KR20180024076A - Manufacturing method of composition for enhancing immuneresponse comprising extract from grifola frondosa - Google Patents

Abstract

The present invention relates to a method of preparing a composition for immunity enhancement using Grifola frondosa, and a soybean paste using the same. More specifically, the present invention relates to a method of preparing a composition for immunity enhancement by preparing an extract of Grifola frondosa through application of a starter production technique, and then using the extract of Grifola frondosa, and to a method of preparing a soybean paste using the same. According to the present invention, the method of preparing a composition for immunity enhancement using Grifola frondosa comprises: a first step in which a hot water extract of Grifola frondosa is prepared, and the solid content of the hot water extract of Grifola frondosa is adjusted to 20 Brix; a second step in which L. plantarum and L. mesenteroides are pre-incubated in an MRS medium at 30°C for one day; a third step in which the 20 Brix hot water extract of Grifola frondosa is sterilized; a fourth step in which the sterilized hot water extract of Grifola frondosa is inoculated with 2 w/w% of a culture broth of the pre-incubated L. plantarum and L. mesenteroides, and cultured in a stationary environment at 30°C for 30 days, to produce fermentation liquid of Grifola frondosa; and a fifth step in which the fermentation liquid of Grifola frondosa is filtered, combined with the same amount of ethanol, precipitated at low temperature for 24 hours, and centrifuged to produce a fermentation extract of Grifola frondosa.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for preparing a composition for enhancing immunity using a mushroom, and a method for preparing the composition for immunity-

The present invention relates to a method for preparing a composition for enhancing immunity using a leaf mushroom and a soybean paste using the same. More specifically, the extract of leaf mushroom is prepared by applying starter production technology, And a method for producing the soybean paste using the same.

The immune response is a mechanism that protects the human body from infectious diseases. It distinguishes itself from the foreign material and neutralizes or removes foreign substances to maintain the homeostasis of the living body. The organs, cells and molecules involved in the immune system constitute the immune system, which cooperates with each other and acts collectively to produce an immune response. The modulation of immune function can be applied to various diseases such as cancer treatment, autoimmune disease and tissue transplantation. Administration of the immunostimulatory agent as an agent of the anticancer effect may increase the antitumor activity by enhancing the bio-defense function against the pathogen or by raising the immune function.

The administration of the immunostimulating agent may reduce the growth and transformation of the cancer cells or increase the immune function against the modified cancer cells. In particular, the immunostimulating substance decreases cancer metastasis by regulating the ability to tolerate cytotoxic treatment, Has been known as a substance capable of causing an immune response.

Therefore, the present invention relates to the effects of the mushroom on nonspecific immune response (measurement of natural killer (NK) cell activity) and macrophage activation and related cytokine production And the like, to thereby prepare a composition for enhancing immunity.

Korean Patent Laid-Open Publication No. 10-2015-0103521 discloses a method for preparing a composition having enhanced beta-glucan content and antioxidative activity by treating the mushroom with ultra high pressure or an enzyme. The present invention relates to a technique for increasing beta-glucan content through high-pressure and enzyme treatment in the prior art, but the above-mentioned technology has been applied to patients who are unable to remove aflatoxin, a liver cancer-causing fungal toxin, Problems can arise.

Korean Patent Publication No. 10-2014-0118324

It is an object of the present invention to provide an immunomodulating composition comprising a mushroom which not only increases anticancer activity but also increases antimicrobial activity.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention as set forth in the accompanying drawings. It will be possible.

The method for preparing a composition for immunity enhancement using a mushroom according to the present invention comprises the steps of: preparing a mushroom hot water extract and adjusting the solid content of the mushroom hot water extract to 20 Brix; Lactobacillus plantarum (L. plantarum) and L. mesenteroides in the MRS medium, A second step of pre-culturing the lactic acid bacteria at 30 占 폚 for 1 day; A third step of sterilizing the 20 Brix mushroom hot water extract; Lactobacillus plantarum (L. plantarum) and L. menesenteroides (pre-cultured) were added to the sterilized hot mushroom hot water extract, A fourth step of inoculating the culture broth of lactic acid bacteria at a concentration of 2 w / w%, culturing the suspension at 30 ° C for 3 days, and preparing a fermented broth mushroom; The fermented broccoli fermentation broth is filtered, and the same amount of ethanol is added thereto, followed by low-temperature precipitation for 24 hours, followed by centrifugation to produce a fermented extract of Lactobacillus acidus.

