KR101886038B1 - Compositions with bioconversion Glycine max for improvement of immunity, diabetes, hyperlipemia or protection against liver damage - Google Patents

Abstract

The present invention relates to a composition for immune enhancement, improvement of diabetes, improvement of hyperlipemia or liver protection composition containing a soybean bioconversion. The soybean bioconversion of the present invention enhances immunity against microbial infection through the activation of macrophages, improves diabetes, improves liver damage or damage of the pancreas to Langerhans islet, and inhibits the production of triglycerides I will exert. It also has the effect of inhibiting alcoholic fatty liver or alcoholic fatty liver disease expressed by alcohol.

Description

TECHNICAL FIELD The present invention relates to a composition for improving immunity, for improving diabetes, for improving hyperlipidemia or for protecting liver, containing a soybean bioconversion, and to a method for improving hyperlipemia or protection against liver damage,

The present invention relates to a composition for immune enhancement, improvement of diabetes, improvement of hyperlipemia or liver protection composition containing a soybean bioconversion.

Immunity is largely divided into innate immunity (aka natural immune) and acquired immunity (aka acquired immunity). The three Nobel laureates of the 2011 Nobel Prize in Physiology awarded the Nobel Prize for medicine for discovering the core principles of the immune system against bacteria, viruses and fungi.

According to these studies, foreign invaders such as pathogenic microorganisms enter our bodies and are first associated with specific receptors on the cell surface of immune cells responsible for innate immunity. At this time, our body discovers external antigens, which are external intruders, and responds quickly. As a result, an inflammatory reaction occurs in cells and tissues, and the body feels fever or body fatigue.

This early immune response occurs immediately, regardless of the identity of the foreign antigen. Congenital immunity reacts nonspecifically to the antigen. In congenital immunity, macrophage, polymorphonuclear leucocyte, and NK cell, which can kill infected cells, are the most common. In fact, most infections are congenital It is defended by immune.

However, when immunity in the human body is weakened for various reasons, many immune enhancers have been developed and used to enhance immunity. Recently, immune activities such as anti-complement activity and lymphocyte division induction activity of electric polysaccharides extracted from fungi including fungi and mushroom have attracted attention. Therefore, in order to develop a new immunomodulating substance, polysaccharides produced in fungi have been intensively studied.

On the other hand, the risk of diabetes is emphasized because it can lead to various complications as well as death itself. The prevalence of diabetes is not only high in the world but also in Korea, the prevalence of diabetes and diabetes complications is higher than the OECD average. Therefore, the social cost is gradually increasing, which is attracting attention as a task to be solved.

Diabetes mellitus is classified into two types. First, the pancreatic beta cell function is broken and insulin is not properly secreted. Therefore, the first type of insulin is not controlled, and there is no problem with insulin secretion, but insulin sensitivity is low. Type 2 diabetes mellitus does not control blood sugar properly because the signal is not properly transmitted.

Type 1 diabetes is mostly congenital, and because insulin secretion is poor, it can be managed by simple treatment with insulin.

On the other hand, type 2 diabetes occurs mainly as an acquired factor, and it is known that type 2 diabetes occurs mainly due to insulin resistance. In the case of normal people, when the blood glucose concentration is increased through digestion and absorption after food intake, insulin secretion is smoothly performed, and cells of each tissue to which glucose is to be absorbed normally recognize insulin and absorb glucose. Absorbed sugars are used for a variety of uses, such as energy production or glycogen storage, thereby lowering blood sugar. However, in a person with insulin resistance, the cells in each tissue are less sensitive to insulin, failing to recognize secreted insulin normally, and not having proper glucose uptake and utilization.

If insulin does not play a role in insulin resistance, glucose can not be absorbed as an energy source by each tissue, or glucose can not be properly decomposed into glycogen, Eventually leading to type 2 diabetes. Such diabetes increases triglycerides, cholesterol, and may also cause liver and pancreatic tissue damage.

On the other hand, alcohol can cause liver damage, which is called alcoholic fatty liver. Currently, the number of chronic liver disease patients in the world reaches about 600 ~ 800 million, and the first cause of death in Korea is the liver disease. However, the currently marketed medicines are not effective in treating liver fibrosis or cirrhosis, and have been making efforts to develop them at home and abroad. However, the results are still insufficient.

On the other hand, soybean ( Glycine max ) refers to herbaceous perennial herb and its fruit. Unlike red beans and peas, the fruit is rich in protein and fat, and contains about 40% protein and 18% fat. Protein is more than 80% glycinin and albumin (regumelin) is only about 5%, but when soybean flour is soaked in water, almost 90% protein is dissolved.

The composition of carbohydrates has different characteristics from other fruits. That is, it contains only about 0.1% of starch, and it contains 7% of sucrose, 4% of stachyose, 4% of arabin, 6% of galactan and 4% of fiber. Soybean contains 18% of fat, more than half of which is unsaturated fatty acid and is used as a source of cooking oil. Soybean fatty acid is mainly semi-drying oil containing a lot of linoleic acid and oleic acid.

In addition, soybean is rich in isoflavone component of unsaturated fatty acid, it increases the absorption rate of calcium in the body to help bone health, and contains saponin component, which also helps prevent aging. It can be used as an excellent protein source instead of meat, so it is said to be good for diet.

However, up to now, it has been confirmed that no composition that exhibits both immune enhancement, diabetic improvement, triglyceride and cholesterol suppression, and hepatoprotective effect, including soybean bioconverted bioconverted organisms into the uppermost mycelium, has not been studied.

Korean Patent No. 10-1084446 discloses a composition for inhibiting cancer metastasis induced by angiogenesis of Angelica keiskei and jujube extract bioconverted by crude enzyme solution of Aspergillus sirousmi strain, Aspergillus usamii var. Shirousamii) and a composition for inhibiting cancer metastasis by neovascularization including a jujube extract. Korean Patent No. 10-1458806, entitled "Root Torula graminis JAG-12 having a catechin bioconverting ability, and a biotransformation method of catechin using the same," converts epigallocatechin gallate to epigallocatechin Rhodotorula graminis JAG-12 (Accession No .: KFCC11539P), which has a biotransformation ability, and a method for producing a fermented green tea extract using the same.

In the present invention, it is intended to develop and provide a novel composition exhibiting an immunity enhancing effect, diabetic improvement, hyperlipidemia improvement or liver protecting effect.

In order to achieve the above object, the present invention provides a process for producing a liquid medium comprising: (a) preparing a liquid medium containing soybean ( Glycine max ); (B) after the step (a), inoculating the mushroom mycelium into the liquid medium and culturing to produce a culture; (C) after the step (b), adding the fibrolytic enzyme to the culture and reacting; The present invention provides a food or pharmaceutical composition for enhancing immunity, which comprises a soybean bioconversion product prepared from a process comprising the steps of:

In the immunoconjugation food or pharmaceutical composition of the present invention, the soybean bioconverting agent is preferably subjected to 'enzyme inactivation and sterilization step (d)' in which the step (c) is followed by treatment at 80 to 100 ° C for 0.5 to 2 hours, May be prepared from the process further comprising. Further, after the 'enzyme inactivation and sterilization step (d)', the 'drying and pulverization step (e)' may be further included.

In the immunoconjugation food or pharmaceutical composition of the present invention, the liquid medium of step (a) is preferably prepared by adding at least one selected from amylase or cellulase to soybean, reacting, . At this time, the amylase or cellulase enzyme is preferably added in an amount of 0.1 to 0.5% (w / v) and reacted for 0.5 to 2 hours. When the amylase or cellulase enzyme is treated, it is possible to decompose starch and cellulose of soybean, and thus it is easy to prepare as a liquid medium.

In the immunoconjugation food or pharmaceutical composition of the present invention, the mushroom mycelium of step (b) may be produced by fermentation to produce a fibrinolytic enzyme and be discharged to the outside. This released fibrinolytic enzyme is capable of hydrolyzing soybeans.

In the immunoconjugation food or pharmaceutical composition of the present invention, the fibrinolytic enzyme of step (c) may be, for example, a cellulase, a hemicellulase, a pectinase, a glucanase, , Beta-glucanase, beta-glucosidase, lysozyme, viscozyme, filtrase, rapidase and sumizyme ). ≪ / RTI >

In the food or pharmaceutical composition for immunostaining according to the present invention, the soybean bioconversion preferably has immunity against microbial infection (activation) through activation of macrophages. The immunity (resistance) refers to resistance to disease.

In the immunomodulating food or pharmaceutical composition of the present invention, the soybean bioconversion may preferably be a polysaccharide. In this case, the polysaccharide is preferably obtained by adding distilled water to the soybean bioconversion obtained through step (c) to extract a water-soluble substance; Removing the residue through centrifugation after extracting the water-soluble substance, and separating the extracted supernatant (B); Adding EtOH to the extracted supernatant, mixing and maintaining (c); Separating the resulting precipitate after said holding (d); Adding distilled water to the separated precipitate to dissolve the precipitate, and then removing undissolved material by centrifugation or filtration; And performing dialysis against the sediment lysis solution after the centrifugation or filtration step (f).

In the pharmaceutical composition for enhancing immunity of the present invention, the pharmaceutical composition preferably contains 0.00001 to 100% by weight of a soybean bioconversion product relative to the whole composition. When the content is less than 0.00001% by weight, the effect is insignificant.

In the pharmaceutical composition for immunostaining according to the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient. Examples of usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and one or more selected from among them may be used. When the pharmaceutical composition is a pharmaceutical, it may further include at least one selected from a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent or an antiseptic agent.

In the pharmaceutical composition for enhancing immunity of the present invention, the formulation of the pharmaceutical composition may be in a desired form depending on the method of use, and may be in the form of a pharmaceutical composition, To be formulated by employing a method known in the art. Examples of specific formulations include, but are not limited to, PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, SYRUPS, OPHTHALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, ), Suspensions, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, SUPPOSITORIES, INJECTIONS, SPIRITS, CATAPLSMA, ), Capsules, CREAMS, TROCHES, TINCTURES, PASTES, PILLS, soft or hard gelatin capsules.

In the pharmaceutical composition for enhancing immunity of the present invention, the dosage of the pharmaceutical composition is preferably determined in consideration of the administration method, the age, sex, weight, and severity of the disease. For example, the pharmaceutical composition of the present invention can be administered at least once per day, 0.00001 to 100 mg / kg (body weight) based on the active ingredient. However, the dose is only an example for illustrative purposes, and may be changed by a physician's prescription depending on the condition of the user.

In the food composition for immunostaining according to the present invention, it is preferable that the food composition contains 0.00001 to 100% by weight of soybean bioconversion to the whole composition. When the content is less than 0.00001% by weight, the effect is insignificant.

In the food composition for enhancing immunity of the present invention, the food composition may be, for example, meat, cereal, caffeinated beverages, ordinary beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, noodles, Alcoholic beverages, alcoholic beverages, vitamin complexes, and other health supplement foods, but the present invention is not limited thereto.

The present invention also relates to a method for producing soybean milk, comprising the steps of: (a) preparing a liquid medium containing soybean; (B) preparing a fermented product by inoculating shiitake mycelia into the liquid medium after the step (a) and culturing the same; (C) a step of adding a fibrinolytic enzyme to the fermentation product after the step (b); A diabetic biologic conversion product produced from a process comprising the steps of: (a) culturing the diabetic microorganism in a culture medium containing the microorganism;

In the 'diabetic food composition for improvement of diabetes' or 'pharmaceutical composition for the prevention or treatment of diabetes' of the present invention, the soybean bioconversion product may be prepared by mixing the enzymatic fire Activation and sterilization step (d) 'are further included. Further, after the 'enzyme inactivation and sterilization step (d)', the 'drying and pulverization step (e)' may be further included.

In the 'diabetic food composition for improving diabetes' or the 'pharmaceutical composition for the prevention or treatment of diabetes' of the present invention, the liquid medium of step (a) preferably contains one or more selected from amylase or cellulase And then sterilization treatment is carried out. At this time, the amylase or cellulase enzyme is preferably added in an amount of 0.1 to 0.5% (w / v) and reacted for 0.5 to 2 hours. When the amylase or cellulase enzyme is treated, it is possible to decompose starch and cellulose of soybean, and thus it is easy to prepare as a liquid medium.

