KR101467903B1 - The preparing method of immune improving agents - Google Patents

Abstract

The present invention relates to an immunologically active substance produced by fermentation and bioconversion of mycelia of mycelia of black rice. The fermentation broth of black rice or black rice is cultivated and fermented into a liquid black rice medium. The mycelia of mycelia (mushroom, Mushroom, thorny mushroom, and snowy mushroom), and a method for producing a bioconversion product that greatly improves immunity through a bioconversion process from a culture produced by a culturing process, The present invention relates to a composition characterized by a macromolecular polysaccharide as an active ingredient having potency.

Description

[0001] The present invention relates to an immune enhancer produced through fermentation and bioconversion of Mycorrhizae mycelium of black rice,

The present invention relates to a method for producing an immunostimulant through the fermentation of mycelium of Mycelia of Mycelia and a bioconversion process, and to an immunostimulant obtained by the method.

Immunity is largely divided into innate immunity (born immunity) and acquired immunity (acquired immunity) which is acquired by adaptation to life and the like. In 2011, three Nobel Prize winners were awarded the Nobel Prize in medicine for discovering key principles of how the body's immune system works against bacteria, viruses and fungi.

Dr. Bruce A. Beutler and Dr. Jules A. Hoffmann have found that the innate immune response occurs at an early stage. According to these studies, foreign invaders such as pathogenic microorganisms enter our body and bind to specific receptors on the cell surface of immune cells responsible for innate immunity. At this time, our body finds external antigens . As a result, an inflammatory reaction occurs in the cells and tissues, and the body feels heat or body fatigue. It informs the invasion of the enemy. This early immune response occurs immediately and rapidly irrespective of the identity of the external antigen. Congenital immunity responds nonspecifically to antigen and has no special memory effect. Congenital immune cells include macrophages, polymorpho-nuclear leucocytes, and NK cells, which can kill infected cells. In fact, most infections are defended by this congenital immunity.

When the initial immune reaction only feels a fight against the external intruder, the dendritic cells that perform the bridging function between the innate immune system and the acquired immune system move in the body's immune system. Dr. Ralph M. Steinmam has identified the role of dendritic cells. Dendritic cells are mainly activated by activating 'T cells', which are the main focus group of the immune defense system. Thereafter, the immune system is fully mobilized to inject antibodies and killer cells, which remove infected cells from outside intruders. This leads to an immune response to eliminate the intruder in earnest, and when the immune system of our body wins, the intruder is gradually removed. Acquired immunity can be remembered for the first-time-invasive antigen as an acquired immune system, and it can be specifically reacted when invading again, thereby effectively removing the external antigen, thereby reinforcing the innate immune system. Dendritic cells are also used to kill cancer cells, and new methods of cancer treatment, such as the introduction of dendritic cells from prostate cancers, have also been introduced to treat cancer.

Acquired immunity is conveniently described as humoral immunity and cell-mediated immunity. Humoral immunity is the secretion of antibodies after B lymphocyte recognizes the antigen, and this antibody shows the ability to remove bacteria or viruses that invade our body. The antibody is present in body fluids and is composed of a glycoprotein called immunoglobulin (Ig). These include IgM, IgG, IgA, IgD, and IgE, each of which performs a unique function, and some functions are duplicated. The IgA antibody is characterized by its transfer to the fetus through the placenta. This type of immunity is called maternal immunity, and it is not well infected for several months after birth. Cell-mediated immunity is a function of the thymus-derived T cells that recognize the antigen and secrete lymphokines or kill directly infected cells. Secreted lymphocytes also activate phagocytosis by activating macrophages. Cellular immunity functions mainly to remove cells infected by bacteria or bacteria that can grow in viral or intracellular cells.

A vaccine is an artificial antigen material that is artificially obtained to acquire acquired immunity. A vaccine, attenuated pathogen, toxin or its toxin is representative vaccine. Immunization vaccine can be obtained by vaccination as an immunogen. The same immunity is called artificial immunity.

Many immune enhancers have been developed and used to enhance the immune response as described above. In general, known immune enhancers are mushroom extract, plant extract, aquatic extract and microbial extract. Mushroom extracts are extracted from mushroom fruiting body or extracted from mycelium produced by culturing mycelium of mushroom, and it is the only product group approved as medicine. It has been developed as a medicine in Japan, and is represented by Krestin (PSK), a polysaccharide extracted from mycelia of cloud mushroom, and Lysinan, a polysaccharide extracted from shiitake fruiting body, and Shizopyran, an extracellular polysaccharide recovered from mycelium culture , Which was developed as a medicinal product in Korea, is a representative product of polysaccharide extracted from mycelium of mushroom. Plant extracts include ginseng and red ginseng products, as well as mixed extracts of Allium gigantis, Angelica gigantosa, and albicans extracts and aloe extracts. Chitosan / chitooligosaccharide, a polysaccharide extracted from crustacean shells such as shrimp and crab, and fucoidan, a polysaccharide extracted pure from brown algae such as seaweed, are examples of aquatic products. Chitosan / It has been difficult to control the quality of fucoidan because the function of enhancing immunity is removed from the function of the functional food because the enhancing function is not recognized. Fucoidan has been purified and sold at a high purity, but has not yet been recognized for its immunostimulatory function. As a microbial extract, chlorella extract together with germanium-yeast has been recognized as a functional food for immunity enhancement. Although the above-mentioned immunity enhancer proved efficacy and functionality, it has been approved and approved as a pharmaceutical and health functional food, but its effect is not high, and therefore, sales are not actively achieved except for red ginseng products.

