KR101814166B1 - Compositions with bioconversion rice bran for improvement of immunity, diabetes, hyperlipemia or protection against liver damage - Google Patents

Abstract

The present invention relates to a composition containing bioconversion of rice bran for reinforcing immunity, alleviating diabetes, alleviating hyperlipemia or protecting liver. The composition containing bioconversion of rice bran of the present invention is effective in reinforcing immunity against microorganism infection through activation of macrophage, alleviating diabetes, repairing damage of liver or damage of the islets of Langerhans in pancreas and inhibiting formation of neutral fats. Also, the composition is effective in inhibiting alcoholic fatty liver or alcoholic steatohepatitis caused by alcohol.

Description

[0001] The present invention relates to a composition for improving immunity, for improving diabetes, for improving hyperlipidemia or for protecting liver,

The present invention relates to a composition for improving immunity, for improving diabetes, for improving hyperlipemia or for protecting liver, which contains a rice bran bioconversion product.

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 responds nonspecifically to antigen, and congenital immune cells include macrophage, polymorphonuclear leucocyte, and NK cell, which can kill infected cells. ), And in fact most infections are defended by congenital immunity.

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, diabetes is not only itself but also various complications that can lead to death, and the danger is emphasized. The prevalence of diabetes mellitus is high throughout the world. 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 mellitus is mostly congenital, and insulin secretion is poor, so it can be managed with simple treatment of insulin.

On the other hand, type 2 diabetes occurs mainly as acquired factors, and it is known that type 2 diabetes occurs mainly due to insulin resistance. In normal people, when the blood glucose concentration is increased through digestion and absorption after food consumption, the insulin secretion is smoothly performed. The cells of each tissue to which glucose is to be absorbed normally recognize insulin, absorbing glucose, Glycogen storage, and so on.

However, in the case of a person with insulin resistance, the cells in each tissue are less sensitive to insulin, failing to recognize secreted insulin normally, and maintaining proper glucose absorption and utilization, resulting in a high blood glucose level.

If insulin does not play a role in insulin resistance, blood glucose levels will increase because each tissue can not absorb sugar and consume energy as a source of energy, or can not adequately inhibit the neogenesis process of degrading glycogen to glucose in the liver. In the end, it progresses to type 2 diabetes, which increases triglycerides, cholesterol, and causes 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.

Managing diabetes more effectively involves additional medication in addition to basic exercise and diet management. Currently, type 1 diabetes therapy can be treated simply by administering insulin itself or by stimulating insulin secretion by stimulating beta cells of the pancreas. However, as a type 2 diabetes therapeutic agent, However, there are a variety of adverse drug reactions that are reported to limit the use of drugs.

Currently, only metformin (Metformin) is prescribed for most patients. Thiazolidinedione and DPP-IV are limited, however, all of them have side effects. New drugs and health functional foods with other effective mechanisms are required.

On the other hand, rice bran refers to the bark powder of rice bran and rice husk separated from the rice husk after rice husk is extracted and then brown rice is cut into white rice. Analysis of the functional components of rice reveals that functional ingredients are distributed in 29% of rice bran, 66% of rice bran, and 5% of rice called rice bran. 95% of rice nutrients are rice bran and rice bran, ).

In general, it is known that the components of rice bran improve blood circulation, activate natural natural healing power of the human body, preserve the blood with proper alkalinity, and exhaust waste materials and toxic metals in the body.

In addition, the rice bran has the effect of revitalizing the internal organs of the internal organs, removing the succulence, reducing cholesterol and triglycerides, healing hypertension and atherosclerosis, activating the functions of the brain cells and enhancing concentration and memory . In addition, it has been reported to be excellent for skin care and allergy improvement, and rice bran contains a large amount of pesticide and heavy metal removal ingredient, 'whipin acid'.

 However, up to now, it has been confirmed that a composition that includes both bioconverted bioconversion products converted into superficial hyphae and exhibits immunity enhancement, diabetic improvement, triglyceride and cholesterol inhibition, and hepatoprotective effect has not been studied.

Korean Patent No. 10-1084446 (entitled "Compositions for inhibiting cancer metastasis by angiogenesis of Angelica gigas Nakai and jujube extract bioconverted by crude enzyme solution of Aspergillus sirousmi strain) A composition for inhibiting cancer metastasis by angiogenesis including angiogenesis extract and jujube extract biotransformed by a strain of Aspergillus usamii var. Shirousamii. Korean Patent Laid-Open No. 10-2015-0118657 (title of the invention: biological conversion method of organic sulfur compounds of garlic) includes the steps of: (a) inoculating yeast into a suspension of garlic pulver or garlic extract; (B) SAC [(+) - S-allyl-1-cystein)] by SPC (S-trans- 1-propenyl-L-cysteine) and SMC (S-methyl-cysteine), which comprises the step of enrichment of at least one organosulfur compound of garlic A biological conversion method is described.

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.

(A) preparing a liquid medium containing rice bran; (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); The present invention provides a food for enhancing immunity or a pharmaceutical composition, which comprises a rice bran bioconversion product prepared from a process comprising the steps of:

In the immunoconjugation food or pharmaceutical composition of the present invention, the microorganism bioconverting agent is preferably subjected to an 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 the rice bran, . 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, rice bran starch and cellulose can be decomposed, which 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. The released cellulolytic enzyme is capable of hydrolyzing rice bran.

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 immunoconjugation food or pharmaceutical composition of the present invention, the above-mentioned microorganism bioconversion preferably has immunity against microbial infection (activation) through activation of macrophages. The immunity (resistance) refers to resistance to disease.

In the immunoconjugation food or pharmaceutical composition of the present invention, the rice bran bioconversion may preferably be a polysaccharide. In this case, the polysaccharide preferably comprises: (a) extracting a water-soluble substance by adding distilled water to the rice bran bioconversion obtained through step (c); 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, it is preferable that the pharmaceutical composition contains 0.00001 to 100% by weight of corpuscle biotransformation as compared with the whole composition. When the content is less than 0.00001% by weight, the effect is insignificant.

In the pharmaceutical composition for enhancing immunity of the present invention, the pharmaceutical composition may further include 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 a rice bran 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 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.

On the other hand, the present invention relates to a process for producing a liquid medium comprising (a) preparing a liquid medium containing rice bran; (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) preparing a diabetic microbial biomarker; and (b) providing a pharmaceutical composition for preventing or treating diabetes mellitus.

In the 'diabetic food composition for improvement of diabetes mellitus' or 'pharmaceutical composition for the prevention or treatment of diabetes' of the present invention, the microorganism bioconversion product may be prepared by mixing the 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 '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) is preferably at least one selected from the group consisting of 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, rice bran starch and cellulose can be decomposed, which 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. The released cellulolytic enzyme is capable of hydrolyzing rice bran.