In the 'diabetic food composition for improving diabetes' or 'the pharmaceutical composition for the prevention or treatment of diabetes' of the present invention, the mushroom mycelium of step (b) may be produced by fermentation to produce a fibrinolytic enzyme and discharge it to the outside. This released fibrinolytic enzyme is capable of hydrolyzing soybeans.

In the 'diabetic food composition for improvement of diabetes' or 'pharmaceutical composition for the prevention or treatment of diabetes' of the present invention, the fibrotic enzyme of step (c) includes, for example, cellulase, hemicellulase, (Eg, pectinase, glucanase, β-glucanase, β-glucosidase, lysozyme, viscozyme, peptase, filtrate, rapidase, and sumizyme.

In the 'diabetic food composition for improving diabetes' or 'the pharmaceutical composition for the prevention or treatment of diabetes' of the present invention, the soybean bioconverting agent may preferably improve liver damage caused by diabetes mellitus or damage of the pancreas to Langerhans islet .

In the 'diabetic food composition for improving diabetes' or 'pharmaceutical composition for the prevention or treatment of diabetes' of the present invention, the soybean bioconversion may preferably be a polysaccharide. In this case, the polysaccharide is preferably obtained by adding distilled water to the soybean bioconversion obtained through step (c) to extract a water-soluble substance; Removing the residue through centrifugation after extracting the water-soluble substance, and separating the extracted supernatant (B); Adding EtOH to the extracted supernatant, mixing and maintaining (c); Separating the resulting precipitate after said holding (d); Adding distilled water to the separated precipitate to dissolve the precipitate, and then removing undissolved material by centrifugation or filtration; And performing dialysis against the sediment lysis solution after the centrifugation or filtration step (f).

In the pharmaceutical composition for the prevention or treatment of diabetes according to the present invention, the pharmaceutical composition preferably contains 0.00001 to 100% by weight of a soybean bioconversion product relative to the whole composition. When the content is less than 0.00001% by weight, the effect is insignificant.

In the pharmaceutical composition for preventing or treating diabetes according to the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient. Examples of usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and one or more selected from among them may be used. When the pharmaceutical composition is a pharmaceutical, it may further include at least one selected from a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent or an antiseptic agent.

In the pharmaceutical composition for the prevention or treatment of diabetes according to the present invention, the formulation of the pharmaceutical composition may be in a desired form depending on the method of use, and in particular, may be formulated so as to provide rapid, sustained or delayed release of the active ingredient It is preferable to employ a method known in the art to formulate it. Examples of specific formulations include, but are not limited to, PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, SYRUPS, OPHTHALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, ), Suspensions, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, SUPPOSITORIES, INJECTIONS, SPIRITS, CATAPLSMA, ), Capsules, CREAMS, TROCHES, TINCTURES, PASTES, PILLS, soft or hard gelatin capsules.

In the pharmaceutical composition for preventing or treating diabetes according to the present invention, the dose of the pharmaceutical composition is preferably determined in consideration of the administration method, the age, sex, weight, and severity of the disease. For example, the pharmaceutical composition of the present invention can be administered at least once per day, 0.00001 to 100 mg / kg (body weight) based on the active ingredient. However, the dosage is only an example and may be changed by a doctor's prescription depending on the condition of the recipient.

In the food composition for improving diabetes mellitus of the present invention, it is preferable that the food composition contains 0.00001 to 100% by weight of the soybean bioconversion product as compared with the whole composition. When the content is less than 0.00001% by weight, the effect is insignificant.

In the food composition for improving diabetes mellitus of the present invention, the food composition may be at least one selected from the group consisting of meat, cereal, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, Alcoholic beverages, alcoholic beverages, vitamin complexes, and other health supplement foods, but the present invention is not limited thereto.

On the other hand, the present invention provides a method for producing soybean milk, comprising the steps of: (a) preparing a liquid medium containing soybean; (B) preparing a fermented product by inoculating shiitake mycelia into the liquid medium after the step (a) and culturing the same; (C) a step of adding a fibrinolytic enzyme to the fermentation product after the step (b); A food composition for improving hyperlipidemia or a pharmaceutical composition for preventing or treating hyperlipidemia, which comprises a soybean bioconversion product prepared from a process comprising the steps of: At this time, the hyperlipemia may be preferably caused by 'diabetes'. In the present invention, the term 'hyperlipidemia' is meant to include hyperlipidemia caused by a rise in triglyceride levels in the body. That is, the hyperlipemia of the present invention is a concept including a disease caused by an increase in the content of triglycerides in the body, including the concept of hypertriglyceridemia.

In the 'food composition for improving hyperlipidemia' or 'pharmaceutical composition for preventing or treating hyperlipidemia' according to the present invention, the soybean bioconverted product may be prepared by mixing the enzymatic flour, which is treated at 80 to 100 ° C. for 0.5 to 2 hours, Activation and sterilization step (d) 'are further included. Further, after the 'enzyme inactivation and sterilization step (d)', the 'drying and pulverization step (e)' may be further included.

In the 'food composition for improving hyperlipidemia' or the 'pharmaceutical composition for the prevention or treatment of hyperlipidemia' according to the present invention, the liquid medium of step (a) preferably contains at least one selected from amylase or cellulase And then sterilization treatment is carried out. At this time, the amylase or cellulase enzyme is preferably added in an amount of 0.1 to 0.5% (w / v) and reacted for 0.5 to 2 hours. When the amylase or cellulase enzyme is treated, it is possible to decompose starch and cellulose of soybean, and thus it is easy to prepare as a liquid medium.

In the 'food composition for improving hyperlipemia' or the 'pharmaceutical composition for preventing or treating hyperlipidemia' according to the present invention, the mushroom mycelium of step (b) may be produced by fermentation to produce a cellulose decomposing enzyme and be discharged to the outside. This released fibrinolytic enzyme is capable of hydrolyzing soybeans.

In the 'food composition for improving hyperlipidemia' or the 'pharmaceutical composition for the prevention or treatment of hyperlipidemia' according to the present invention, the fibrotic enzymes of step (c) include, for example, cellulase, hemicellulase, pectinase, glucanase, beta-glucanase, beta-glucosidase, lysozyme, viscozyme, filtrase, Rapidase and Sumizyme may be used.

In the 'food composition for improving hyperlipidemia' or the 'pharmaceutical composition for the prevention or treatment of hyperlipidemia' according to the present invention, the soybean bioconversion inhibits the production of triglyceride and LDL-cholesterol and the production of high-density cholesterol (HDL- cholesterol. < / RTI >

In the 'food composition for improving hyperlipemia' of the present invention or the pharmaceutical composition for the prevention or treatment of hyperlipidemia, the soybean bioconversion may be polysaccharide. In this case, the polysaccharide is preferably obtained by adding distilled water to the soybean bioconversion obtained through step (c) to extract a water-soluble substance; Removing the residue through centrifugation after extracting the water-soluble substance, and separating the extracted supernatant (B); Adding EtOH to the extracted supernatant, mixing and maintaining (c); Separating the resulting precipitate after said holding (d); Adding distilled water to the separated precipitate to dissolve the precipitate, and then removing undissolved material by centrifugation or filtration; And performing dialysis against the sediment lysis solution after the centrifugation or filtration step (f).

In the pharmaceutical composition for preventing or treating hyperlipidemia according to the present invention, the pharmaceutical composition preferably contains 0.00001 to 100% by weight of a soybean bioconversion product relative to the whole composition. When the content is less than 0.00001% by weight, the effect is insignificant.

In the pharmaceutical composition for preventing or treating hyperlipidemia of the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient. Examples of usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and one or more selected from among them may be used. When the pharmaceutical composition is a pharmaceutical, it may further include at least one selected from a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent or an antiseptic agent.

In the pharmaceutical composition for the prevention or treatment of hyperlipidemia according to the present invention, the formulation of the pharmaceutical composition may be in a desired form depending on the method of use, and in particular, may be formulated so as to provide rapid, sustained or delayed release of the active ingredient It is preferable to employ a method known in the art to formulate it. Examples of specific formulations include, but are not limited to, PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, SYRUPS, OPHTHALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, ), Suspensions, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, SUPPOSITORIES, INJECTIONS, SPIRITS, CATAPLSMA, ), Capsules, CREAMS, TROCHES, TINCTURES, PASTES, PILLS, soft or hard gelatin capsules.

In the pharmaceutical composition for preventing or treating hyperlipidemia of the present invention, the dose of the pharmaceutical composition is preferably determined in consideration of the administration method, the age, sex, weight, and severity of the disease. For example, the pharmaceutical composition of the present invention can be administered at least once per day, 0.00001 to 100 mg / kg (body weight) based on the active ingredient. However, the dosage is only an example and may be changed by a doctor's prescription depending on the condition of the recipient.

On the other hand, in the food composition for improving hyperlipemia according to the present invention, it is preferable that the food composition contains 0.00001 to 100% by weight of the soybean bioconversion product as compared with the whole composition. When the content is less than 0.00001% by weight, the effect is insignificant.

In the food composition for improving hyperlipidemia according to the present invention, the food composition may be at least one selected from the group consisting of meat, cereal, caffeinated beverages, ordinary beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, noodles, Alcoholic beverages, alcoholic beverages, vitamin complexes, and other health supplement foods, but the present invention is not limited thereto.

On the other hand, the present invention provides a method for producing soybean milk, comprising the steps of: (a) preparing a liquid medium containing soybean; (B) preparing a fermented product by inoculating shiitake mycelia into the liquid medium after the step (a) and culturing the same; (C) a step of adding a fibrinolytic enzyme to the fermentation product after the step (b); , Or a " pharmaceutical composition for prevention or treatment of liver disease ", which is characterized by containing a soybean bioconversion product prepared from a process comprising the steps of:

In the 'liver protecting food composition' or the 'pharmaceutical composition for preventing or treating liver disease' of the present invention, the soybean bioconverting agent may be treated with an enzymatic fire, which is treated at 80 to 100 ° C. for 0.5 to 2 hours, Activation and sterilization step (d) 'are further included. Further, after the 'enzyme inactivation and sterilization step (d)', the 'drying and pulverization step (e)' may be further included.

In the 'liver protecting food composition' or the 'pharmaceutical composition for preventing or treating liver disease' according to the present invention, the liquid medium of step (a) is preferably one or more selected from amylase or cellulase And then sterilization treatment is carried out. At this time, the amylase or cellulase enzyme is preferably added in an amount of 0.1 to 0.5% (w / v) and reacted for 0.5 to 2 hours. When the amylase or cellulase enzyme is treated, it is possible to decompose starch and cellulose of soybean, and thus it is easy to prepare as a liquid medium.

In the 'liver protecting food composition' or the 'pharmaceutical composition for prevention or treatment of liver disease' of the present invention, the mushroom mycelium of step (b) may be produced by fermentation to produce a cellulase. This released fibrinolytic enzyme is capable of hydrolyzing soybeans.

In the 'liver protecting food composition' or the 'pharmaceutical composition for the prevention or treatment of liver disease' of the present invention, the fibrinolytic enzyme of step (c) includes, for example, cellulase, hemicellulase, pectinase, glucanase, beta-glucanase, beta-glucosidase, lysozyme, viscozyme, filtrase, Rapidase and Sumizyme may be used.

In the 'liver protecting food composition' or the 'pharmaceutical composition for preventing or treating liver disease' of the present invention, the soybean bioconduct may preferably inhibit alcoholic fatty liver or alcoholic fatty liver hepatitis expressed by alcohol.

In the 'liver protecting food composition' or 'pharmaceutical composition for preventing or treating liver disease' of the present invention, the soybean bioconverting agent may be a polysaccharide. In this case, the polysaccharide is preferably obtained by adding distilled water to the soybean bioconversion obtained through step (c) to extract a water-soluble substance; Removing the residue through centrifugation after extracting the water-soluble substance, and separating the extracted supernatant (B); Adding EtOH to the extracted supernatant, mixing and maintaining (c); Separating the resulting precipitate after said holding (d); Adding distilled water to the separated precipitate to dissolve the precipitate, and then removing undissolved material by centrifugation or filtration; And performing dialysis against the sediment lysis solution after the centrifugation or filtration step (f).

In the pharmaceutical composition for preventing or treating liver diseases according to the present invention, the pharmaceutical composition preferably contains 0.00001 to 100% by weight of a soybean bioconversion product relative to the whole composition. When the content is less than 0.00001% by weight, the effect is insignificant.