In the case of polysaccharide products extracted from medicinal mushroom mycelium, the immune activity is already well known, such as being sold as a medicament. However, since the immune activity is low enough to show the effect in our body, it is urgent to develop a product capable of realizing immune enhancement effect upon ingestion It is true. Oryza sativa L. is an abundance of anthocyanins and polyphenols. Black rice contains nutrients such as starch, protein, and fatty acid, and the anthocyanins and In addition to polyphenolic compounds, it contains fiber, minerals essential for human body, vitamin B group, vitamin C, niacin, carotene and so on. I eat a lot. According to the main rice gangmok black rice, stomach and strengthen the function of the kidneys to strengthen the spleen and liver, brighten the eyes and let the blood to go well, eat every day to improve the overall control of the body and strengthen the immune function to improve disease Prevention is good for the treatment of anemia of the elderly and pregnant women, and it is effective for prevention of aging of skin and skin beauty of women and makes hair black. Antioxidative, anti - cancer, anti - allergic, anti - inflammatory and anti - hyperlipidemic effects of anthocyanin pigments are known to be medicinal effects of black rice. However, the anthocyanin compounds of black rice have been actively developed except for the processed products which are extracted at low temperature due to the instability of anthocyanin compounds, even though the anthocyanin compounds of black rice are destroyed and the black rice is expected to have good efficacy when subjected to heat treatment or fermentation I can not.

Accordingly, it is an object of the present invention to provide a method for producing an immunostimulating agent having excellent immunological activity as compared with various natural immune enhancers and a method for preparing the same.

The present inventors have found that, in the development of an immunologically active natural fermentation material, (1) it induces the production of a material having immunoactivity which is remarkably improved by the bioconversion of a natural plant ingredient during the fermentation process, As a result of efforts to develop an immunologically active material exhibiting a dramatically improved immunological activity by maximizing the immunological activity by improving the immunological activity of the present invention, the inventors' unique technology of cell wall bioconversion (Bioconversion) technology, it has been confirmed that the immune activity of medicinal mushroom mycelium can be improved remarkably even if the anthocyanin component of black rice is destroyed.

Therefore, the inventors of the present invention have found that a culture of medicinal and edible bacterium mycelium (mushroom, chaga mushroom, shiitake mushroom, mushroom, mushroom, snow mushroom, skirt mushroom, etc.) produced by culturing black rice as a culture and fermentation medium The insoluble complex consisting of a complex polysaccharide of the β-Glycan structure, which is a cell wall structure of the mycelial mushroom mycelium, which is a cultured product produced through cultivation and fermentation, (Bioconversion Product), which has a dramatically improved immune activity, was obtained as a water soluble immunoconjugate polysaccharide of the present invention.

In addition to activating macrophages, which are cells responsible for innate immunity, the bioconversion products induce maturation of dendritic cells that play a role of bridging between innate immunity and acquired immunity, thereby activating dendritic cells, Ability and dendritic cell activation ability were remarkably improved more than 100 times as compared with the simple extract of mycelium. Therefore, the above-described black rice / basidiomycete (fermentation) / biotransformation products are very useful as an immunity enhancer.

Fig. 1 shows the cell mass
FIG. 2 shows the results of HPLC analysis of polysaccharide fraction powder of various saccharides and black rice / mushroom mycelium (fermentation) / biotransformation product, wherein FIG. 2a shows glucose, FIG. 2b shows Mesima capsule 550, It is the result of HPLC analysis of the polysaccharide fraction powder of the mushroom mycelium (fermentation) / biotransformation product.
Fig. 3 shows the results of macrophage activation of black rice / mushroom mycelium (fermentation) / biotransformation products
Fig. 4 shows the result of macrophage activation of black rice / chaga mushroom mycelium (fermentation) / biotransformation product
Fig. 5 shows the result of macrophage activation of black rice / shiitake mycelium (fermentation) / biotransformation product
Fig. 6 shows the results of macrophage activation of the black rice / thistle mushroom mycelium (fermentation) / biotransformation product
Fig. 7 shows the result of macrophage activation of mycelium (fermentation) / biotransformation product of black rice / snow blossom mushroom
Figure 8a shows the results of macrophage activation on the polysaccharide fraction powder of black rice / situ mushroom mycelium (fermentation) / biotransformation product
FIG. 8b shows the result of macrophage activation of the black rice / thistle mushroom mycelium (fermentation) / biotransformation product
Figure 9 shows the ratio of mononuclear cells in human peripheral blood (PBMC) to the purity of mononuclear cells
Figure 10 shows the phenotypic analysis of dendritic cells differentiated and matured from monocytes
Fig. 11 shows the effect of treatment concentration of black rice / thistle mushroom mycelium (fermentation) / biotransformation product powder on the phenotype of mature dendritic cells (muture DC) derived from human blood mononuclear cells
Fig. 12 is a graph comparing the macrophage activation efficacy of polysaccharide fraction powder of black rice / situation mushroom mycelium (fermentation) / biotransformation product and conventional immunostimulating substance
13 to 15 show the results of anticancer efficacy in a multiple-cancer animal model transplanted with cancer cells
Figure 16 shows the results of an improved mortality rate for bacterial infections in an animal model of Salmonella infection

The configuration of the present invention in the present invention will be described in detail as follows.