According to the solution of the above-mentioned problems, the present invention can produce an immunomodulating composition including a mushroom which not only increases the anti-cancer immunity activity but also increases the antimicrobial activity at the same time.

FIG. 1 is a flow chart showing a method for preparing a composition for enhancing immunity using a mushroom of the present invention
FIG. 2 is a photograph showing the amount of FITC-E. coli particles captured inside macrophages using fluorescence
FIG. 3 is a graph showing the activity of macrophage-
4 is a graph showing the ability of macrophage activation-related cytokine expression
5 is a graph showing the effect on natural killer (NK) cell activity

The present invention relates to a method for preparing a composition for enhancing immunity using a leaf mushroom and a soybean paste using the same. More specifically, the extract of leaf mushroom is prepared by applying starter production technology, And a method for producing the soybean paste using the same.

The above and other objects, features and advantages of the present invention will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: FIG. BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention and the manner of achieving them will become apparent by reference to an embodiment which will be described in detail below with reference to the accompanying drawings.

Hereinafter, a method for preparing a composition for enhancing immunity using the mushroom described above will be described in detail with reference to the drawings.

≪ Preparation method of composition for enhancing immunity using mushroom on leaves >

FIG. 1 is a flow chart showing a method for preparing a composition for enhancing immunity using the leaf mushroom of the present invention.

First, the first step (S100) is to prepare a hot water extract of Leaf mushroom, and the solid content of the prepared Leaf mushroom hot water extract is adjusted to 20 Brix. When the solids content of the leaf mushroom hot-water extract is less than 20 Brix, there is a fear that the concentration of the hot-water extract of the leaf mushroom is too low in the step of lactic acid germ inoculation in the second step (S200), so that fermentation does not occur after inoculation of the lactic acid bacteria. When the solids content of the extract is more than 20 Brix, the concentration of the lactic acid bacteria is relatively low, which may cause decay. Therefore, it is preferable to prepare the solid of the mushroom hot water extract under the above conditions.

In addition, the above-mentioned hot-water extract of Leaf mushroom is preferably prepared by the following method.

First, in Step 1-1 (S101), 14 parts by weight of distilled water is mixed with 1 part by weight of Leaf mushroom, and the mixture is stirred at 95 ° C for 12 hours to prepare a Leaf mushroom extract.

The above mushroom ( Grifola frondosa) is equally a taxonomically as a kind of edible Basidiomycetes belonging to the Democratic reummok (Aphyllophorales) holes Zhang mushroom (polyporaceae), fragrance and excellent taste, essential amino acids including beta-glucan, vitamins, minerals, acids and dietary fiber, etc. It is also called "king of mushroom" because it contains a large amount. In Japan, it is called "dancing mushroom" or "mai tak" when it is found that it is good to dance. One of the greatest features of leaf mushrooms is the structural difference of beta-glucan, the pharmacological component of most mushrooms. Most of the edible and medicinal mushroom beta-glucan is β-1.3-glucose, but the mushroom is a cyclic polymer having β-1.6-glucose structure with β-1.3-glucose polymer. to be.

It is preferable that the dried mushroom used in the step 1-1 (S101) is dried. Specifically, the leaf mushroom is dried in a food drier in a fermentation vessel at a temperature of 45 ° C for 1 to 1.5 days, dried at a temperature of 10 to 15 ° C for 2 to 2.5 days at a wind speed of 2 to 5 m / s, / w% relative humidity to increase the flavor and flavor of the dried leaf mushroom.

In addition, when mixing less than 14 parts by weight of distilled water with respect to 1 part by weight of the mushroom in the step 1-1 (S101), the concentration of the distilled water may be lowered, which may make it difficult to produce the mushroom extract. The concentration of the mushroom may be lowered and the effect of the mushroom extract may be insufficient.

