KR20130077803A - The preparing method of agents for improving liver function - Google Patents

Abstract

PURPOSE: A physiologically active substance which protects liver and improves liver function is provided to enhance the effect and to be used as a composition for preventing or treating diseases caused by liver damage and liver function deterioration. CONSTITUTION: A method for preparing a physiologically active substance which protects liver and improves liver function comprises the steps of: making a liquid medium using black rice (Oryza sativa L.); inoculating Basidiomycetes to the medium and culturing; and providing a bioconverted product with improved efficacy for protecting liver and improving liver function through bioconversion. The bioconverted product is a polymeric polysaccharide with immunity. A pharmaceutical or health food composition contains the physiologically active substance. [Reference numerals] (1) Normal liver tissue; (2) Alcoholic hepatotoxicity-induced liver tissue; (3) Black rice bran/Lentinus edodes mycelium (fermentation)/bioconverted product powder

Description

Liver protection and liver function improving agent produced through fermentation and bioconversion process of basidiomycetes mycelium of black rice

The present invention relates to a bioactive substance that protects the liver and improves liver function produced through fermentation and bioconversion process of basidiomycete mycelium of black rice. Liver protection and liver function-improving effect through the bioconversion process from the cultures produced by the inoculation process by inoculating mycelia of situation mushrooms, chaga mushrooms, shiitake mushrooms, leaf mushrooms, thirsty mushrooms, snow mushrooms, and skirt mushrooms Method for producing a greatly improved bioconversion product and a bioconversion product relates to a composition characterized by a polymeric polysaccharide as an active ingredient having an effect of protecting the liver and improving liver function. In particular, the bioconversion process is characterized by a bioconversion process by enzyme treatment and a bioconversion process by microbial fermentation, the enzyme treatment is by a fibrinolytic enzyme that can break down the cell wall components, the microbial fermentation is fibrinolytic enzyme It relates to a manufacturing method characterized in that by the microorganisms that can produce.

In general, the liver, which can be regarded as the most important of the organs of the human body, is also called the liver, and its function is mainly due to the metabolism of carbohydrates, proteins, fats, etc. by the catalysis of many enzymes present in the liver cells. It performs four functions. First, it is secretion function. It is composed of bilirubin, cholesterol, bile acid, sodium, chlorine, calcium, minerals (mineral), endogenous estrogen and androgen. It makes bile juice and drains it out of the liver through bile ducts and bile ducts.

The second function is the synthesis of protein such as albumin and biosynthesis and decomposition of glycogen, lipids, nucleic acids, vitamins, etc. The third function is to convert ammonia absorbed from the intestine into urea, and the fourth function is fibrinogen, prothrombin, Heparin is made and functions to destroy red blood cells, hematopoiesis and blood clotting.

Many liver protection and liver function improvement products have been developed and used to improve the liver function and protect the liver from liver toxic substances. In general, drugs known to be used for the treatment of liver damage include soymarin and biphenyl dimethyl dicarboxylate (BDD), which are herbal medicines for treating liver disease. It is used to treat early liver damage or early hepatitis because it is effective in preventing liver cell destruction, stabilizing changes in cell membranes, protecting the liver from harmful substances and normalizing damaged liver cells. In addition, antiviral agents for viral hepatitis are being used for treatment. In the development of health functional food materials, the development of products has been focused on simple extracts of herbal medicines that have been proven to improve liver function, and the representative materials include hawthorn fruit extract, shiitake mushroom mycelium extract, milkseed extract (silinine preparation), and bokbunja. Extracts, and lactic acid bacteria fermented shimashima extract, etc. have been developed and used as individual certified health functional food materials. In addition, there are fermented rice bran extracts and ungerminated germ and soybean extracts developed in Japan for improving liver function related to natural fermentation.They are inoculated under alkaline conditions by inoculating natto bacteria, which are alkaline alkaline microorganisms, in medium containing rice bran (soy embryos) and soybeans. It is a fermented material and is used as a hangover relief material. However, many kinds of compositions related to liver function improvement and hangover relief are being marketed and marketed, but the situation is not great.

Black rice (Oryza sativa L.) is a rice rich in anthocyanins and polyphenols. Black rice contains starch, protein, and fatty acids as nutrients. In addition to phenolic compounds, fiber, minerals essential for the human body, vitamin B group, vitamin C, niacin, carotene, etc. are contained. It is ingested. Black rice, according to the herbaceous tree, said that it supports the stomach, strengthens the function of the kidneys, activates the spleen and liver, brightens the eyes and makes blood flow better.Eating daily improves the body's overall regulation and strengthens immune function. It is effective in preventing anemia of the elderly and pregnant women, and it is effective in preventing skin aging and skin care of women and makes hair black. The medicinal effects of black rice are known as antioxidant, anti-cancer, anti-allergic, anti-inflammatory, and anti-hyperlipidemic effects due to anthocyanin-based pigment components, and have recently been processed and developed as functional foods. However, the anthocyanin compound of black rice is actively developed except for processed products extracted at low temperature due to instability of the anthocyanin compound, although the anthocyanin compound is destroyed when heat treated or fermented. I can't.

At present, the number of chronic liver disease patients in the world is about 600 million to 800 million, and in Korea, liver disease is serious enough to be the number one cause of death in the 40s. However, drugs currently on the market have a low effect of treating liver fibrosis or cirrhosis, so that they are trying to develop at home and abroad, but they are still insufficient.

It is excellent in protecting liver and improving liver function, and it is effective in preventing early liver disease and improving liver function by preventing liver disease and improving liver function by effectively reviving damaged liver cells and improving liver function. There is a need and urgent need to develop curable materials.

Accordingly, it is an object of the present invention to provide a method of preparing a bioprotective and liver function improving bioactive material having excellent efficacy.

Another object of the present invention is to provide a liver protection and liver function improving bioactive material prepared by the above method.

Still another object of the present invention is to provide a pharmaceutical and a functional food composition comprising the liver protection and liver function improving bioactive substances as active ingredients.

The present inventors induce the production of a substance having significantly improved liver protection and liver function improvement by the bioconversion of natural plant components in the development of the natural foot enzyme in the liver protection and liver function improvement. Also, by maximizing the protection of liver and improving the function of liver that fermented microorganism itself has, the protection of liver and the improvement of liver function are maximized. As a result of our efforts, we compared cell extract bioconversion technology, which is our own technology, compared to simple extraction of medicinal mushroom mycelium and black rice, to protect liver and improve liver function of medicinal mushroom mycelium. Even if destroyed, the black rice will protect liver and improve liver function That could have been improved to make sure the results of ongoing research.

