KR20120132646A - Liquid cultivating method of Phellinus linteus increasing antioxidant and immunostimulating activity - Google Patents

Abstract

The present invention relates to a new situation mushroom mycelium liquid culture method, and more particularly, to a situation mushroom mycelium culture method that can greatly increase the antioxidant and anti-aging effect and the immunity enhancing effect. To this end, the present invention is to prepare a new medium by adding the acid extract (e) extract from the various natural products to the existing culture medium mushroom mycelium culture, incubated by inoculating the situation mushroom mycelium after sterilization of the prepared medium at high temperature and high pressure It is done. In addition, the extract of the cultured mushroom mushroom mycelium has an antioxidant and anti-aging effect, and immune function is higher than the existing mushroom mushroom mycelium, it provides a health food manufacturing method using the mushroom mushroom mycelium culture extract.

Description

Liquid cultivating method of Phellinus linteus increasing antioxidant and immunostimulating activity

The present invention relates to a new situation mushroom mycelium liquid culture method, and more particularly, to a situation mushroom mycelium culture method that can greatly increase the antioxidant and anti-aging effect and the immunity enhancing effect. To this end, the present invention is to prepare a new medium by adding the acid extract (e) extract from the various natural products to the existing culture medium mushroom mycelium culture, incubated by inoculating the situation mushroom mycelium after sterilization of the prepared medium at high temperature and high pressure It is done.

In addition, the extract of the cultured mushroom mushroom mycelium has an antioxidant and anti-aging effect, and immune function is higher than the existing mushroom mushroom mycelium, it provides a health food manufacturing method using the mushroom mushroom mycelium culture extract.

Situation mushroom is a fungus belonging to the family Hypnenochaetaceae and is called Phellius Linteus. It is a fungal fungus that grows in old trees such as mulberry, oak, chestnut and oak.

Situation mushrooms are classified into five types, such as woody mud mushrooms, dry mud mushrooms, horse mud mushrooms, black mud mushrooms, and deciduous fungus mushrooms.

Mushrooms contain a protein called polysaccharide that boosts immunity. Among the various mushrooms, the woody condition called mulberry is known to contain the most protein.

Mushrooms have attracted much attention as a development material for pharmaceuticals not only for edible but also for their taxonomic characteristics. As a result, various biologically active compounds, such as antibiotics, anti-mutagenic substances and anti-cholesterol substances, have been reported. In addition, active researches have been conducted to develop anticancer drugs such as Lentinan, Krestin, and Schizophyllan, which are polysaccharides having low molecular weight and anticancer activity, and these fungi are attracting attention as a source of bioactive substances.

In particular, P. linteus has been the subject of great interest since the Japanese National Cancer Institute announced in 1968 that the antitumor activity reached 96.7% and the highest activity among edible mushrooms and medicinal mushrooms. As a result of the successful commercialization of mass culture technology of mycelium, many studies have been conducted to reveal various physiological activities of mushrooms.

In the last decade, much research has been conducted on the functions of cancer suppression and human immunity in the linteus mushrooms, and prospects for the development of new anticancer substances from natural resources are brightening. There is also increasing interest in improving cancer treatment when situation mushrooms are used in combination with existing chemotherapy drugs.

As with other traditional Asian medicines, the general prescription of situational mushrooms is based on empirical use and lacks data on specific ingredients and functions, making them difficult to apply to Western medical systems based on scientific evidence and safety. In the end, many scientists sought to uncover the secrets of situational mushrooms using state-of-the-art biopharmaceuticals to combat various diseases. These studies suggest that linteus circulating mushrooms stimulate cell-mediated or innate immunity, activate T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, and macrophages to enhance immune or immune regulation, as well as many types of experiments. It has been demonstrated to have direct cytotoxicity against animal or human tumors.

From these results, the anti-metastatic, anti-angiogenic, and antioxidant properties of Linteus S. mushrooms were demonstrated, and Linteus S. mushrooms were potent chemo-preventive agents or additional agents. Chemotherapy has shown the potential of anticancer drugs.