If the mixing temperature is less than 95 ° C in the step 1-1 (S101), the temperature is too low to be extracted, and the effect of the mushroom on the mushroom extract may be insignificant. If the mixing temperature exceeds 95 ° C The fragrance of the leaf mushroom may be altered due to the influence of temperature, or a problem due to the evaporation of the distilled water may occur.

If the mixing time is less than 12 hours in the first step (S101), the extraction may be insignificant and the efficacy may be lowered. If the mixing time is more than 12 hours, distillation water may be evaporated. .

Next, in step 1-2 (S102), the leaf mushroom extract is filtered and concentrated under reduced pressure to 40 Brix. Specifically, the reduced-pressure concentration is preferably performed by rotating at a pressure of 760 mmHg and a temperature of 50 DEG C at 100 to 200 rpm.

When the pressure is less than 760 mmHg and the pressure is reduced to less than 50 캜, the temperature may be too low to concentrate. If the pressure exceeds 760 mmHg or exceeds 50 캜, bubbling may occur and overflow may occur. Therefore, it is preferable to concentrate under reduced pressure at the above temperature.

Further, when the reduced pressure concentration in the step 1-2 (S102) is lower than 100 rpm, there is a problem that the rotation speed is low and the concentration is difficult, and when the concentration exceeds 200 rpm, bubbling occurs. desirable.

In addition, when the extract is concentrated by filtration under reduced pressure of less than 40 Brix, the effect of the mushroom may be insignificant. When the filtrate is concentrated under reduced pressure exceeding 40 Brix, freeze drying may be difficult in the step 1-3 (S103) Therefore, it is preferable to execute it under the above conditions.

Next, in step 1-3 (S103), the decompressed concentrated mushroom extract is lyophilized. Specifically, it is preferable that the freeze-drying is performed by freeze-drying at a temperature of -15 to -25 캜 to a final moisture content of 1.5 to 2.0 w / w%.

It is preferable that the beta-carotene component of the Leaf mushroom extract is included in the highest amount by freeze-drying under the above conditions.

Next, the second step (S200) is carried out in the medium of L. plantarum (L. plantarum) and L. menesenteroides in the MRS (de Man, Rogosa and Sharp) The lactic acid bacteria are pre-cultured at 30 占 폚 for 1 day.

Lactobacillus plantarum (L. plantarum) plays an important role in the fermentation of kimchi in Korea. It produces antimicrobial substances to suppress herpes virus and regulate immune function. Especially, it has excellent ability to remove intestinal toxicity. It is also less destroyed by acid and bile than other fungi.

The Lukono Stokes meencenteroides is a lactic acid bacterium that is produced at the early stage of fermentation of kimchi and leads to fermentation until kimchi is found to be delicious. The Lukono Stokes meencenteroides has a blood pressure lowering effect and a thrombolytic effect.

Next, in the third step (S300), the 20Brix mushroom hot water extract is sterilized. Specifically, the 20 Brix mushroom hot water extract was sterilized by mixing 1 part by weight of Aspergillus oryxae spore suspension with 1 part by weight of Bacillus subtilis culture medium, adding 20 parts by weight of the 20 Brix mushroom hot water extract It is preferable to spray and sterilize.

The Bacillus subtilis is a non-pathogenic bacterium widely distributed in nature and exists in air, dry grass, sewage and soil. It is a rod-like bacterium that is actively actuated by a flagella and forms circular or ovoid apoploids at the center of the cells. By inhibiting the activity of intestinal decongestants in our body, it inhibits the carcass, which is produced by the decaying bacteria, and reduces the carcinogenic substances such as ammonia, indole and amine, and has antimicrobial action against the pathogens.

The Aspergillus oryxae is generally referred to as a yeast mold, and is also referred to as Hwanggukjun. It has the property of decomposing proteins into amino acids and is also used in the production of soybean paste and soy sauce. It also has the property of decomposing starch into glucose, and is used for making sake, sikhye, potato, mirin. In Korea, soy sauce, soybean paste, miso soup, and rice wine such as makgeolli is used in the manufacture of general fermented foods.

