was repeatedly used for washing and regeneration in a conventional manner. Elution was carried out at a rate of 4 ml per minute with a gradient of NaCl and fractions of 10 ml. The polysaccharide concentration of each fraction was measured by phenol-sulfuric acid method. In other words, the color of the sugar in the fractions by phenol and sulfuric acid, the absorbance was measured at 490 nm, 750 nm by spectrophotometer to measure the concentration of the sugar according to the fraction number is shown in FIG. At this time, the desired fractions were collected and repeated several times to confirm reproducibility and (a) and (b) were presented as representative drawings.

<43-2> gel chromatography

Neutral and acidic polysaccharides separated through ion exchange chromatography were separated by gel chromatography to prepare a final polysaccharide. At this time, the column was Sepharose (Sepharose) CL-4B was used, elution was 1 mL per minute. The molecular weight of the standard polysaccharide was analyzed in FIG. 12, the neutral polysaccharide was shown in FIG. 13, and the acidic polysaccharide was shown in FIG. 14. The molecular weight was analyzed using FIGS. 12 to 14 and shown in FIG. 15. It summarized in Table 12. At this time, the desired fractions were collected and repeated several times to confirm reproducibility, and (a) and (b) were presented as representative drawings, respectively.

Standard polysaccharides

As shown in FIG. 12 and Table 12, the material having a molecular weight of 2000 k in the standard elutes at fractions 18-30 and has the highest concentration in fraction 22. A substance having a molecular weight of 500 k elutes in fractions 20-37 and has the highest concentration in fractions 27 and 200 k elutes in fractions 27-40 and has the highest concentration in fractions 32. Finally, the material having a molecular weight of 40 k elutes in fractions 40-50 and has the highest concentration in fraction 43.

As shown in FIG. 13 and Table 12, the neutral polysaccharide eluted at fractions 30 to 38 and eluted at the highest concentration in fractions 34, and eluted at fractions 39 to 52 and at the highest concentration in fractions 45. Consists of eluted material. Thus, based on the function shown in Figure 15, it can be seen that the neutral polysaccharide consists of a polysaccharide having a molecular weight of 232,273 and a polysaccharide of 33,573.

In addition, as shown in FIG. 14 and Table 12, the acidic polysaccharide is composed of a substance eluting at fractions 18 to 38 and eluting at the highest concentration in fractions 25. Thus, based on the function shown in Figure 11, it can be seen that the neutral polysaccharide consists of a polysaccharide having a molecular weight of 990,832.

Sample Name Molecular Weight Elution volume log (molecular weight) Molecular Weight Standard material 40KDa 210 4.602 39995 200 KDa 170 5.301 199,986 500 KDa 125 5.699 500,035 2000KDa 105 6.301 1,999,862 Heavy Ingredients 1 F-N1 170 5.2133 163,418 Barite fraction 2 F-N2 230 4.3193 20,859 Acid fraction 1 F-A1 125 5.966 990,832

Accordingly, it was confirmed that the polysaccharides produced by the fungus in liquid culture were composed of neutral polysaccharides having a molecular weight of 20 KDa, 163 KDa and acidic polysaccharides of 990 KDa, respectively.

Per composition  analysis

In order to determine the components of the polysaccharide separated through the above steps, Glucose, Mannose, Galactose, Fucose and neutral polysaccharides and acidic polysaccharides were analyzed by high performance liquid chromatography (HPCL, Young Lin MP 30) as a standard. 16 to 18 and Table 13 are shown.

HPCL analysis conditions are shown in Table 13 below.

column Agilent Zorbox Carbohydrate Flow rate 1.4 per minute Oven temperature 60 Injection volume 20 Mobile phase Acetonitrile: Distilled water = 85:15 Detector ELSD ZAM 3000

As shown in FIG. 16, glucose (a) contained in the standard material was 15.40 minutes, mannose (b) was 16.25 minutes, galactose (c) 13.80 minutes, fucose (d) 6.65 It was detected in minutes.