In the pharmaceutical composition for preventing or treating liver disease according to the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient. Examples of usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and one or more selected from among them may be used. When the pharmaceutical composition is a pharmaceutical, it may further include at least one selected from a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent or an antiseptic agent.

In the pharmaceutical composition for the prevention or treatment of liver disease according to the present invention, the formulation of the pharmaceutical composition may be in a desired form depending on the method of use, and can be used to provide rapid, sustained or delayed release of the active ingredient It is preferable to employ a method known in the art. Examples of specific formulations include, but are not limited to, PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, SYRUPS, OPHTHALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, ), Suspensions, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, SUPPOSITORIES, INJECTIONS, SPIRITS, CATAPLSMA, ), Capsules, CREAMS, TROCHES, TINCTURES, PASTES, PILLS, soft or hard gelatin capsules.

In the pharmaceutical composition for preventing or treating liver disease according to the present invention, the dose of the pharmaceutical composition is preferably determined in consideration of the administration method, the age, sex, weight, and severity of the disease. For example, the pharmaceutical composition of the present invention can be administered at least once per day, 0.00001 to 100 mg / kg (body weight) based on the active ingredient. However, the dosage is only an example and may be changed by a doctor's prescription depending on the condition of the recipient.

On the other hand, in the food composition for liver protection of the present invention, the food composition preferably contains 0.00001 to 100% by weight of a soybean bioconversion product relative to the whole composition. When the content is less than 0.00001% by weight, the effect is insignificant.

In the food composition for liver protection of the present invention, the food composition may be, for example, meat, cereal, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, noodles, , An alcoholic beverage, a drink, a vitamin complex, and other health supplement foods, but the present invention is not limited thereto.

The soybean bioconversion of the present invention enhances immunity against microbial infection through the activation of macrophages, improves diabetes, improves liver damage or damage of the pancreas to Langerhans islet, and inhibits the production of triglycerides I will exert. It also has the effect of inhibiting alcoholic fatty liver or alcoholic fatty liver disease expressed by alcohol.

BRIEF DESCRIPTION OF THE DRAWINGS FIG.
FIG. 2 is a process chart of post-treatment and recovery of the soybean bioconversion product of the present invention.
FIG. 3 is a graph showing NO production results of macrophage cells which are remarkably enhanced by NO production ability and concentration-dependently increased by the soybean bioconversion of the present invention.
4 is a graph showing the cytokine expression potential of soybean and the soybean bioconversion of the present invention.
5 is a graph showing the antimicrobial activity of salmonella in the soybean bioconversion product of the present invention.
FIG. 6 is a graph showing the effect of the present invention soybean bioconversion on the salmonella colonizing ability of macrophages.
FIG. 7 is a graph showing the effect of the present invention soybean bioconversion on the inhibition of salmonellosis proliferation in macrophages.
8 is a graph showing the delayed effect of the present invention soybean bioconverting agent in the mortality rate caused by Salmonella infection of a mouse.
FIG. 9 is a graph showing the results of inhibiting salmonella in the abdominal cavity of the present invention.
FIG. 10 is a graph showing the penetration inhibiting activity of the present soybean bioconverting agent in intestinal cell infiltration of Salmonella using Caco-2 cell line. FIG.
11 is a graph showing the effect of the present soybean bioconverting agent on the death of INS-1 cell line induced by alloxan and production of reactive oxygen species (ROS).
Fig. 12 (A) is a graph showing the protective effect of hepatic injury protection of the soybean bioconversion of the present invention in a type 1 diabetes mouse induced by alloxan.
Fig. 12 (B) is a photograph of the hepatocyte protective effect of the present soybean bioconversion in a type 1 diabetes mouse induced by alloxan. Fig.
Fig. 13 is a photograph showing the protective effect of pancreatic damage of the soybean bioconversion of the present invention in a type 1 diabetic mouse induced by alloxan.
14 is a graph showing the effect of the soybean bioconversion of the present invention in a type 2 diabetic mouse.
Fig. 15 (A) is a photograph showing the protective effect of the liver tissue of the soybean bioconversion product of the present invention in a type 2 diabetic mouse. Fig.
Fig. 15 (B) is a photograph of the protective effect of the pancreatic tissue of the soybean bioconversion of the present invention in a type 2 diabetic mouse. Fig.
16 is a graph showing an inhibitory effect of the present soybean bioconversion on alcohol-induced hepatocyte death.
17 is a graph showing the inhibitory effect of the present soybean bioconversion on the generation of ROS in alcohol-induced hepatocyte death.
18 is a graph showing an effect of inhibiting liver weight increase due to the administration of the soybean bioconversion of the present invention in an alcohol-induced liver injury mouse.
FIG. 19 is a photograph showing the protective effect of liver damage caused by the administration of the soybean bioconversion of the present invention in an alcohol-induced liver injury mouse.
Figure 20 shows a manufacturing process diagram and a manufacturing process for obtaining a polysaccharide fraction from the soybean bioconversion product of the present invention.
FIG. 21 shows the chromatogram results of the HPLC analysis of the polysaccharide fractions of the soybean bioconversion of the present invention.
22 shows the HPLC chromatogram results of the polysaccharide fractions of the present invention soybean bioconversion.
23 shows the results of evaluation of macrophage activation ability of the polysaccharide fractions of the soybean bioconverted product of the present invention.
24 shows the results of the cytokine secretion pattern of the soybean bioconversion product and soybean bioconversion polysaccharide fraction of the present invention.
25 shows the result of inhibition of the secretion amount of cytokines in the polysaccharide fractions of soybean bioconverted product and soybean bioconverted product of the present invention at the time of TLR4 inhibitor treatment.

(A) preparing a liquid medium containing soybean ( Glycine max ); (B) a step (b) of adding a fibrinolytic enzyme to the culture after the step (a), and then adding a fibrinolytic enzyme to the culture after the step (c); A soybean bioconversion product prepared from a process comprising the steps of:

According to the experiment of the present invention, it was confirmed that the soybean bioconversion obtained from the process of the present invention can increase immunity against microbial infection through activation of macrophages. In addition, it has been confirmed that diabetes can be improved, hepatic damage or damage of the pancreas to Langerhans islet can be prevented, and the production of triglyceride is inhibited, thereby improving or preventing (or treating) hyperlipemia. In addition, it has been confirmed that the effect of protecting the liver such as alcoholic fatty liver or alcoholic fatty liver disease expressed by alcohol or preventing (or treating) liver disease can be exerted.

Meanwhile, in the present invention, step (a) of adding a distilled water to the soybean bioconversion product obtained through the step (c) to extract a water-soluble substance; Removing the residue through centrifugation after extracting the water-soluble substance, and separating the extracted supernatant (B); Adding EtOH to the extracted supernatant, mixing and maintaining (c); Separating the resulting precipitate after said holding (d); Adding distilled water to the separated precipitate to dissolve the precipitate, and then removing undissolved material by centrifugation or filtration; And performing dialysis against the sediment lysis solution after the centrifugation or filtration. The polysaccharide fraction obtained by the process further comprises:

In the present invention, after obtaining the polysaccharide fraction from the soybean bioconversion, the soybean bioconversion and the polysaccharide fraction isolated therefrom were recognized in a pattern recognition receptor which is a specific receptor of immune cells, and it was confirmed whether signal transduction is mediated.

As a result of the following experiments, it was confirmed that the TNF-α, IL-6, IL-10 and IFN-β were expressed in the soybean bioconversion product and the polysaccharide fractions thereof. The activity was confirmed to be due to the polysaccharide fraction isolated therefrom.

In the following experiment, it was confirmed that the polysaccharide fraction isolated from the soybean bioconversion was similar to the lipopolysaccharide (LPS) and cytokine secretion pattern. Thus, it was confirmed that the polysaccharide fraction of the present invention is an antioxidant of LPS Which is an antagonist.

On the other hand, TAK-242, a specific inhibitor for TLR4, was used to confirm whether the soybean bioconversion product and its polysaccharide fraction of the present invention were specifically recognized and mediated by TLR4. As a result of the following experiment, it was confirmed that the reactions induced by the treatment of the soybean bioconversion product or its polysaccharide fraction were all inhibited by the TLR4-specific inhibitor TAK242. From this, it was confirmed that TLR4 is a receptor for the soybean bioconversion product and its polysaccharide fraction I could confirm.

From these results, it was confirmed that the soybean bioconversion product and its polysaccharide fraction produced by the bioconversion process pursued in the present invention have TLR4 agonist activity.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[Example 1: Preparation of soybean biotransformation]

 (1) Pretreatment process (foreign body and mold washing process)

In order to produce a soybean bioconversion, a washing process was performed to remove pollutants such as foreign matter and mold of soybean ( Glycine max ).

First, foreign matter and dust attached to the surface of soybeans were removed with a high-pressure air gun. Secondly, the sieve was sieved to remove other foreign matter. Thirdly, water or ethanol was treated to remove contaminants such as mold spores.

The soybean which had been subjected to the third washing process was used as a raw material after the microbial contamination test.

 (2) Enzymatic treatment with hydrolytic enzymes for culture

After removing the foreign matter and fungal contamination, the soybean was pulverized and enzymatic treatment was carried out using various hydrolytic enzymes. The hydrolytic enzymes were tested using enzymes of the amylase family and the cellulase family which can decompose starch and cellulose of soybean.

Each enzyme was treated with 0.3% (w / v), reacted for 1 hour at the temperature showing optimal activity, and sterilized to prepare a liquid medium.

 (3) Fermentation and enzyme treatment Bioconversion process

Soybean was cultured at 30 ° C inoculated with a mycelium of liquid culture medium. At the time when the residual carbon source was depleted, the cells were cultured for 8 days in a liquid phase process in which a concentrated liquid medium was added. The soybean hyphae can be hydrolyzed by cultivating the enzymes to produce cellulose decomposing enzymes. After cultivation, the fermented soybeans produced by the fermentation process were further subjected to an enzyme treatment process, which is a secondary bioconversion process.

The above-mentioned fermentation and enzymatic treatment bioconversion process is an effective production of cell wall-derived immunologically active substance from soybean fermentation substrate fermentation product, which is a cultured fermentation product, and is characterized in that the structural change of a specific component , Which is an effective method for extracting and efficiently extracting an immunologically active substance.

Accordingly, the present inventors have found that a cell wall lytic enzyme complex (cellulase, hemi-cellulase, pectinase, and beta-glucanase, which is a cell wall hydrolase capable of degrading a cell wall, The enzyme / substrate reaction was optimized by shaking at 250 rpm at 50 to 60 ° C using an enzyme conjugate of β-glucanase (see FIG. 1). BRIEF DESCRIPTION OF THE DRAWINGS FIG.

 (4) Post-treatment process

The soybean bioconversion product produced by the secondary biotransformation process (enzyme reaction) was subjected to 'enzyme inactivation and sterilization' process at 90 ° C for 1 hour, followed by drying and pulverization, and used in the following experimental examples 2). Fig. 2 is a flow chart of the post-treatment and recovery operation of the soybean bioconversion product.

[ Experimental Example 1: Measurement of in vitro NO production ability in macrophages ( macrophages ) by soybean and soybean biotransformation ]

In order to confirm macrophage activation effect of soybean and soybean bioconversion, NO production ability of macrophage (macrophage), RAW264.7 cell line was examined.

100 μl of soybean and soybean biotransformers and standards diluted with a soybean biotransformant in a 96-well plate was dispensed into a 96-well plate and the mixture was treated with a Griess reagent (N- (1-naphtyl) ethylene diamine dihydrochloride: 0.5 g / 500 ml ② Sulfanilamide: 5 g / 85% H ₃ PO ₄ 29.5 ml / 470.5 ml) was added and reacted for 1 minute. Then, the immunoreactivity of the sample was measured by measuring the abundance of macrophage NO by measuring the absorbance at 540 nm (or 550 nm) using an ELISA leader.

The results showed that the soybean bioconversion showed NO production ability at a concentration lower than 1 / 1,000 ~ 1 / 10,000 compared with that of soybean, and the NO production ability of the macrophage by the soybean bioconversion was remarkably improved, (Fig. 3). FIG. 3 is a graph showing the NO generation ability and macrophage NO production of macrophage which are increased in a dose-dependent manner by the soybean bioconversion.