The preparation and spraying of the Bacillus subtilis pure culture medium and the Aspergillus oryxae spore suspension is based on the starter technology and the detection of aflatoxin in the 20Brix hot water extract of mushroom It can be sterilized and sterilized.

The starter technology is a microorganism culture liquid used for fermented milk products, and is a technique used to determine the characteristic quality of dairy products and to produce butter, cheese, yogurt, etc., mainly using lactic acid bacteria alone or in combination.

When 1 part by weight of the suspension of Aspergillus oryxae spore is mixed with 1 part by weight of the culture medium of Bacillus subtilis, protein decomposition does not take place effectively and the bactericidal effect may be insignificant. When the Bacillus subtilis When 1 part by weight of Aspergillus oryxae spore suspension is mixed with 1 part by weight of Bacillus subtilis culture medium, the concentration of Bacillus subtilis is low and the activity of the spore bacteria is inhibited The effect may be insignificant. Therefore, it is preferable to mix under the above conditions.

Also, it is preferable to spray the above-mentioned mixture of Bacillus subtilis culture solution and Aspergillus oryxae spore suspension onto the 20Brix mushroom hot water extract.

Next, in a fourth step (S400), the pre-cultured Lactobacillus plantarum (L. plantarum) and L. menesenteroides are added to the sterilized mushroom hot water extract, The cultured lactic acid bacterium is inoculated and cultured at 30 ° C for 3 days to prepare a fermented mushroom fermentation broth. Specifically, the pre-cultured Lactobacillus plantarum (L. plantarum) and L. menesenteroides It is preferable to inoculate 2% w / w% of the cultured lactic acid bacteria.

When the culture of the lactic acid bacteria is cultured in the above-mentioned sterilized hot mushroom hot water extract at a temperature lower than 30 ° C or a temperature higher than 30 ° C, the Lactobacillus plantarum (L. plantarum) and L. menesenteroides Since it may not be efficiently cultured differently from the initial conditions of survival of the lactic acid bacteria, it is preferable to culture at the above temperature.

In addition, when the incubation time is less than 3 days, the contact of nutrients with the mycelium may not be uniform and the culture may be insignificant. When the incubation time exceeds 3 days, oxygen may be insufficient to cause contamination of the lactic acid bacteria. .

In addition, the pre-cultured Lactobacillus plantarum (L. plantarum) and L. menesenteroides It is preferable to inoculate 2w / w% of each of the cultures of lactic acid bacteria. If the inoculation is less than 2w / w%, the concentration of the lactic acid bacteria may be low and the fermentation may not occur or may be insignificant. When inoculated, since the efficacy of the sterilized hot mushroom hot water extract may be insignificant, it is preferable to inoculate under the above conditions.

Next, in the fifth step S500, the fermented broccoli fermentation broth is filtered, and the same amount of ethanol is added thereto, followed by low-temperature precipitation for 24 hours, followed by centrifugation to produce a fermented extract of Lactobacillus acidus lactis.

Specifically, it is preferable that the fermented broccoli fermentation broth is filtered with 200-300 mesh filter paper and then 70-75 w / w% ethanol is used. To 1 part by weight of the filtered fermented mushroom fermentation broth, 2 to 3 parts by weight of ethanol Lt; / RTI > It is also preferable to carry out low-temperature precipitation at a temperature of -20 to -70 DEG C for 24 hours, followed by centrifugation at 200 to 300 rpm.

When the fermented broccoli fermentation broth is filtered to less than 200 mesh, the filtration time may be too long. When the filtrate is filtered more than 300 mesh, there is a possibility that the fermented broth is not filtered. Filtration is preferred.

In addition, when less than 2 parts by weight of the ethanol is added to 1 part by weight of the filtered fermented mushroom fermentation broth, there is a possibility that the non-polar substances are not sufficiently dissolved in the ethanol than water. When mixing more than 3 parts by weight of ethanol, the above-mentioned low-temperature precipitation may not effectively take place.

The low-temperature precipitation may be slight if the precipitation is carried out for less than 24 hours, and the fermentation broth of the leaf mushroom may solidify when precipitated for more than 24 hours.