Therefore, the inventors of the present invention in the medicinal and edible basidiomycete mycelium (seed mushroom, chaga, shiitake mushroom, leaf mushroom, myrtle mushroom, snow mushroom, skirt mushroom, etc.) produced using black rice as a culture and fermentation medium The insoluble binder composed of the complex polysaccharide of β-Glycan structure, which is the cell wall structure of the black rice, which is a culture and fermentation medium component, and the medicinal mushroom mycelium, which is produced through the culture process Water-soluble polysaccharide of 'Bioconversion' to obtain a bioconversion product (Bioconversion Product) significantly improved the efficacy of liver protection and liver function improvement.

Ammonia becomes ammonia poisoned when more than 60mM in human blood, causing lethargy, tremor, speech impairment, visual impairment, and even death. The presence of a large amount of ammonia, a liver toxic substance, is subject to strict control of the urea circuit, and when liver detoxification is reduced, liver failure (liver failure) is placed. When the liver is in normal condition, ammonia in blood can be detoxified by hepatic urea synthesis or muscle glutamine systhesis. The hepatocyte cells isolated from the liver of rats, which are produced by the method of the present invention, which is a macromolecular polysaccharide, a biodegradation product of hepatoprotective and liver function improving effect, induce hepatocyte damage by ammonia (NH4Cl) (FIG. 3), the survival rate of the hepatocyte was increased when the cells were treated under the same conditions. Therefore, it was confirmed that it protects the liver cells from liver damage caused by ammonia and raises the ammonia degrading capacity of the liver, thereby effectively affecting ammonia reduction.

In addition, the biotransformation product was treated with ammonia-damaged hepatocytes, and then the activity of glutathione S-transferase (GST), one of the activating enzymes that performs detoxification in hepatocytes, was measured. Glutathione S-transferase forms a glutathione thioester (RSG) by combining reduced glutathione with an electrophilic substance among pathogens, carcinogens, metabolites such as toxic substances and endogenous toxins. It is an enzyme that catalyzes the reaction. As a result of measuring glutathione S-transferase activity, glutathione S-transferase activity was increased for detoxification in the ammonia-treated group, and glutathione S-transferase activity was proportional to the concentration in the group treated with the bioconversion product. Decreased. Therefore, it was confirmed that the bioconversion product effectively affected the reduction of hepatotoxicity.

Superoxide dismutase (SOD), an antioxidant enzyme, is mainly distributed in the tissues of the liver, adrenal glands, kidneys, etc., and is one of the most important enzymes in the antioxidant system. It is the first product of the aerobic exercise process. It converts radicals into hydrogen peroxide (H2O2). As a result of measuring the activity of peroxidase removal enzyme for the evaluation of antioxidant activity, it was decreased in the group treated with ammonia, but recovered in the group treated with the bioconversion product. Therefore, it was confirmed that the bioconversion product effectively inhibits oxidative damage of liver.

In vivo experiments, the biotransformation product was administered to rats that caused liver damage with anti-inflammatory acetaminophen, and aspartate aminotransferase (AST: Aspartate aminotransferase or GOT: Glutamic oxalacetic transferase) was measured. Alanine aminotransferase (ALT: Glutamic pyruvate transferase) and lactic acid dehydrogenase (LDH: Lactate dyhydrogenase) were measured. Aspartate aminotransferase and alanine aminotransferase are enzymes that catalyze the transfer of amino groups between amino acids and keto acids. When various types of liver disease occur, they are released from destroyed hepatocytes and serum aspartate aminotransferase and alanine. The level of aminotransferase rises. When the levels of aspartate aminotransferase and alanine aminotransferase rise significantly, acute hepatitis increases alanine aminotransferase levels higher than that of aspartate aminotransferase, whereas in fatty liver, chronic hepatitis, cirrhosis, liver cancer, etc. Aspartate aminotransferase levels rise more than alanine aminotransferase levels. Alanine aminotransferase is widely distributed in hepatocytes, whereas aspartate aminotransferase is present in myocardium, skeletal muscle, and red blood cells. Therefore, when alanine aminotransferase level is not changed and only aspartate aminotransferase level is high, myocardial infarction and myopathy may be suspected. In vivo experiments showed that aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase levels were measured after administration of a bioconversion product to rats that caused liver damage with anti-inflammatory acetaminophen. It was confirmed that the elevated level by the biotransformation product significantly decreased. Therefore, the effect of restoring liver damage induced by the toxic substance acetaminophen was confirmed.

In general, alcohol replaces other calories and you lose weight, but if alcohol adds calories, it can cause weight gain. Alcohol consumption affects metabolism and energy metabolism, causing damage to the liver, digestive tract mucosa, and other organs and tissues. Secondary nutritional disorders increase nutrient digestion, malabsorption, decreased utilization, and nutrient excretion. Let's do it. In the present invention, the liver was extracted after administration of the bioconversion product to the rats that caused liver damage with alcohol, and liver tissue was observed using hematoxylin and Eosin staining (H & E). As a result, alcohol vaginal necrosis and inflammation, infiltration and lipid degeneration of lipid vacuoles were scattered around the portal vein, whereas those treated with bioconversion products showed less destruction of hepatocytes. The cell structure is also well maintained, and fatty degeneration is also weakly observed, confirming the protective effect of the liver.

Lipid peroxidation reaction promotes lipid peroxidation through a series of chain reactions with unsaturated fatty acids in the cell membrane and increases the concentration of malondialdehyde, the end product of lipid peroxidation, resulting in oxidative damage to cells. It is known that the physiological function caused by various diseases such as liver disease, causing aging or genetic disorders. This lipid peroxidation reaction is the most important process of liver damage caused by various toxic compounds and drugs, and is attributed to the increase in oxidative stress caused by the increase of free radical production and the decrease of antioxidant defense. In addition, liver tissue has a major role in alleviating toxicity by decomposing exogenous substances including all nutrients absorbed into the body and exogenous substances including alcohol and drugs, so that there are many opportunities to be exposed to various toxic substances in the process, It is known as an accumulation site. Therefore, the antioxidant system is degraded to provide a good environment for the production of lipid peroxides, which may cause tissue damage. When exposed to such an environment, a defense system is established to protect itself. The defense mechanism is not only antioxidant enzymes, but also antioxidants such as glutathione and tocopherol. Glutathione is a nonproteinaceous thiol in animal tissues. It occupies most of the nonprotein thiols, and is a free radical scavenger of free radicals and plays an important role in intracellular antioxidants as an important substrate for metabolizing lipid peroxide. It has been reported that lipid peroxidation is increased in liver tissue and plasma of humans and rats along with chronic bile secretion. Fatty liver is caused by excessive drinking, obesity, diabetes, etc., and decreases lipid binding protein synthesis, inhibits fat migration, and lipid peroxide. It accumulates and is reported to progress to cirrhosis through fatty liver, hepatitis, and liver fibrosis. As a result of measuring the change in the level of fat peroxidation of the bioconversion product of the present invention, the observation of liver histological change showed no decrease in fat peroxidation level compared to the alcohol group in the positive control group (Fructaceae extract group) in which no fat degeneration was observed. . In the alcohol-administered group, the fat peroxidation level increased to a significant level, whereas in the bio-converted product group, the fat peroxidation level was significantly decreased compared to the alcohol-administered group. Therefore, it can be seen that the bioconversion product of the present invention is excellent in the liver protection effect by the antioxidant power, and the results such as the report that the MDA value is effectively reduced when applied to the human body.