Immunity is a biological defense force for the human body to effectively prevent external invasion, and the treatment of various diseases including cancer using the defense system in vivo is called immunotherapy. Cells that perform immune function include T-cells that support the immune response and inhibit the destruction of foreign cells such as cancer cells and suppress the immune response, B-cells that produce antibodies to counteract foreign substances when invaded, viruses from outside Macrophage, which directly digests and digests small foreign bodies, and NK cells, which directly recognize or destroy viruses and certain cancer cells.

Biological Response Modifiers (BRMs) as nonspecific immune enhancers are substances that exert a variety of physiological effects by stimulating the immune system to alter the biological response of the host. BRM is a substance that acts to enhance, modulate or repair the host's immune capacity, reduces the malignant call of tumor cells, promotes differentiation, enhances the effects of anticancer agents and reduces side effects, and acquires cancer cell resistance. It is included in the category of BRMs with a wide range of actions, such as those that reduce toxicities and those that inhibit tumorigenization or metastasis.

The antitumor mechanism of representative mushroom-derived polysaccharides of BRM has been suggested in various theories, but until now, it has been reported to be caused by the activation of B cells, T cells, macrophages and NK cells. IL-1 and TNF-alpha Th progenitor and NK cells induced by beta-glucan are matured and differentiated. IL-2, IFN-gamma macrophage activator (MAF), etc. produced by T cell activation mature or activate cytotoxic T cells (Tc), NK cells, and macrophages, which target or target cells. Destroy nonspecifically.

Another pathway is known to attack tumor cells by inducing cytotoxic macrophages nonspecifically through the increase of serum proteins and activation of the complement system by IL-1 and TNF-alpha isolated from macrophages or helper T cells.

Linteus Situation mushrooms induce chemotherapy and radiotherapy to revive the function of various organs in the body weakened by chemotherapy and radiotherapy, activate immune function, vomiting, weight loss, nausea, and hair loss following chemotherapy treatment. The same side effects are minimized, and surgical treatments such as resection of cancerous sites are reported to play an important role in strengthening the weakened immune function in vivo and combating foreign substances and cancer cells that have been invaded from the outside.

Recent studies have shown that when taking chemotherapy after liver cancer, especially gastric cancer, esophageal cancer, duodenal cancer, colon cancer, and rectal cancer, which are cancers of the gastrointestinal system, taking a combination of situational mushrooms increases immune function, activates and detoxifies intestinal bleeding and intestinal function. It is reported that it works.

In addition, there was no direct cytotoxicity against cancer cell lines, but the test group administered with the situation mushroom extract to solid and ascites-induced mice showed increased tumor suppression rate, survival time, and white blood cell chemotactic performance, and immune cell proliferation. And cytokine production capacity has been increased, and this anticancer activity has been explained to be due to the increase of IL-6 and TNF-alpha production.

In mice transplanted with fibrosarcoma cancer cells (MCA-102), the administration of the protein polysaccharide isolated from the linteus circulating mushroom fruiting body promoted the differentiation of dendritic cells derived from bone marrow into mature state. It has been demonstrated to migrate to increase the anticancer immune response mediated by T cells.

Protein polysaccharides derived from Linteus Situation mushrooms increase the production of Th1-dependent cytokines such as IFN-gamma and inhibit the production of Th2-regulated cytokines IL-4, leading to Th1-driven immune states, and side effects such as weight loss. It showed no effect of inhibiting the growth of cancer cells.

Through many studies, the linteus mushrooms increase the activity of natural killer cells, aid the immune response, destroy the antigen-bearing cells, such as cancer cells, and increase the activity of T cells that direct the immune response, as well as invading foreign substances. When it does, it acts to increase the activity of B cells that produce antibodies to produce substances to cope with this by several orders of magnitude. In addition, the invasion of foreign bodies in the body also increases the activity of the macrophages fighting in the forefront. Therefore, the linteus situation mushroom acts to inhibit the growth or metastasis of cancer cells by activating immune cells that attack cancer.