The low temperature precipitation may cause precipitation due to the high temperature when the precipitation is performed at a temperature higher than -20 ° C. When the low temperature precipitation is performed at a temperature lower than -70 ° C, It is preferable that the above conditions are satisfied.

In addition, when the centrifugation is performed at a temperature of less than 200 rpm after the low-temperature precipitation, the centrifugation may not be effectively performed because the separation rate is too low. When the centrifugation is performed at a rate of more than 300 rpm, Bubbles may be generated or deterioration may occur. Therefore, it is preferable that the above conditions are satisfied.

Further, another composition for enhancing immunity can be prepared by mixing natural extracts of the mushroom hot water extract prepared in the first step (S100). Specifically, the natural complex includes 1 part by weight of ginseng, 1 part by weight of ginseng, 1 part by weight of licorice, and 1 part by weight of visible fiber, and then subjected to hot extraction.

The mistletoe is one of the strongest anticancer plants, and it has been known as a plant that has a very excellent effect on cancer treatment in Europe, such as Germany and England. It is also known to exert excellent effects on old blood pressure, neuralgia, arthritis, diuretic action and anesthetic action.

The ginseng is rich in saponin and plays a role similar to the hormone secreted in the body, so it is excellent in brain development and has a very high anticancer effect.

It is said to be the roots of halibut, which can alleviate headaches and reduce body swelling. In addition, it is effective for chronic gastritis.

The licorice has the role of neutralizing gastrointestinal protection and toxicity. In particular, it neutralizes the poison of other medicinal materials and alleviates the efficacy, so that the effect of the natural compound is appropriately compounded.

This visibility is mainly found in southern Vietnam, also called graviola. The above-mentioned adonacin component has the effect of preventing cancer cells from being nourished by self-destructing by blocking ATP which is a nutritional supply channel of cancer cells.

It is preferable that the natural complexes are mixed in the same weight parts, respectively. However, since the anticancer efficacy can be most effectively exhibited if they are mixed in the same amount, mixing is preferably carried out under the above conditions.

The hot water extraction of the natural complex is preferably performed in the same manner as the method for preparing the hot water extract of Leaf mushroom in steps 1-11 (S101) to 1-3 (S103).

In addition, it is preferable that 1 part by weight of the natural complex is mixed with 1 part by weight of the hot mushroom extract to prepare an anti-cancer immunostimulating agent. When the natural complex is mixed in an amount of less than 1 part by weight, The anticancer effect may be insignificant. When the natural complex is mixed in an amount of more than 1 part by weight, the concentration of the hot mushroom extract is low and the effect is not sufficiently manifested and the taste and flavor are poor.

In addition, yeast, aloe, and garlic may be mixed with the hot-water extract of Leaf mushroom to prepare another composition for enhancing immunity. Specifically, 0.2 part by weight of yeast, 0.1 part by weight of aloe, and 0.1 part by weight of garlic are preferably mixed with 1 part by weight of the hot mushroom extract.

Preferably, the yeast is fermented using a pure culture medium of Bacillus subtilis used in the third step (S300) and a suspension of Aspergillus oryxae spores.

The aloe is mixed with the hydrothermal extract of the mushroom to further enhance the anticancer effect and inhibit the secretion of gastric acid, thereby killing the germs of the gastric ulcer and raising the freshness.

The garlic may be mixed with the mushroom hot water extract to enhance the anti-cancer effect. The allysine contained in the garlic has a strong antibacterial action and protects against bacteria and fungi in the body.

It is preferable that 0.2 part by weight of yeast, 0.1 part by weight of aloe and 0.1 part by weight of garlic are mixed with 1 part by weight of the hot mushroom hot water extract. When 0.2 part by weight of yeast, 0.1 part by weight of aloe and 0.1 part by weight of garlic are mixed, , Aloe and garlic may be difficult to act due to the addition of the hot-water extract of Leaf mushroom. When the yeast is mixed with more than 0.2 part by weight of yeast, 0.1 part by weight of aloe and 0.1 part by weight of garlic, the yeast, aloe and garlic- And the flavor and flavor of the mushroom hot water extract may be lowered.

The composition for enhancing immunity using the mushroom produced by the present invention can be mixed together at the time of preparing meju, and then fermented to prepare meju for immune enhancement using the mushroom.