The method for producing an immunity enhancer through mycelium fermentation and biotransformation of bacillus meats of the present invention

(a) In order to facilitate the utilization of nutritional components such as starch and proteins such as bacillus mycelia, a liquid medium containing black rice powder obtained by pulverization of black rice or black rice produced from rice black rice is added with starch degrading enzyme and protein degradation Adding an enzyme to the reaction mixture to cause a black rice to be cultured and fermented;

(b) inoculating a mycelium of mycelia into a liquid black broth culture obtained by cultivating a black rice of the step (a) and culturing it in a fermentation medium, culturing the mycelium of mycelia with bioconversion of black rice;

(c) adding a cellulolytic enzyme to the cultured product produced in step (b) to conduct a secondary bioconversion process, and then adding the cellulolytic enzyme to the cellulase;

(d) For culturing the culture produced in step (b), a microorganism producing a fibrinolytic enzyme after hot water extraction and a culture solution containing a fibrinolytic enzyme produced by the microorganism are added ;

(e) heating the final bioconversion product produced in the step (c) or (d) to inactivate and sterilize the enzyme, followed by lyophilization to obtain a final product of the bioconversion product;

(f) dissolving the biotransformation product powder in water to remove insoluble residues, lyophilizing the supernatant to obtain a water-soluble powder, or adding ethanol to the supernatant to induce precipitation, thereby obtaining a polymeric polysaccharide having immunological activity .

The black rice powder obtained by pulverizing the black rice in the step (a) or the black rice produced from the black rice may be a medium containing 1 to 10% (w / v). To the medium, 0.1 to 5% (w / v) of soybean powder and 0.1 to 0.5% (w / v) of KH2PO4 may be further added. Examples of the starch degrading enzyme added to the medium for culture and fermentation medium include an amylase and an enzyme such as a protease as a protease (or a complex enzyme) in a suitable amount Optimal amount).

In step (b), the mycelium of mycelia may be selected from mycelia of mushroom, edible mushroom, mushroom, shiitake mushroom, mushroom mushroom, mushroom mushroom, These mycelia of mycelia belong to the Korean Microorganism Conservation Center (KCCM), Korea Biotechnology Research Institute (KCTC), Korea Cell Line Bank (KCLB), Korea Agricultural Microbiological Resource Center (KACC) ATCC), and the like. The mycelium of mycelia may be inoculated in the culture fermentation medium of step (a), and cultured for 1 to 10 days at 25 to 40 ° C and pH 4.5 to 7. At this time, in the mycelial culture of basidiomycetes, it is possible to carry out a batch culture in which the culture of the basidiomycete culture is continued for a certain period of time until the concentration of the residual carbon source is depleted to a certain concentration or less, and the concentration of the residual carbon source is depleted to a certain concentration And the concentrated liquid black rice rice medium is added at a point of time when the concentration of the residual carbon source is exhausted to a certain concentration or less. have.

The cell wall component and the cell wall component of the medicinal mushroom mycelium produced in the step (b) are composed of an insoluble complex composed of polysaccharides such as cellulose, hemicellulose, pectin, glucan, chitin and mannan. In step (c) Enzymatic treatment for the tea bioconversion process is carried out by adding various amounts of cellulose decomposing enzyme enzymes such as cellulase, hemicellulase, pectinase and glucanase in an appropriate amount And the like). As the above-mentioned fibrinolytic enzyme, commercially available enzymes can be used, but not limited thereto, but the fibrinolytic enzyme Viscozyme (a compound derived from Aspergillus), cellulase (derived from Aspergillus niger), Filtrase (A complex derived from Tricoderma reesei), Rapidase (a complex derived from Aspergillus niger), and Sumizyme (a complex containing Aspergillus niger derived pectinase).

In step (d), the microorganism producing the fibrinolytic enzyme may be microorganisms such as lactic acid bacteria, Bacillus bacteria, yeast bacteria, Aspergillus strains, and Trichoderma regies which are known to produce fibrinolytic enzymes. In addition, a microbial culture solution containing a fibrotic enzyme produced by the microorganism may be added.

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

The biotransformation product powder obtained in the step (f) is dissolved in water at a concentration of 10% (w / v) to remove insoluble residues, and the supernatant is lyophilized to obtain a water-soluble powder, or 2 to 10 times of ethanol To induce precipitation, from which a macromolecular polysaccharide having immunological activity can be obtained.