As a result of comparing liver function values between the liver protecting and liver function improving physiologically active substance of the present invention and conventional liver protecting and liver function improving products, it was found that the liver protecting and liver function improving physiologically active substances of the present invention are more effective Was better.

Therefore, the bioactive substance according to the present invention may be provided as a composition showing the effect of protecting the liver and improving liver function.

In addition, the bioactive substance according to the present invention can be provided as a composition for the prevention or treatment of diseases caused by liver damage and liver function decline.

1 is a graph showing the cell production according to the medium and basidiomycetes
FIG. 2 shows the results of HPLC analysis of various sugars and the polysaccharide fraction powder of black rice bran / situation mushroom mycelia (fermentation) / bioconversion product. FIG. 2A is glucose, FIG. 2B is mesima capsule 550, and FIG. 2C is black rice / HPLC analysis of the polysaccharide fraction powder of the situation mushroom mycelium (fermentation) / bioconversion product.
3 is a graph showing the results of measuring cell viability
Figure 4 is a graph showing the results of measuring glutathione S-transferase (GST; glutathione s-transferase)
5 is a graph showing the results of the superoxide dismutase (SOD) measurement
6 shows alanine aminotransferase (ALT: Alanine aminotransferase or
Graph showing results of GPT: Glutamic pyruvate transferase activity
Figure 7 is a graph showing the results of assay for aspartate aminotransferase (AST: Aspartate aminotransferase or GOT: Glutamic oxalacetic transferase) activity
8 is a graph showing the results of lactic acid dehydrogenase (LDH: Lactate dyhydrogenase) activity measurement
Figure 9 is a photograph showing the liver tissue observation results for alcohol-induced hepatotoxicity
10 is a graph showing the measurement results of liver fat peroxidation level for alcohol-induced hepatotoxicity
Figure 11 is a photograph showing the visual observation results of sepsis-induced liver

Details of the present invention in the present invention are as follows.

The method of preparing a bioactive substance for protecting liver and improving liver function according to the present invention

(a) culturing and fermenting black rice by adding starch dehydrogenase and protease to a liquid medium containing black rice powder powdered black rice or black rice bran from black rice milling process;

(b) inoculating and culturing basidiomycete mycelium on the liquid black rice medium in which the black rice of step (a) is cultured and fermented, and culturing basidiomycete mycelium with bioconversion of black rice;

(c) performing a secondary bioconversion reaction by adding fibrinase after hot water extraction to the culture produced in step (b);

(d) performing a secondary bioconversion reaction by adding a culture solution containing the microorganism producing fibrinase and fibrinase produced by the microorganism after hot water extraction with respect to the culture produced in step (b);

(e) heating the final bioconversion product produced in step (c) or (d) to undergo enzyme inactivation and sterilization, followed by lyophilization to finally obtain a powder of the bioconversion product; And

(f) dissolving the bioconversion product powder of step (e) in water to remove insoluble residues and freezing-drying the supernatant to obtain water-soluble powders or adding ethanol to the supernatant to induce precipitation to improve liver protection and liver function It is characterized by comprising the step of obtaining a polymeric polysaccharide having efficacy.

It may be a medium containing 1 ~ 10% (w / v) of the black rice powder powdered black rice in the step (a) or black rice river coming out from the black rice milling process. In addition, soybean powder 0.1-5% (w / v) and KH 2 PO 4 0.1-0.5% (w / v) may be further added to the medium. The appropriate amount of enzymes (or complex enzymes) such as amylase as amylase and protease as protease added to the medium for culture and fermentation medium (provided in the product description of each enzyme) Optimal amount).

Basidiomycete mycelium in step (b) can be used to specifically select any of the mycelium, such as physiological mushrooms, chaga, shiitake mushrooms, leaves, mushrooms, shiitake mushrooms, snow mushrooms, skirt mushrooms, etc. These fungal myceliums include the Korea Microorganism Conservation Center (KCCM), the Korea Research Institute of Bioscience and Biotechnology (KCTC), the Korea Cell Line Bank (KCLB), the Korea Agricultural Microbial Resource Center (KACC), and the US Standard Bacteria Culture Preservation Center ( ATCC) can be used by receiving a strain from an institution such as. In addition, by inoculating the soybean fungus mycelia to the culture and fermentation medium of step (b) can be incubated for 1 to 10 days under the conditions of 25 ~ 40 ℃ and pH 4.5 ~ 7. At this time, in the culture of basidiomycete mycelium, batch culture may be performed for a certain period of time until the concentration of residual carbon source of the basidiomycete mycelium culture is depleted below a certain concentration, and the concentration of residual carbon source is depleted below a certain concentration. It is possible to improve productivity by repeating the fed-batch process of adding concentrated liquid black rice medium at a time point and adding concentrated liquid black rice medium at a time when the concentration of residual carbon source is depleted below a certain concentration. have.

The cell wall component of the medicinal mushroom mycelium produced in step (b) and the cell wall component of black rice are insoluble conjugates composed of polysaccharides such as cellulose, hemicellulose, pectin, glucan, chitin, and mannan, and in step (c) 2 Enzymatic treatment for the secondary bioconversion process involves appropriate amounts of various fibrinolytic enzymes, such as cellulase, hemicellulase, pectinase, and glucanase (in the product description of each enzyme). Can be carried out by adding). As the fibrinase, commercially available enzymes may be used, that is, without limitation, but not limited thereto, the fibrinase biscozyme (complex derived from Aspergillus) and cellulase (Cellulase: derived from Aspergillus niger) and filtase (Filtrase) (Tricoderma reesei-derived complex), rapidase (Rapidase: aspergillus niger-derived complex), and Sumizyme (composite containing Sumymeme: Aspergillus niger-derived pectinase).