[ Experimental Example 2: Analysis of cytokine secretion ability of soybean and soybean bioconversion in macrophages ]

To confirm the innate immune response activation effect of soybean and the soybean bioconversion of the present invention, cytokine secretion ability in macrophages (macrophages) and RAW264.7 cell lines was examined.

Soybean and the soybean bioconduct of the present invention were treated at a concentration of 1, 10, and 100 / / ml, respectively. After 16 hours of sample treatment, the supernatant of the culture was taken and TNF-α, IL-1β, IL-4, IL-5, (Interleukin-6), IL-10 (Interlukine-10), IL-12p70 (Interlukine-12p70), IFN- Respectively.

As a result of the experiment, it was confirmed that TNF-α, IL-6, IL-10 and IFN-β were expressed when the soybean bioconversion was treated (FIG. 4 is a graph showing the cytokine expression potential of soybean and soybean transformants.

TNF-α is secreted from macrophages and mononuclear cells during phagocytosis, and is known to have an antiviral effect against various viruses such as influenza virus along with 'inducible nitrogen monoxide (NO)'.

IL-6 is a major mediator in antigen-specific immune responses, inflammatory responses, and acute responses, and is a representative cytokine that plays a key role in the in vivo defense system.

IFN-β is a type I interferon with IFN-α, and has antiviral and antiviral effects. It is a typical cytokine that induces Th1 immune response by contributing to Th1 cell differentiation, inducing Th1 immune response, while Th2 And Th17 immune response.

The culture supernatant was taken every 6 hours, 12 hours, and 18 hours after the sample treatment time, and the cytokine secretion was examined. The experiment was repeated three times.

Therefore, the soybean bioconversion of the present invention is a material capable of specifically activating the Th1 immune response, and it was judged that the Th2 immune response at the same time can be suppressed. It is also expected that it may be effective in diseases where it is necessary to improve the Th1 immune response or that the Th2 immune response is exaggerated.

Test Example 3: Soybean biotransformation of water, in vitro evaluation jeneung million salmonella infections in macrophages;

In this experiment, we investigated the effect of soybean bioconversion on the phagocytosis and inhibition of microbial survival by inducing macrophage activation in an in vitro system using macrophages.

 (1) Measurement of antibacterial activity of soybean bioconversion

In order to evaluate the inhibitory effect of soybean bioconversion on Salmonella infection, we tried to confirm whether soybean bioconversion has direct antimicrobial activity.

Salmonella typhimurium ATCC14028 strain was used as the indicator strain and nutrient broth (NBGP), which is a general salmonella culture medium, and phenol red and glucose for fermentation, was used as an indicator strain. The growth of salmonella and color change due to sugar fermentation were measured.

In the culture of Salmonella, it was confirmed that the soybean bioconversion was inhibited by 1, 10, and 100 ㎍ / mL, respectively. The survival rate was measured at 0, 2, 4 and 8 hours after incubation. Respectively.

As a result of the experiment, it was confirmed that the soybean bioconversion does not show direct antimicrobial activity (FIG. 5). 5 is a graph showing antimicrobial activity of salmonella in a soybean bioconversion product.

 (2) Measurement of Macrophage Phagocytosis of Soybean Bioconversion

The macrophage RAW 264.7 cell line was treated with soybean bioconducts at concentrations of 1, 10, and 100 ㎍ / mL for 4 hours, followed by Salmonella typhimurium ATCC14028 was infected for 30 minutes and 60 minutes.

As a result of the experiment, it was confirmed that the increase in the phagocytosis due to the treatment with the soybean biotransformant increased to a maximum of 3.49-fold at 30 minutes of infection with Salmonella, and increased up to 9.05-fold at 60-minute infection with Salmonella (FIG. 6). FIG. 6 is a graph showing the effect of concentration-based soybean conversion on macrophage salmonellosis.

 (3) Measurement of inhibitory effect of soybean bioconversion on salmonella growth

The macrophage RAW 264.7 cell line was treated with soybean bioconverters at a concentration of 1, 10, and 100 μg / mL for 4 hours, followed by Salmonella typhimurium ATCC14028) was infected for 2 hours, 4 hours, and 8 hours, and then an increase in Salmonella was observed.

As a result of the experiment, the increase of intracellular salmonella occurred for 2 hours, and then decreased. In the group treated with 100 μg / mL of soybean bioconversion and infected with Salmonella, it was confirmed that salmonella present in cells significantly decreased after a maximum of 8 hours (FIG. 7). 7 is a graph showing the effect of the concentration-based soybean bioconversion on inhibition of salmonellosis in macrophages.

[ Experimental Example 4: Evaluation of modulating efficacy against mouse mortality caused by Salmonella infection ]

As a result of the above experiment, it was confirmed that the soybean bioconversion product induces macrophage activation in macrophage in vitro to increase phagocytosis and suppress the survival of intracellular microorganisms after phagocytosis, Salmonella typhimurium ATCC14028) 'strains were used to test the in vivo antimicrobial activity of soybean biotransformants.

In order to determine if the administration of the soybean conversion could reduce the mortality rate of the mice due to Salmonella infection, 10 ⁵ CFU of salmonella ( Salmonella typhimurium ATCC14028) strain was injected intraperitoneally to induce the death of the mice by infection.

As a result, the control mice showed that all mice died at 7 days after the infection, whereas when the soybean bioconversion was administered at a dose of 10 mg / kg, the mouse survived up to 18 days, (Fig. 8). FIG. 8 is a graph showing the delayed effect of the soybean biotransformation on the mortality caused by Salmonella infection of the mouse.

Experimental Example 5: Soybean bioconversion of water, in vivo live in Salmonella infection mice infected with Monel called inhibitory ability evaluation;

As a result of the above experiment, it was confirmed that the administration of the soybean biotransformant reduced the mortality rate of the mice due to Salmonella infection at the lethal concentration. Thus, in this Example, the in vivo antimicrobial activity and the soybean biotransformation Induced activation of the immune response.

Soybean bioconver- sion was fed at a dose of 10 mg / kg for 2 weeks, and then ' Salmonella typhimurium ATCC14028 'strain was intraperitoneally injected at a concentration of ¹ × 10 ⁴ CFU to induce intraperitoneal Salmonella infection.

Two days after infection, the salmonella in the abdominal cavity of the mice was recovered using PBS, and the number of Salmonella surviving in the abdominal cavity was measured by spreading on an LB plate.

As a result of measurement, it was confirmed that salmonella surviving in the group treated with the soybean bioconversion was significantly reduced compared with the control group (FIG. 9). 9 is a graph showing a result of inhibiting salmonella in the abdominal cavity of a soybean bioconversion product.

[ Experimental Example 6: Induction of lymphocyte proliferation and evaluation of interferon-gamma production activation in Salmonella-infected mice ]

After the intraperitoneal salmonellar experiment, the spleen of the mouse was excised and splenic lymphocytes were isolated. The isolated lymphocytes were stimulated with concanavalin A, which is a T cell proliferation inducing substance, and LPS, which is a B cell proliferation inducing substance, to determine how cell proliferation is induced. The results are shown in Table 1 below.

Sample Lymphocyte proliferation (fold-increase) IFN-y (pg / mL) vehicle 1.38 + - 0.12 287.204 ± 13.586 10 mg / kg soybean biotransformation 1.77 + - 0.12 337.596 ± 30.048

Experimental results showed that mouse spleen lymphocytes were increased more in the soybean conversion-treated mice than in the control group. In addition, it was confirmed that the expression level of interferon-gamma (IFN-y), a representative Th1 cytokine, was increased.

Test Example 7: Evaluation Th1 cytokine (cytokine) activation efficacy in mice infected with Salmonella;

In order to confirm whether the administration of the soybean bioconversion induces the activation of the Th1 immune response, the amount of Th1 cytokine expressed in the spleen of the mouse infected with salmonella at the above-mentioned concentration of saliva was measured to determine the Th1 activation effect Respectively. Four cytokines, IL-1β, IL-2, IL-6 and IL-12, were measured in the Th1 cytokine using ELISA.

Activation effects were compared between the mice infected with Salmonella and the mice infected with Salmonella by the soybean bioconversion, respectively. The results are shown in Table 2 below.

sample cytokines (pg / mL) IL-1? IL-2 IL-6 IL-12 salmonella (-) vehicle 171 ± 12 41.7 ± 3.7 57.3 ± 3.2 240 ± 13 10 mg / kg
Soybean biotransformation 183 ± 15 42.1 ± 3.0 57.7 ± 4.0 250 ± 16 salmonella (+) vehicle 239 ± 13 55.7 ± 3.8 73.4 ± 5.4 284 ± 13 10 mg / kg
Soybean biotransformation 251 ± 17 60.3 ± 2.7 76.9 ± 5.3 320 ± 27

As a result, there was no significant difference in the expression level of cytokine except that the expression level of IL-1β was slightly increased when the soybean transformant was administered to normal mice. On the other hand, when infected with Salmonella, the expression level of cytokines such as IL-1β, IL-2, IL-6 and IL-12 is significantly increased by Salmonella infection, In the case of simultaneous administration, it was confirmed that more cytokine production was induced. In particular, IL-12, which is a representative Th1 cytokine, showed the most remarkable increase in the expression level.

These results show that the administration of the soybean bioconversion does not induce unnecessary activation of the immune response in the normal living body and induces the activation of the Th1 immune response upon microbial infection, thereby effectively controlling the infected microorganisms in vivo.

Test Example 8: Th2 cytokine (cytokine) expression efficacy evaluation in Salmonella infected mice;

As a result of evaluating the cytotoxic activity of Th1 cytokine, it was confirmed that Th1 immune response, which is an antagonistic reaction of Th1 immune response, is activated by the administration of the soybean bioconversion activates the expression of Th1 cytokine To investigate the expression level of Th2 cytokine,

Three cytokines, IL-4, IL-5 and IL-10, which are representative Th2 cytokines, were detected. Similar to Th1 cytokine, it was identified in both normal and Salmonella-infected mice not infected with Salmonella. The results are shown in Table 3 below.

sample cytokines (pg / mL) IL-4 IL-5 IL-10 salmonella (-) vehicle 52.1 ± 3.4 294 ± 17 44.7 ± 2.3 10 mg / kg
Soybean biotransformation 51.4 ± 2.7 289 ± 19 44.5 ± 2.7 salmonella (+) vehicle 56.8 ± 4.3 289 ± 17 48.7 ± 3.3 10 mg / kg
Soybean biotransformation 56.9 ± 4.1 292 ± 13 49.6 ± 4.3

As a result of the experiment, it was confirmed that the administration of the soybean biotransformant did not increase the production of Th2 cytokine in both the normal mouse not infected with Salmonella and the infected mouse

These results indicate that the administration of the soybean biotransformant does not induce activation of unnecessary immune responses in normal organisms and does not induce activation of Th2 immune response upon microbial infection.

Experimental Example 9: Soybeans in bioconversion of water, the intestinal cells Evaluation of inhibition of penetration of Salmonella in vitro ]

In this experiment, we evaluated the effect of soybean bioconversion on the intestinal cell adhesion and penetration inhibition of Salmonella using Caco-2 cell line. Since most of the Salmonella infections occur through intestinal infections, we wanted to see if the soybean bioconversion could inhibit salmonella intestinal cell adhesion and invasion. Who was evaluated in colon cancer derived cell line, Caco-2 cell line and in vitro experimental system using the Salmonella typhimurium SL1344 strain.

Caco-2 cell line was treated with 100 μg / mL of soybean bioconversion for 4 hours or with Salmonella typhimurium SL1344, and the number of Salmonella infiltrated into cells was measured.

As a result of the experiment, it showed 7.1% inhibition of salmonella penetration by 4 hours pretreatment, and 52.4% inhibition of salmonella penetration by simultaneous treatment. Since the soybean bioconversion was confirmed to have no antimicrobial activity in the above experiment, it was judged that it acted directly on the Caco-2 cell line and inhibited microbial penetration (FIG. 10). 10 is a graph showing the intestinal permeation inhibitory activity of a soybean bioconversion product using a Caco-2 cell line.

[ Experimental Example 10: Effect of Soybean Biotransformation on Salmonella Infected Mice in vivo inhibition of Salmonella penetration]

In this experiment, the inhibition of Salmonella permeation in vivo of soybean bioconversion was investigated.