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

In the 'diabetic food composition' or 'pharmaceutical composition for the prevention or treatment of diabetes' of the present invention, the microorganism bioconverting agent may be a polysaccharide. In this case, the polysaccharide preferably comprises: (a) extracting a water-soluble substance by adding distilled water to the rice bran bioconversion obtained through step (c); 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 diabetes according to the present invention, the pharmaceutical composition preferably contains 0.00001 to 100% by weight of corpuscle biotransformers 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 diabetic food composition for improving diabetes mellitus of the present invention, the food composition preferably contains 0.00001 to 100% by weight of a rice bran 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 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 relates to a process for producing a liquid medium comprising (a) preparing a liquid medium containing rice bran; (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 preventing or treating hyperlipidemia, which comprises a microorganism 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 'composition for improving hyperlipidemia' or 'the composition for preventing or treating hyperlipidemia' of the present invention, the corpuscle bioconverting product may further comprise an enzymatic fire treatment for treating the composition 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 hyperlipemia' or 'pharmaceutical composition for the prevention or treatment of hyperlipidemia' according to the present invention, the liquid medium of step (a) is preferably 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, rice bran starch and cellulose can be decomposed, which 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. The released cellulolytic enzyme is capable of hydrolyzing rice bran.

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 'composition for improving hyperlipidemia' or 'the composition for preventing or treating hyperlipidemia' according to the present invention, the corpuscle bioconversion product inhibits the production of triglyceride and LDL-cholesterol, and the HDL- cholesterol. < / RTI >

In the 'food composition for the improvement of hyperlipemia' or the 'pharmaceutical composition for the prevention or treatment of hyperlipidemia' according to the present invention, the microorganism transformant may be a polysaccharide. In this case, the polysaccharide preferably comprises: (a) extracting a water-soluble substance by adding distilled water to the rice bran bioconversion obtained through step (c); 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 of the present invention, it is preferable that the pharmaceutical composition contains 0.00001 ~ 100% by weight of corpuscle biotransformer as compared with 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 99% by weight of corpuscle biotransformation 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 relates to a process for producing a liquid medium comprising (a) preparing a liquid medium containing rice bran; (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 comprises a rice bran bioconversion product produced from a process comprising the steps of:

In the present invention, the food composition for protecting the liver is preferably a food composition for protecting liver against hepatitis or hepatitis, more specifically, an alcoholic fatty liver or alcoholic fatty liver hepatitis expressed by alcohol or obesity, Non-alcoholic fatty liver disease or non-alcoholic fatty liver disease.

In the present invention, the liver disease may be, for example, fatty liver or hepatitis, and more particularly, alcoholic fatty liver or alcoholic fatty liver hepatitis or obesity expressed by alcohol, nonalcoholic fatty liver or nonalcoholic fat expressed by diabetes It can be hepatitis.

In the 'liver protecting food composition' or the 'pharmaceutical composition for prevention or treatment of liver disease' of the present invention, the corpuscle bioconverting product 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 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, rice bran starch and cellulose can be decomposed, which 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. The released cellulolytic enzyme is capable of hydrolyzing rice bran.

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 the prevention or treatment of liver disease' of the present invention, the corpuscle bioconverting agent may be a polysaccharide. In this case, the polysaccharide preferably comprises: (a) extracting a water-soluble substance by adding distilled water to the rice bran bioconversion obtained through step (c); 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).

Meanwhile, in the pharmaceutical composition for preventing or treating liver disease according to the present invention, it is preferable that the pharmaceutical composition contains 0.00001 ~ 100% by weight of corpuscle biotransformation as compared with 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, it is preferable that the food composition contains 0.00001 to 100% by weight of corpuscle biotransformation 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 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 microorganism bioconversion of the present invention can increase the immunity against microbial infection through the activation of macrophages, improve diabetes, improve liver damage or damage of the pancreas to Langerhans islet, and inhibit the production of triglyceride 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 microorganism bioconversion product of the present invention.
FIG. 3 is a graph showing NO production results of macrophage cells which are remarkably improved by NO production ability and concentration-dependently increased by the microorganism bioconversion of the present invention.
4 is a graph showing the cytokine expression potential of rice bran and microorganism transformants of the present invention.
5 is a graph showing the antimicrobial activity of salmonella in the microorganism transformant of the present invention.
FIG. 6 is a graph showing the effect of the present inventive microorganism conversion on concentration of Salmonella sp.
FIG. 7 is a graph showing the effect of the inventive microorganism transformed by concentration on the inhibition of salmonellosis in macrophages.
8 is a graph showing a delayed effect of the microorganism transformant of the present invention in the mortality rate caused by Salmonella infection of a mouse.
9 is a graph showing a result of inhibiting salmonella in the abdomen of the microorganism transformant of the present invention.
FIG. 10 is a graph showing the penetration inhibiting activity of the microorganism transformant of the present invention upon intestinal permeation of salmonella using Caco-2 cell line. FIG.
11 is a graph showing the effect of the microorganism transformant of the present invention 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 effect of protecting the liver damage of the microorganism transformant of the present invention in a type 1 diabetes mouse induced by alloxan.
Fig. 12 (B) is a photograph showing a liver damage protection effect of the microorganism transformant of the present invention in a type 1 diabetes mouse induced by alloxan. Fig.
FIG. 13 is a photograph showing the protective effect of pancreatic damage of the microorganism transformant of the present invention in a type 1 diabetic mouse induced by alloxan.
FIG. 14 is a graph showing the effect of the microorganism transformant of the present invention in a type 2 diabetic mouse. FIG.
Fig. 15 (A) is a photograph showing the liver tissue protective effect of the microorganism transformant of the present invention in a type 2 diabetic mouse. Fig.
Fig. 15 (B) is a photograph showing the protective effect of the pancreatic tissue of the biotransformation product of the present invention in the type 2 diabetic mouse. Fig.
FIG. 16 is a graph showing the inhibitory effect of the microorganism transformant of the present invention on alcohol-induced hepatocyte death. FIG.
FIG. 17 is a graph showing the effect of inhibiting the generation of ROS by the microorganism transformant of the present invention in alcohol-induced hepatocyte death. FIG.
18 is a graph showing an effect of inhibiting liver weight increase due to the administration of the microorganism transformant of the present invention in an alcohol-induced liver damaged mouse.
FIG. 19 is a photograph showing the protective effect of liver damage caused by the administration of the microorganism transformant of the present invention in an alcohol-induced liver damaged mouse.
20 shows a production process chart and a manufacturing process for obtaining a polysaccharide fraction from the microorganism bioconversion of the present invention.
FIG. 21 shows the chromatogram results of HPLC analysis of polysaccharide fractions of the microorganism transformant of the present invention.
22 is a chromatogram of the HPLC analysis of the polysaccharide fractions of the microorganism bioconversion of the present invention.
23 shows the results of evaluation of macrophage activation ability of the polysaccharide fractions of the microorganism bioconverted product of the present invention.
Fig. 24 shows the results of the cytokine secretion pattern of the microorganism transformant of the present invention and the polysaccharide fractions of the microorganism transformant.
Fig. 25 shows the result of evaluation of suppression of the secretion amount of cytokine in the microorganism transformant of the present invention and the polysaccharide fractions of the microorganism transformant in the TLR4 inhibitor treatment.