The immunopotentiating substance of the polymeric polysaccharide of the present invention activates macrophages which are immune cells, and the concentration of the EC100 is not more than 1 μg / ml in the measurement of macrophage activation as well as the EC50 concentration (FIGS. ), And also affects the maturation of dendritic cells, which are immune cells. In the measurement of dendritic cell activation in which immature dendritic cells are matured into mature dendritic cells, the immune activity effect is dramatically improved to an effective concentration of EC50 of 1 μg / ml or less (Fig. 11).

In addition, the macrophage activation ability of the immunostimulatory substance of the present invention and the conventional immunostimulating product was compared. As a result, it was confirmed that the immunostimulatory substance of the present invention is remarkably superior in the effect of promoting macrophage activation as compared with conventional immunostimulating products (FIG. 12).

In addition, in the cancer cell transplantation rat (ICR mouse) transplanted with the sarcoma 180 into the abdominal cavity of the rat, the immunosuppressive substance of the present invention was intraperitoneally administered to the abdomen, The average survival days were 10.7 days in the test period, whereas in the group to which the immunostimulatory substance of the present invention was administered, the average survival days were 28.5 days, and only one out of 10 animals died within the test period, .

In addition, the immunologically active substance of the present invention was orally and intraperitoneally administered for 14 days, and the bacterial infection was observed in a rat (Balb / c mice) infected with the pathogenic bacterium Salmonella enterica serovar Typhimurium (ATCC # 14028) In the experiment observing the effect of improving the mortality rate, Salmonella-infected control group died at 7 days after the infection, whereas the group to which the immunopotentiating substance of the present invention was orally administered survived to 23 days, Survival and mortality improvement.

Therefore, the physiologically active substance according to the present invention can be provided as a composition showing immunity enhancement and regulating effect with an immunosuppressive substance.

In addition, the physiologically active substance according to the present invention may be provided as a composition for the prevention or treatment of diseases and infectious diseases caused by immunosuppression as immunologically active substances.

As a result, it is a main object of the present invention to provide a pharmaceutical, functional food and feed additive composition, which comprises the immunologically active substance as an active ingredient.

Another object of the present invention is to provide a vaccine adjuvant composition containing the immunologically active substance as an active ingredient.

Still another object of the present invention is to provide a cosmetic composition containing the above immunoactive substance.

≪ Example 1 >

Cultivation of black rice for fermentation of fermentation broth and mycelia culture

In order to facilitate utilization of nutrients such as starch and protein in black rice or black rice, 2% (w / v) [medium 1] or black rice in black rice powder obtained by pulverization of black rice powder or black rice Amylase and proteolytic degradation were carried out with starch hydrolyzate in [medium 2] liquid medium supplemented with 2% (w / v) steel, 0.1-0.5% (w / v) KH2PO4 and 1% Protease was treated with the enzyme, reacted at 60 ° C for 1 hour, and sterilized at a high temperature for 30 minutes to carry out culture fermentation of black rice powder or black rice.

The cultured fermentation broth was inoculated with 10% (v / v) of mycelia of mushroom mycelia, mushroom mycelium, mushroom mycelia, mushroom mycelium, mycelium of mycelia, At the time when the concentration of the residual carbon source was exhausted to a certain concentration or less at ~ 30 ° C, the amount of the produced bacteria was compared by culturing for 7-10 days in a liquid phase process in which concentrated liquid black rice medium was added. The results are shown in Fig.

≪ Example 2 >

Enzymes and Enzymes for Bioconversion Process of Enzyme Treatment in Mycelial Culture of Basidiomycetes

Cell wall components of medicinal mushroom mycelium and black rice such as bacillus species mushroom, chaga mushroom, shiitake mushroom, mushroom mushroom, mushroom mushroom, snow mushroom mushroom and skirt mushroom have cellulosic components such as cellulosic, hemicellulosic, pectin, glucan, chitin, It is composed of complex polysaccharides. The enzymatic treatment of the secondary biotransformation step of the culture of Example 1 produced by culturing mycelium mycelium in a liquid black rice mead culture obtained by cultivating black rice or black rice in a fermentation broth of a black rice or black rice was carried out in the same manner as in Example 1, Various kinds of enzymes such as cellulase, hemicellulase, pectinase and glucanase were added in an appropriate amount (the optimum amount shown in the product manual of each enzyme preparation). Examples of enzymes include commercially available enzymes, such as the cellulase (derived from Aspergillus niger), the cellulase (Viscozyme: a complex derived from Aspergillus), the fibrinolytic enzyme (Filtrase: a complex derived from Tricoderma reesei) The complexes containing Rapidase (a composition derived from Aspergillus niger) and Sumizyme (a complex containing Aspergillus niger derived pectinase) were used at the recommended concentrations given in the product manual of each enzyme preparation.

≪ Example 3 >

Fermentation for Bioconversion Process of Microbial Fermentation in Basidiomycetes 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 fibrinolytic enzymes for carrying out a biotransformation process through fibrinolytic enzymes, namely lactic acid bacteria, bacillus bacteria , Yeast, Aspergillus, and Trichoderma rejay were identified and used. The secondary bioconversion process of microbial fermentation was performed by subjecting the culture of Example 1 or Example 2 to hot water extraction and then adding 10% (v / v) of the culture medium in which the fibroblast-producing microorganism was cultured.

