KR20120095753A - Culture media for mushroom cultivation comprising herbal slurgies and mushroom having the activity of anticancer and immunological enhancement produced thereby - Google Patents

Abstract

PURPOSE: A mushroom cultivation medium containing waste medical herb sludge, and functional mushrooms using thereof are provided to offer the antitumor activity and immune-potentiating activity of the mushroom to users. CONSTITUTION: Functional mushrooms using a mushroom cultivation medium containing waste medical herb sludge is obtained by the following steps: mixing the 50-150 meshes dried waste medical herb sludge with medium materials; sterilizing the mixture to obtain the medium having the pH of 6.5-6.8 and the water content of 64-68%; injecting mushroom fungi into the obtained medium; and cultivating the mushroom fungi at 20-26 deg C for 5-10 days. The mushrooms are flammulina velutipes, lentinus edodes, or ganoderma lucidum. The waste medical herb sludge is sleeted from scutellaria baicalensis, eleutherococcus senticosus, eucommia ulmoides, ligusticum acutilobum, cnidium officinale, astragalus membranaceus, paeonia lactiflora, atractylodes japonica, poria cocos, rehmannia glutinosa, cinnamon, glycyrrhiza uralensis, or polygonatum sibiricum.

Description

Mushroom media for mushroom cultivation comprising herbal slurgies and mushroom having the activity of anticancer and immunological enhancement produced according to the present invention.

The present invention provides a mushroom cultivation medium containing lung herbal sludge and a method for cultivating functional mushrooms having enhanced immunopotentiating and antitumor activity, and functional mushrooms having increased immunopotentiating and antitumor activity cultivated therefrom. And a glycoside extract thereof.

Mushrooms have excellent aroma and taste, and are known to have good healing and suppression effects in cancer, as well as improving and preventing effects on adult diseases such as biofunction control, heart disease and diabetes. Polysaccharides derived from mushrooms are known to have excellent anticancer effects, such as inhibiting the proliferation of breast cancer and colon cancer when used with their own and other substances. In particular, the enoki mushroom is known to be excellent in anti-cancer effect, hypertension treatment, as well as immune enhancing effect. Enoki mushrooms, also called oyster mushrooms, are a type of edible mushroom that has grown to become a mass-consumer mushroom since its commercial production began in Japan in 1945. It belongs to the family Maresmiaceae and is one of the white viscous bacteria with strong surface viscosity.

Recent studies on mushrooms have been actively conducted to investigate the anticancer activity and physiological activity of edible mushrooms and medicinal mushroom extracts. However, as the artificial cultivation of mushrooms has been expanded, In addition to research, high-quality mushrooms and high yields are being actively researched for the improvement of medium materials by investigating the effects of various nutrient sources on the cultures and the changes in the chemical composition of the medium during the cultivation process.

For example, Korean Patent Laid-Open Publication No. 2003-0067131 discloses a mushroom cultivation medium in which a common mushroom cultivation medium is mixed with an additive consisting of tobacco powder with bactericidal components, eggshell containing high calcium, and charcoal that neutralizes. In addition, Korean Patent Publication No. 2011-0006267 discloses a raw material composition for cultivating oyster mushroom and a method of cultivating oyster mushroom using flower mushroom patch, shiitake mushroom badge, citrus gourd, green tea gourd, and barley fermentation products. In addition, Korean Patent Publication No. 2004-0061528 discloses a culture medium composition for mushroom cultivation containing saponin.

However, the main ingredients of mushroom medium, sawdust and corn cop, are dependent on imports, and the rise in raw material prices not only adds to the mushroom industry, but also causes foreign currency leakage.

On the other hand, many herbal medicines used in oriental medicine, such as golden, thorny scabies, tofu, wagong, cheongung, and huanggi, include carbohydrates, fats, proteins, alkaloids, terpenoids, carotenoids, etc. It contains a variety of bioactive substances such as vitamins, steroids, flavonoids and antibiotics. Therefore, traditionally, herbal medicines have been used in the form of herbal extracts for the maintenance and action of in vivo adaptation and defense mechanisms.