As shown in FIG. 13, in the neutral fraction 1 (a), a sugar substance was detected at 13.80 and 16.25 minutes, and the peak of mannose was very large, indicating that the neutral fraction 1 was composed of mannose 95.16% and galactose 4.84%. It was. In neutral fraction 2 (b), peaks were detected at 6.65 minutes, 13.8 minutes, and 16.25 minutes, with the maximum peak at 16.25 minutes. The neutral fraction 2 was found to be composed of mannose 78.72%, galactose 14.18%, fucose 7.10%.

As shown in FIG. 14, the acidic fractions showed peaks at 15.40 minutes and 16.25 minutes, and the largest peaks appeared at 16.25 minutes, constituting 81.13% mannose and 18.87% glucose.

< Experimental Example  1> Measurement of hypoglycemic properties of mycelium mycelium

<1-1> diabetes mellitus

In order to determine the effect of the mycelium mushroom mycelium liquid culture dry matter on the blood glucose of the present invention, diabetes was induced in mice as follows.

Streptozotocin (STZ) dissolved in 0.01 M citrate buffer solution at pH 4.5 was intraperitoneally injected at 60 mg / kg in Sprague Dawley male rats weighing 140 ± 10 g of the same age. Two days after the intraperitoneal injection, orbital venous body blood was checked and the blood glucose was checked by a liver glucose meter, and mice having a blood glucose level of 300 mg / ㎗ or more were selected.

During the experiment, the rats were kept at a temperature of 23 ° C. and a humidity of 40 to 60%, and the contrast cycle was maintained at 12 hours. Basic feed and drinking water were supplied without restriction, but only a day before blood collection.

<1-2> reagent addition

Example 38 in the diabetic rats were prepared orally administered in a concentration of 500 mg / kg (Experimental Group I) and 1000 mg / kg (Experimental Group II) in 2 ml of distilled water, respectively.

The control group was compared with the rats that did not induce diabetes (control I) and the rats that did not enter the reagents after the diabetes induction (control II).

Each group consisted of 10 rats, averaged to derive results.

<1-3> Weight Change Survey

 To investigate the weight change caused by streptozotocin (STZ, Streptozotocin) -induced diabetes and oral administration of the liquid culture dried mycelium mycelium mycelium, the weight change of each group was measured at weekly intervals from the purchase date. And FIG. 19.

Week 0 (g)  1 week (g) 2 weeks (g) 3 g (g) 4 g (g) Control group (I) 144.1667 176.5444 198.8889 255.7556 294.7556 Control group (II) 145.6889 176.3667 191 154.0778 176.6 Experimental group (I) 146.0222 177.9778 198.8889 162.9333 208.8889 Experimental group (II) 146.7 177.1222 198.7778 161.4556 184.4667

As shown in Table 14, the rats in each group gained weight regularly during the initial 2 weeks of adaptation after purchase, but all the rats with diabetes tended to lose weight and then increase again.

<1-4> liver Wet weight survey

In order to investigate the wet weight of the liver according to the mycelium liquid culture dried product of the present invention, the liver was extracted two weeks after the administration of the mycelium, and the wet weight was measured. A wet weight means the weight of the liver containing water. Liver extraction in all rats was performed under ether anesthesia after 24 hours fasting. After the blood was collected from the abdominal aorta, a syringe was inserted into the portal vein, and then perfused with saline to remove blood remaining in the liver. The extracted liver was pressed evenly with a cotton cloth to remove all the saline remaining in the liver as much as possible, and then weighed using a microbalance.

Control group (I) Control group (II) Experimental group (I) Experimental group (II) Average weight 9.67 6.97 7.70 6.753 Standard Deviation 0.690724 0.921253 0.817408 0.729182 g / 100 g equivalent 3.2 3.95 3.69 3.66

As shown in Table 15, the wet weight of the control group (II) was 3.95 g / 100 g, which was about 20% higher than the 3.2 g / 100 g of the control group (I) that did not induce diabetes, but converted to the experimental group (I). The amount was 3.69 g / 100 g, which was about 10% higher than the 3.95 g / 100 g of the control group (II) which did not receive the mycelium, and the conversion amount of the experimental group (II) was 3.66 g / 100 g, which was about 11% higher than the control group (II). Decreased.