 (1) Identification of salmonella excreted in feces

As a result of the experiment using the cell line, it was confirmed that the soybean bioconversion inhibits intestinal permeability of salmonella, so that the salmonella infestation inhibitory effect of the salmonella food poisoning mouse model was evaluated.

The soybean bioconver- sion was fed at a dose of 10 mg / kg for 2 weeks, followed by oral administration of 10 ⁸ CFU of ' Salmonella typhimurium SL1344' to induce Salmonella infection. The feces were sampled 24 hours and 48 hours after infection, and the viable cell counts of Salmonella in feces were measured. The results are shown in Table 4 below.

Sample Salmonella in feces (x10 ⁴ CFU / g) 1 day 2 day normal mice 0.00 ± 0.00 0.00 ± 0.00 SL1344 infection only 338 ± 18 420 ± 31 SL1344 infection +
10 mg / kg soybean biotransformation 613 ± 42 665 ± 36

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As a result of the experiment, it was confirmed that a large amount of Salmonella did not penetrate into the intestinal tract and escaped into the feces when all of the soybean transformants were administered.

 (2) Identification of Salmonella infiltrated into tissues

In the results of Table 4, it was confirmed that when the soybean bioconversion was administered, Salmonella entered into the organism could not penetrate into the tissues and was discharged to the outside of the body. Thus, the fecal release experiment mice were administered to the cecum and intestinal membrane lymph nodes, spleen, liver And the number of Salmonella infiltrated into the tissues was measured.

① Cecum and intestinal membrane lymph nodes

After the infection of salmonella, salmonella in the intestinal membrane lymph node, the cecum, which is the primary infiltrating tissue, and the lymphatic tissue connected to it, were measured and the effect of inhibiting tissue infiltration of the soybean bioconversion was examined. The results are shown in Table 5 below.

Sample Salmonella in tissue Cecum (x10 ⁵ CFU / g) Mesenteric lymph node (x10 ³ CFU / organ) normal mice 0.00 ± 0.00 0.00 ± 0.00 SL1344 infection only 489 ± 32 109 ± 8 SL1344 infection +
10 mg / kg soybean biotransformation 257 ± 16 40.8 ± 3.2

The results showed that the soybean biotransformation inhibited the penetration of salmonella into the cecum, and the soybean bioconversion showed 47.4% inhibition. Soybean biotransformation was found to inhibit Salmonella in 62.5% of intestinal membrane lymph nodes, and it was found that the inhibition rate in intestinal membrane lymph nodes was greater than in cecum.

These results suggest that the immune cells in the intestinal membrane lymph node were activated by the soybean biotransformant and could be effectively removed even if Salmonella infiltrated.

② spleen and liver

The amount of Salmonella in the spleen and liver which can penetrate into tissues and move through body fluids was measured.

Sample Salmonella in tissue Spleen (x10 ² CFU / organ) Liver (x10 ² CFU / organ) normal mice 0.00 ± 0.00 0.00 ± 0.00 SL1344 infection only 129 ± 11 77.4 ± 5.6 SL1344 infection +
10 mg / kg soybean biotransformation 29.4 ± 1.9 24.3 ± 1.5

The results showed that 77.2% of salmonella in the spleen was inhibited when the soybean bioconversion was administered. In addition, soybean bioconversion against Salmonella present in the liver showed a 68.6% inhibition rate. Both the spleen and liver showed a higher inhibition rate than the cecum, and these results were also attributed to the activation of various immune cells.

[ Experimental Example 11: Synthesis of Soybean Biotransformant in Beta Cells Evaluation of inhibition of type 1 diabetes in vitro ]

In order to confirm whether the soybean bioconversion product has an inhibitory activity against type 1 diabetes, the activity of the soybean bioconversion product was evaluated in an in vitro system using the INS-1 cell line, a mouse-derived pancreatic beta cell line.

 (1) Effect on beta-cell cytotoxicity and reactive oxygen production

Pancreatic beta cells and beta-cell specific reactive oxygen species (ROS) through INS-1 cell line through 'Glucose transporter 2 (GLUT2)', and treatment with alloxan, And inhibited cell death by treating soybean bioconversion.

The INS-1 cell line was dispensed into a 96-well plate at a concentration of ⁵ × 10 ⁵ cells / well, treated with 10 mM alloxan and 1, 10 and 100 μg / mL of soybean bioconversion And cultured for 48 hours. After incubation, cell viability was measured by MTT assay.

As a result of confirming the ability of the soybean biotransformation to inhibit beta cell death and inhibit ROS formation, it was found that the inhibition of cell death and ROS generation by alloxan was dose dependent, and the cell death rate by alloxan To a maximum of about 30.4%, and ROS production was inhibited up to about 46.2% (FIG. 11). 11 is a graph showing the effect of soybean bioconversion on the death of INS-1 cell line induced by alloxan and production of reactive oxygen species (ROS).

 (2) Effect on beta -cellular NO production and insulin secretion

Increased ROS and cell death in the beta cell, INS-1 cell line by treatment with alloxan increase the amount of nitric oxide (NO) produced and secreted by the cell and decrease the secretion of insulin do.

The amount of nitric oxide (NO) secreted by the INS-1 cell line and the amount of insulin secreted by the INS-1 cell line were significantly decreased by the simultaneous treatment of the soybean biotransformation with the increase of intracellular ROS and the cell death rate by alloxan treatment The amount was also measured. The results are shown in Table 7 below.

Sample Nitric oxide
(arbitrary fluorescent unit) Insulin release (ng / mL) vehicle 19.784 ± 1.253 1.809 ± 0.057 10 mM Alloxan only 82.479 ± 5.261 0.821 + 0.034 10 mM Alloxan +
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Bioconversion 1 / / mL 77.883 + 5.100 0.859 + 0.042 10 [mu] g / mL 54.128 ± 3.821 0.956 + 0.063 100 / / mL 37.771 ± 1.914 1.196 + 0.073

As a result of the experiment, it was found that the secretion of NO increased by the treatment of alloxan was inhibited by the simultaneous treatment of the soybean bioconversion and the amount of insulin secreted by the treatment of alloxan decreased Water, and it was confirmed that soybean bioconversion inhibited NO production and secretion by 54.2% at the maximum concentration of 100 ㎍ / mL.

In the case of insulin, the amount of secretion was recovered to 45.7% at the maximum concentration of 100 ㎍ / mL.

[ Experimental Example 12: Effect of Soybean Biotransformation on Type 1 Diabetes Mellitus-induced Mice in vivo Type 1 diabetes inhibiting ability evaluation]

In the above-mentioned in vitro experiment, it was confirmed that the treatment with soybean biotransformant had an antidiabetic effect on type 1 diabetes, so that the antidiabetic effect of the soybean bioconversion in the mouse model of type 1 diabetes was tested .

(1) The effect of the type 1 diabetic mouse model on blood glucose, serum insulin, and hepatic glycogen level

Type 1 diabetes mellitus was induced by intraperitoneal injection of 100 mg / kg of alloxan in a diet of 10 mg / kg for 2 weeks. After one week of diabetes induction, blood glucose level, blood insulin concentration, and hepatic glycogen concentration were measured as an indicator of diabetes, and the results are shown in Table 8 below.

Sample Blood glucose (mg / dL) Serum insulin
(ng / mg protein) Glycogen
(mg / g liver wt.) vehicle 127.0 ± 8.1 59.204 + - 4.283 3.041 + - 0.213 Alloxan only (100 mg / kg) 308.4 ± 15.7 9.407 ± 0.821 2.427 ± 0.154 Alloxan +
10 mg / kg soybean biotransformation 247.3 ± 12.0 30.749 ± 2.109 2.724 + 0.231

As a result, the blood glucose level was increased to 308.4 mg / dL in the control group in which the diabetes was induced, but the blood glucose level was 247 mg / dL in the mouse treated with the soybean conversion.

In addition, blood insulin concentration decreased to 15.9% compared to normal mouse when diabetic induced, and recovered to 51.9% when the soybean bioconversion was administered. The glycogen level in the liver, which is decreased due to the reduction of blood insulin, was also recovered to 89.6% when the soybean bioconversion was administered, while the diabetic control group was reduced to 79.8%.

 (2) Evaluation of the ability to regulate glucose metabolism enzymes

To investigate the effect of soybean bioconversion on the activity of glucose-6-phosphatase (G6pase), phosphoenolpyruvate carboxykinase (PEPCK), and glucokinase (GCK) .

When the insulin concentration in the blood is reduced by diabetes, G6pase and PEPCK are activated to promote glucose synthesis, and inhibition of glycogen synthesis is inhibited by inhibition of GCK. Therefore, it was confirmed that the activity of these three enzymes can be controlled as the insulin secretion is restored upon administration of the soybean bioconversion. The results are shown in Table 9 below.

Sample enzyme activity (nmol / min / mg protein) G6pase PEPCK GCK Vehicle 71.422 + - 4.231 17.540 ± 1.161 10.840 ± 0.893 Alloxan only (100 mg / kg) 172.559 ± 12.549 33.426 + 2.076 5.224 + 0.335 Alloxa +
10 mg / kg soybean biotransformation 115.696 ± 7.501 25.528 ± 2.007 8.493 + 0.710

As a result, the activity of G6pase was increased to 2.4 times as much as that of the normal mouse when diabetic induction was performed, but the activity increase was suppressed to 1.6 times that of the normal mouse when the soybean bioconversion was administered. In the case of PEPCK, the activity was increased about 1.9 fold in diabetic mice, but it was inhibited to about 1.5 fold in each case when the soybean bioconversion was administered. In the case of GCK, the activity was reduced to 48.2% of the normal mice when diabetic was induced, but the activity was increased to 78.3% of the normal mice when the soybean bioconversion was administered.

 (3) Protection effect of liver tissue and pancreatic tissue

GOT / GPT levels were measured and tissue staining was performed to determine if liver damage induced by type 1 diabetes could be inhibited by soy bioconversion.

In the case of diabetic induction, GOT increased 1.8 times and GPT was increased 3.1 times, but when the soybean bioconversion was administered, the increase was 1.4 times and 1.7 times, respectively, (Fig. 12 (A)). Fig. 12 (A) is a graph showing the protective effect of hepatoprotective effect of a soybean bioconversion in a type 1 diabetes mouse induced by alloxan.

In addition, liver tissue was extracted, and paraffin blocks were stained with 'hematoxylene' and 'eosin Y' to examine the degree of tissue damage. As a result, it was shown that when alloxan induced diabetes, , The degree of liver damage was alleviated upon administration of the soybean bioconversion (Fig. 12 (B)). Fig. 12 (B) is a photograph showing the protective effect of hepatocyte conversion of a soybean biotransformant in a type 1 diabetic mouse induced by alloxan. Fig.

As shown in FIG. 13, when the pancreatic tissue was stained in the same manner, the size of the Langerhans islet in the pancreas tissue of the mouse induced diabetes was reduced due to the destruction of the? -Cells, In mice treated with the diets, it was found that the tissue damage of the pancreatic islets was alleviated. FIG. 13 is a photograph showing the protective effect of pancreatic injury of a soybean biotransformant.

[ Experimental Example 13: Effect of soybean biotransformation on type 2 diabetes induced mice in vivo Type 2 diabetes inhibiting ability evaluation]

In this experiment, we evaluated the antidiabetic effect of soybean bioconversion in obesity-induced type 2 diabetic mouse model induced by high fat diet.

(1) Effect of soybean biotransformation on weight, liver weight and white fat weight change in type 2 diabetic mouse model

The high fat diet for diabetes induction was manufactured by Dyet Company and the composition of the diet is shown in Table 10 below.

component normal diets (g / kg diet) high-fat diets (g / kg diet) casein 200.0 200.0 DL-methionine 3.0 3.0 corn starch 150.0 150.0 sucrose 500.0 150.0 cellulose 50.0 50.0 corn oil 50.0 salt mix # 200000 ^a 35.0 35.0 vitamin mix # 310025 ^b 10.0 10.0 choline bitartrate 2.0 2.0 beef tallow 400.0

(* ^A Dyets cat No. 200000, ^b Dyets cat.