Linteus Situation Mushroom has the ability to regulate excessive immune function as well as immune function to overcome cancer cells. The water extract of Linteus S. aureus fruiting body inhibited the release of histamine in peritoneal mast cells, either IgE dependent or independent, in response to allergic stimulation and reported an increase in intracellular cAMP concentration or calcium uptake. .

These results indicated that Linteus hydroponic hydrothermal extracts regulated passive cutaneous anaphylaxis and histamine-induced skyrocketing vascular permeability and lowered TNF-a-induced ear swelling in the mouse allergic response model. It is the same as the result of calming the irritability of sexual rhinitis.

Linteus situation mushrooms have a hypoglycemic effect, which has been recognized in mouse experiments, and is rich in polysaccharides, protein polysaccharides, and RNA complexes that increase the function of the pancreas to promote insulin secretion. In addition, when there is an immune dysfunction in pancreatic dysfunction, the immune function is restored by beta-glucan and the interleukin produced by an excessive immune response is regulated, resulting in an autoimmune response to beta cells in the pancreas. Studies have been reported to be effective for type diabetes.

Hispidine isolated from Linteus S. mushroom was very effective in removing free radicals such as superoxide anion radical and hydrogen peroxide, while hot water extraction and ethanol extract of fruiting bodies and cultured mycelium were excellent in inhibiting lipid peroxidation. It has been reported to be effective.

Linteus S. mushroom extract was found to inhibit lipid peroxidation in animal experiments using carbon tetrachloride (CCl4), a liver damage substance, and reported to improve liver lipid metabolism and protect liver function. In liver-induced liver injury model animal experiments, liver function index was significantly decreased by the administration of Linteus S. mushroom extract, and the expression of COX-2, iNOS and cytokines related to inflammatory response was decreased. It is known to be effective in protective activity.

In addition, it has been shown to be effective in preventing and treating cerebral nervous system diseases such as stroke and dementia because it has an inhibitory effect on harmful enzymes of intestinal bacteria, which cause colorectal cancer and bladder cancer, and has neuroprotective activity. Research has been published that is effective in treating and preventing arthritis.

The ethanol extracts of linteus circumferential mushrooms were administered at different concentrations and the analgesic effects were observed. The high concentrations of analgesic effects were similar to those of the control phenylbutazone.

In addition, papers have been reported on antiallergic effects, hypotensive effects, antibacterial and antiviral effects, and anti-mutant effects.

Oxygen is essential to life and is used by the body to create energy in the body through the process of breathing, but oxygen does not only give us energy. Oxygen creates harmful oxygen called free radicals during the process of binding to food in the body, which is responsible for causing diseases in our body, and the medical community has recently pointed to free radicals as the main culprit of human aging.

Normal oxygen stays in our body for about 100 seconds or more, but unstable free radicals occur for 1 million to 1 billionth of a second, and then disappear quickly, and have a very reactive characteristic. These free radicals attack the cell membranes of cells, the basic unit of our body, and cause the loss of proto-cell function. Eventually, this will damage the signaling system or compromise the immune system, leading to body diseases such as diabetes, arteriosclerosis and cancer. It also plays a role in inducing or promoting aging because it prevents cell regeneration. As such, free radicals generated during respiration are rapidly increased in the body when exposed to ultraviolet rays, radiation, pesticides, insecticides, and tobacco smoke.

Because reactive oxygen is highly reactive, it easily reacts with any substance around it. Especially, the major components of the living body, such as lipids, proteins, and nucleic acids (DNA, RNA), are subject to active oxygen attack and are easily oxidatively damaged. . In addition, active oxygen blocks cell regeneration, and therefore, is particularly associated with degenerative diseases such as cardiovascular disease, dementia, arthritis, and cataracts. Therefore, free radicals are the source of all diseases and the main culprit of promoting aging, and 90% of modern diseases are known to be related to free radicals.