The Meju for enhancing immunity using the mushroom of the present invention can produce hygienic doenjang by not detecting aflatoxin when compared with the meju produced by the conventional technology and shortening the period of manufacturing the doenjang by one third, It can be seen that this is a useful technique.

The anticancer activity of the soybean paste prepared by the prior art is 18% or more. However, the soybean paste prepared by the present invention has a salt content of 15%.

Hereinafter, the immunity enhancing activity of the composition for enhancing immunity using the mushroom produced by the present invention is examined.

In general, the anti-cancer immune function is characterized by a nonspecific immune response using natural killer (NK) cells based on innate immune response and a specific immune response using cytotoxic T lymphocytes (CTL) cells based on acquired immune response .

Macrophages are distributed in almost all tissues in the body, and they have primary defense against bacterial and viral infectious pathogens, as well as phagocytosis against aging normal cells and cancer cells, thereby eliminating unnecessary elements in the human body have. The physiologically active substances secreted from the activated macrophages are known as cytokines and interleukins, which are directly toxic to cancer cells, thereby maximizing the secondary immune response.

Natural killer cells (NK-cells) are nonspecific immune cells that attack virus-infected cells or cancer cells and exhibit injurious effects.

Therefore, in order to investigate the activity of the composition for enhancing immunity using the mushroom with enhanced anti-cancer immunity, the present invention was conducted to investigate the effect of nonspecific immune response and macrophage activation.

[Comparative Example 1]

I have not processed anything.

[Comparative Example 2]

Comparative Example 1 (LPS) is a positive control treated with lipopolysaccharide (LPS).

[Example 1]

Example 1 (GW) is a hot-water extract of Leaf mushroom prepared by the method of the first step (S100).

[Example 2]

Example 2 (GE) was prepared by the method of the first step (S100), except that ethanol was used instead of the distilled water.

[Example 3]

Example 3 (GF) is a fermented mushroom fermentation broth prepared by the method of the first step (S100) to the fourth step (S400), wherein 1% of the lactic acid bacteria is inoculated.

[Example 4]

Example 4 (GG) is a fermented mushroom fermentation broth prepared by the method of the first step (S100) to the fourth step (S400), in which the lactic acid bacterium was inoculated at 2%.

[Example 5]

Example 5 (GHF) was prepared by the method of the first step (S100) to the fourth step (S400), except that 1 part by weight of the culture medium of Bacillus subtilis was added to Aspergillus oryxae spore suspension in an amount of 1 part by weight.

[Example 6]

Example 6 (GCF) was prepared by the method of the first step (S100) to the fourth step (S400), except that 1 part by weight of the natural complex compound was further mixed with 1 part by weight of the hot water extract of Leaf mushroom to be.

[Example 7]

Example 7 (GCF-1) was prepared by the first step (S100) to the fourth step (S400), except that 0.2 part by weight of yeast, 0.1 part by weight of aloe, 0.1 part by weight of a mixture of the mushroom extract.

A. Measurement of phagocytosis activity in macrophages

Macrophages associated with congenital immune responses are distributed in almost all tissues of the body, and have phagocytosis to infectious pathogens such as germs and viruses, and have the ability to remove bacteria, viruses and human unnecessary elements.

In the immune response, macrophages are activated by a variety of stimuli. Activation is initiated by stimulation of the antigen or by stimulation of some of the molecules derived from the pathogenic microorganism into the receptors of macrophages. After that, macrophages are activated T cells or It is activated more strongly by cytokines secreted by other immune system cells.

In activated macrophages, phagocytosis can be more effectively removed than in the period of resting, because macrophage killing activity, inflammatory mediator secretion, and T cell activation are greatly increased, and the activated macrophages are various Cytotoxic proteins are secreted to remove various types of antigenic targets such as virus-infected cells, tumor cells, and intracellular infected bacteria.

Macrophages can digest and digest all the microorganisms and insoluble particles that are exogenous antigens, as well as endogenous substances such as damaged or dead human cells, cell debris, and activated blood coagulation factors.