On the other hand, the soybean bioconversion was dietary supplemented with 10 mg / kg of high-fat diet and fed for 7 weeks to induce type 2 diabetes. After 7 weeks, mouse weights, liver weights, and white fat weights were measured, and the results are shown in Table 11 below.

Sample initial weights (g) final weights (g) liver weights (g) white adipose tissue weights (g) feed intakes (g / day) normal control 23.15 ± 1.75 31.16 + - 2.24 1.79 ± 0.12 0.41 + 0.03 3.44 ± 0.22 HFD-control 23.54 + 1.43 35.58 ± 2.18 2.63 ± 0.16 1.77 + - 0.12 3.01 ± 0.19 HFD-10 mg / kg
Soybean biotransformation 23.61 ± 1.86 34.02 + - 2.73 2.11 + - 0.17 1.18 ± 0.10 3.01 ± 0.21

As a result, weight gain of 12.04 g in the type II diabetic mouse was observed in normal mice compared with 8.01 g in the weight gain at 7 weeks.

In the high fat diet, when the soybean bioconversion was administered to the mice, the body weight gain was 10.41 g, indicating a decrease in the body weight gain relative to the control. Liver weight and white fat weight were also inhibited compared to diabetic control group. However, since the difference in the average food intake was not observed, it was confirmed that this effect was not due to unhealthy food.

 (2) Measurement of effect on blood glucose and insulin in serum

The effects of the administration of the soybean bioconversion on the blood glucose level and the blood insulin concentration in the type 2 diabetic mouse model were measured and the results are shown in Table 12 below.

Sample Blood glucose (mg / dL) Serum insulin (ng / mL) normal control 113.5 ± 9.7 0.805 ± 0.053 HFD-control 216.7 ± 10.8 0.412 + 0.027 HFD-10 mg / kg soybean bioconversion 161.7 ± 11.6 0.517 + 0.033

As a result of blood glucose measurement, blood glucose level was increased about 1.9 times in the high fat diet mouse, and about 1.4 times in the high fat diet mouse.

In the case of insulin concentration in blood, it was confirmed that the high-fat diet decreased to 51.1% of the normal level in the mouse, and the type 2 diabetes was induced, and the high fat diet recovered to 64.2% .

(3) Effect on glucose tolerance

Oral glucose tolerance test was performed to measure blood glucose control disorder by type 2 diabetes. Seven-week high-fat diet-fed mice were fasted for 16 hours, and glucose was orally administered. Blood was collected from mouse tail vein at intervals of 30 minutes and blood glucose level was measured.

As a result, in the case of normal mice, the blood glucose level increased until 30 minutes after the administration of glucose, and then decreased after 120 minutes. However, in the case of mice in which type 2 diabetes was induced, It was confirmed that the descent effect was greatly reduced.

It was confirmed that when the soybean biotransformant was administered to the high-fat diet mice, the overall blood glucose increase was reduced, and the blood glucose increase inhibition and control ability was excellent (Fig. 14). 14 is a graph showing the effect of a soybean biotransformation in a type 2 diabetic mouse.

(4) Lipid analysis

Analysis of serum lipids increased by high fat diet showed that the amount of HDL decreased and the amount of triglyceride, cholesterol and LDL increased in type 2 diabetic mice. The high fat diet regenerated the reduced amount of HDL and inhibited the increase of triglyceride, cholesterol and LDL when the soybean biotransformant was administered to mice. This is shown in Table 13 below.

Sample triglyceride (mg / dL) total cholesterol (mg / dL) HDL (mg / dL) LDL (mg / dL) normal control 112 ± 9 141 ± 8 84.2 ± 5.6 28.3 ± 1.7 HFD-control 163 ± 12 169 ± 12 61.9 ± 6.0 35.7 ± 2.3 HFD-10 mg / kg
Soybean biotransformation 133 ± 10 155 ± 9 68.3 ± 5.1 31.2 ± 1.9

 (5) Assessment of metabolic enzyme-regulating ability

Activity of G6pase, PEPCK, and GCK was measured as the soybean bioconversion regained the reduced amount of insulin secreted by type 2 diabetes. The results are shown in Table 14 below.

Sample enzyme activity (nmol / min / mg protein) G6pase PEPCK GCK Vehicle 62.419 + 5.056 22.908 ± 1.087 11.524 ± 0.993 HFD control 183.442 ± 10.115 41.438 ± 3.240 5.113 + 0.427 HFD-10 mg / kg
Soybean biotransformation 113.176 ± 9.591 34.241 ± 2.754 8.508 0.624

The results showed that soybean bioconversion inhibited the activity of G6pase and PEPCK as well as that of type 1 diabetes, and had the effect of restoring the activity of GCK. From these results, it was concluded that the soybean bioconversion product had an excellent effect of controlling the enzyme activity.

 (6) Inflammatory cytokine analysis

Although the induction mechanism of type 2 diabetes due to obesity is not yet known, inflammatory cytokines secreted from adipose tissue have been reported to play an important role. Therefore, we examined the effect of soybean bioconversion on the expression of cytokines in adipose tissue and the cytokines secreted into blood. The results are shown in Table 15 below.

Sample serum (pg / mL) adipose tissue (pg / mL) TNF-a IL-1? IL-6 TNF-a IL-1? IL-6 normal control 2.87 ± 0.13 13.2 ± 1.1 16.9 ± 1.3 23.0 ± 1.1 58.3 ± 4.2 77.3 ± 5.8 HFD-control 15.2 ± 1.2 109 ± 8 58.9 ± 4.0 74.9 ± 5.6 175 ± 9 212 ± 16 HFD-10 mg / kg
Soybean biotransformation 9.23 + - 0.56 70.3 ± 4.1 42.4 ± 3.9 50.8 ± 3.6 135 ± 8 153 ± 12

As a result, the expression of TNF-α, IL-1β, and IL-6 in adipose tissue and serum was increased when type 2 diabetes was induced, and the expression of all three cytokines Lt; / RTI >

In the case of IL-1β, which is known to play the most important role in the insulin resistance of the beta cells, both the serum and the adipose tissue showed a suppressive effect when the soybean conversion was administered.

 (7) Protection effect of liver tissue and pancreatic tissue

When liver and pancreatic tissues were extracted and stained in mice induced type 2 diabetes, tissue damage and fat accumulation were observed in the liver during diabetic induction, and Langerhans' islet was observed in the pancreas. On the other hand, when the soybean bioconversion was administered, the hepatic tissue damage and fat accumulation were alleviated, and the pancreatic Langerhans is also protected (FIGS. 15 (A) and 15 (B)). Fig. 15 (A) is a photograph of the liver tissue protective effect of a soybean biotransformant in a type 2 diabetic mouse, and Fig. 15 (B) is a photograph showing a pancreatic tissue protective effect .

[Experimental Example 14: Evaluation of in vitro liver protective function of soybean biotransformants in hepatocytes ]

In this experiment, HepG2 cell line, a human-derived liver cancer cell line, was used to examine the alcohol-induced hepatotoxicity of soybean bioconversion.

 (1) Cytotoxicity measurement using HepG2 cell line

HepG2 cells were seeded in a 96 well plate at a density of 1 × 10 ⁵ cells / well and treated with 1, 10, 100 μg / mL of soybean bioconversion and 200 mM of ethanol to kill the hepatocytes Respectively. After 24 hours of culture, cell viability was measured by MTT assay.

As a result, the survival rate of the soybean biotransformant was increased in a concentration-dependent manner, and the survival rate was found to be 74.4% at the concentration of 100 μg / mL (FIG. 16). 16 is a graph showing an inhibitory effect of soybean bioconversion on alcohol-induced hepatocyte death.

 (2) ROS scavenging activity induced by ethanol treatment

It was confirmed that the generation of ROS, which is one of the mechanism of cell death induced by hepatocyte ethanol treatment, can be inhibited by the administration of soybean bioconversion. Soybean bioconverts were treated at concentrations of 1, 10, and 100 ㎍ / mL for 30 minutes, followed by treatment with 200 mM ethanol for 30 minutes to induce the development of ROS. Then, DCFH-DA, a fluorescent marker of ROS, was treated and labeled, and the fluorescence generated by ROS was measured.

As a result, the soybean bioconversion showed a concentration-dependent ability to inhibit ROS formation, and it was confirmed that ROS inhibition ability was 30.1% at a concentration of 100 ㎍ / mL (FIG. 17). 17 is a graph showing the inhibitory effect of soybean bioconversion on the generation of ROS in alcohol-induced hepatocytes.

[ Experimental Example 15: Effect of soybean biotransformation Evaluation of in vivo liver protection]

In this experiment, the inhibitory effect of soybean bioconversion on alcoholic fatty liver was investigated in vivo .

(1) Changes in liver weight in alcoholic fatty liver mouse models induced by alcohol administration

The ethanol-induced liver mouse model using the 'Liber-Decarli' liquid basic diet was used in the in vivo experimental system. The composition of the 'Liber-Decarli' liquid basic diet is shown in Table 16 below, and the same calories as the ethanol contained in the normal diets (AIN-93G) and the ethanol diets fed with the normal diet were substituted with maltodextrin Control diet (control diet).

component control diets (g / kg diet) ethanol diets (g / kg diet) casein (100 mesh) 41.4 41.4 L-cysteine 0.5 0.5 DL-methionine 0.3 0.3 corn oil 8.5 8.5 olive oil 28.4 28.4 safflower oil 2.7 2.7 dextrin maltose 115.2 25.6 cellulose 10.0 10 choline bitartrate 0.53 0.53 xanthan gum 3.0 3.0 salt mix ^a 8.75 8.75 vitamin mix ^b 2.5 2.5 ethanol 0 48

(* ^A salt mix (g / kg mix): calcium phosphate, dibasic, 500; sodium chloride, 74; potassium citrate, monohydrate, 220; potassium sulfate, 52 magnesium oxide, 24 manganous sulfate, , 4.95; zinc carbonate, 1.6; cupric carbonate, 0.3; potassium iodate, 0.01; sodium selenite, 0.01; chromium potassium sulfate, 0.55; sodium fluoride, 0.06; sucrose, finely powdered, 117.92./ b vitamin mix (g / kg mix ), vitamin A12 (0.1%), vitamin A acetate (500,000 IU / ml), thiamine HCl, 0.6, riboflavin 0.6, pyridoxine HCl 0.7, niacin 3.0, calcium pantothenate 1.6, folic acid 0.2, biotin 0.02, g), 4.8, vitamin D3 (400,000 IU / g), 24 menadione sodium bisulfite, 0.08, p-aminobenzoic acid, 5, inositol, 10, dextrose, 939.)

Soybean bioconverted diets were added to ethanol diet at a dose of 10 mg / kg for a total of 8 weeks and induced fatty liver and hepatitis by ethanol.

After 8 weeks, the weight and liver weight of the mice were measured. As a result, it was confirmed that the liver weight / body weight ratio was increased by 13.0% compared to that of the normal mice due to inflammation and fatty liver in the ethanol diet group. However, when the soybean bioconversion was administered, it was confirmed to increase by 2.9%, and thus it was judged to inhibit the inflammatory reaction and fatty liver formation in the liver (FIG. 18). 18 is a graph showing an effect of inhibiting liver weight increase due to administration of a soybean bioconversion in an alcohol-induced liver damaged mouse.

 (2) Measurement of serum biochemical indicators

After isolating the mouse serum, biochemical markers present in the serum were measured to confirm liver damage. The results are shown in Table 17 below.

Sample Serum index (U / L) GOT GPT γ-GTP billirubin normal control 63.7 ± 5.2 22.1 + 1.7 5.56 + - 0.47 0.49 + 0.03 control diet 58.9 ± 4.9 26.7 ± 1.5 5.53 + - 0.39 0.51 + 0.04 Ethanol diet-control 75.5 ± 5.9 45.1 ± 3.6 6.38 + - 0.42 0.81 ± 0.05 Ethanol diet-10 mg / kg
Soybean biotransformation 63.2 ± 4.4 30.8 ± 3.0 5.77 + - 0.49 0.69 ± 0.03

Experimental results showed that all of the four biochemical markers were increased in ethanol - induced liver injury, but they were found to be close to the normal values when the soybean bioconversion was administered together.

 (3) Changes in antioxidants in liver tissues

Reduced GSH shows cytoprotective effect against oxidative stress, but it is known that when alcohol induced liver toxicity, glutathione is oxidized to GSSG and it does not control liver toxicity. Therefore, the amount and type of glutathione (GSH), a representative antioxidant in liver tissue, were measured. The results are shown in Table 18 below.