(A) preparing a liquid medium containing rice bran in the present invention; (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); The present invention provides a food or pharmaceutical composition comprising a rice bran bioconversion product produced from a process comprising the steps of:

According to the experiment of the present invention, it was confirmed that the microorganism transformant 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 rice germ 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 the polysaccharide fraction was obtained from the microorganism bioconverted product, the microorganism bioconversion product and the polysaccharide fraction separated therefrom were recognized in a pattern recognition receptor which is a specific receptor of immune cells, and it was confirmed whether signal transduction was 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 immunocompetent cells of the rice bran bioconversion product and the polysaccharide fractions thereof. The activity was confirmed to be due to the polysaccharide fraction isolated therefrom.

In the following experiments, it was confirmed that the polysaccharide fraction isolated from the rice bran bioconverting product was similar to that of LPS (lipopolysaccharide) and the 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 microorganism 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 rice bran bioconversion product or its polysaccharide fraction were all inhibited by the TLR4-specific inhibitor TAK242. From this, it was confirmed that TLR4 was a receptor for the rice bran bioconversion product and its polysaccharide fraction I could confirm.

From these results, it was confirmed that the microorganism 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 rice bran bioconversion product]

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

In order to produce microorganism bioconversion, a washing process was carried out to remove contaminants such as foreign matter and mold of brown rice.

First, foreign matter and dust on the surface of brown rice were removed with a high-pressure air gun. Secondly, the sieve was squeezed to remove any foreign matter that might be present. The third step was to treat water or ethanol to remove contaminants such as mold spores.

Brown rice, which had been subjected to the third washing process, was subjected to a microbial contamination test, followed by corrugation to obtain rice bran, which was used as a raw material.

 (2) Enzymatic treatment with hydrolytic enzymes for culture

Enzyme treatment was carried out using various hydrolytic enzymes in rice bran. The hydrolytic enzymes were amylase-based and cellulase-based enzymes capable of degrading rice starch and cellulose.

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

* Rice bran culture was cultured at 30 ℃ 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 mycelium of the oviduct is capable of hydrolyzing the rice gut by cultivating the cellulolytic enzyme. After the incubation, the fermented rice husk produced by the fermentation process was further subjected to an enzyme treatment process, which is a secondary bioconversion process.

The fermentation and enzymatic treatment bioconversion process is intended to effectively produce the cell-wall-derived immunologically active substance from the fermentation product, the fermentation product, of the fermentation product, together with the microorganism fermenting the fermentation substrate. , 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). Fig. 1 is a flow chart of a process for producing a rice germ bioconversion product.

(4) Post-treatment process

The microorganism bioconversion product produced by the secondary bioconversion process (enzyme reaction) was subjected to 'enzyme inactivation and sterilization' process at 90 ° C for 1 hour, followed by freeze-drying and pulverization, and used in the following experiment 2). Fig. 2 is a view showing a process of post-treatment and recovery of rice bran bioconversion products.

[ Experimental Example 1: Measurement of in vitro NO production ability in macrophages ( macrophages ) by microbial and microorganism bioconversion ]

In order to confirm the macrophage activation effect of the biotransformation of rice bran and rice bran, the NO production ability of macrophages (macrophages) and RAW264.7 cell lines was examined.

Griess reagent (1) (N- (1-naphtyl) ethlene diamine dihydrochloride: 1-naphthyl) ethylenediamine dihydrochloride was prepared by dispensing 100 μl of a micro- 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.

As a result, it was confirmed that the microorganism bioconversion showed a NO production ability at a concentration lower than 1/256 of that of the rice bran, and that the NO production ability of the macrophages by macrophage bioconversion was remarkably improved and also increased in a concentration-dependent manner (Fig. 3). FIG. 3 is a graph showing the NO generation ability and macrophage NO production of macrophage cells which are increased in a concentration-dependent manner, which is remarkably improved by the microorganism bioconversion.

Test Example 2: in the macrophages, between the rice bran and rice bran biotransformation water cytokine (cytokine) secretion analysis;

In order to confirm the innate immune response activation activity of rice bran and the microorganism bioconversion of the present invention, the secretory activity of cytokine in macrophages (macrophages) and RAW264.7 cell lines was examined.

The rice bran and the rice bran bioconversion product 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-a, IL-6, IL-10 and IFN-? Were expressed when the rice microbial transformant was treated (Fig. FIG. 4 is a graph showing the cytokine expression potential of rice bran and rice bran bioconverted products.

TNF-α is secreted from macrophages and mononuclear cells during predation, 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, it was concluded that the microorganism bioconversion of the present invention can specifically activate the Th1 immune response and simultaneously suppress the enhanced Th2 immune response. 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.

Experimental Example 3: in vitro evaluation inhibiting Salmonella infection ability of rice bran in the biotransformation of water, macrophages;

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

 (1) Measurement of antimicrobial activity of rice bran bioconversion product

In order to evaluate the ability of rice microbial transformants to inhibit Salmonella infection, we sought to determine whether rice microbial 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.

The viability of salmonella cultured at 0, 2, 4, and 8 hours after culturing was determined by incubating the microorganisms with 1, 10, and 100 ㎍ / mL, respectively. Respectively.

As a result of the experiment, it was confirmed that the microorganism bioconversion does not show direct antimicrobial activity (FIG. 5). 5 is a graph showing the antimicrobial activity of Salmonella in rice bran bioconversion.

(2) Measurement of Macrophage Phagocytosis of Rice Biotransformants

The macrophages bioconverted to macrophage RAW 264.7 cells at a concentration of 1, 10, and 100 ㎍ / mL for 4 hours, respectively, and then 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 microorganism transformant was increased up to 3.65 times at the time of infection with Salmonella 30 minutes, and increased up to 10.06 times at the time of infection with Salmonella 60 minutes (FIG. 6). FIG. 6 is a graph showing the effect of the concentration of RGCs on the salmonellosis of macrophages.