As such, the ability to protect them from the toxicity of free radicals is the antioxidant defense ability, the substance that has the ability is an antioxidant. In other words, antioxidants block, inhibit or delay the damage caused by free radicals from oxygen in living organisms and the damage of lipids, proteins, nucleic acids, and carbohydrates by non-active oxygen (e.g., single oxygen, ozone). Can be defined as a substance.

All living things grow and mature with birth, and over time, their physiological function decreases, their ability to adapt to external changes and their resistance to stress falls, eventually leading to death. Called aging. The process of aging, as a biologically inherent process in vivo, is facilitated by an inappropriate hazardous environment and can be further facilitated by the occurrence of disease.

With the advent of an aging society, many companies are conducting research and development of new silver functional foods, as the silver food industry for the health and disease prevention and treatment of old age is expected to become a future blue ocean capable of rapid growth. . The most notable physiologically active functions in silver foods are immune function enhancement, antioxidant activity, and antibacterial function.

 Phellinus linteus belongs to the genus Mud (Phellinus linteus) is a very rare species in nature, it is very difficult to obtain the fruiting body, it is also very difficult to separate the mycelium and artificial culture. Recently, it has been reported that the fruiting body has succeeded in artificial cultivation, but its authenticity is unclear, and although the effect as an anticancer enhancer has been revealed earlier, the development of mass production technology is still inadequate at home and abroad. Development of production process technology is urgently required.

Horses, also called mountain drugs, have been widely used for medicinal purposes since they have dizziness, headache, sedation, stamina reinforcement, and phlegm removal.

The horse has a special effect on nourishing tonic and is effective in indigestion, gastrointestinal disorders, diabetes, cough, and lung diseases. In particular, the kidney function is strong, strong people with weakened energy is good to take longer.

The perennial herb is a perennial herb that is native to China and is distributed in Korea, Japan, and Taiwan. Although roasted, they are eaten, but it is common to chop or grind raw food. You can also dry it and eat it. Enzymes contained in hemp are weak to heat, so it is better to eat it with fresh juice.

Hemp is a good food that prevents the accumulation of cholesterol in blood vessels. Saponin in horses is known to lower blood pressure by lowering cholesterol content.

In terms of nutrition, hemp is rich in starch and sugar, and also contains vitamins B, B2, C, and saponin. Hemp mucosa contains mucin, a protein that helps digestive enzymes and proteins absorb. Muchin is also secreted from the human gastric mucosa, and its deficiency causes gastric ulcers. Therefore, the intake of hemp helps to prevent stomach ulcers and improve digestion.

In addition, hemp is rich in potassium, which plays a great role in improving the health of the entire body, as it dissolves the poisons of salt and works with other ingredients in the hemp to promote pancreatic insulin secretion to nourish the cells.

In particular, because diabetes is caused by insulin deficiency, horses that promote insulin secretion play a great role in preventing and treating diabetes. To put it simply, the food we eat is made from soy sauce, made from a variety of nutrients, which are solidified and stored. When its nutrients are supplied to cells, they are converted to liquid glucose, which requires insulin from the pancreas to be absorbed by the cells.

However, if the pancreas weakens or becomes sick, insulin is not secreted, so the body is not supplied with nutrients, and the body is weakened and the liquid (glucose) is not absorbed by the cells. That is diabetes.

Therefore, when you eat horses, the pancreas is strengthened with various ingredients of horses, potassium of horses promotes insulin secretion, and nutrients are well absorbed by each cell of the body.

Recent studies have shown that the roots of hemp contain Diogenin, a source of DHEA, which is known to be very effective in preventing aging. Accordingly, the development of health food using this ingredient is also in progress.

An object of the present invention is to provide a method for cultivating a situation mushroom mycelium improved antioxidant and anti-aging function, and immune enhancement function,

Another aspect is to provide a method for producing a health food using the situation mushroom mycelium.