Sample Total GSH
([Mu] g / mg tissue) Reduced GSH
([Mu] g / mg tissue) GSSG / GSH ratio
([Mu] g / mg tissue) normal control 4.27 ± 0.28 3.75 ± 0.27 0.16 ± 0.01 control diet 4.95 + - 0.45 4.30 0.38 0.17 ± 0.01 Ethanol diet-control 3.82 ± 0.29 3.01 ± 0.21 0.22 0.02 Ethanol diet-10 mg / kg
Soybean biotransformation 4.25 0.41 3.70 ± 0.26 0.18 ± 0.01

In the alcohol-fed mice, both the total amount of GSH and the amount of reduced GSH were decreased, and the ratio of GSSG / GSH was 1.38 times higher than the normal value. On the other hand, when the soybean biotransformants were treated, the total GSH amount and the reduced GSH amount were increased, and the GSSG / GSH ratio was found to be decreased to the normal value.

 (4) Protection effect of liver tissue

The mice were sacrificed and liver was extracted and stained with 'hematoxylene' and 'eosin Y'.

Experimental results showed that normal mice and control mice had no hepatic lesions and no fat accumulation, whereas hepatocyte damage, hemorrhage, and fat accumulation occurred in ethanol - fed mice. However, when the soybean bioconversion was administered, liver damage by ethanol was strongly inhibited, and fat accumulation was also found to be very low. Thus, it was confirmed that the soybean bioconversion was excellent in liver protection (Fig. 19) . FIG. 19 is a photograph showing the protective effect of liver damage due to the administration of a soybean biotransfer in an alcohol-induced liver damaged mouse.

[Example 2: Isolation and purification of immunological active ingredient (polysaccharide)] [

(1) Separation and purification process

The production process for obtaining the polysaccharide fraction from the soybean bioconversion is shown in Fig. To the pulverized soybean bioconversion (Example 1), about 20 times more of the third distilled water was added to extract the water-soluble substance. After extracting the water soluble substance, the residue was removed by centrifugation and the extract supernatant was separated. To the extract supernatant was added EtOH in an amount corresponding to four times that of the supernatant, mixed well, and stored at 4 ° C overnight.

The resulting precipitate is separated by centrifugation, and the supernatant liquid is removed by adding tertiary distilled water to the precipitate, and the undissolved material is removed by centrifugation or filtration. Dialysis is performed using a dialysis tube for the filtered precipitate solution. The third distilled water was changed every 2 hours, and this was repeated 10 times or more. After completion of dialysis, it is pulverized by freeze drying.

(2) Analysis of polysaccharide fractions

The instrument used for the analysis of the polysaccharide fractions was Shimadzu LC 010Avp (Shimadzu Co., Japan), and the analysis conditions were as follows.

device Condition Instrument SHIMADZU_HPLC, SEDEX 75_ELSD Column Ultrahydrogel (TM) 1000 (7.8 x 300 mm) Waters, WAT011535 Column oven 45 ° C Flow rate 0.6 ml / min Solvent HPLC grade water Elution Isocratic, HPLC water 100% Run time 40 min Injection 20 μl Sample extraction 100 mg of the sample was shaken with 10 ml of HPLC water at 250 rpm for 1 hour,
Analysis after centrifugation

Repeated experiments were conducted to confirm the uniformity of the polysaccharide fractions isolated and purified using the first batch of soybean bioconducts produced in the 50 L fermenter. The polysaccharide fractions were repeated twice per batch.

Analysis of the polysaccharide fraction of the soybean bioconverted product in each batch by HPLC revealed that all of the low molecular weight substances were well removed and the fractions of the immunologically active polysaccharide were well formed (FIG. 21).

In the purification of the polysaccharide fractions, the recovery rates of the polysaccharide fractions of the first batch of 50 L were found to be about 10.36% and 9.96%, respectively, with an average of 10.16% and a deviation of 0.28.

As a result, it was confirmed that there was no difference in application of the polysaccharide fractions according to batches, and it was confirmed that the polysaccharide fractions were produced with excellent reproducibility.

Average polysaccharide fraction recovery (%) Deviation Soybean Biotransformation 50L 1st batch 10.16 0.28 n = 2

On the other hand, a sample was taken for each polysaccharide fraction production process of the soybean bioconversion, and the chromatogram pattern change according to the process was analyzed. As a comparative substance, Mesima Capsule 550, which is a special medicine used for chemotherapy, was used as an immunosuppressive polysaccharide derived from mycelia of Satsuma mushroom.

The supernatant from which the insoluble matter of the soybean biotransformant was removed was analyzed by gel filtration HPLC. As shown in FIG. 22, the peak of the low molecular weight substance was observed at 18.7 minutes and the peak of the polysaccharide fraction of the polymer at 10.5 minutes Was detected.

As a result of measuring the macrophage NO production ability of the eluate of the small molecule molar peak and the eluate of the polysaccharide fraction peak of the polymer, macrophage NO production ability was observed only in the polysaccharide fraction peak of the polymer.

After the EtOH precipitation process and the dialysis process, the low molecular weight material was removed and the peak of the low molecular weight material was decreased. Also, it was confirmed that no significant change was observed after freeze drying.

[Experimental Example 16: Assessment of macrophage activation ability by measurement of macrophage NO production]

Repeated experiments were conducted to confirm the uniformity of separation and purification of the polysaccharide fractions using the first batch of soybean bioconducts produced in the 50 L fermenter. Polysaccharide fractions were prepared twice per batch, and their macrophage NO production ability was measured to evaluate their ability to activate macrophages.

As a result of the measurement, there was no significant difference in the NO production ability between the polysaccharide fraction 1 and the polysaccharide fraction 2 prepared from the soybean biotransformant of the 50 L first batch. It was confirmed that the immunostimulatory activity MEC ₁₀₀ of the polysaccharide fractions 1 and 2 prepared from the soybean bioconversion produced in the first batch was approximately 1/4 ug / ml (FIG. 23).

[ Experimental Example 17: Analysis of cytokine secretion pattern in macrophages and analysis of receptor solubility ]

(1) Outline of experiment

Recently, research results on various physiological activities such as macrophage activation, NK cell stimulating activity and stimulation of cytokine production by polysaccharide showing immunological activity have been continuously announced.

In addition, the specific structure of the polysaccharide exhibiting immunological activity is known to be a pattern recognition receptor (PRR), which is a specific receptor expressing on the cell surface, such as macrophages, and it is known that it mediates signal transduction.

Based on these results, it was concluded that the polysaccharide fractions of the soybean bioconverts prepared by the separation and purification process established above were recognized in pattern recognition receptors, which are specific receptors of immune cells, The specificity of the specific receptor was determined by comparing the secretion patterns of cytokines. In order to confirm this, specific experiments were carried out to confirm that signaling is blocked by treating inhibitors of specific receptors.

The TLR4 receptor (Toll-like receptor4) was identified as a specific receptor for the soybean bioconversion. TLR4, an TLR4 agonist, was identified as a TLR4 ligand. Immunoreactivity of soybean bioconversion was investigated by comparison with MPLA, a proven immunomodulatory agent.

First, the secretory pattern of cytokines in macrophages was examined in order to confirm the immunological activity of the polysaccharide fraction of soybean bioconversion. As a control, LPS (Lipopolysaccharide) was treated at concentrations of 0.01 ㎍ / mL, 0.1 ㎍ / mL and 1 ㎍ / mL, respectively, and the soybean bioconverts were treated with 0.1 ㎍ / mL, 1.0 ㎍ / mL and 10 ㎍ / mL , While the polysaccharide fraction of soybean bioconversion was tested at the same concentrations as LPS (0.01 ㎍ / mL, 0.1 ㎍ / mL, 1 ㎍ / mL). The concentrations of the polysaccharide fractions were determined on the basis of the recovery rate of the polysaccharide fractions obtained from the soybean biotransformations by about 10% in repeated experiments.

(2) Evaluation of TNF-α secretion amount

Sixteen hours after the sample treatment, the supernatants of the macrophage culture were taken and the secretion and concentration of TNF-α (Tumor necrosis factor-α) were analyzed by ELISA (enzyme linked immuno solvent assay).

The results of the analysis are shown in FIG. In the case of soybean bioconversion, it was confirmed that TNF-α was secreted from the treatment concentration of 0.1 μg / mL. As the treatment concentration increased, the amount of TNF-α secretion was also increased. In the case of the soybean bioconversion polysaccharide fraction, the secretion amount of TNF-α increased as the treatment concentration increased from 0.1 μg / mL to 0.1 μg / mL and 1 μg / mL at the treatment concentration of 0.01 μg / mL And the level of secretion was similar to that of TNF-α in the soybean bioconversion at the concentration determined based on the recovery rate. In addition, it was confirmed that the TNF-α secretion amount was similarly increased with increasing concentration in both soybean bioconversion and soybean bioconversion polysaccharide fractions.

It was confirmed that polysaccharide component of various substances contained in soybean bioconversion is recognized as a specific receptor of macrophage, and thus, it is an effective one for mediating signal transduction to secrete TNF-α.

When LPS was secreted at 0.01 ㎍ / mL, the amount of LPS secreted by the polysaccharide fraction of soybean bioconverted polysaccharide was higher than that of LPS. However, at 0.1 ㎍ / mL and 1 ㎍ / mL, TNF- Respectively.

(3) Evaluation of IL-1β secretion amount

16 hours after the sample treatment, the supernatant of macrophage culture was taken and the secretion and concentration of IL-1β (Interlukine-1β) were analyzed by ELISA (enzyme linked immuno solvent assay).

As a result, the LPS, soybean bioconversion, and soybean bioconversion polysaccharide fractions showed a remarkably low value or no value at all, regardless of their concentrations (not shown).

(4) Evaluation of IL-4 secretion amount

At 16 hours after the sample treatment, the supernatant of macrophage culture was taken and the secretion and concentration of IL-4 (Interlukine-4) were analyzed by ELISA (enzyme linked immuno solvent assay).

As a result, the LPS, soybean bioconversion, and soybean bioconversion polysaccharide fractions showed a remarkably low value or no value at all, regardless of their concentrations (not shown).

(5) Evaluation of IL-5 secretion amount

The supernatant of macrophage culture was taken 16 hours after the sample treatment, and the secretion and concentration of IL-5 (Interlukine-5) were analyzed by ELISA (enzyme linked immuno solvent assay).

As a result, the LPS, soybean bioconversion, and soybean bioconversion polysaccharide fractions showed a remarkably low value or no value at all, regardless of their concentrations (not shown).

(6) Evaluation of IL-6 secretion amount

Sixteen hours after the sample treatment, the culture supernatant of macrophages was collected and the secretion and concentration of IL-6 (Interlukine-6) were analyzed by ELISA (enzyme linked immuno solvent assay).

The results of the analysis are shown in FIG. In soybean biotransformation, IL-6 secretion started from 1.0 ㎍ / mL at the treatment concentration, and the secretion of IL-6 increased at 10 ㎍ / mL.

In the case of soybean bioconversion polysaccharide fractions, the secretion of IL-6 was increased as the concentration of IL-6 started to be secreted from the concentration of 0.1 ㎍ / mL and the concentration of treatment increased. As the concentrations of soybean bioconversion and soybean bioconversion polysaccharide fractions were increased, the amount of IL-6 secretion in the soybean bioconversion was higher.

It was confirmed that the polysaccharide component of various substances contained in the soybean bioconversion was recognized as a specific receptor of macrophages, and thus, it was effective to secrete IL-6 by mediating signal transduction.

When IL-6 secretion levels of soybean bioconverted polysaccharide fractions were compared with those of LPS, IL-6 secretion by LPS was low at concentrations of 0.01 ㎍ / mL and 0.1 ㎍ / mL, but the concentration of 1 ㎍ / mL IL-6 levels were similar to each other.

(7) Evaluation of IL-10 secretion amount

16 hours after the sample treatment, the supernatant of macrophage culture was taken and the secretion and concentration of IL-10 (Interlukine-10) were analyzed by ELISA (enzyme linked immuno solvent assay).