The reason for the increase in the weight of the above-mentioned wet weight affects the immune function during diabetes-induced diabetes using STZ, which causes insulin secretion to be lowered, resulting in abnormal glucose metabolism, and consequently the accumulation of lipid components in the liver. However, the experimental group (I) and the experimental group (II) to which the mycelium liquid culture dried product according to the present invention were administered were confirmed that the liver wet weight was reduced by alleviating abnormal glucose metabolism.

<1-5> cholesterol ( cholesterol )Measure

The amount of change in cholesterol, the main cause of macrovascular complications, which is one of the main complications of diabetes mellitus, according to the mycelium liquid culture dry matter of the present invention was measured by enzyme method using a V-cholesterol kit, and is shown in Table 16 and FIG. 20 (V- cholesterol kit, Asan Pharmaceutical Co., Ltd., Korea).

Control group (I) Control group (II) Experimental group (I) Experimental group (II) Average amount (mg / ㎗) 99.24658 158.8063 130.3327 122.8767 Standard Deviation 9.493161 14.2219 19.45219 18.01253

As shown in Table 16 and FIG. 20, the experimental group (I) (130.3348 mg / dL) and the experimental group (II) (122.8767 mg / d) in which diabetes was induced compared to the control group (I) (99.24658 mg / dL) which did not induce diabetes. Iii) and control (II) (158.8063 mg / dL) total cholesterol was generally high. Furthermore, the cholesterol of the experimental group (I) was reduced by about 18%, and the experimental group (II) was reduced by about 22% compared to the control group (II) which did not receive the mycelia liquid culture dried product, and the mycelia liquid culture dry powder according to the present invention was cholesterol. It confirmed that it was effective in the fall.

<1-6> Triglyceride ( Triglyceride ) Measure

The amount of triglycerides, which is a major cause of macrovascular complications, which is one of the main complications of diabetes mellitus, according to the mycelium liquid culture dry matter of the present invention, was measured by enzyme method using TG-S kit, and is shown in Table 17 and FIG. 21 (TG). -S kit, Asan Pharmaceutical Co., Ltd., Korea).

Control group (I) Control group (II) Experimental group (I) Experimental group (II) Average value (mg / ㎗) 139.9632 203.729 177.7311 175.3676 Deviation 15.75701 18.7036 16.94883 21.38682

As shown in Table 17 and FIG. 21, the experimental group (I) (177.7311 mg / dL) and experimental group (II) (175.3676 mg / d) that induced diabetes compared to the control group (I) (139.9632 mg / dL) that did not induce diabetes. Iii) and control (II) (203.729 mg / dl) triglyceride levels were generally high. Furthermore, the amount of triglycerides in the experimental group (I) was reduced by about 12% and the experimental group (II) was reduced by about 13% compared to the control group (II) which did not receive the mycelia liquid culture dried product. It was confirmed that it is effective in reducing triglycerides.

<1-7> glucose ( Glucose ) Measure

Changes in the amount of glucose, one of the blood sugars according to the mycelium liquid culture dry matter of the present invention, were measured by enzyme method using a V-glucose kit, and are shown in Table 18 and FIG. 22 (V-glucose kit, Asan Pharmaceutical Co., Ltd., Korea). ).

Control group (I) Control group (II) Experimental group (I) Experimental group (II) Average value (mg / ㎗) 140.2082 299.9681 193.6059 182.5428 Deviation 11.41025 18.11295 15.84742 15.44723

As shown in Table 18 and FIG. 22, the experimental group (I) (193.6059 mg / dL) and the experimental group (II) (182.5428 mg / d) that induced diabetes compared to the control group (140.2082 mg / dL) which did not induce diabetes. Iii) and control (II) (299.9681 mg / dL) glucose levels were generally high. Further, compared to the control group (II) not administered the mycelia liquid culture, the amount of glucose in the experimental group (I) was reduced by about 35%, and the experimental group (II) was reduced by about 39%, so the mycelia liquid culture dried powder according to the present invention It confirmed that it was effective in the fall.