One aspect of the present invention provides a situation mushroom mycelium culture method excellent in antioxidant and aging inhibitory function, and immuno-promoting function.

To this end, the present invention is to prepare a situation mushroom culture medium by mixing the acid powder extract prepared by extracting the hot water with the existing mushroom mushroom mycelium culture medium composition, and then inoculated with mushroom mushroom spawn seed culture method To provide.

In addition, water is added to the cultured mushroom mycelium culture medium prepared by the above method, extracted by heating, the concentrate is prepared through filtration and concentration steps or powdered through a drying process to prepare a raw material of health food, powder, beverage, It provides a method for processing into food forms such as pills, granules, capsules.

Situation mushroom mycelium cultured by the above method can be used as a raw material of health food for the prevention and treatment of various diseases because the antioxidant and anti-aging, and immune function enhancement effect is higher than the existing situation mushroom mycelium. In particular, it has an excellent effect in preventing diseases such as diabetes, cancer, arthritis, heart disease caused by aging and free radicals, and infectious diseases caused by bacteria and viruses, and can be usefully used for silver food for the elderly.

1 is a process mushroom culture method of the situation mushroom mycelium of the present invention.
Figure 2 is a comparison of the antioxidant-free radical scavenging effect of the situation mushroom mycelium
Figure 3 is a comparison of anti-complement activity of the situation mushroom mycelium
Figure 4 is a comparison of macrophage Lysosomal enzyme activity of the situation mushroom mycelium

Hereinafter, the present invention will be described in more detail.

To this end, the present invention is to prepare a situation mushroom culture medium by mixing the acid powder extract prepared by extracting the hot water with the existing mushroom mushroom mycelium culture medium composition, and then inoculated with mushroom mushroom spawn seed culture method To provide.

The method of preparing the extract of the acid extract is washed with water and collected in a suitable size of about 2 ~ 10cm in length and put into the extraction container, 1 to 30 times the weight, preferably 5 to 15 times the volume (w / v%) a solvent selected from water containing purified water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably water, at a temperature of 30 to 120 캜, preferably 80 to 100 캜 for 0.5 to 20 hours. Preferably, 2 to 15 hours of extraction; Repeating the extraction step about 1 to 10 times, preferably 2 to 7 times; Through this step of filtration concentration and drying it can be prepared a powder extract to add to the situation mushroom mycelium culture of the present invention.

Situation mushroom mycelium cultures include YM, Malt yeast extract, MCM, Synthetic? A medium containing an organic nitrogen source or a complex nitrogen source such as a medium can be used.

Carbon source is an essential source of nutrients for the growth and growth of carbohydrates, proteins, lipids, nucleic acids, etc. in fungi, and the monosaccharides D-glucose, D-mannose, disaccharides Maltose, ginseng extract, molasses, beer dipped food, etc. The same food by-products are also available.

Nitrogen sources are essential nutrients for the synthesis of the major components of the cytoplasm. Cassamino acid and Amino acids, L-alanine and L-glutamic acid, complex nitrogen sources, Peptone, Yeast extract, Tryptone, and inorganic nitrogen sources, Potassium nitrate, Sodium nitrate, CSL (corn starch leachate) beer yeast extract, cottonseed extract, whey, etc. can be used.

The carbon source concentration and ratio with nitrogen source (Carbon / Nitrogen) suitable for mycelial growth of woody mud mushrooms are appropriate in the range of 2 ~ 3% of carbon source (D-Glucose) and C / N ratio of 15: 1 ~ 30: 1. In particular, mycelial growth and density are good at C / N ratio of 20: 1 when the concentration of D-Glucose is 2%.

The optimum temperature for mycelial growth is 25 ~ 37 °, and the optimum pH for mycelial growth is 5.0 ~ 8.0, 0.5 ~ 2.0 vvm for aeration, and 50 ~ 200 rpm for agitation speed.

The present invention provides a health functional food having antioxidant and aging inhibitory effects and immune function enhancement effect, which contains the situation mushroom mycelium extract obtained as the active ingredient.