 (3) Measurement of inhibition of salmonella proliferation of rice bran bioconverted material

After treatment of macrophage RAW 264.7 cells with macrophage bioconducts at concentrations of 1, 10, and 100 μg / mL for 4 hours, 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 ㎍ / mL of rice bran bioconversion and infected with Salmonella, it was confirmed that Salmonella present in the cells was decreased as compared with the control group after a lapse of 8 hours (FIG. 7). FIG. 7 is a graph showing the effect of the RGMs on the 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 experiments, it was confirmed that the microorganism bioconverted product induces macrophage activation in an in vitro experimental system using macrophages to increase phagocytosis and inhibit survival of intracellular microorganisms after phagocytosis, ( Salmonella typhimurium ATCC14028) strains were used to test the in vivo antimicrobial activity of rice microbial transformants.

In order to confirm whether the administration of microorganism bioconversion can reduce the mortality rate of mice due to Salmonella infection, 10 ⁵ CFU of salmonella ( Salmonella typhimurium ATCC14028) strain was injected intraperitoneally to induce mouse death by infection.

As a result, in the control mice, all mice died at 7 days after the infection, whereas when the ruminal bioconversion was administered at a dose of 10 mg / kg, mice survived up to 21 days, (Fig. 8). FIG. 8 is a graph showing the delayed effect of the microbial bioconversion on the mortality rate caused by Salmonella infection of a mouse. FIG.

* Test Example 5: Evaluation of Rice Bran Oil biotransformation of water, in vivo live Salmonella infection in mice infected with La Monel inhibitory ability;

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

After the rice microbial conversion was administered at a dose of 10 mg / kg for 2 weeks, the ' 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 the measurement, it was confirmed that surviving salmonella was significantly reduced in the group treated with the rice bran bioconversion compared to the control group (FIG. 9). 9 is a graph showing a result of inhibiting salmonella in the abdominal cavity of a rice bran 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 Rice Biotransformation 1.89 + 0.13 359.725 ± 22.614

As a result, it was confirmed that mouse spleen lymphocytes were more increased in the case of mice administered with the microbial bioconversion than in the control. 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 microorganism bioconversion induces the activation of the Th1 immune response, the amount of Th1 cytokine expressed in the mouse spleen infected with salmonella at the above-mentioned concentration of saliva was measured to evaluate the Th1 activation effect . Four cytokines, IL-1β, IL-2, IL-6 and IL-12, were measured in the Th1 cytokine using ELISA.

Activation effects were compared between mice immunized with Salmonella and those infected with Salmonella, and 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
Rice microbial conversion product 186 ± 14 43.3 ± 2.8 58.0 ± 3.9 253 ± 17 salmonella (+) vehicle 239 ± 13 55.7 ± 3.8 73.4 ± 5.4 284 ± 13 10 mg / kg
Rice microbial conversion product 256 ± 11 63.9 ± 4.3 77.3 ± 4.8 325 ± 29

* As a result of the experiment, there was no significant difference in the expression level of cytokine, except that the expression level of IL-1β was slightly increased when the rice microbial transformant was administered to normal mice. On the other hand, when infected with Salmonella, the amount 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 indicate that the administration of microorganism bioconversion does not induce activation of unnecessary immune responses in a normal organism, and induces activation of Th1 immune response upon microbial infection, thereby effectively controlling 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 the Th1 cytokine, it was confirmed that the administration of the microorganism bioconversion activates the expression of Th1 cytokine, thereby activating the Th2 immune response, which is an antagonistic reaction of the Th1 immune response To investigate the expression level of Th2 cytokine,

Representative Th2 cytokines showed three cytokine expression levels: IL-4, IL-5 and IL-10. 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
Rice microbial conversion product 50.9 ± 3.6 290 ± 22 45.1 ± 3.1 salmonella (+) vehicle 56.8 ± 4.3 289 ± 17 48.7 ± 3.3 10 mg / kg
Rice microbial conversion product 57.2 ± 3.8 296 ± 15 49.2 ± 3.8

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

These results show that the administration of microorganism bioconversion does not induce the activation of unnecessary immune responses in normal organisms and does not induce the activation of Th2 immune response upon microbial infection.

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

In this experiment, we investigated whether intestinal bioconversion of Salmonella can inhibit intestinal cell adhesion and invasion by using Caco-2 cell line. Since most of the Salmonella infections are caused by intestinal infections, we wanted to see if the Rice 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 rice bran 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, the pretreatment for 4 hours showed 10.7% inhibition of salmonella penetration, and simultaneous treatment showed 58.4% inhibition of salmonella penetration. In the above experiment, it was confirmed that the microorganism bioconversion product did not have antimicrobial activity, and thus it was judged that it acted directly on the Caco-2 cell line and inhibited microbial infiltration (FIG. 10). 10 is a graph showing the intestinal permeation inhibitory activity of rice germ bioconversion using Caco-2 cell line.

[Example 10: Salmonella infection in mice, rice bran bioconversion of water in vivo inhibition of Salmonella penetration]

In this experimental example, the ability to inhibit Salmonella infiltration in vivo in rice germ 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 microorganism transformant had an effect of inhibiting intestinal permeation of salmonella, so that the salmonella infestation inhibitory effect was evaluated in the mouse model of Salmonella food poisoning.

The microbial bioconver- sion was fed at a dose of 10 mg / kg for 2 weeks, and then 10 ⁸ CFU of ' Salmonella typhimurium SL1344' was orally administered 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 Rice biotransformation 662 ± 48 729 ± 53

* 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 microorganism transformants were administered.

 (2) Identification of Salmonella infiltrated into tissues

From the results of Table 4, it was confirmed that salmonella entered into the organism was not able to penetrate into the tissues during the administration of the microorganism biotransformant and was discharged to the outside of the body. Therefore, the fecal release experiment mice were examined for cecal and intestinal membrane lymph nodes, 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 infiltrate tissue, and the lymphatic tissue connected with it, were examined to confirm the tissue infiltration inhibition effect of the microorganism. 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 Rice biotransformation 201 ± 12 26.8 ± 1.9

As a result, it was confirmed that the microorganism bioconversion inhibited the penetration of Salmonella into the cecum and the microorganism bioconversion showed a suppressive effect of 58.9%. The microbial bioconversion was found to inhibit salmonella in 75.4% of intestinal membrane lymph nodes, and it was found that the inhibition rate in intestinal membrane lymph nodes was greater than in the cecum.