In the step (d), the microorganism producing the fibrinase may be a conventional microorganism known to produce fibrinase, that is, microorganisms such as lactic acid bacteria, Bacillus bacteria, yeast, Aspergillus genus, Trichoderma lyse and the like. Microbial culture solution containing the fibrinolytic enzyme produced by the microorganism can be added.

In the step (e), the enzyme inactivation and sterilization process may be performed at 90 to 121 ° C. for 5 to 120 minutes, and lyophilized to obtain a powder of the bioconversion product.

In step (f), the bioconversion product powder is dissolved in water at 10% (w / v) to remove insoluble residue, and the supernatant is lyophilized to obtain an aqueous powder, or 2 to 10 times ethanol is added to the supernatant. Precipitation can be induced, from which a polymeric polysaccharide having immunological activity can be obtained.

Induced hepatocyte damage with ammonia (NH4Cl) in hepatocyte cells isolated from rat liver, a bioconversion product, which is a polymeric polysaccharide of liver protection and liver function improving efficacy, prepared according to the above method. When treated under the conditions it was confirmed that the liver cell survival rate is improved. In addition, peroxide depleting enzymes for the evaluation of the activity and antioxidant activity of glutathione S-transferase (GST), one of the activating enzymes that perform detoxification in hepatocytes after treating the bioconversion product with ammonia-damaged hepatocytes. Inhibition of liver oxidative damage was confirmed by measuring the activity of (SOD: superoxide dismutase).

Aspartate aminotransferase or GOT: Glutamic oxalacetic transferase (AST) in liver function test levels after bioconversion products were administered to rats that caused liver damage with anti-inflammatory acetaminophen under in vivo conditions, Alanine aminotransferase (ALT) or GPT: glutamic pyruvate transferase (LDT) and lactate dehydrogenase (LDH: Lactate dyhydrogenase) were measured to determine the recovery effect against toxic liver damage.

In addition, after administration of the bioconversion product to rats that caused liver damage with alcohol, livers were extracted, and liver tissues were observed using hematoxylin and Eosin staining (H & E), and lipid peroxidation levels of liver cells were measured. By doing so, hepatic necrosis, alcohol-induced histologic damage, such as inflammation and fat degeneration, and hepatic protective effects due to antioxidant activity were confirmed.

≪ Example 1 >

Cultivation and Fermentation of Black Rice for Cultivation of Basidiomycetes and Cultivation of Basidiomycetes

Black rice powder 2% (w / v) [medium 1] or black rice from black rice powder or black rice milled in order to facilitate the use of nutrients such as starch, protein, etc. Amylase and proteolysis of starch degrading enzymes in liquid medium containing 2% (w / v) steel, 0.1-0.5% (w / v) of KH2PO4 and 1% (w / v) of soybean powder. The enzyme was treated with protease (Protease) and reacted at 60 ° C. for 1 hour, and sterilized at high temperature for 30 minutes to perform culture and fermentation medium of black rice powder or black rice bran.

10% (v / v) inoculation of the spawn mushroom mycelium, chaga mushroom mycelium, shiitake mushroom mycelium, shiitake mushroom mycelium, snow mushroom mycelium spawn culture medium into the culture and fermentation broth, and the residual carbon source at 28 ~ 30 ℃ When the concentration was exhausted below a certain concentration, the cell mass produced by culturing for 7 to 10 days in a fed-batch process of adding the concentrated liquid black rice medium was compared. The results are shown in FIG.

≪ Example 2 >

Enzyme and Enzyme Treatment for Bioconversion Process of Enzymatic Treatment in Basidiomycete Mycelia Culture

Cell wall components of medicinal mushroom mycelium and black rice such as shiitake mushrooms, chaga, shiitake mushrooms, shiitake mushrooms, shiitake mushrooms, snow-blowing mushrooms and skirt mushrooms include cellulose, hemicellulose, pectin, glucan, chitin and mannan. It is composed of complex polysaccharides. Enzymatic treatment of the second bioconversion process of the culture of Example 1 produced by the cultivation process was carried out by culturing basidiomycete mycelium in liquid black rice medium cultured and fermented medium of black rice or black rice bran. That is, various enzymes such as cellulase, hemicellulase, pectinase, and glucanase were added in an appropriate amount (the optimal amount suggested in the product description of each enzyme). Enzymes include commercially available enzymes such as biszyme (Viscozyme: Aspergillus niger complex), cellulase (from Cellulase: Aspergillus niger), filtase (Filtrase: complex from Trichoderma reesei), and rapidase. Aspergillus niger-derived complex) and Sumizyme (complex containing Aspergillus niger-derived pectinase) were used at the recommended concentrations indicated in the product description of each enzyme.

≪ Example 3 >

Fermentation for Bioconversion Process of Microbial Fermentation in Basidiomycetes Mycelial Culture

Among the various enzymes produced by microbial fermentation in the cultures of <Example 1> or <Example 2>, in particular, conventional microorganisms known to produce fibrinase for performing a bioconversion process through fibrinase, Fibrinase production was confirmed and used against microorganisms such as lactic acid bacteria, Bacillus bacteria, yeast, Aspergillus genus and Trichoderma liz. The second bioconversion process of microbial fermentation was carried out by adding 10% (v / v) of the culture medium of the fibrinolytic enzyme-producing microorganisms after hot water extraction of the culture of Example 1 or Example 2.

<Example 4>

Bioconversion Product Recovery Process

The bioconversion product produced by the bioconversion process of <Example 3> was powdered by lyophilization after 90 ℃, 1 hour enzyme inactivation process and sterilization process. In addition, the bioconversion product powder having the above immunological activity was dissolved in 10 times of water, centrifuged at 10,000 rpm for 30 minutes to remove insoluble residue, and the supernatant was freeze-dried to obtain an aqueous powder or 5 times of ethanol in the supernatant. Addition induced precipitation at 4 ° C. for at least 24 hours and the precipitate was lyophilized to obtain a powder of polymeric polysaccharide fraction with immunological activity.