<Example 4>

Bioconversion product recovery process

The bioconversion product produced by the bioconversion process of Example 3 was subjected to enzyme inactivation and sterilization at 90 ° C for 1 hour and then lyophilized and powdered. The biotransformation product powder having the above immunoactivating activity was dissolved in 10 times of water and centrifuged at 10,000 rpm for 30 minutes to remove insoluble residues. The supernatant was lyophilized to obtain a water-soluble powder, or 5-fold ethanol was added to the supernatant Followed by induction of precipitation at 4 DEG C for 24 hours or longer, and the precipitate was lyophilized to obtain a powder of a polymeric polysaccharide fraction having immunological activity.

&Lt; Example 5 >

Analysis of the immunoactive substance prepared according to the method of the present invention

The polysaccharide fraction of the black rice / situ mushroom mycelium (fermentation) / biotransformation product prepared in Example 4 was analyzed by HPLC using a gel chromatography column (UltrahydroTM 1000, Waters, USA). In order to confirm the molecular weight of the polysaccharide, retention time of the polysaccharide was determined by using starch (starch), which is one of commonly known polysaccharides. As the monosaccharide, the retention time of the monosaccharide Respectively. In addition, by purchasing the [new] drug [Mesima Capsule 550], a special medicine licensed by Korea Food and Drug Administration, by the effect of digestive cancer, liver cancer, and immunosuppression by combination with chemotherapy after surgery, Respectively. [Mesima Capsule 550] is currently being sold as an immunosuppressive agent to aid in cytotoxic chemotherapy. An acid polysaccharide (153 kD) having an anticancer immune activity including a mycelium extract (Phellinus linteus polysaccharide) . As a result of the analysis, the immunostimulatory substance of the polysaccharide fraction powder of black rice / mushroom mycelium (fermentation) / biotransformation product prepared in Example 4 was observed at a time similar to the retention time of the starch and the polysaccharide peak of [Mesima Capsule 550] (Fig. 2).

The constituent monosaccharides of the polymeric polysaccharide fractions were analyzed. The experimental method is as follows. For the extraction and pretreatment of this sample, the neutral sugars were hydrolyzed at 100 ° C for 4 hours using trifluoroacetic acid (2M) at a concentration of 10 mg / ml. ICS-5000 with electrochemical detection (Dionex, Sunnyvale, Calif., USA) was used as the analysis equipment. The column was CarboPac PA1 (4) × 250 mm, Dionex) with a CarboPac PA1 cartridge (4 × 50 mm). The analytical solvent was analyzed by isocratic composition using sodium hydroxide (NaOH, 16 mM), and the flow rate was set at 1 ml / min and 10 μl of sample was injected. As a result of analyzing the constituent monosaccharides of the polymeric polysaccharide fraction, the monosaccharide of the polysaccharide fraction was mainly composed of glucose and galactose, and mannose, rhamnose, arabinose, , Fructose, xylose, and the like (FIG. 2).

&Lt; Example 6 >

In vitro activity assay of the immunoactive substance prepared according to the method of the present invention

[6-1] Measurement of macrophage activation

The macrophage activation ability of each sample prepared in Example 4 was measured. The samples used for the macrophage activation assay were black rice / mushroom mycelium (fermentation) / biotransformation products (experimental group A), black rice mushroom mycelium (fermentation) / biotransformation products (experimental group B), black rice mushroom / (Fermentation) / biotransformation products (experimental group C), black rice / thirsty mushroom mycelium (fermentation) / biotransformation products (experimental group D), black rice / snowflake mushroom mycelium (fermentation) Respectively.

Macrophage activation of the samples was measured by the following method using mouse peritoneal macrophages. 2.5 ml of 5% thioglycollate medium was injected into the abdominal cavity of ICR mice (8 weeks old, male, purchased from BioLink Co., Ltd.), and the peritoneal macrophages induced in 72-96 hours were recovered and resuspended in RPMI 1640 And washed 2 to 3 times. The number of macrophages obtained above was adjusted to 1 * 10 &lt; 6 &gt; cells / ml, and 200 [mu] l was dispensed into 96 well plates. The cells were cultured in a 5% CO 2 incubator at 37 ° C for 2 hours to form macrophage monolayers. The concentrations of the samples (experimental groups A to E) were 1 μg / ml, 10 μg / ml and 100 μg / ml And further cultured for 24 hours. The negative control group was treated with physiological saline and the positive control group was treated with LPS. It was then washed with RPMI 1640 and Triton X-100 was added to dissolve the cell membrane. The liberated lysosomal phosphatase was reacted with ρ-NPP, a substrate, and absorbance was measured at 405 nm using a microplate reader. At this time, the absorbance of wells containing no cells was also measured and used to calibrate the data of the experimental group.

The results are shown in Figs. 3 to 7. Fig.

Experimental results showed that all the experimental groups were more effective in stimulating macrophage activation than the negative control using saline. In addition, the effect of stimulating macrophage activation in all experimental groups was similar to that of the positive control group treated with LPS at a concentration of 1 μg / ml, which is the lowest in the sample treatment concentration, and it was confirmed that the macrophage activation effect was excellent. As a result, the concentration of EC50, which is the effective concentration of physiological activity, as well as the minimum concentration of EC100 of 1 μg / ㎖ or less were enough to be defined for the bioconversion product polysaccharide fraction powder sample of each experimental group.