<1-8> GOT ( Glutamic oxaloacetic transaminase ) Measure

In order to examine the toxicity of the mycelium liquid culture dry matter of the present invention, the amount of GOT generated when the liver tissue is damaged by drugs or external stress is measured using the Reitman-Franke method using the GOT, GPT kit, which is a reagent for measuring serum transaminase. Measured as shown in Table 19 and Figure 23 (GOT, GPT kit, Asan Pharmaceutical Co., Ltd., Korea).

Control group (I) Control group (II) Experimental group (I) Experimental group (II) Average value (mg / ㎗) 26.7553 80.382 47.3918 44.8309 Deviation 2.33 6.434 4.0217 8.2951

As shown in Table 19 and FIG. 23, the experimental group (I) (47.3918 mg / dL) and the experimental group (II) (44.8309 mg / d) in which diabetes was induced compared to the control group (I) (26.7553 mg / dL) that did not induce diabetes. Iii) and control (II) (101.808 mg / dl) GOT amounts were generally high. Further, the GOT of the experimental group (I) was reduced by about 53%, and the experimental group (II) was greatly reduced to about 55% compared to the control group (II) which did not receive the mycelia liquid culture dried product, and the mycelia liquid culture dried powder according to the present invention. Has been found to be non-toxic and has the effect of restoring liver tissue damaged by STZ.

<1-9> GPT ( Glutamic pyruvic transaminase )Measure

In order to examine the toxicity of the mycelium liquid culture dried product of the present invention, the amount of GPT generated when the liver tissue is damaged by drugs or external stress is measured by the Reitman-Franke method using GOT, GPT kit, which is a reagent for measuring transaminase. The measured values are shown in Table 20 and FIG. 24 (GOT, GPT kit, Asan Pharmaceutical Co., Ltd., Korea).

Control group (I) Control group (II) Experimental group (I) Experimental group (II) Average value (mg / ㎗) 26.9596 80.382 58.908 50.1431 Deviation 2.13 6.434 3.6166 3.5998

As shown in Table 20 and FIG. 24, the experimental group (I) (58.908 mg / dL) and the experimental group (II) (50.1431 mg / d) that induced diabetes compared to the control group (I) (26.9596 mg / dL) that did not induce diabetes. Iii) and control (II) (80.382 mg / dL) GPT amounts were generally high. Furthermore, the cholesterol of the experimental group (I) was reduced by about 26%, and the experimental group (II) was reduced by about 37% compared to the control group (II) which did not receive the mycelia liquid culture dried product. It was confirmed that it is not toxic and has an effect of restoring liver tissue damaged by STZ.

<1-10> β- Glucan (β- glucan ) analysis

In Table 21, the content of β-glucan, which is estimated to be the main functional component of blood sugar drop in the dried liquid mycelium mycelium liquid culture confirmed the hypoglycemic effect through the animal experiment.

For analysis, Megazyme (Island) Mushroom and Yeast assay kit was used, and the relative concentration was measured by measuring the absorbance using an infrared spectrometer.

The content of β-glucan was calculated by the following formula.

β-glucan content = total glucan content- α-glucan content

Total Glucan (%) a-glucan (%) β-glucan (%) Example 38 18.86 ± 0.46 17.19 ± 0.28 1.68 ± 0.18

As shown in Table 21, it was confirmed that the mycelium mushroom mycelium liquid culture dried according to the present invention contained about 1.7% of β-glucan.

<1-11> Toxicity Assessment

Example 38 was prepared at a concentration of 50 mg / ml in 0.5% tween 80, then 0.04 ml (100 mg / kg), 0.2 ml (500 mg / kg), 0.4 ml (1,000 mg) per 20 g of mouse body weight. / Kg) and 0.8 ml (2,000 mg / kg). Samples were administered orally once and observed for side effects or lethality for 7 days after administration. That is, on the day of administration, changes in general symptoms and the presence or absence of dead animals were observed at least 1 hour, 4 hours, 8 hours, 12 hours after the administration, and at least once every morning and afternoon from the day after the administration. In addition, on the 7th day of administration, animals were killed and dissected, and the internal organs were visually examined. The change in body weight was measured at one day intervals from the day of administration, and the weight loss phenomenon of the animal according to Example 38 was observed.