 &Lt; Example 5 >

Analysis of physiologically active substances prepared according to the method of the present invention

The polysaccharide fraction of the black rice bran / situation mushroom hyphae (fermentation) / bioconversion product prepared in <Example 4> was analyzed by HPLC using a gel chromatography column (UltrahydrogelTM 1000, Waters, USA). In order to confirm the molecular weight of polysaccharides, the retention time of polysaccharides was confirmed using starch, one of the well-known polysaccharides, and the retention time of monosaccharides was commonly used as a monosaccharide. Confirmed. In addition, after purchasing gastrointestinal cancer, liver cancer, and patient resection surgery, it is possible to increase the immune function by combination with chemotherapy, and purchase and compare [Meshima capsule 550] of Korea New Medicine Co., Ltd. Analyzed. [Meshima capsule 550] is currently sold as an immune function enhancer to assist in the treatment of cytotoxic anti-cancer, and is an active ingredient of anti-cancer immunity acidic polysaccharide (153kD) including the situation mushroom mycelium extract (polysaccharide of Shellinus linteus) It is known to have. As a result of analysis, the immunoactive substance of the polysaccharide fraction powder of black rice bran / situation mushroom mycelia (fermentation) / bioconversion product prepared in <Example 4> was similar to the retention time of the starch and the polysaccharide peak of [Meshima capsule 550]. It was confirmed that it is detected in the time zone [Fig. 2].

The constituent monosaccharides of the polymeric polysaccharide fraction were analyzed. The experimental method is as follows. As a sample extraction and pretreatment method, neutral sugar was hydrolyzed at 100 ° C. for 4 hours using trifluoroacetic acid (2M) at a concentration of 10 mg / ml. The analysis was commissioned by the Korea Institute of Basic Science and Support. As an analytical instrument, ICS-5000 with electrochemical detection (Dionex, Sunnyvale, CA, USA) was used as the equipment of the Korea Institute of Basic Science. The column was CarboPac PA1 (4). × 250 mm, Dionex) was analyzed by connecting a CarboPac PA1 cartridge (4 × 50 mm). The analytical solvent was analyzed by isocratic composition using sodium hydroxide (NaOH, 16 mM), the flow rate was set to 1 ml / min and 10 μl sample was injected. As a result of analyzing the constituent monosaccharide of the polymer polysaccharide fraction, the constituent monosaccharide of the polysaccharide fraction is mainly composed of glucose and galactose, in addition to mannose, rhamnose, and arabinose. , Fructose (fructose), xylose (xylose) was found to be included (Figure 2).

&Lt; Example 6 >

In vitro analysis of physiologically active substances prepared according to the method of the present invention

[6-1] Rat Hepatocyte Isolation

Rat hepatocyte isolation was performed using the following method. Isolation of hepatocytes from rats was based on Seglen's method and a modified method was performed in two stages by digesting the liver substrate with collagenase in situ. Sprague-Dawley rats weighing 180-200 g were injected with 0.1 mg / kg rumoon (Xylazine, Bayer Korea Co., Ltd.) and 0.58 mg / kg ketamine (ketamine hydrochloride, 1 ug / kg chlorine chloride, Yuhan) Anesthetized and opened the rat's abdomen. Find the portal vein and plug in the catheter, followed by 1st stage perfusion solution (NaCl 8g / L KCl 0.4g / L, HEPES (4- (2-HydroxyEthyl) -1-PiperazineEthane-Sulfonic acid, Sigma Chem Co.) 2.38g / L, EDTA (Ethylenediaminetetraacetic Acid, Gibco Co) 0.19g / L, Sodium bicarbonate 0.35g / L, Glucose 0.9g / L, Penicillin 100Unit / mL.Streptomycin 10mg / mL, Amphotericin B 25ug / mL) Rinse 10 minutes at speed and wash thoroughly. At this time, the temperature of the perfusion solution was maintained at 37 ° C., and the silicon gas was passed through the silicon tube and equilibrated with a mixture of 90% oxygen (O 2) and 10% carbon dioxide (CO 2), and immediately before entering the liver to prevent air bubbles from entering the liver. The bubble removal device was installed in the. In the second step, collagenase solution (collagenase (Sigma Chem Co.) 0.5g / L, trypsin inhibitor (Sigma Chem Co.) 0.05g / L, sodium chloride (NaCl) 8g / L, potassium chloride (KCl) 0.4 g / L, calcium chloride (CaCl) 20.56 g / L, hepes (HEPES) 2.381 g / L, sodium bicarbonate 0.35 g / L, penicillin 100 Unit / mL, streptomycin 10 mg / mL, amphotericin B (Amphotericin B) 25ug / mL) was flowed through the catheter in the same manner as in step 1 for 10 minutes at a flow rate of 20mL / min. As the perfusion solution begins to flow, the dark red liver turns yellow immediately as the blood drains, and when the perfusion solution containing collagenase begins to flow, the liver's substrate begins to degrade and the liver begins to swell little by little. By the end of the perfusion, it will be about twice as large as the beginning. Remove these livers, transfer them to Petri dishes, cut liver capsules with a scalpel to release the liver cells, wash the loose tissue with William's Medium E (Wellzine), filter it with gauze, filter it with 100um pore nylon mesh, and then 4 °. Centrifugation was performed at C (500 rpm, 50 g, 2 minutes). Under these centrifugation conditions, most of the liver cells sink and most of the other nonparenchymal cells of the liver remain suspended. The above centrifugation was repeated three times to obtain only hepatocytes. Survival of hepatocytes was measured by trypan blue staining method. Normally, about 2 to 3 X 108 hepatocytes were recovered from 200 g of rats, and the survival rate was more than 85%.

[6-2] Rat Primary Hepatocyte Culture

Rat liver cell culture was performed as follows. The medium used for the culture of mouse hepatocytes was hormonaly defined media (HDM), and William's Medium E (Wellzine) was used as the basic medium, and epidermal growth factor (20ug / L, Daewoong Pharmaceutical), Insulin (10 mg / L, lily), hepes (HEPES) (1.19 g / L, Sigma Chem. Co), penicillin- streptomycin (100 Unit / mL, Gibco), amphotericin The ratio (amphotericin B) (25ug / mL, Gibco) was added and used. After 4 hours of inoculation, the medium was changed, and after 24 hours, the medium was changed and used for the experiment. By treatment with medicinal herbs (1mg / mL) at each concentration of 5, 10, 20, 50, 100mM of ammonium chloride (concentration) for 24 hours, the cell viability was confirmed using MTT and selected for medicinal herbs. The selected medicinal herbs were treated by concentration (0.25, 0.5, 1, 2.5mg / mL) and incubated for 1 hour, then treated with 100mM ammonium chloride (ammonium chloride) and incubated for 24 hours.