These results suggest that the immune cells in the intestinal membrane lymph node were activated by the microorganism bioconversion, and thus 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 Rice biotransformation 21.0 + 1.7 18.9 ± 1.3

As a result, 83.8% of salmonella in the spleen was inhibited when the microorganism bioconversion was administered. In addition, for the salmonella present in the liver, the microorganism bioconversion showed a suppression rate of 75.6%. 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.

Test Example 11: in beta cells, rice bran biotransformation water In vitro inhibition of type 1 diabetes evaluation]

In order to confirm that the rice microbial bioconversion product has an inhibitory activity against type 1 diabetes, the activity of the microbial bioconversion product was evaluated in an in vitro assay system using 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 it was confirmed whether or not cell death was inhibited by treating the microorganism bioconversion product.

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

 Experimental results showed that about 62.5% of the cells died when treated with alloxan, and the mortality was reduced by up to 38.6% when the microorganism transformants were treated together.

In addition, when the amount of intracellular ROS produced by alloxan was measured by labeling with DCF-DA, the amount of ROS produced was reduced to 34.9% up to 34.9% And this effect was found to increase in a concentration-dependent manner (Fig. 11). 11 is a graph showing the effect of microorganism conversion 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.

Since the amount of intracellular ROS and cell death rate increased by simultaneous treatment of corpuscle bioconversion in the above experiment, the amount of NO (nitric oxide) secreted by the INS-1 cell line and the amount of insulin Respectively. 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 < RTI ID = 0.0 > 1 / / mL 80.219 + - 6.463 0.830 0.077 10 [mu] g / mL 66.890 + - 5.437 0.921 + - 0.059 100 / / mL 50.429 ± 2.106 1.079 + 0.086

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 microorganism bioconversion and the amount of insulin secreted by alloxan treatment Water at the maximum concentration of 100 ㎍ / mL, and it was confirmed that the microorganism bioconversion inhibited NO production and secretion by 38.8%.

In the case of insulin, the amount of secretion was recovered up to 31.4% at the maximum concentration of 100 ㎍ / mL.

Test Example 12:, rice bran biotransformation of water in the type 1 diabetes induced mouse in vivo Type 1 diabetes inhibiting ability evaluation]

As a result of confirming the antidiabetic effect on the type 1 diabetes mellitus by the treatment with the microorganism bioconverted product in the above in vitro experimental system, the antidiabetic effect of the microorganism transformant in the mouse model of the type 1 diabetes mellitus .

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

After a 2-week dietary dose of 10 mg / kg of rice bran bioconversion, 100 mg / kg of alloxan was administered intraperitoneally to induce type 1 diabetes. 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 Rice Biotransformation 263.0 ± 17.8 24.221 + 1.556 2.613 + 0.179

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 it was confirmed that the blood glucose level was suppressed by 263.0 mg / dL in the mice administered with the microbial bioconversion.

In addition, blood insulin concentration was decreased to 15.9% compared with that of normal mouse when diabetic induction was performed, but it was recovered to 40.9% when the microbial bioconversion was administered. The glycogen level in the liver, which is decreased due to the decrease of blood insulin, was also recovered to 85.9% when the rice microbial conversion was administered, while the diabetic control was reduced to 79.8%.

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

To investigate the effect of microorganism conversion 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 microorganism bioconversion product. 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 Alloxan + 10 mg / kg
Rice microbial conversion product 128.304 + - 7.203 26.736 ± 1.948 7.990 ± 0.507

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 was suppressed to 1.8 times as much as that of the microbial conversion product. 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 the case of administration of microbial bioconversion. In the case of GCK, the activity was decreased to 48.2% of normal mice when diabetic induction was induced, but the activity was increased to 73.7% when the rice microbial conversion 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 caused by type 1 diabetes could be inhibited by microbiotics.

In the case of diabetic induction, GOT increased 1.8 times and GPT increased 3.1-fold compared with the normal, but decreased to 1.4-fold and 1.8-fold, respectively, when administered with a microbial bioconversion product, thereby effectively suppressing liver damage caused by diabetes (Fig. 12 (A)). Fig. 12 (A) is a graph showing the protective effect of liver biopsy of corpuscular bioconversion in alloxan-induced type 1 diabetic mice.

In addition, liver tissue was extracted, and paraffin blocks were stained with 'hematoxylene' and 'eosin Y' to examine the extent of damage to the tissues. As a result, it was shown that when alloxan induced diabetes, , It was confirmed that the degree of liver damage was alleviated upon administration of the rice bran bioconversion (Fig. 12 (B)). Fig. 12 (B) is a photograph of the effect of protecting the liver damage of corpuscular bioconversion in a type 1 diabetes mouse induced by alloxan.

The pancreatic tissue was stained in the same manner and as a result, as shown in FIG. 13, the size of the Langerhans islet in the pancreatic tissue of the mouse induced diabetes was decreased due to the breakdown 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 damage on the biomass bioconversion product.

[ Experimental Example 13: In type 2 diabetes induced mice, Rice Biotransformation in vivo SecondType diabetes control ability evaluation]

In this experiment, we aimed to evaluate the antidiabetic effect of corpuscle bioconversion in a type 2 diabetic mouse model induced by obesity induced by high fat diet.

*

(1) Effect of administration of microorganism bioconversion on the weight, liver weight, and white fat weight change in the 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, rice microbial bioconver- sion was dietary supplemented with high-fat diet at a dose of 10 mg / kg and fed type-2 diabetes for 7 weeks. 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
Rice microbial conversion product 23.48 ± 1.52 34.39 + - 2.56 2.20 ± 0.09 1.28 ± 0.09 3.03 ± 0.28

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 rice microbial conversion product was administered to mice, weight gain was 10.91 g, which was lower than that of 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 administration of the microbial bioconverting agent on the blood glucose level and 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 Rice Biotransformation 168.0 ± 8.9 0.489 + 0.038

As a result of blood glucose measurement, blood glucose level was increased about 1.9 times in the case of the high fat diet mouse, but the increase of the blood glucose level was suppressed about 1.5 times in the high fat diet when the microorganism bioconversion was administered to the 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 60.7% .

(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 the whole blood glucose increase was reduced when the rice microbial biotransformant was administered to the high fat diet mouse, and it was confirmed that the blood glucose increase inhibition and control ability was excellent (Fig. 14). Fig. 14 is a graph showing the effect of corpuscle biotransformation in a type 2 diabetic mouse. Fig.