After homogenizing the situation mushroom mycelium culture medium prepared by the above method with a homogenizer, adding water, heating and extracting, preparing a concentrated solution through filtration and concentration steps or powdering it through a drying process as a raw material of health food use.

Antioxidant and anti-aging, immune function enhancing health food containing the situation mushroom mycelium extract of the present invention comprises 0.1 to 50% by weight of the composite food composition based on the total weight. However, the composition as described above is not necessarily limited thereto, and may vary depending on the type and progression of the condition and disease of the user.

Food containing the mushroom mushroom mycelium extract of the present invention may further comprise a suitable carrier, excipient and diluent commonly used in the preparation of the composition. The food composition containing the situation mushroom mycelium extract according to the present invention can be used in the form of pills, granules, tablets, capsules, beverages and the like according to conventional methods, respectively.

Carriers, excipients, and diluents that may be included in the composition containing the situation mushroom mycelium extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch , Acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Can be mentioned. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

The amount of the situation mushroom mycelium extract of the present invention may vary depending on age, sex, and weight, but may be 0.1 to 100 mg / kg, preferably 1 to 10 mg / kg once or several times daily. The dosage may also be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Accordingly, the dosage is not limited in any way to the scope of the present invention.

Since the composition itself of the present invention has little toxicity and side effects, it is a medicament that can be used safely even when taken for a long time for the purpose of prevention.

The health functional beverage composition containing the mycelium mushroom mycelium extract of the present invention is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated ratio, and various flavors or natural carbohydrates, such as ordinary drinks, may be used. It may contain as an additional component. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the composite food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, and alginic acid. And salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extracts of the present invention may contain pulp for the production of natural fruit juices and vegetable drinks.

These components may be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Situation mushroom mycelium culture

After washing with dry water cultivated in Andong, Gyeongbuk, dried, weigh 100 g, put it in the extraction container, add 10 L of purified water and extract it for 4 hours at 100 ℃. Repeat this process three times. The extract is collected, filtered through filter paper, and spray dried to produce a mixed extract powder. 1, 2, 4, and 5 g of the powder of the extract powder obtained through the above process, 4 g of glucose, 4 g of yeast extract, and 10 g of malt extract are dissolved in 1 L of purified water.

After sterilizing the medium prepared as described above at 121 ° C. and 1.2 atm for 15 minutes, the cultured mushroom mycelium seedlings were inoculated to 5% (v / v) and cultured at 30 ° C. for 7 days to prepare a situation mushroom mycelium culture medium. .

For comparison, inoculated with mycelium mushroom mycelium under the same conditions in the medium without adding the extract, and used as a comparison group.

Situation Mushroom Mycelia Extract Powder Preparation

After homogenizing the cultured mycelium mycelium culture medium prepared by the above method with a homogenizer, water was added in the same amount as the culture medium and heated and extracted at 100 ° C. for 3 hours, followed by filtration and concentration, followed by vacuum drying to obtain powder Make up.

Determination of Beta-Glucan Content of Situary Mushroom Mycelia Extract Powder

Beta-glucan was measured by using a Megazyme β-glucan assay kit (Megazyme International Ireland Ltd., Ireland) to determine the sugar content other than total glucan and glucan, and then excluding alpha-glucan and glucan. Obtained by subtracting the value of sugar content.

To 100 mg of mycelial powder of 0.5 mm or less, 1.5 mL of hydrochloric acid (37%, v / v) was added, and then the contents were mixed well, followed by mixing at 30 ° C. for 60 minutes at 15 minutes intervals. In order to decompose and remove starch and protein, 10 mL of distilled water was added thereto and then left in boiling water (100 ° C.) for 2 hours.