[6-3] Cell viability measurement

Ammonia was used to induce hepatocellular damage in in vitro cell experiments. Cytotoxicity was apparent in 100 mM ammonia, and 100 mM ammonia was injected into the experimental group of hepatocyte damage. The samples used in the experiment were each sample prepared in Example 4, that is, black rice / situation mushroom hyphae (fermentation) / water-soluble powder of the bio-conversion product (experimental group 1), black rice / chaga mushroom (fermentation) / bio conversion product Water soluble powder of experimental group 2, black rice / shiitake mycelia (fermentation) / water soluble powder of biological conversion product (experimental group 3), black rice / wood mushroom mycelia (fermentation) / water soluble powder of biological conversion product, black rice / snow A water-soluble powder of fermented mushroom mycelium (fermentation) / biotransformation product (Experimental Group 5) was used, and screening experiments were carried out with a substance (Experimental Group 6 ~ 11) with similar expected effects in order to compare the protection effect of hepatocytes. The sample was treated with ammonia, incubated for 24 hours, and then treated with MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl-2H-tetrazolium bromide) at a concentration of 0.5 mg / ml for 4 hours. After the reaction, the solution was dissolved and treated with dimethyl sulfoxide (DMSO), and the absorbance was measured at 540 nm. Cell viability is expressed as a relative percentage (%) based on normal cells treated with nothing. As a result of cell viability test, it was confirmed that the cell survival rate was improved to about 90% when the water-soluble powder of the bioconversion product was treated. The results are shown in Fig.

[6-4] Glutathione s-transferase (GST) measurement

Glutathione S-transferase activity was measured using a Glutathione S-Transferase Assay kit (Sigma Chem. Co). Calculate glutathione S-transferase activity by measuring the absorbance at 340 nm by mixing the sample well with Dulbecco's Phosphate buffer saline, L-glutathione reduce and CDNB. (FIG. 4).

Glutathione S-transferase activity was increased in the rat ammonia-treated group for detoxification. Glutathione S-transferase was in the group treated with black rice / shiitake mycelia (fermentation) / bioconversion product soluble powder. Activity decreased proportionally in a concentration dependent manner.

[6-5] Determination of superoxide dismutase (SOD)

After 24 hours of the black rice bran / shiitake mushroom mycelia (fermentation) / bioconversion product water-soluble powder prepared in Example 4 together with ammonia, the cells collected by trypsin-EDTA treatment were freeze-thawed. It was dissolved to determine the peroxidase activity. As an indirect method using nitroblue tetrazolium, an assay kit (Superoxide dismutase Assay kit, Sigma Chem. Co) was used. After the sample and the enzyme was added to the WST working solution (WST working solution) and reacted at 37 ° C. for 20 minutes, measured by 450nm absorbance and using the sample and the reagent blank (inhibition rate) to calculate the inhibitory activity (inhibition rate).

Peroxide scavenging enzyme activity was decreased in ammonia treatment group, but increased in proportion to concentrations in the group treated with black rice / shiitake mushroom mycelium (fermentation) / bioconversion product water-soluble powder, and increased in proportion to the concentration. High activity was shown (FIG. 5).

Example 7

In vivo analysis of physiologically active substances prepared according to the method of the present invention

[7-1] Acetaminophen-induced hepatotoxicity animal experiment

Acetoaminophen-induced hepatotoxicity animal experiments were performed in rats by in vivo experiments to observe the effect on hepatotoxicity of the black rice / shiitake mushroom mycelia (fermentation) / bioconversion product powder prepared in Example 4.

Sprague-Dawley male rats, 8 weeks of age, were distributed from Orient Co., Ltd. (Guide for Care and Use of Laboratory Animals (Guide for the Care and Use of Laboratory). Animals, Institute of laboratory Animal Resources, Commission on Life Sciences, National Research Council, USA; National Academy Press: Washington DC, 1996). The conditions were maintained in the photoperiod at 12-hour intervals at the humidity of 12. The experimental procedure was carried out according to the animal control guidelines of the Korea Centers for Disease Control and Prevention, and was approved by the Animal Experimental Ethics Committee of Dongguk University.

Prior to the experiment, all experimental animals were fed with an unlimited amount of water and solid feed, adjusted to the environment for one week, and the experimental groups were classified according to their weight.

Acetaminophen was administered for 4 days at a concentration of 1 mg / kg and the normal group received water instead of acetaminophen.

The animals were then sacrificed and examined. That is, blood was collected directly from the heart under anesthesia, and the liver was stored in liquid nitrogen immediately after the body anesthesia. Plasma was centrifuged at 1000 × g in blood at 4 ° C for 15 minutes, and all samples were stored at -70 ° C until the experiment.

The sample black rice / shiitake mushroom mycelia (fermentation) / bioconversion product powder and the control group were all administered 250 mg / kg. In the administration of the black rice / shiitake mushroom mycelium (fermentation) / bioconversion product powder was first weighed according to the dosage, and then the supernatant was dissolved in the powder and the precipitate was removed.

[7-1-1] Alanine aminotransferase or GPT: Glutamic pyruvate transferase activity measurement

In order to measure alanine aminotransferase activity, a kit prepared by the Reitman-Frankel method (GOT kit, Asan pharmaceutical, Korea) was used. After reacting at 37 ° C using L-alanine and alpha-ketoglutaric acid as substrates, color reagent (2,4-dinitrizine hydrazine) and 0.4N sodium hydroxide (NaOH) Colorimetry was measured at 505 nm to measure absorbance. The unit of activity was expressed as Kamen unit / ml.

The effect of alanine aminotransferase activity on blood was significantly increased in acetinaminophen intake control group compared to normal control group. In the positive control group, no statistically significant decrease was observed when compared with the acetaminophen intake control group, but the alanine aminotransferase group was observed in the experimental group treated with black rice, shiitake mushroom mycelium (fermentation) and bioconversion product powder. It was observed that the enzyme activity decreased to a statistically significant level (FIG. 6).

[7-1-2] Aspartate aminotransferase (AST: Aspartate aminotransferase or GOT: Glutamic oxalacetic transferase) activity measurement

In order to measure aspartate aminotransferase activity, a kit (GOT kit, Asan pharmaceutical, Korea) prepared by Reitman-Frankel method was used. After reacting at 37 ° C using L-aspartic acid and alpha-ketoglutaric acid as substrates, color reagent (2,4-dinitrizine hydrazine) and sodium hydroxide (NaOH) Colorimetry was measured at 505 nm to measure absorbance. The unit of activity was expressed as Kamen unit / ml.