(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 decreased amount of HDL and decreased the amount of triglyceride, cholesterol and LDL when the rice microbial conversion was administered to the 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
Rice microbial conversion product 135 ± 8 154 ± 10 69.2 ± 5.3 31.0 ± 1.8

 (5) Assessment of metabolic enzyme-regulating ability

The activity of G6pase, PEPCK, and GCK was measured as the administration of microbial bioconversion restored insulin secretion reduced 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
Rice microbial conversion product 125.019 8.436 37.109 ± 2.053 7.426 0.632

As a result of the experiment, it was confirmed that the microorganism bioconversion inhibits the activities of G6pase and PEPCK and restores the activity of GCK. From these results, it was concluded that the effect of controlling the enzyme activity of the rice bran bioconverting product was excellent.

 (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 administration of the microorganism 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
Rice microbial conversion product 10.2 ± 0.8 78.9 ± 5.2 47.1 ± 2.5 56.0 ± 4.1 143 ± 11 155 ± 9

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 cytokine expression was suppressed Respectively.

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

 (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 rice microbial transformant was administered, such liver tissue damage and fat accumulation were alleviated, and the pancreatic Langerhans isomer was also protected (FIG. 15 (A) and FIG. 15 (B)). Fig. 15 (A) is a photograph of the liver tissue protective effect of a corpuscle biotransformation product in a type 2 diabetic mouse, Fig. 15 (B) is a photograph showing a pancreatic tissue protective effect .

[Experimental Example 14: Experimental Example 14: in vitro  Liver protection function evaluation]

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

 (1) Cytotoxicity measurement using HepG2 cell line

HepG2 cells were seeded in a 96 well plate at a density of 1x10 ⁵ cells / well and treated with 1, 10 and 100 μg / mL of rice bran 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 was increased in a concentration-dependent manner when the microorganism transformant was treated, and the survival rate was 80.1% at the concentration of 100 μg / mL (FIG. 16). FIG. 16 is a graph showing an inhibitory effect of a microorganism convertor on alcohol-induced hepatocyte death. FIG.

 (2) ROS scavenging activity induced by ethanol treatment

It was confirmed that the generation of ROS, one of the mechanisms of cell death induced by hepatocyte ethanol treatment, can be inhibited by the administration of microorganism bioconversion. Rice transformants were treated at 1, 10, and 100 ㎍ / mL for 30 minutes, respectively, and 200 mM ethanol was treated 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 of the experiment, it was confirmed that ROS was generated in ethanol treatment of HepG2 cell line and 1.8 times of ROS was generated by fluorescence measurement. In addition, the rice microbial bioconversion showed a concentration-dependent ability to inhibit ROS formation, and a 30.6% ROS inhibitory ability at 100 μg / mL concentration (FIG. 17). FIG. 17 is a graph showing the effect of inhibiting the generation of ROS by the microbial bioconversion in alcohol-induced hepatocytes. FIG.

Test Example 15: in the mouse liver damage induced by alcohol, bran biotransformation water Evaluation of in vivo liver protection]

In this experiment, we investigated the inhibitory effect of rice bran bioconversion on alcoholic fatty liver 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.)

The microbial bioconverts were added to the ethanol diet at a dose of 10 mg / kg and fed for 8 weeks to induce 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 rice microbial conversion product was administered, it was confirmed that it increased by 3.8%, 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 the administration of a rice bran 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
Rice microbial conversion product 62.9 ± 5.1 31.2 ± 2.9 5.80 0.37 0.66 + 0.04

Experimental results showed that all four biochemical markers of liver injury induced by ethanol were increased, but they were decreased to the normal values when they were administered together with the bioconversion product.

*

 (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
Rice microbial conversion product 4.22 0.30 3.71 ± 0.39 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 microorganism was treated with the bioconversion product, 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 liver was extracted from the mice to which the rice bran bioconversion was administered, and the liver was stained with 'hematoxylene' and 'eosin Y', and the degree of tissue damage and fat accumulation was confirmed.

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 rice microbial conversion product was administered, it was found that the ethanol-induced liver damage was strongly inhibited, and that the fat accumulation was also very low, . FIG. 19 is a photograph showing the protective effect of liver damage due to the administration of a rice bran bioconversion 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 rice bran bioconverts is shown in FIG. To the pulverized rice germ bioconversion (Example 1), about 20 times 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 was separated by centrifugation, and the supernatant was removed. The precipitate was dissolved by adding tertiary distilled water, and the undissolved material was removed by centrifugation or filtration. Dialysis was carried out 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 the dialysis, it was pulverized through lyophilization.

(2) Analysis of polysaccharide fractions

The apparatus used for the analysis of the polysaccharide fractions was Shimadzu LC 010Avp (Shimadzu Co., Japan), and the analysis conditions were as shown in Table 19 below.

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 carried out to confirm the uniformity of the polysaccharide fractions separated and purified using two batches produced in the 50 L fermenter and one batch of the microorganism bioconversion produced in the two-ton fermenter. The polysaccharide fractions were repeated twice per each batch.

HPLC analysis of the polysaccharide fractions of each batch of microorganism bioconverts showed that all of the low molecular weight substances were well removed and that the fractions of the immunologically active polysaccharide were well formed (FIG. 21).

In the purification of polysaccharide fractions, the recovery rates of the polysaccharide fractions of the 50 L first batch and the 50 L second batch were about 10.74% and 3.31%, respectively, and the recovery rate of the first two batches was 10.92% (Table 20). However, this is due to the excess of loss due to the refining purity as well as the purity of the tablets. In the separation and purification of the two repeated polysaccharide fractions in each batch, there is almost no deviation of 2.52, 0.86, and 0.00, respectively Respectively.

As a result, it was confirmed that there was no difference in application of the polysaccharide fraction according to the batch, and it was confirmed that the process of the present invention is a process for producing a polysaccharide fraction having excellent reproducibility.

Recovery of polysaccharide fractions (%) Deviation Rice Biotransformation 50L 1st batch 10.74 2.52 Rice Biotransformation 50L 2nd batch 3.31 0.86 1st batch of rice bran biota conversion 2 tons 10.92 0.00 n = 2

On the other hand, samples of polysaccharide fractions of microorganism bioconverted product were collected by the manufacturing process, 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 obtained by removing the insoluble materials of the microorganism bioconverted material was analyzed by gel filtration HPLC. As shown in FIG. 22, the peak of the low molecular material was observed at the 16.7 minutes, and the peak of the polymer at the range of 8.6 to 9.7 minutes Of the polysaccharide fraction was detected.

As a result of measuring the macrophage NO production ability of the eluate of the peak of the low molecular substance and the eluate of the polysaccharide fraction peak of the polymer, only macrophage NO production ability was observed 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 reduced in the analysis, and it was confirmed that there was no significant change even after freeze drying.