In order to set the lowest concentrations of EC50 and EC100, which are effective concentrations of all the black rice / basidiomycetes (fermentation) / biotransformation products prepared in Example 4 for the polysaccharide fraction powder, all the polysaccharide fraction powders (Macrophage cell line, RAW 264.7), macrophage activation was measured by the following method.

The macrophages were cultured at a concentration of 1 × 10 4 cells / ml in each well of a 96-well plate. After culturing in an incubator at 37 ° C, 5% CO 2 for 24 hours, the samples were treated by concentration. The negative control group was treated with physiological saline and the positive control group was treated with LPS. The 96-well plate treated with the sample is incubated for 18 hours to measure nitric oxide produced from macrophages. After completion of the incubation, 100 μl of the culture solution of each well was dispensed into a 96-well plate, 100 μl of a Griess reagent was dispensed, and the absorbance was measured at 540 nm using a microplate reader.

The lowest concentration of effective concentration EC50 and EC100 for black rice / bacillus mycelium (fermentation) / biotransformation products for polysaccharide fraction powder of black rice / situation mushroom mycelium (fermentation) / biotransformation product is below 1 μg / And the activation effect was excellent (FIG. 8A). In addition, the same results were obtained for the results of all polysaccharide fraction of black rice / bacillus mycelium (fermentation) / biotransformation product.

[6-2] Dendritic cell activation assay

[6-2-1] Analysis of the effect of differentiation and maturation from monocytes to dendritic cells

The fermentation broth of Japanese black rice throat / fermentation product / biotransformation product powder in the sample prepared in Example 4 was selected as an experimental material and activation of macrophages was measured to determine the experimental concentration (FIG. 8B) In order to evaluate the effect on the activation, we analyzed the effect on the dendritic cells which play a bridge role between the congenital and acquired immune system. For the dendritic cell activation assay, human blood was supplied from the central blood circulation and the cells corresponding to each immune system were separated. The supernatant obtained by dissolving the black mushroom mycelium (fermentation) / biotransformation product powder in an aqueous solution was treated On the activation and proliferation of dendritic cells.

The same amount of PBS (phosphate buffered saline) was added to the peripheral blood supplied from the central blood circulation and mixed well. Then, the blood was diluted in a 50 mL container containing 15 mL of a density gradient medium for treating mononuclear cells (Ficoll-Paque plus; GE Healthcare) 35 mL of blood was added to maintain the Ficoll layer and the peripheral blood mononuclear cells were separated by centrifugation at 2,500 rpm for 20 minutes. Separated mononuclear cells were washed three times with PBS and monocytes were separated using CD14 antibody (BD Sciences) according to the manufacturer's instructions. Separated mononuclear cells were stained with a fluorescent antibody and analyzed by FACS (Fluorescence Activated Cell Sorter (BD Sciences) instrument) to confirm that the proportion of CD14 + CD83- 9).

6 days after culture of immature dendritic cells in culture medium supplemented with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) , And LPS were added to induce differentiation into mature dendritic cells (mutated DCs), and their phenotype was analyzed by an automatic cell analyzing and separating apparatus. As a result, the expression of CD80 and CD1a from mononuclear cells was differentiated into monocytes as immature dendritic cells (DCs) and increased expression of CD83 and CD86 induced immature DCs to mature into mature dendritic cells ) Was confirmed (FIG. 10). Based on the results of this experiment, we investigated the effect of black rice / thistle mushroom mycelium (fermentation) / biotransformation product powder on dendritic cell activation. In addition, CD80 is a co-stimulatory molecule of dendritic cells involved in the activation of T cells through the interaction of dendritic cells with T cells, while CD1a is an immune dendritic cell (immuture DC) It is well known as a related factor.

In order to investigate the effect of black rice / thistle mushroom mycelium (fermentation) / biotransformation product powder on dendritic cell activation through immature dendritic cell maturation, CD14 + CD83-mononuclear cells were treated with granulocyte macrophage colony stimulating factor and interleukin-4 (ImDC) in the presence of the same amount of the granulocyte macrophage-colony stimulating factor and the interleukin-4 (IL-4) on the fourth day of culture in the RPMI-1640 medium containing 37% (ImDC) was treated with LPS as a positive control. After 6 days of differentiation, LPS was treated as a positive control for immature dendritic cells (imDC), treated with concentration of black rice / thistle mushroom mycelium After inducing maturation into dendritic cells, the phenotype was analyzed by an automated cell analyzer (Fig. 11).

The results showed that maturation of immature dendritic cells to mature dendritic cells was observed when LPS was treated as a positive control. In the case of black rice / thrips mushroom mycelium (fermentation) / biotransformation product powder, It was confirmed that dendritic cells were matured into mature dendritic cells, and that the concentration required for the maturation of mussel-to-mushroom mycelium (fermentation) / biotransformation product powder was matched with that of macrophage activation (Fig. 8B, 11).