In order to measure the activity of macrophages, the macrophage cell line RAW264.7 was treated with 50 μg / ml of each sample and then cultured with FITC-labeled E. coli particles. Then, the phagocytic activity of macrophages was measured The amount of FITC-E. coli particles captured in the medium was measured using fluorescence.

As shown in Fig. 3, macrophage activity of macrophages for Comparative Examples 1 to 2 and Examples 1 to 7 was examined. When the LPS of Comparative Example 2 was used as a positive control, it was confirmed that significant macrophage effects were increased in all of the test groups of Examples 1 to 7 (FIG. 3).

In particular, Example 4 (GG) exhibited the highest macrophage morphological activity among the above-mentioned mushroom extracts, followed by Example 3 (GF), Example 2 (GE), and Example 1 (GW). In addition, Example 5 (GHF) showed the highest macrophage morphological activity, followed by Example 6 (GCF) and Example 7 (GCF-1).

Therefore, the fermented mushroom fermented by the first step (S100) to the fourth step (S400) was found to have the highest macrophage morphological activity, and the anticancer activity was very high.

N. Measurement of macrophage activation and related cytokine expression

Cytokine is a protein secreted by cells involved in innate immunity and adaptive immunity. It modulates various functions of these cells, stimulates the growth and differentiation of lymphocytes in the activation phase of adaptive immune response, and activates innate immunity and adaptive immunity Activates several kinds of working cells to remove microorganisms and antigens.

Immunity against cancer cells in vivo is enhanced through mechanisms that act on macrophages, which are important cells of the innate immune system, and produce cytokines such as TNF-α (tumor necrosis factor-α) .

In order to investigate the gene expression of various cytokines related to the activation of macrophages, RAW264.7 cells, which are macrophages, were treated with 50 μg / ml of each sample to collect total RNA from the cells, Expression of TNF-α, IL-6 and IL-1β, which are representative cytokines involved in the activation of phagocytes, was measured using real-time PCR.

4 (A), IL-6 (FIG. 4B), TNF (FIG. 4A), and the like were examined for the expression ability of various cytokines in Comparative Examples 1 to 2 and Examples 1 to 7, -α (FIG. 4C) was significantly increased in all test groups.

C. Natural kill (NK) cell activity measurement

Natural killer (NK) cells, unlike T or B cells, do not have antigen - specific receptors and thus are classified as involved in the innate immune response. Natural killer (NK) cells have granules and are large in size and exist in small numbers in the human body. They have a cytotoxic effect that circulates the whole body through the blood and removes cells showing abnormalities such as tumor cells and virus-infected cells .

Natural killer (NK) cells have natural killer (NK) cell receptors that can differentiate abnormalities of other cells, recognize abnormal surface antigens expressed in tumors and virus-infected cells, Reduced can also be perceived. Some tumor cells and cells infected with a specific virus express an antigen capable of antibody deficiency on the surface to induce an immune response, in which multiple antibodies bind to the surface of the antigen-expressing cells.

Since natural killer (NK) cells have a receptor (CD16) that recognizes a specific site of an antibody molecule on the cell membrane, it binds to the antibody and subsequently destroys the target cell.

Therefore, natural killer (NK) cells and their activities were investigated for extracts of Leaf mushroom and their compositions. The spleen was excised from the mouse, and the cell suspension was centrifuged at 300 xg for 5 minutes, and red blood cells were removed from the blood for red blood cells to separate the spleen cells. The YAC-1 cells were treated with 50 [mu] g / ml of each sample at a concentration of 50 [mu] g / ml for 30 minutes, and the fluorescence stained with Calcein-AM was adjusted so that the ratio of splenocytes: YAC- 1 cells were added and cultured for 4 hours.

As shown in FIG. 5, the activity of natural killer (NK) cells was measured for Comparative Examples 1 to 2 and Examples 1 to 7, and the activity of NK cells was significantly increased in all the sample groups Respectively.

Thus, it can be seen from the above examples that the present invention can produce an immunomodulating composition including a mushroom which not only increases the anticancer activity but also increases the antimicrobial activity.

As described above, it is to be understood that the technical structure of the present invention can be embodied in other specific forms without departing from the spirit and essential characteristics of the present invention.