The results of the analysis are shown in FIG. The concentration of IL-10 was measured at 10 ㎍ / mL for the soybean bioconversion. For the soybean bioconversion polysaccharide fraction, the amount of IL-10 secretion was measured at 1 ㎍ / mL.

It was confirmed that the IL-10 secretion level of the polysaccharide fraction of soybean bioconversion was measured at a high concentration. It was confirmed that polysaccharide component of various substances contained in soybean bioconversion was recognized as a specific receptor of macrophage, and thus, it was effective for mediating signal transduction and secretion of IL-10.

When IL-10 secretion level of LPS was compared, IL-10 secretion increased from 0.01 ㎍ / mL to 0.1 ㎍ / mL and 1 ㎍ / mL, and IL-10 secretion increased . Compared with the polysaccharide fraction of soybean bioconversion, the amount of IL-10 secreted in the polysaccharide fraction of LPS and soybean bioconverted polysaccharide fraction was similar at 1 ㎍ / mL.

(8) Evaluation of secretion of IL-12p70

16 hours after the sample treatment, the supernatant of the macrophage culture was taken and the secretion and concentration of IL-12p70 (Interlukine-12p70) were analyzed by ELISA (enzyme linked immuno solvent assay).

As a result, the LPS, soybean bioconversion, and soybean bioconversion polysaccharide fractions showed a remarkably low value or no value at all, regardless of their concentrations (not shown).

(9) Evaluation of IFN-beta secretion amount

At 16 hours after the sample treatment, the supernatant of the macrophage culture was taken and the secretion and concentration of IFN-β (Interferon-β) were analyzed by ELISA (enzyme linked immuno solvent assay).

The results of the analysis are shown in FIG. In the case of soybean bioconversion, IFN-β was secreted from the treatment concentration of 0.1 μg / mL. As the treatment concentration increased, the secretion of IFN-β also increased. In the case of the soybean bioconversion polysaccharide fraction, the secretion of IFN-? Started to increase from 0.01 占 퐂 / mL to 0.1 占 퐂 / mL and 1 占 퐂 / mL, , And the amount of secretion was similar to that of soybean bioconversion at the concentration determined based on the recovery rate. In addition, it was confirmed that IFN-β secretion levels of the soybean bioconversion and soybean bioconversion polysaccharide fractions increased similarly as the concentration increased.

It was confirmed that the polysaccharide component of various substances contained in the soybean bioconversion was recognized as a specific receptor of macrophages, and thus, it was effective for mediating signal transduction to secrete IFN-β.

When the amount of IFN-beta secreted by LPS was compared, it was confirmed that the amount of secretion increased in a concentration-dependent manner. Compared with the polysaccharide fraction of soybean bioconversion, LPS was relatively high.

(10) Analysis of results

As a result of analysis of immunological activity-related cytokines of soybean bioconverts, it was confirmed that TNF-α, IL-6, IL-10 and IFN-β were expressed and that the immunological activity of the soybean bioconversion was attributed to the polysaccharide fraction .

In comparison with LPS, the cytokine secretion patterns of TNF-α, IL-6, and IFN-β were similar to those of soybean bioconversion polysaccharide fractions, as well as IL-1β, IL-4 and IL-5 , And IL-12p70 were not secreted together.

LPS is well known as a substance recognized by TLR4 (Toll-lokreceptor4), and it can be inferred that polysaccharide fraction of soybean bioconverted polysaccharide is recognized in TLR4 and mediates signal.

On the other hand, TNF-α is secreted from macrophages and mononuclear cells during phagocytosis and is known to have an antiviral effect against various viruses such as influenza virus along with inducible nitric oxide (NO). In addition, IL-6 is a major mediator in antigen-specific immune responses, inflammatory responses, and acute responses, and is a representative cytokine that plays a key role in the in vivo defense system. In addition, IFN- [beta] is a type I interferon with IFN- [alpha] and has an antibacterial and antiviral effect. In particular, it induces Th1 immune response as a typical cytokine which induces Th1 immune response by contributing to Th1 cell differentiation, And immunomodulatory cytokines that inhibit the Th17 immune response.

In conclusion, it was confirmed that the production of interferon-β was induced in the activated macrophages, and it was judged that the Th1 immune response could be specifically activated. All of these immunological activities were confirmed by the polysaccharide fraction of the polymer.

[Experimental Example 18: Identification of receptor]

(1) Outline of experiment

Many of the immunomodulators developed so far have not been identified with receptors, and only some beta-glucan products are known to bind to receptors called dectin-1. However, even beta-glucan products are known to bind to deutin in the case of insoluble materials, while water-soluble materials do not bind to deutin-1.

If the receptor is known, a biomarker can be established, and on the basis of this, there is an advantage that biomarker can be used in the clinical test for the development of pharmaceuticals and veterinary drugs.

Based on the results of previous experiments, it was concluded that TLR4 is a receptor for the soybean bioconversion and soybean bioconversion polysaccharide fractions, and that the soybean bioconversion and soybean bioconversion polysaccharide fractions are specific to TLR4 and mediate signals TAK-242, a specific inhibitor for TLR4, was used for the final confirmation.

(2) Evaluation of TNF-α secretion amount

Sixteen hours after the sample treatment, the supernatants of the macrophage culture were taken and the secretion and concentration of TNF-α (Tumor necrosis factor-α) were analyzed by ELISA (enzyme linked immuno solvent assay).

The results of the analysis are shown in FIG. In the case of LPS, the level of TNF-α secretion was completely blocked by treatment with TAK-242, a TLR4 inhibitor, at a concentration of 0.01 μg / ml and 0.1 μg / ml, and at a high concentration of 1 μg / ml, It can be confirmed that the decrease is greatly reduced. In the case of the soybean bioconversion, it was also confirmed that the TNF-α secretion level was completely blocked by treatment with the TLR4 inhibitor TAK-242 at a concentration of 0.1 μg / ml. At a high concentration of 1 μg / ml and 10 μg / ml It can be confirmed that the amount greatly decreases greatly. In the case of the soybean bioconversion polysaccharide fraction, it was also confirmed that when TAK-242 was treated at a concentration of 0.01 μg / ml and 0.1 μg / ml, the secretion amount of TNF-α was completely blocked, and 1 μg / ml It was confirmed that the concentration at the high concentration was greatly reduced greatly.

Based on the results of completely blocking or greatly reducing the secretion of TNF-α upon treatment with the TLR4 inhibitor TAK-242, polysaccharides of soybean bioconverters and soybean bioconverters were specifically bound to TLR4 and recognized TNF-α secretion signal was mediated.

(3) Evaluation of IL-6 secretion amount

Sixteen hours after the sample treatment, the culture supernatant of macrophages was collected and the secretion and concentration of IL-6 (Interlukine-6) were analyzed by ELISA (enzyme linked immuno solvent assay).

The results of the analysis are shown in FIG. In the case of LPS, IL-6 secretion was completely blocked by treatment with the TLR4 inhibitor TAK-242 at all three concentrations. In the case of soybean transformants, it was also confirmed that the secretion of IL-6 was completely blocked by treatment with the TLR4 inhibitor TAK-242 at all three concentrations. In the case of soybean bioconversion polysaccharide fractions, it was also confirmed that when TAK-242 was treated at all three concentrations, the secretion of IL-6 was completely blocked as well.

Based on the result that the secretion of IL-6 was completely blocked during treatment with the TLR4 inhibitor TAK-242, the polysaccharide of the soybean bioconversion product and soybean bioconversion was specifically bound to TLR4 like IL-4 and IL-6 secretion signal mediated .

(4) Evaluation of IL-10 secretion amount

16 hours after the sample treatment, the supernatant of macrophage culture was taken and the secretion and concentration of IL-10 (Interlukine-10) were analyzed by ELISA (enzyme linked immuno solvent assay).

The analysis result was as shown in Fig. In the case of LPS, treatment with TAK-242, a TLR4 inhibitor, at all three concentrations showed complete secretion of IL-10. In the case of the soybean bioconversion, it was also confirmed that when the TLR4 inhibitor TAK-242 was treated at all three concentrations, the secretion of IL-10 was completely blocked, and in the case of the soybean bioconversion polysaccharide fraction, the TLR4 inhibitor TAK -242, it was confirmed that secretion of IL-10 was completely blocked as well.

Based on the result that the secretion of IL-10 was completely blocked by the TLR4 inhibitor TAK-242, the polysaccharide of the soybean bioconversion product and soybean bioconversion was specifically bound to TLR4 like IL-10 and IL-10 secretion signal Respectively.

(5) Evaluation of IFN-beta secretion amount

At 16 hours after the sample treatment, the supernatant of the macrophage culture was taken and the secretion and concentration of IFN-β (Interferon-β) were analyzed by ELISA (enzyme linked immuno solvent assay).

The analysis result was as shown in Fig. In the case of LPS, when the TLR4 inhibitor TAK-242 was treated at all three concentrations, the secretion of IFN-β was completely blocked. In the case of soybean transformants, it was also confirmed that IFN-β secretion was completely blocked by treatment with the TLR4 inhibitor TAK-242 at all three concentrations. In the case of soybean bioconversion polysaccharide fractions, it was also confirmed that when the TLR4 inhibitor TAK-242 was treated at all three concentrations, the secretion amount of IFN-β was completely blocked.

Based on the result that the secretion of IFN-? Is completely blocked during the treatment with the TLR4 inhibitor TAK-242, the polysaccharide of the soybean bioconversion product and the soybean bioconversion is specifically bound to TLR4 similarly to LPS, .

(6) Analysis of results

The macrophage immunoactivity of the soybean bioconversion was confirmed by the polysaccharide fraction. The responses induced by macrophage activation were all inhibited by the TLR4-specific inhibitor TAK242, confirming that TLR4 is a receptor for the soybean bioconversion, The soybean bioconversion product and the soybean bioconversion polysaccharide fraction produced by the bioconversion process pursued in the present invention had TLR4 agonist activity.

Claims (6)

(A) adding at least one selected from amylase or cellulase to soybean, reacting, and sterilizing to prepare a liquid medium;
(B) after the step (a), inoculating the mushroom mycelium into the liquid medium and culturing to produce a culture;
(C) after the step (b), adding the fibrolytic enzyme to the culture and reacting; And a soybean bioconversion product produced from a process comprising the steps of:
Wherein the soybean bioconversion product is a polysaccharide.
The method according to claim 1,
The soybean biotransformation may comprise,
Wherein the liver damage caused by diabetes or the damage of the pancreas to Langerhans isomer is improved.
The method according to claim 1,
The polysaccharide,
(A) adding distilled water to the soybean bioconversion obtained through step (c) to extract a water-soluble substance;
Removing the residue through centrifugation after extracting the water-soluble substance, and separating the extracted supernatant (B);
Adding EtOH to the extracted supernatant, mixing and maintaining (c);
Separating the resulting precipitate after said holding (d);
Adding distilled water to the separated precipitate to dissolve the precipitate, and then removing undissolved material by centrifugation or filtration; And
And (ii) dialyzing the sediment lysis solution subjected to the centrifugation or filtration. The composition for improving diabetes as recited in any one of claims 1 to 3,
(A) adding at least one selected from amylase or cellulase to soybean, reacting, and sterilizing to prepare a liquid medium;
(B) after the step (a), inoculating the mushroom mycelium into the liquid medium and culturing to produce a culture;
(C) after the step (b), adding the fibrolytic enzyme to the culture and reacting; And a soybean bioconversion product produced from a process comprising the steps of:
Wherein the soybean bioconversion product is a polysaccharide.
5. The method of claim 4,
The soybean biotransformation may comprise,
A pharmaceutical composition for the prophylaxis or treatment of diabetes, which is characterized in that liver damage caused by diabetes or damage of the pancreas to Langerhans isomer is improved.
5. The method of claim 4,
The polysaccharide,
(A) adding distilled water to the soybean bioconversion obtained through step (c) to extract a water-soluble substance;
Removing the residue through centrifugation after extracting the water-soluble substance, and separating the extracted supernatant (B);
Adding EtOH to the extracted supernatant, mixing and maintaining (c);
Separating the resulting precipitate after said holding (d);
Adding distilled water to the separated precipitate to dissolve the precipitate, and then removing undissolved material by centrifugation or filtration; And
And (ii) performing dialysis against the sediment lysis solution after centrifugation or filtration. The pharmaceutical composition for preventing or treating diabetes mellitus according to any one of

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