The effect of aspartate aminotransferase activity on the serum aspartate aminotransferase activity was significantly increased in the acetaminophen intake control group compared to the normal control group. The positive control group showed a statistically significant decrease compared to the acetaminophen intake control group, and the aspartate aminotransferase activity was observed in the experimental group treated with black rice, shiitake mushroom mycelium (fermentation) and bioconversion product powder. A further decrease was observed to this statistically significant level (FIG. 7). Therefore, it was confirmed that the enzyme activity level increased due to the liver damage was significantly decreased by the black rice / shiitake mushroom mycelia (fermentation) / bioconversion product powder.

[7-1-3] Lactate Dehydrogenase (LDH: Lactate dyhydrogenase) Activity

Lactate dehydrogenase cytotoxicity kit (LDH Cytotoxicity Detection Kit; Takara Bio Inc., Atsu, Shiga, JAPAN) was used to measure lactate dehydrogenase activity. Lactic acid dehydrogenase is an enzyme distributed in all tissues and cells in the body and has a function of catalyzing the interconcentration between pyruvate and lactate. As a result of cytotoxicity and apoptosis, lactate dehydrogenase levels are increased in the same way as AST and ALT.

The effect of lactate dehydrogenase activity on blood serum was significantly increased in acetaminophen intake control group compared to normal control group, and decreased in the positive control group compared to acetaminophen intake control group. In the experimental group of this experiment, the black rice bran / shiitake mushroom mycelia (fermentation) / bioconversion product powder was also found to decrease the lactate dehydrogenase activity to a significant level when compared to the acetaminophen intake control (Fig. 8). Therefore, it was confirmed that the enzyme activity level increased due to the liver damage was significantly decreased by the black rice / shiitake mushroom mycelia (fermentation) / bioconversion product powder.

[7-2] Alcohol-induced hepatotoxicity animal experiment

Alcohol-induced liver damage in rats treated with black rice / shiitake mycelium (fermentation) / bioconversion product powder, followed by extraction of liver and liver tissue using hematoxylin and Eosin staining (H & E) Was observed. The experimental method is as follows.

After sufficient fixation in formalin, the extracted livers were transferred to 70% ethanol solution and stored at 4 ° C. until analysis. Paraffin processing of tissues was first immersed in 70% methanol solution for 1 hour, then 1 hour in 90% methanol solution, 1 hour in 95% methanol solution, 1 hour in 100% methanol solution, 1 hour in 100% ethanol solution, xyl A total of 1 hour soaking in ren was repeated. It was then soaked in paraffin (65 ° C.) for 30 minutes and then paraffin blocks of tissues were prepared. The blocks are cut into 4 μm tissue sections using a thin slicer and soaked in a water bath and fixed on a clean glass slide. The glass slide is heated for 20 minutes at 65 ° C. open to secure the tissue to the glass slide. The glass slide was repeated three times of dipping for 2 minutes in xylene and then immersed in 100% ethanol solution for two times.Then, the glass slide was dehydrated in the order of 95% ethanol solution, 80% ethanol solution, and finally water Paraffin (deparaffinization) and rehydration (rehydration) will go through.

Hematoxylin & Eosin (H & E) tissue staining is first soaked in hematoxylin for 2 minutes and then rinsed with running water. After immersing in eosin for about 15 seconds, immersed in 95% ethanol solution twice, and then immersed in 100% ethanol solution twice. After 2 minutes dipping in xylene a total of two times and covered with a cover glass. The prepared slides were observed under a microscope and each image was captured.

[7-2-1] Liver Tissue Observation for Alcohol-induced Hepatotoxicity

Alcohol-induced liver damage in rats treated with black rice / shiitake mycelium (fermentation) / bioconversion product powder, followed by extraction of liver and liver tissue using hematoxylin and Eosin staining (H & E) In the alcohol administration group, lipid vacuoles caused by fatty degeneration were scattered around the portal vein along with inflammation and infiltration with necrosis of hepatocytes, whereas black rice / shiitake mushroom mycelium (fermentation) / bioconversion product powder In the group treated together, less destruction of hepatocytes was observed, relatively well maintained cell structure, and fat degeneration was also observed weakly, confirming the protective effect of the liver (FIG. 9).

[7-2-2] Measurement of Liver Fat Peroxidation Levels for Alcohol-induced Hepatotoxicity

Hepatic lipid peroxidation level measurements were expressed in MDA using the STA-330 OxiSelect ™ TBARS Assay Kit (Cell Biolabs, Inc., San Diego, Calif., USA). First, the liver was resuspended at 100 mg / mL phosphate buffer solution containing 1X BHT, an antioxidant solution kit. The tissue was homogenized on ice and the resulting homogenate was centrifuged at 10,000 g for 5 min at 4 ° C. Supernatants were carefully separated and immediately examined for TBARS level determination.

Hepatic fat peroxidation levels were significantly increased in the alcohol intake control group compared to the normal control group. On the other hand, there was no change in the positive control group compared to the alcohol intake control group, but the black rice / situation mushroom hyphae (fermentation) / bioconversion product powder, the black rice / shiitake mushroom mycelia (fermentation) / bioconversion product powders The black rice bran / myrtle mushroom mycelia (fermentation) / bioconversion product powder intake group all confirmed that the liver fat peroxidation level was lower than the ethanol intake group and the positive control group (Fig. 10).

In the positive control group where no fat degeneration was observed compared with the observation of liver tissue, the fat peroxidation level did not decrease as compared with the alcohol intake group. In the powder, black rice / shiitake mycelia (fermentation) / bioconversion product powder and the black rice / thirsty mushroom mycelia (fermentation) / bioconversion product powder, the fat degeneration was observed in comparison with the alcohol intake group, Fat peroxidation levels were found to be significantly reduced. Therefore, hepatoprotective effect of black rice / situation mushroom hyphae (fermentation) / bioconversion product powder, black rice / seed mushroom mycelium (fermentation) / bioconversion product powder and black rice / thirst mushroom hyphae (fermentation) / bioconversion product powder It was confirmed excellent (Fig. 10).

[7-3] Observation of inhibition of liver damage in sepsis animal model induced by LPS (Lipopolysaccharide (LPS))

In order to observe the effect of inhibiting liver damage in the sepsis animal model of the black rice / situation mushroom hyphae (fermentation) / bioconversion product powder prepared in Example 4 in the in vivo experiments in rats (LP) ( LPS) induced liver injury Animal experiments were performed. The experimental method is as follows.