Therefore, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than the foregoing description, All changes or modifications that come within the scope of the equivalent concept are to be construed as being included within the scope of the present invention.

S100. A first step of preparing a hot water extract of Leaf mushroom and adjusting the solid content of the hot water extract of Leaf mushroom to 20 Brix;
S200. Lactobacillus plantarum (L. plantarum) and L. mesenteroides in the MRS medium, A second step of pre-culturing the lactic acid bacteria at 30 占 폚 for 1 day;
S300. A third step of sterilizing the 20 Brix mushroom hot water extract;
S400. Lactobacillus plantarum (L. plantarum) and L. menesenteroides (pre-cultured) were added to the sterilized hot mushroom hot water extract, A fourth step of inoculating the culture broth of lactic acid bacteria at a concentration of 2 w / w%, culturing the suspension at 30 ° C for 3 days, and preparing a fermented broth mushroom;
S500. The fermented broccoli fermentation broth is filtered, and the same amount of ethanol is added thereto, followed by low-temperature precipitation for 24 hours, followed by centrifugation to produce a fermented extract of Lactobacillus acidophilus.
S101. 1-1) mixing 14 parts by weight of distilled water with 1 part by weight of mushroom and stirring at 95 ° C for 12 hours to prepare a mushroom extract;
S102. A step (1-2) of filtering the leaf mushroom extract and concentrating it under reduced pressure to 40 Brix;
S103. Step 1-3 of freeze-drying the decompressed concentrated mushroom extract

Claims (6)

A first step of preparing a hot water extract of Leaf mushroom and adjusting the solid content of the hot water extract of Leaf mushroom to 20 Brix;
L. plantarum and L. menesenteroides in the MRS (de Man, Rogosa and Sharpe) A second step of pre-culturing the lactic acid bacteria at 30 占 폚 for 1 day;
A third step of sterilizing the 20 Brix mushroom hot water extract;
Lactobacillus plantarum (L. plantarum) and L. menesenteroides (pre-cultured) were added to the sterilized hot mushroom hot water extract, A fourth step of inoculating the culture broth of lactic acid bacteria at a concentration of 2 w / w%, culturing the suspension at 30 ° C for 3 days, and preparing a fermented broth mushroom;
The fermented broccoli fermentation broth is filtered, and ethanol is added thereto, followed by low-temperature precipitation at -20 to -70 ° C for 24 hours, followed by centrifugation to produce a fermented extract of Lactobacillus acidophilus Lactobacillus. For preparing immunostimulating composition The method according to claim 1,
The above-mentioned hot-water extract of mushroom
1-1) mixing 14 parts by weight of distilled water with 1 part by weight of mushroom and stirring at 95 ° C for 12 hours to prepare a mushroom extract;
A step (1-2) of filtering the leaf mushroom extract and concentrating it under reduced pressure to 40 Brix;
And a third step of lyophilizing the reduced-pressure concentrated concentrated mushroom extract to prepare a composition for enhancing immunity using the mushroom The method according to claim 1,
1 part by weight of the natural complex was mixed with 1 part by weight of the above-mentioned hot water extract of Leaf mushroom,
Wherein the natural compound is prepared by mixing 1 part by weight of ginseng, 1 part by weight of ginseng, 1 part by weight of licorice, 1 part by weight of licorice and 1 part by weight of viscous oil, followed by hot water extraction to prepare a composition for immunity enhancement using leaf mushrooms The method according to claim 1,
To 1 part by weight of the hot water extract of Leaf mushroom, 0.2 part by weight of yeast, 0.1 part by weight of aloe, and 0.1 part by weight of garlic were further mixed to prepare a composition for improving immunity The method according to claim 1,
The sterilization of the 20Brix leaf mushroom hot-
1 part by weight of an Aspergillus oryxae spore suspension was mixed with 1 part by weight of a culture medium of Bacillus subtilis culture,
A method for manufacturing a composition for enhancing immunity using a mushroom prepared by spraying the 20Brix mushroom hot water extract A miso for enhancing immunity using a leaf mushroom produced by the method according to any one of claims 1 to 5

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