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

Repeated experiments were carried out to confirm the uniformity of the separation and purification of the polysaccharide fractions by using a total of three batches of microorganism bioconverted products produced in the 50 L fermenter and the 2 ton fermenter respectively. Polysaccharide fractions were prepared twice per each batch and their macrophage NO production ability was measured to evaluate macrophage activation ability.

As a result of the measurement, the polysaccharide fractions (RB_50L first batches-1 and 2) prepared from the microorganism bioconversion of the 50L first batch and the polysaccharide fractions (RB_50L second batch-1 , 2), and the NO production ability of the polysaccharide fractions (RB_2 tonnal batches - 1 and 2) prepared from the first batch of microorganism biotransformation of 2 tons. It was confirmed that the polysaccharide fractions prepared from the microorganism bioconverts produced in each batch had an immunostimulatory activity MEC100 of about 1/4 μg / ml (FIG. 23).

The results show that the recovery rate of the polysaccharide fractions of the rice germ bioconverts in the second batch of 50L is 3.1%, the recovery rate of the polysaccharide fractions of the rice germ bioconverts in the first batch is 10.74% 10.92%, but it was confirmed that there was no significant difference when comparing the above NO production capacities.

[ Experimental Example 17: Investigation of cytokine secretion pattern and receptor expression in macrophages ]

(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, we tried to confirm whether the polysaccharide fractions of the microorganism bioconverts prepared by the separation and purification process established above were recognized in pattern recognition receptors, which are specific receptors of immune cells, and mediated signal transduction. First, a specific receptor was deduced from the comparison of cytokine secretion patterns in macrophages. In order to confirm this, an experiment was carried out to examine whether signaling is blocked by treating an inhibitor against the specific receptor expressed .

The TLR4 (Toll-like receptor4), a specific receptor for the rice germ bioconversion, was confirmed by the inhibitor treatment experiments. TLR4 (TLR4), a ligand for TLR4, and TLR4 agonist ), The immunoreactivity of the microorganism bioconverted product was investigated by comparison with MPLA, a proven immune modulating material.

First, the secretion patterns of cytokines in macrophages were examined in order to confirm the immunological activity of the polysaccharide fractions of rice bran bioconverted product. As a control, LPS (lipopolysaccharide) was treated at concentrations of 0.01 ㎍ / mL, 0.1 ㎍ / mL and 1 ㎍ / mL, respectively. The microorganism bioconversion was performed at concentrations of 0.08 ㎍ / mL, 0.8 ㎍ / mL, and 8 ㎍ / mL, while the polysaccharide fractions of the microorganism bioconverted were treated with 0.01 ㎍ / mL, 0.1 ㎍ / mL, / mL. < / RTI > The concentrations of the polysaccharide fractions were determined on the basis of the recovery rate of the polysaccharide fractions from the microorganism bioconverts, which was about 12% through 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 the rice bran bioconversion, it was confirmed that TNF-α was secreted from the treatment concentration of 0.08 μg / mL. As the treatment concentration increased, the secretion amount of TNF-α also increased. In the case of the rice germ bioconversion polysaccharide fraction, the secretion amount of TNF-α increased as the treatment concentration was increased from 0.1 μg / mL to 0.1 μg / mL and 1 μg / mL from the start of secretion of TNF-α , And the amount of secretion was similar to the amount of TNF-α secreted by the bioreactor 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 the microorganism bioconversion and the microbial bioconversion polysaccharide fraction.

It was confirmed that the polysaccharide component was recognized as a specific receptor of macrophages among the various substances contained in the microorganism bioconversion, and thus it was confirmed that TNF-α is secreted by mediating signal transduction.

In addition, LPS was found to be secreted at 0.01 ㎍ / mL when compared with the TNF-α secretion fraction of the microorganism-bioconverted polysaccharide fraction. However, similar concentrations of TNF-α were observed at 0.1 ㎍ / mL and 1 ㎍ / -α secretion.

(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 of the analysis, all of the LPS, corpuscle biotransformant and microorganism bioconversion polysaccharide fractions showed a remarkably low value or no numerical value at all (regardless of the concentration) (not shown).

(4) Evaluation of IL-4 secretion amount

At 16 hours after the sample treatment, the supernatant of the 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 of the analysis, all of the LPS, corpuscle biotransformant and microorganism bioconversion polysaccharide fractions showed a remarkably low value or no numerical value at all (regardless of the concentration) (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 of the analysis, all of the LPS, corpuscle biotransformant and microorganism bioconversion polysaccharide fractions showed a remarkably low value or no numerical value at all (regardless of the concentration) (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 the case of rice microbial transformants, the secretion of IL-6 started from the treatment concentration of 0.8 ㎍ / mL, and the secretion of IL-6 increased at 8 ㎍ / mL.

In the case of the polysaccharide fractions of rice bran bioconverted polysaccharide fractions, the amount of IL-6 secretion was increased as the concentration of IL-6 started to increase from 0.01 ㎍ / mL. In addition, it was confirmed that the IL-6 secretion amount showed a similar amount of secretion as the concentration of the rice bran bioconversion and the rice bran bioconversion polysaccharide fraction increased.

It was confirmed that the polysaccharide component was recognized as a specific receptor of macrophages among the various substances contained in the microorganism bioconversion, and it was confirmed that it is an effective one for mediating signal transduction and secreting IL-6.

When LPS-induced IL-6 secretion levels were compared between the microbial bioconverted polysaccharide fractions and IL-6 at the concentration of 0.01 μg / mL and 0.1 μg / mL, the level of IL-6 secretion by LPS was low, 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. In the case of rice microbial transformants, the secretion amount of IL-10 was measured at 8 ㎍ / mL. In the case of the polysaccharide fraction of rice bran bioconversion, the secretion amount of IL-10 was measured at 1 / / mL.

The amount of IL-10 secretion in the rice bran bioconversion and the rice bran bioconversion polysaccharide fractions was measured at high concentrations. It was confirmed that the polysaccharide component was recognized as a specific receptor of macrophages among the various substances contained in the microorganism bioconversion, and it was confirmed that it is an effective one 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 rice bran bioconversion, IL-10 secretion was higher in the polysaccharide fraction of rice bran bioconversion than LPS 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 of the analysis, all of the LPS, corpuscle biotransformant and microorganism bioconversion polysaccharide fractions showed a remarkably low value or no numerical value at all (regardless of the concentration) (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 microorganism bioconversion, IFN-β was secreted from the treatment concentration of 0.08 ㎍ / mL, and the secretion amount of IFN-β was also increased as the treatment concentration was increased. In the case of the rice germ bioconversion polysaccharide fraction, the secretion amount of IFN- [beta] was increased as the treatment concentration was increased from 0.1 [mu] g / mL to 0.1 [mu] g / mL and 1 [ , And the amount of secretion was similar to the amount of IFN-β secreted from the microorganism bioconverted at the concentration set based on the recovery rate. In addition, it was confirmed that the IFN-β secretion amount of the rice germ bioconversion and the rice germ bioconversion polysaccharide fraction increased similarly as the concentration increased.