As a result, no significant clinical symptoms were observed in all the mice treated with the sample, no mice died, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc.

Therefore, the compounds of the present invention did not show a toxicity change in all mice up to 3,000 mg / kg, it was determined that oral administration minimum dose (LD ₅₀ ) is a safe substance of more than 3,000 mg / kg.

As seen in the above experiments, the mycelia liquid culture dry matter of the present invention was administered to the diabetic rats showed significant (0p <0.05) hypoglycemic effect, and the experimental group with twice the concentration of the mycelia liquid culture dry matter (II) showed the best hypoglycemic effect, but not significantly different from experimental group (I). In addition, the mycelium liquid culture dry matter of the present invention reduced the amount of total cholesterol and triglycerides, and it was confirmed that it contains no β-glucan, which is not toxic and effective in lowering blood sugar, even when used medicinally, and damaged by STZ. The effect of restoring liver tissue can also be estimated.

Examples of preparations for the compositions of the present invention are illustrated below.

< Formulation example  1> Preparation of Pharmaceutical Formulations

<1-1> Powder  Produce

2 g of mycelium mushroom mycelium liquid culture dried powder according to the present invention

1 g lactose

After mixing the above components, the airtight cloth was filled to prepare a powder.

<1-2> Preparation of Tablet

Wood mushroom mycelium liquid culture dry matter powder 100 mg according to the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

<1-3> Preparation of Capsule

Wood mushroom mycelium liquid culture isolated polysaccharide 100 mg according to the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

<1-4> Preparation of Injection Solution

Throat mushroom mycelium liquid culture polysaccharide 10 ㎍ / ㎖ according to the present invention

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

Dissolve the wood mushroom mycelium powder according to the present invention in an appropriate volume of sodium chloride BP for injection, adjust the pH of the resulting solution to pH 3.5 with dilute hydrochloric acid BP, and adjust the volume with sodium chloride BP for injection and sufficiently Mixed. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

< Formulation example  2> Manufacture of food

Food comprising a dry mushroom mycelium liquid culture dry powder of the present invention according to the present invention was prepared as follows.

<2-1> Preparation of Flour Food

Thirst mushroom mycelium liquid culture dry powder 0.1 ~ 10.0 parts by weight of the present invention was added to the flour, and using this mixture to prepare bread, cake, cookies, crackers and noodles in a conventional manner to prepare a food for health promotion.

<2-2> soup  And juicy ( gravies Manufacturing

Thirst mushroom mycelium liquid culture polysaccharide of the present invention was added to 0.1 ~ 1.0 parts by weight to soup and broth to prepare a health-promoting meat products, noodles soup and gravy in a conventional manner.

<2-3> Preparation of Ground Beef

10 parts by weight of the dry mushroom mycelium liquid culture dry powder of the present invention was added to the ground beef to prepare a ground beef for health promotion in a conventional manner.

<2-4> dairy products ( dairy products Manufacturing

Thirst mushroom mycelium liquid culture dry powder of the present invention 0.1 to 1.0 parts by weight was added to the milk, using the milk to prepare a variety of dairy products such as butter and ice cream in a conventional manner.

<2-5> Linear  Produce

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The mycelium mycelium mycelium culture solution of the present invention was decompressed and concentrated in a vacuum concentrator, and the dried product obtained by drying with a spray and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and the mycelium mycelium mycelium powder according to the present invention were prepared in the following ratio to prepare a conventional method.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Wood mushroom mycelium powder of the present invention (1 part by weight),

Ganoderma lucidum (0.5 parts by weight),

Foxglove (0.5 part by weight)

< Formulation example  3> Manufacture of beverage

The beverage containing the dry mushroom mycelium liquid culture dry powder of the present invention was prepared as follows.

<3-1> Preparation of health drink

Homogeneously mixes dry ingredients such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%) and dry mushroom mycelium liquid culture dry powder according to the present invention. After instant sterilization, it was packaged in small packaging containers such as glass bottles and plastic bottles to prepare healthy drinks.