Sprague-Dawley male rats, 6 weeks of age, were distributed from Samtaco (Osan, Kyunggido, Korea), and the Guidelines for Care and Use of Laboratory Animals (Guide for the Care and Use of Laboratory Animals, Institute of laboratory Animal Resources, Commission on Life Sciences, National Research Council, USA; National Academy Press: Washington DC, 1996). Constant conditions were maintained with photoperiods at 12-hour intervals at% humidity The experimental procedure was conducted according to the Animal Care Guidelines of the Centers for Disease Control and Prevention, with the approval of the Animal Experimental Ethics Committee of Ajou University.

Prior to the experiment, all experimental animals were fed with an unlimited amount of water and solid feed, adjusted to the environment for one week, and the experimental groups were classified according to their weight.

As a control group, sepsis was induced by intraperitoneal administration at the concentration of 5 g / kg LPS and 700 mg / kg of galactosamine (Galactosamine). The product powder was ingested orally (feed diet) and intraperitoneally for 14 days at a concentration of 10 mg / kg body weight / day, and sepsis was induced.

At the end of the experiment animals were sacrificed and examined. That is, blood was collected directly from the heart under anesthesia, and the liver was immediately stored in liquefied nitrogen after taking pictures. Plasma was centrifuged at 1000 × g in blood for 15 minutes at 4 ° C, and all samples were stored at -70 ° C until the experiment.

[7-3-1] Alanine aminotransferase or GPT: Glutamic pyruvate transferase activity measurement

In order to measure alanine aminotransferase activity, a kit prepared by the Reitman-Frankel method (GOT kit, Asan pharmaceutical, Korea) was used. After reacting at 37 ° C using L-alanine and alpha-ketoglutaric acid as substrates, color reagent (2,4-dinitrizine hydrazine) and 0.4N sodium hydroxide (NaOH) Colorimetry was measured at 505 nm to measure absorbance. The unit of activity was expressed as Kamen unit / ml.

The effect of alanine aminotransferase activity on blood was significantly increased in alanine aminotransferase activity in the sepsis-induced control group compared to the normal control group. On the other hand, the oral (feed diet) and intraperitoneal administration of the black rice / strain mushroom mycelia (fermentation) / bioconversion product powders, which were experimental groups of this experiment, showed that the alanine aminotransferase activity decreased to a statistically significant level. (Table 1).

[7-3-2] Aspartate aminotransferase (AST: Aspartate aminotransferase or GOT: Glutamic oxalacetic transferase) activity measurement

In order to measure aspartate aminotransferase activity, a kit (GOT kit, Asan pharmaceutical, Korea) prepared by Reitman-Frankel method was used. After reacting at 37 ° C using L-aspartic acid and alpha-ketoglutaric acid as substrates, color reagent (2,4-dinitrizine hydrazine) and sodium hydroxide (NaOH) Colorimetry was measured at 505 nm to measure absorbance. The unit of activity was expressed as Kamen unit / ml.

The effect of aspartate aminotransferase activity on blood serum was significantly increased in sepsis-induced control group compared with normal control group. It was confirmed that aspartate aminotransferase activity was reduced to a statistically significant level in the group ingested black rice bran / situation mushroom hyphae (fermentation) / bioconversion product powder of the experiment (Table 1).

The results of alanine aminotransferase and aspartate aminotransferase activity in sepsis-induced animal models of black rice bran / situation mushroom mycelia (fermentation) / bioconversion product powders showed that they have an excellent effect on inhibiting liver damage (Table 1). .

<Table 1>

In addition, the visual observation of the collected liver showed that the sepsis-induced liver color was very darkly damaged as compared to the normal control group, and the oral solution of the black rice bran / situation mushroom mycelia (fermentation) / bioconversion product powder of the experiment group was Feed diet) and intraperitoneally administered intake groups showed very little liver damage, so that the color of the liver appeared bright red ( FIG. 11 ).

Therefore, it was confirmed that the rice bran / situation mushroom hyphae (fermentation) / bioconversion product powder had an excellent effect on inhibiting liver damage in the sepsis-induced animal model.

Claims (14)

(a) culturing and fermenting black rice into liquid phase
(b) inoculating C. mycelia on the cultured black fermented broth of culture and fermentation medium in step (a)
(c) liver protection from the culture produced by the culturing process of step (b), comprising the step of producing a bioconversion product which improves liver protection and liver function improving efficacy through a bioconversion process; Method of producing a bioactive substance for improving liver function.
The method of claim 1, wherein the bioconversion product of step (c) is characterized in that the high molecular weight polysaccharide having immunological activity.
The method of claim 1, wherein the culture and fermentation medium of the black rice of step (a) is powdered black rice
The black rice powder or the black rice river from the black rice milling process is treated with starch degrading enzyme and protease, and then sterilized to make liquid black rice medium.
The method of claim 3, wherein the soybean powder is added to the black rice powder or the black rice bran to make a liquid black rice medium.
According to claim 1, wherein the fungal fungus mycelium of step (b) is characterized by consisting of mycelia of the fungus selected from the group consisting of mushrooms, chaga, shiitake mushrooms, leaf mushrooms, sour mushrooms, snow mushrooms and skirt mushrooms How to.
The method of claim 1, wherein the culturing step (b) includes a fed-batch culture step of additionally culturing the black rice medium at the time when the concentration of the residual medium component is depleted below a certain concentration together with the batch culture step. Characterized in that the method.
The method of claim 1, wherein the step (c) of the bioconversion process is characterized in that the bioconversion process by the enzymatic treatment after the hydrothermal extraction and the bioconversion process by microbial fermentation after the hot water extraction.
8. The method of claim 7, wherein the enzymatic treatment is by fibrinolytic enzymes capable of breaking down cell wall components.
9. The method of claim 8, wherein the fibrinolytic enzyme is selected from the group consisting of cellulase, hemicellulase, glucanase.
8. The method according to claim 7, wherein the microorganism in the bioconversion process by microbial fermentation is caused by a microorganism capable of producing fibrinase.
The method of claim 10, wherein the microorganism capable of producing fibrinase is characterized in that it is selected from the group consisting of lactic acid bacteria, Bacillus bacteria, yeast, Aspergillus, Trichoderma lyse.
The method of claim 2, wherein the polysaccharide is separated by inducing precipitation by adding ethanol to the supernatant from which the insoluble residue was removed from the culture medium subjected to the bioconversion step of step (c).
Liver protection and liver function improving bioactive material prepared by the method of claim 1.
A pharmaceutical or health food composition comprising the bioactive substance of claim 13.

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