By the way, the leftover Chinese herbal sludge produced after manufacturing these herbal medicines in the form of Chinese medicine extract is generated more than 10,000 tons per year on average, but most of them are sent to waste incinerators, and only partially used by composting companies or feed mills. There is almost no place for systematic use.

The inventors of the present invention, while studying the use of these discarded waste herbal sludge, when used in the culture medium for mushroom cultivation not only increases the growth rate of the mushroom by the nutrients contained in the waste herbal sludge, especially the mushroom The present invention has been found to further increase the immunopotentiating activity and tumor suppression effect.

One object of the present invention is to provide a mushroom fungus culture medium for producing functional mushrooms enhanced in vivo immuno-enhancing activity and anti-tumor activity by using the lung herbal sludge.

Another object of the present invention is to provide a method for cultivating functional mushrooms having increased immunopotentiating and antitumor activity using the medium.

It is another object of the present invention to provide a functional mushroom and its glycoside extracts which are enhanced in vivo immuno-enhancing and anti-tumor activity as mushrooms grown using waste herbal sludge.

In one embodiment, the present invention contains 5 to 15% by weight of the dried Chinese herbal sludge having a particle size of 50 to 150 mesh based on the dry weight of the medium, the pH is 6.5 to 6.8 after sterilization in the sterilizer, the water content is 64 to 68%, and relates to a functional mushroom fungi culture medium characterized by enhancing the immuno-enhancing activity and anti-tumor activity of the cultivated mushrooms.

The medium of the present invention contains 85 to 95% by weight of the conventional mushroom fungal culture medium components and 5 to 15% by weight of dried waste herbal sludge having a particle size of 50 to 150 mesh based on the dry weight of the medium.

Waste herbal sludge of the present invention refers to the residue left after extracting the herbal medicine extract. Here, the herbal medicine refers to a conventional herbal medicine used in oriental medicine, examples of which include golden, thorny ogapi, tofu, Korean herb, cheongung, astragalus, peony, baekryeok, bokyeong, sujiji, cinnamon, licorice, brown root, etc. It is not limited.

Waste herbal sludge of the present invention should be contained in the medium in an amount of 5 to 15% by weight, preferably 8 to 12% by weight based on dry weight. If the content of waste herbal sludge is less than 5% by weight, it cannot be grown as a functional mushroom because it does not sufficiently express immune function enhancement and anti-tumor inhibitory effects, and if the content of waste herbal sludge is more than 15% by weight The growth of mushrooms is delayed, there are some deaths, and the effect of expression relative to its content is also uneconomical.

In addition, the waste herbal sludge of the present invention is dried to have a moisture content of 10% or less, and then ground to a particle size of 50 to 150 mesh, preferably 50 to 100 mesh. If the moisture content of the waste herbal sludge exceeds 10%, the growth of the mushroom is bad, and there is some undesirable killing phenomenon, and if the particle size of the waste herbal sludge is out of the above range, it is difficult to mix when preparing the medium, Due to the uneven supply of the mycelial culture, it is difficult to grow mushrooms in good yield.

In the medium of the present invention, in addition to the waste herbal sludge, the common mushroom fungus culture medium component included in the medium of the present invention is well known in the art, and preferred examples thereof include corn cob, rice bran, bran, feldspar, rice pine sawdust, Beet pulp, cotton wool and the like. These conventional ingredients may be appropriately selected depending on the type of mushroom for the purpose of cultivation. As an example, the fungus culture medium of the fungus is preferably corn cob, rice bran, bran, calcite, and the culture medium of fungus is preferably beet pulp, corn cob, rice bran, bran. In addition, the conventional medium component may further include sugar, glucose, maltose, maple sugar and the like for the purpose of promoting growth, enhancing functionality, strengthening the hardness of the fruiting body in addition to the components described above.

Mushrooms cultivated by the medium of the present invention include all of them if the mushroom can be grown on a fungus, but particularly preferably, mushrooms, top mushrooms, shiitake mushrooms.

Mushrooms cultivated by the medium of the present invention had better mycelial growth rate and mycelial density than the mushrooms cultivated by the normal cultivation medium, and the average yield was superior, and the rate of malformed mushrooms and the production of second-class mushrooms and The incidence of mushroom disease was significantly reduced (see results of Example 1).