<3-2> Preparation of Vegetable Juice

0.5 g of the wood mushroom mycelium liquid culture dry powder of the present invention was added to 1,000 ml of vegetable juice such as tomatoes or carrots to prepare vegetable juice for health promotion in a conventional manner.

<3-3> Fruit juice  Produce

0.1 g of the mycelium mushroom mycelium liquid culture dry powder of the present invention was added to 1,000 ml of fruit juice such as apple or grape to prepare fruit juice for health promotion in a conventional manner.

Figure 1 is a photograph of the passage cultured thirsty mushroom mycelium:

(a) MML-00118;

(b) MML-01055; And

(c) North Korean strains.

Figure 2 is a photograph of the mycelium mushroom mycelium in the incubation culture:

(a) North Korean strains;

(b) MML-01055; And

(c) MML-00118.

Figure 3 is a photograph of one embodiment according to the present invention in liquid culture.

4 is a graph showing the change according to the incubation time of the North Korean strain according to the present invention L

(a) dry mycelium weight;

(b) pH; And

(c) extracellular copolysaccharide.

5 is a graph showing the change according to the incubation time of the MML-00118 strain according to the present invention:

(a) dry mycelium weight;

(b) pH; And

(c) extracellular copolysaccharide.

Figure 6 is a graph showing the change according to the incubation time of the MML-01055 strain according to the present invention:

(a) dry mycelium weight;

(b) pH; And

(c) extracellular copolysaccharide.

7 is a graph showing the change according to the incubation time of the North Korean strain according to the present invention:

(a) dry mycelium weight;

(b) pH; And

(c) extracellular copolysaccharide.

8 is a graph showing the change according to the incubation time of the MML-00118 strain according to the present invention:

(a) dry mycelium weight;

(b) pH; And

(c) extracellular copolysaccharide.

9 is a graph showing the change according to the incubation time of the MML-01055 strain according to the present invention:

(a) dry mycelium weight;

(b) pH; And

(c) extracellular copolysaccharide.

10 is a flowchart illustrating a process of separating and purifying an embodiment according to the present invention.

Figure 11 is a graph showing the color absorbance by the phenol sulfate method of the concentration of the polysaccharide according to the fraction number of one embodiment according to the present invention.

12 is a graph showing the absorbance of the color standard by the phenol sulfate method according to the fraction number of the embodiment of the present invention:

(a) 2,000 K;

(b) 40 K to 500 K; And

(c) 40 K ~ 200 K.

Figure 13 is a graph showing the color absorbance by the phenol sulfate method of the concentration of the polysaccharide according to the fraction number of one embodiment according to the present invention.

14 is a graph showing the color absorbance of the polysaccharide according to the fraction number of the embodiment of the present invention by the phenol sulfate method.

15 is a graph showing the log value of the molecular weight according to the elution volume of one embodiment according to the present invention.

16 is a graph of sugar components detected through HPCL on a standard substance:

(a) glucose;

(b) mannose;

(c) galactose; And

(d) Fucose.

17 is a graph of sugar components detected through HPCL in one embodiment according to the present invention:

(a) F-N1; And

(b) F-N1.

18 is a graph of a sugar component detected through HPCL in one embodiment of the present invention.

19 is a graph showing the weight change of the rat administered one embodiment according to the present invention:

(a) control group I;

(b) control group II;

(c) experimental group I; And

(d) Experiment group II.

20 is a graph showing changes in cholesterol concentration in blood of rats administered with one embodiment according to the present invention:

(a) control group I;

(b) control group II;

(c) experimental group I; And

(d) Experiment group II.

21 is a graph showing the change in the concentration of triglycerides in the blood of the rat administered one embodiment according to the present invention:

(a) control group I;

(b) control group II;

(c) experimental group I; And

(d) Experiment group II.

22 is a graph showing changes in glucose concentrations in blood of rats administered with one embodiment according to the present invention:

(a) control group I;

(b) control group II;

(c) experimental group I; And

(d) Experiment group II.

FIG. 23 is a graph showing changes in GOT levels in blood of rats administered with one embodiment according to the present invention:

(a) control group I;

(b) control group II;

(c) experimental group I; And

(d) Experiment group II.