&Lt; Example 7 >

Comparison of macrophage activation of the immunostimulatory substance prepared according to the method of the present invention with the conventional immunostimulating product

The polysaccharide fraction powder of the black rice / mushroom mycelium (fermentation) / biotransformation product prepared according to the method of the present invention and the polysaccharide fraction of the biosynthetic product produced by the method of the present invention, AHCC produced from polysaccharide derived from shiitake mushroom, medicinal product produced from mycelia of mushroom, and fucoidan, a polysaccharide derived from brown algae, from the mycelia of mycelia, Were measured and compared in the same manner. The results are shown in Fig.

As a result, the activity of macrophage activation of the polysaccharide fraction powder of black rice / mushroom mycelium (fermentation) / biotransformation product prepared according to the method of the present invention was remarkably high.

&Lt; Example 8 >

In vivo activity analysis of the immunoactive substance prepared according to the method of the present invention

[7-1] Verification of anticancer efficacy in an animal model of multiple cancers implanted with cancer cells

The effect of prolonging the life span was examined in a multicarrier animal model in which cancer cells were transplanted into the black rice / pine mushroom mycelium (fermentation) / biotransformation product prepared in Example 4 above.

The effect of extending the lifetime of the sample was carried out in the following manner. Black rice / thistle mushroom mycelium (fermentation) / biotransformation products were dissolved in distilled water and administered intraperitoneally. Cells (Sarcoma-180) for culturing multiple cancer cells were purchased from Korean Cell Line Bank (Seoul, Korea). Sarcoma-180 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS), 100 units / mL penicillin and 100 μg / mL streptomycin in a CO2 incubator % CO2, / 95% air). The cells were washed with phosphate-buffered saline (PBS, pH 7.4) and the cells were removed. The cells were collected by centrifugation, and the medium was changed every 2 days.

Five-week-old male ICR mice without specific pathogen free were purchased from Orient Bio Inc. (Seongnam, Korea). After a week of quarantine and adaptation, healthy animals without weight loss were selected and used in the experiment. The animals were housed in a breeding environment set at a temperature of 23 ± 3 ° C, a relative humidity of 50 ± 10%, a ventilation frequency of 10-15 times / hour, a lighting time of 12 hours (08:00 - 20:00) Respectively. During the 1-week adaptation period, the experimental animals were fed free of drinking water with the solid feed for experimental animals (Cargill Agri Purina, Gunsan, Korea). After a week of adaptation period, healthy animals were selected and classified into three groups based on the nodule method.

In the experimental group, Sarcoma-180 cells (1 × 10 6 cells / mouse) were transplanted into the normal control group and abdominal cavity to induce multiple cancers. In the experimental group, Sarcoma-180 cells cells / mouse). After the transplantation, the experiment was conducted by dividing the black rice / thistle mushroom mycelium (fermentation) / biotransfer products into 10 mg / kg body weight / After 24 hours from the injection of the cancer cells, the test substance was dissolved in distilled water and 0.2 ml was administered intraperitoneally once a day for 14 days. The control group was treated with the same amount of distilled water for the same period, and the average life span was calculated by observation for 30 days after the final administration of the sample. The increase of life span (ILS) was calculated as follows.

{ILS = [(mean life span of test group - mean life span of control group) / mean life span of control group] x 100}

All animal studies in this study were conducted with the approval of the Animal Experimental Ethics Committee of Hallym University (Hallym 2011-86).

All analytical values for statistical analysis were expressed as mean ± SEM. Statistical analysis was performed using the SAS 9.1 program (SAS Institute, Cary, NC, USA). The significance of each test group was analyzed by analysis of various and Duncan's multiple range test at α = 0.05 level.

The results are shown in Figs. 13, 14 and 15.

In order to examine the effect of extending the life span, Sarcoma-180 cells were transplanted into the normal control and abdominal cavity to induce multiple cancers. In the test group, The experimental group was divided into the experimental group treated with 10 mg / kg body weight / day intraperitoneal product of mycelia (fermentation) / biotransformation product of black rice and throat. FIG. 13 shows the results of evaluating the effect of prolonging the life of the test substance by intraperitoneal administration once a day for 14 days, and observing the test substance for 30 days after the final administration of the test substance. And the site where cancer was induced is shown in Fig. The experimental group of black rice / thistle mushroom mycelium (fermentation) / biotransformation product was found to be effective in prolonging the life span of mice in the intraperitoneal administration group of 10 mg / kg body weight / day. The mean survival days of the control group were 10.7 days. The mean survival days of the experimental group treated with 10 mg / kg body weight / day of black rice / thistle mushroom mycelium (fermentation) / biotransformation products were 28.5 days Only one of 10 dogs died and the life extension effect was 166.4%, showing an excellent life extension effect (FIG. 13).

In FIG. 14, the Sarcoma-180 cells used in this experiment were confirmed to be multiple (abdominal cavity) cancer cells and injected into the abdominal cavity. In the positive control group, In the abdomen group, the abdomen seemed to be normal as in the normal control group, and the test substance was not administered until the dissection during the test substance administration period and the cancer mass was not found at the time of the dissection. It is judged to be an excellent effect of the test substance.