The contents were then cooled to room temperature, adjusted to pH by addition of 2N KOH, and diluted with 200 mM sodium acetate buffer (pH 5.0). The diluted contents were obtained by centrifugation to obtain supernatant, and then, 100 μL of supernatant, exo-1,3-β-Glucanase (100 U / mL) + β-glucosidase (β-Glucosidase; 20 unit / mL) solution was added at 100 μL and left at 40 ° C. for 1 hour, and 3 mL of glucose determination reagent was added and then left at 40 ° C. for 20 minutes. Thereafter, absorbance was measured at 510 nm to obtain a total glucan and sugar content values other than glucan. In addition, sugar content values other than alpha-glucan and glucan were added to 2 mg KOH 2mL in 100mg of mycelium powder, and stirred for 20 minutes in an ice-filled water bath using a magnetic bar, followed by 8mL of 1.2M sodium acetate buffer (pH 3.8). Added.

In addition, 200 μL of amyloglucosidase (1,630 unit / mL) + Invertase (500 unit / mL) solution was added, and then left at 40 ° C. for 30 minutes, and 200 mM sodium acetate buffer (sodium acetate buffer) ; pH 5.0). Then, 3 mL of glucose determination reagent was added and left at 40 ° C. for 20 minutes. The absorbance was then measured at 510 nm to obtain sugar content values other than alpha-glucan and glucan.

Beta-glucan content was obtained by the following equation.

Beta-Glucan (%) = (Total Glucan (%) + Sugars other than Glucan (%))-(Alpha-Glucan (%) + Sugars other than Glucan (%))

The results of measuring the beta-glucan content of the situation mushroom mycelium cultured in the medium of the extract powder of 0, 1, 2, 4, 5 g / L, respectively are shown in [Table 1].

The content of beta-glucan in the mushroom mycelium increased in proportion to the concentration until 4 g / L of the extract was added.

division
Strain Herbicide Extract (g / L) 0 One 2 4 5 Beta Glucan Content (%) 17.8 20.8 22.4 26.8 26.4

Antioxidant Activity

Antioxidant activity was measured using the situation mushroom mycelium extract prepared under each medium condition of Example 1. It has been found that free radicals produced by various factors in oxidative biological damage become direct mediators. Therefore, the electron donating action is used as a measure of the action of inhibiting bio damage by the active radicals by donating electrons to the active radicals generated in vivo and converting them into stable compounds.

Therefore, to assess the free radical scavenging effect of a particular substance, the substance is measured for its ability to donate electrons to diphenylpicrylhydrazyl (DPPH; Sigma) molecules. DPPH is a very stable free radical compound that absorbs light at 520 nm by non-covalent electrons in a molecule and is dark purple. When a substance capable of providing electrons from the outside is added to the pair of non-shared electrons of DPPH, the dark purple disappears. Therefore, the antioxidant effect can be measured using the difference in absorbance, which is widely used in antioxidant activity assays.

In order to measure the electron donating ability of the composite food composition of the present invention, 600 μl of 1.5 × 10 ⁻⁴ M DPPH according to the method of Blosi so that the concentration of the sample was 100, 50, 25 and 12.5 μl / ml, respectively. After addition, methanol solution was added to bring the final volume to 3 ml. After shaking for 10 seconds to mix well, absorbance was measured at 520 nm, and the scavenging activity of the DPPH free group was calculated by Equation 2 below. The results are shown in FIG.

As shown in FIG. 2, the mycelial mushroom mycelium extract cultured in a medium having an added concentration of the extract of the fungus extract showed the highest antioxidant activity.

Immune function enhancement effect

To understand the effects on the activation of complements, the complement test was performed using an antibody test called the complement fixation test, which uses the property that complement can dissolve erythrocytes in the presence of specific anti-hemolysin. The degree of activation is being measured. Normal human serum, GVB ⁺⁺ and the same amount of sample was added to the primary reaction (37 ℃, 30 min) and diluted to 10-160 magnification, sensitized blood cells were added and the secondary reaction (37 ℃, 1 h) to sensitized blood cells The degree of hemolysis inhibition was measured at absorbance 412 nm.