24 is a graph showing changes in GPT levels in blood of rats administered with one embodiment according to the present invention:

(a) control group I;

(b) control group II;

(c) experimental group I; And

(d) Experiment group II.

Claims (20)

Auricularia auricula (Hook.) Underw) A pharmaceutical composition for lowering blood sugar, which contains a mycelia liquid culture dried product as an active ingredient. The pharmaceutical composition for lowering blood sugar according to claim 1, wherein the thirsty mushroom mycelium is cultured for 5 to 15 days at 20 to 30 ° C. using a culture medium composed of a carbon source, an organic nitrogen source, and an inorganic ion source. The method of claim 2, wherein the carbon source is any one selected from the group consisting of glucose, D-fractose, D-galactose, D-arabinose, D-xylose, sucrose, mannitol, starch or dextrin. A pharmaceutical composition for lowering blood sugar, which is cultured. The pharmaceutical composition for lowering blood sugar according to claim 2, wherein the carbon source is cultured using glucose. The pharmaceutical composition for lowering blood sugar according to claim 2, wherein the concentration of the carbon source is 0.1 to 5% by weight / volume. The method of claim 2, wherein the organic nitrogen source is potassium nitrate (KNO ₃ ), ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), ammonium phosphate (NH ₄ H ₂ PO ₄ ), ammonium nitrate (NH ₄ NO ₃ ), nitric acid Any one selected from the group consisting of sodium (NaNO ₃ ), sodium nitrite (NaNO ₂ ), ammonium chloride (NH ₄ Cl), yeast extract, casein peptone, soy flour, soybean meal (ISP) or skim milk (skim milk) Pharmaceutical composition for lowering blood sugar, characterized in that one or a mixture thereof. According to claim 2, wherein the organic nitrogen source is any one or a mixture thereof selected from the group consisting of yeast extract, casein peptone, soy flour, isolated soy protein (ISP) or skim milk (skim milk). Pharmaceutical composition for. The pharmaceutical composition for lowering blood sugar according to claim 6, wherein the organic nitrogen source is 0.01 to 5% by weight / vol%. The pharmaceutical composition for lowering blood sugar according to claim 2, wherein the inorganic ion source is cultured using potassium phosphate monobasic, potassium diphosphate or magnesium sulfate, or a mixture thereof as an inorganic ion source. 10. The pharmaceutical composition for lowering blood sugar according to claim 9, wherein the inorganic ion source is 0.01 to 1 weight / vol%. The pharmaceutical composition for lowering blood sugar according to claim 1, wherein the thirsty mushroom mycelium contains 0.1 to 99.5% of β-glucan. The pharmaceutical composition for lowering blood sugar according to claim 1, wherein the thirsty mushroom mycelium liquid culture dried product is in a powder form obtained by drying and pulverizing a culture solution. A pharmaceutical composition for lowering blood sugar, comprising a polysaccharide isolated from a liquid culture medium of mycelium mycelium. The pharmaceutical composition for lowering blood sugar according to claim 13, wherein the polysaccharide is composed of glucose, mannose, galactose, and fucose. The pharmaceutical composition for lowering blood sugar according to claim 14, wherein the polysaccharide has a molecular weight of 10 k to 1000 k. A hypoglycemic agent containing the pharmaceutical composition of claim 1 or 13. A blood sugar-lowering health food containing the pharmaceutical composition of claim 1 or 13. i) inoculating a mycelium ( Auricularia auricula (Hook.) Underw) mycelium on the culture medium (step 1); ii) incubating the inoculated medium of step 1) at 20 to 30 ° C. for 5 to 15 days (step 2); and iii) a method for mass production of a dry mushroom mycelium culture medium comprising drying and pulverizing a culture solution containing the cultured soybean mushroom mycelium and extracellular polysaccharide of step 2). A method for treating hypoglycemic diseases comprising administering the pharmaceutical composition for lowering blood sugar of claim 1 as an active ingredient to a disease generator due to hypoglycemic activity. 20. The method of claim 19, wherein the hypoglycemic disease is selected from the group consisting of type 1 diabetes, type 2 diabetes, arteriosclerosis, heart disease, and renal failure.

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