[7-2] Improvement of mortality rate for bacterial infection in Salmonella infected animal models

The morphological improvement effect of bacterial infection on the Salmonella-infected animal model against the black rice / mushroom mycelium (fermentation) / biotransformation product prepared in Example 4 was examined.

The mortality improvement effect of the bacterial infection in the Salmonella infected animal model for the above sample was tested in the following manner. The experimental animals were housed in Sprague-Dawley male rats at 6 weeks of age, from Samutako (Osan, Kyunggido, Korea) and were classified as' Guide for Care and Use of Laboratory Animals and the National Academy Press: Washington, DC, 1996). The breeding chamber was maintained at a temperature of 25 ± 2 ° C The experiment was carried out in accordance with the Animal Care Guidelines of the Disease Control Headquarters and conducted with the approval of Animal Experimental Ethics Committee of Ajou University.

Prior to the experiment, all experimental animals were fed with water and solid feed without restriction, and were administrated to the environment for one week. Salmonella was purchased from Salmonella enterica serovar Typhimurium (ATCC # 14028) from Korea Microorganism Conservation Center. To investigate the effect of Salmonella typhimurium infection on the abdominal cavity at a concentration of 1 × 10 5 cfu / ml, the control group and the sample of this experiment, The experimental group was divided into two groups.

In order to observe the mortality improvement effect of Salmonella infection, the samples of this experiment were applied to oral and intraperitoneal for 14 days at a concentration of 10 mg / kg body weight / day, , Salmonella typhimurium was infected into abdominal cavity at a dose of 1 × 10 5 cfu / ml at the level of lethal dose, and the infectious mortality was observed by continuously administering the black rice / mushroom mycelium (fermentation) Were evaluated.

The results are shown in Fig.

Effect of Salmonella Infection on Mortality Improvement As a result of the experiment, Salmonella typhimurium infection control group died 7 days after the microbial infection, while oral administration group of black rice / situ mushroom mycelium (fermentation) / biotransformation product survived to 23 days , And black rice / situation mushroom mycelium (fermentation) / biotransformation product of the peritoneal administration group showed excellent mortality improvement effect surviving to 27 days (FIG. 16).

Claims (17)

(a) a step of cultivating black rice into a liquid fermentation medium
(b) a step of inoculating and cultivating mycelium mycelium in a liquid black rice medium obtained by culturing the fermentation medium of step (a)
(c) producing a biotransformation product having improved immunological activity from a culture product produced by the culturing step of (b) through a biotransformation process.
The method according to claim 1, wherein the bioconversion product of step (c) is a polymeric polysaccharide having an immunological activity.
[7] The method according to claim 1, wherein the culture of fermentation broth of black rice in step (a)
Wherein the black rice obtained from one black rice powder or black rice is processed with starch hydrolyzate and proteolytic enzyme and sterilized to prepare a liquid black rice medium.
4. The method according to claim 3, wherein soybean powder is added to the black rice powder or black rice to make a liquid black rice medium.
[3] The method according to claim 1, wherein the basidiomycetes hyphae in step (b) are composed of hyphae of mycelia selected from the group consisting of a mushroom, a mushroom, a shiitake, a mushroom, a mushroom, Lt; / RTI &gt;
[2] The method according to claim 1, wherein the culturing step (b) includes an oil-in-water culture step in which a black rice medium is further added and cultured at the time when the concentration of the residual medium component is exhausted to a certain concentration or less, &Lt; / RTI &gt;
The method according to claim 1, wherein the biological conversion process of step (c) is a biotransformation process by hydrothermal extraction, followed by a biotransformation process by hot water extraction, and a microorganism fermentation process by hot water extraction.
The method according to claim 7, wherein the enzyme treatment is by a fibrinolytic enzyme capable of degrading the cell wall component.
9. The method according to claim 8, wherein the cellulolytic enzyme is selected from the group consisting of cellulase, hemicellulase, and glucanase.
The method according to claim 7, wherein the microorganism in the bioconversion process by microbial fermentation is a microorganism capable of producing a fibrinolytic enzyme.
11. The method according to claim 10, wherein the microorganism capable of producing the fibrinolytic enzyme is selected from the group consisting of lactic acid bacteria, Bacillus bacteria, yeast, aspergillus, and trichomatase.
[3] The method according to claim 2, wherein the polysaccharide is isolated by inducing precipitation by adding ethanol to a supernatant from which an insoluble residue has been removed from the culture broth after the bioconversion step of step (c).
The immune enhancer produced by the method of claim 1, wherein the concentration of the effective concentration EC50 in the measurement of macrophage activation is 1 占 퐂 / ml or less.
The immune enhancer produced by the method of claim 1, wherein the concentration of the effective concentration EC50 in the dendritic cell activation assay for immature dendritic cells, which are immune cells, is matured into mature dendritic cells is 1 占 퐂 / ml or less.
3. The method according to claim 2, wherein the polysaccharide has an effective concentration EC100 of 1 μg / ml or less in the measurement of the macrophage cell activation.
A physiologically active substance having immunological activity produced by the method of claim 1.
A health food comprising the physiologically active substance of claim 16.

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