Anti-complement activity is an antibody test called the complement fixation test that uses the property that complement can hemolyze red blood cells in the presence of specific anti-hemolysin antibodies in order to determine the effect on complement activation. The degree of activation of complement is measured.

In Example 1 of the present invention, the results of measuring the anti-complement effect according to the sample concentration of the situation mushroom extract cultured in 4g / L medium of the acid extract are as shown in FIG. It was found that anti-complement activity was increased in a concentration-dependent manner, and showed maximum activity at 1000 ㎍ / mL. Compared to the LPS (lipopolysaccharide) used as a positive control, it showed low activity at low concentrations (100, 500 mg / mL), but higher activity than the positive control at high concentrations (1000 mg / mL). As a result, the composite composition is considered to have a very high anticomplement activity.

Measurement of Lysosomal Enzyme Activity of Macrophages

Lysosomal enzymes and phagocyte activity are important factors in evaluating macrophage function, and the production of lysosomal enzymes by monocytes occurs in response to numerous foreign stimulants (Page et al ., 1978).

Lysosomal enzyme activity was analyzed using 96-well flat-bottomed tissue culture plates (Suzuki et al., 1990). 25 μl of 0.1% Triton X-100 was added to lyse the macrophage monolayer in the microplate (2 × 10 ⁵ cells / well). 150 μl of 10 mM PNPP (p-nitrophenyl phosphate) solution was added as a reductant of lysosomal enzyme (acid phosphatase) of macrophages. Then, 50 μl citrate buffer was added to each well, followed by incubation at 37 ° C. for 1 hour, and then 25 μl of 0.2 M borate buffer (pH 9.8) was added to the reaction mixture, and the absorbance was measured at 405 nm.

The results of measuring the lysosomal enzyme activity of the macrophages according to the sample concentration of the mushroom extract incubated in 4g / L medium of the extract of the present invention in Example 1 are as shown in FIG. The lysosomal enzyme activity of macrophages was higher at higher concentrations of mycelial extract, and compared to the negative control (NC), respectively, at the concentrations of 10, 50 and 100 ㎍ / ml Activity increased to 59.3, 68.9 and 102.9%, respectively.

Claims (4)

Situation mushroom mycelium culturing method excellent in antioxidant and immune function enhancement effect, characterized by cultivating the situation mushroom mycelium in the medium to which the acid extract is added
According to claim 1, the collected powder is washed with water, cut into a suitable size of about 2 ~ 10cm in length and put into the extraction container, the volume of 1 to 30 times, preferably 5 to 15 times the weight of ( w / v%) a solvent selected from water containing purified water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably water, at a temperature of 30 to 120 ° C., preferably at 80 to 100 ° C. for 0.5 to 20 hours, Preferably extracting for 2 hours to 15 hours; Repeating the extraction step about 1 to 10 times, preferably 2 to 7 times; Situation mushroom mycelium culturing method excellent in antioxidant and immune function enhancement effect using the powder extract obtained through the step of filtration concentration and drying
The method of claim 1, as a carbon source, monosaccharides, disaccharides, and food by-products such as ginseng extract, molasses, beer immersion, etc. As a nitrogen source, organic nitrogen source, complex nitrogen source, inorganic nitrogen source, and food byproduct CSL (corn starch leachate) beer yeast Use at least one carbon source and nitrogen source material selected from extracts, cottonseed extracts, whey, etc. The concentration of carbon source and nitrogen source is 2 ~ 3% of carbon source (D-Glucose) and C / N ratio 15: 1 ~ Situation mushroom mycelium culture method excellent in antioxidant and immune function enhancement effect, characterized in that 30: 1 range
The optimum temperature for mycelial growth of the situation mushroom is 25-37? Range, optimum pH for mycelial growth is 5.0 ~ 8.0 range, the aeration amount is 0.5 ~ 2.0 vvm, the stirring speed is 50 ~ 200 rpm excellent anti-oxidation and immune function, characterized in that the mushroom fungus culture method

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