It was confirmed that the polysaccharide component was recognized as a specific receptor of macrophages among the various substances contained in the microorganism bioconversion, and thus it was confirmed to be 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 rice bran bioconversion, LPS was slightly higher at all concentrations.

(10) Analysis of results

Analysis of immunological activity-related cytokines of rice bran bioconductors showed that TNF-α, IL-6, IL-10 and IFN-β were expressed, and that the immunological activity of the rice bran bioconversion was due to the polysaccharide fraction .

In comparison with LPS, the secretory pattern of TNF-α, IL-6, and IFN-β in the LPS was similar to that of the bioconversion polysaccharide fractions of IL-1β, IL-4 and IL-5 , And IL-12p70 were not secreted together.

LPS is well known as TLR4 (Toll-lokreceptor4), and it can be interpreted as a polysaccharide fragrance of rice gut bioconversion is recognized in TLR4 and mediated 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, Lt; RTI ID = 0.0 > Th2 < / RTI > and Th17 immune responses.

As a result, it was confirmed that the intestinal bioconversion of the present invention induces the production of interferon-beta in activated macrophages, and thus it is considered that the immune activity of Th1 immune response can be specifically activated. And it was confirmed that the fractions appeared.

[Experimental Example 18: Identification of receptor]

(1) Outline of experiment

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

If the receptor is known, a biomarker can be established, and on the basis of this, there is an advantage that biomarkers can be used in the clinical tests 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 microorganism bioconversion and microorganism bioconversion polysaccharide fraction, and that the microorganism bioconversion and microorganism bioconversion polysaccharide fraction are specifically recognized and mediate TLR4 We used TAK-242, a specific inhibitor for TLR4, to confirm the results.

(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, it was confirmed that when the TLR4 inhibitor 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. At a high concentration of 1 μg / ml And it was confirmed that the decrease greatly greatly. It was also confirmed that the TNF-α secretion level was completely blocked by treatment with the TLR4 inhibitor TAK-242 at concentrations of 0.08 μg / ml and 0.8 μg / ml, It can be confirmed that the amount greatly decreases greatly. Similarly, when TAK-242 was treated at concentrations of 0.01 μg / ml and 0.1 μg / ml, the amount of TNF-α secreted was completely blocked in the case of the polysaccharide fractions of rice bran bioconversion, and 1 μg / ml At a high concentration of < RTI ID = 0.0 > 0. < / RTI >

Based on the results of completely blocking or greatly reducing the secretion of TNF-α when treated with the TLR4 inhibitor TAK-242, polysaccharides of rice bran bioconverters and rice bran bioconverts are 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, the TLR4 inhibitor TAK-242 was completely blocked at all three concentrations, confirming that IL-6 secretion was completely blocked. In the case of the microorganism bioconversion, it was also confirmed that when the TLR4 inhibitor TAK-242 was treated at all three concentrations, the secretion of IL-6 was completely blocked. In the case of the rice bran bioconversion polysaccharide fraction, it was 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 microorganism bioconversion product and the microorganism bioconversion product was specifically bound to TLR4 like IL-4, .

(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, when the TLR4 inhibitor TAK-242 was treated at all three concentrations, the secretion of IL-10 was completely blocked. In the case of the microorganism 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. In the case of the rice germ bioconversion polysaccharide fractions, it was confirmed that when the TLR4 inhibitor TAK-242 was treated at all three concentrations, the secretion of IL-10 was completely blocked as well.

The TLR4 inhibitor, TAK-242, completely blocked the secretion of IL-10, and the polysaccharide of microorganism bioconversion and microorganism 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 the rice microbial transformants, it was confirmed that the secretion of IFN-β was completely blocked when the TLR4 inhibitor TAK-242 was treated at all three concentrations. In the case of the rice germ bioconversion polysaccharide fractions, it was confirmed that when the TLR4 inhibitor TAK-242 was treated at all three concentrations, the secretion amount of IFN-β was completely blocked.

On the basis of the result that the secretion of IFN-? Is completely blocked during treatment with the TLR4 inhibitor TAK-242, the polysaccharide of the microorganism bioconversion product and the microorganism bioconversion product is specifically bound to the TLR4 like IFN-? .

(6) Analysis of results

The macrophage immunoactivity of rice bran bioconversion was confirmed by the polysaccharide fraction, and TLR4 was confirmed to be a receptor for rice bran bioconversion by confirming that the reactions induced by macrophage activation were all inhibited by TAK242, a TLR4 specific inhibitor there was. From the above results, it was confirmed that the biomass conversion product and the polysaccharide fraction of rice bran bioconversion produced by the bioconversion process pursued in the present invention have TLR4 agonist activity.

Claims (6)

(A) adding at least one selected from amylase or cellulase to rice bran, reacting the same, and sterilizing the mixture 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 microorganism bioconversion product produced from a process comprising the step of:
Wherein the rice bran bioconversion product is a polysaccharide.
The method according to claim 1,
The corpuscle biotransformation product may contain,
Wherein the composition inhibits alcoholic fatty liver or alcoholic fatty liver hepatitis or obesity caused by alcohol, and nonalcoholic fatty liver or nonalcoholic lipid hepatitis expressed by diabetes.
The method according to claim 1,
The polysaccharide,
(A) extracting a water-soluble substance by adding distilled water to the rice bran bioconversion obtained through step (c);
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 the centrifugation or filtration process.
(A) adding at least one selected from amylase or cellulase to rice bran, reacting the same, and sterilizing the mixture 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 microorganism bioconversion product produced from a process comprising the step of:
Wherein the biomass bioconversion product is a polysaccharide. ≪ RTI ID = 0.0 > 11. < / RTI >
5. The method of claim 4,
The polysaccharide,
(A) extracting a water-soluble substance by adding distilled water to the rice bran bioconversion obtained through step (c);
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 prevention or treatment of fatty liver or hepatitis according to any one of
5. The method of claim 4,
The fatty liver or hepatitis,
A pharmaceutical composition for the prevention or treatment of fatty liver or hepatitis characterized by inhibiting alcoholic fatty liver or alcoholic fatty liver hepatitis or obesity caused by alcohol, nonalcoholic fatty liver expressed by diabetes or nonalcoholic fatty liver disease.

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