In particular, mushrooms grown by the medium of the present invention have been found to have at least two-fold enhanced effects on immune function and tumor suppression effect known as efficacy of conventional mushrooms. Therefore, the immuno-enhancing activity and anti-tumor activity used in the present invention means increased by at least 2 times, preferably 3 times, more preferably 5 times, even more preferably 10 times compared to the conventional mushroom. . More specifically, the mushrooms grown by the medium of the present invention induced the proliferation of splenocytes compared to the mushrooms grown by the normal fungal culture medium, IL-6 is about 27.3 times, TNF-α is about 33 times, IFN-γ induced secretion by about 15.5 times, induced B cell proliferation more than 2 times, increased the production of immunoglobulins by B cells, induced the release of nitric oxide from macrophages, and secreted cytokines. The production of caine was increased, and the measurement of tumor suppression effect on ascites cancer showed that the mushroom of the present invention had an increased inhibitory effect of about 16.11% by the common mushroom. Therefore, the mushrooms cultivated by the medium of the present invention have a significantly increased immune function activity and anti-tumor effect compared to the conventional mushrooms and will be useful as a functional mushroom.

In another aspect, the present invention provides a method for cultivating functional mushrooms having a markedly increased immune function activation and antitumor effect compared to conventional mushrooms.

Specifically, the cultivation method of the functional mushroom of the present invention

(a) mixing a medium component containing 5 to 15% by weight of dry dry herbal sludge having a particle size of 50 to 150 mesh based on the dry weight of the medium;

(b) sterilizing the mixture obtained from step (a) to prepare a medium having a pH of 6.5 to 6.8 and a water content of 64 to 68%;

(c) inoculating mushroom spawn on the medium prepared from step (b); And

(d) culturing the inoculated mushroom spawn of step (c) at a temperature of 20 to 26 ° C. for 5 to 10 days, and then cultivating to a predetermined size.

The step (a) is a step of mixing with the mushroom culture medium component of the normal mushroom to contain 5 to 15% by weight dry dry sludge of the particle size 50 to 150 mesh based on the dry weight of the medium.

The culture medium component for the fungus culture of the conventional mushroom is as described above.

In addition, the waste herbal sludge is prepared by using the herbal medicine extract and dried the remaining residues to 10% or less moisture content and then pulverized to a size of 50 to 150 mesh after the content of 5 to 15% by weight based on the dry weight of the medium Will be mixed.

Next, step (b) is to sterilize the medium mixture to prepare a medium having a pH of 6.5 to 6.8 and a water content of 64 to 68%.

The sterilization may be performed by a known sterilization method. For example, sterilization by a high temperature and high pressure sterilizer, sterilization by an ultraviolet sterilizer, sterilization by a microwave sterilizer, and the like. By this sterilization, by killing the mold and the like present in the medium mixture, the growth of the mushroom spawn by the toxin ejected by the mold and the like is not hindered and growth of the mushroom alone is possible.

In addition, the growth of the mushroom spawn can be made smoothly only in the pH and water content range, and in particular, it is possible to produce mushrooms with enhanced immune and antitumor activity by the lung herbal sludge contained in the medium of the present invention.

Next step (c) is a step of inoculating mushroom spawn in the prepared medium.

Herein, the mushroom seedlings to be inoculated may be various seedlings depending on the type of mushrooms to be cultivated, and are preferably seedlings of mushrooms, green mushrooms or shiitake.

Such inoculation is preferably carried out in a sterile state in the inoculation room (inoculation temperature 15 ℃).

Step (d) is a step of culturing and culturing the inoculated mushroom spawn.

The conditions of the culture may be different depending on the type of inoculated mushrooms, but preferably 5 to 10 days of incubation at a P temperature of 20 to 26 ℃.

After the cultivation is carried out according to the conventional method, the bacteria are cultivated, ie, grown, harvested by culturing, curing, inhibiting and growing the mushrooms.

The mushrooms harvested by the method described above had a much higher average yield than the mushrooms harvested by the conventional method, resulting in an excellent yield. Also, the yield of the deformed mushrooms and the production of second-class mushrooms were markedly reduced. Can be produced. In addition, the mushrooms harvested by the method of the present invention by the conventional method to form a glycoside extract by the conventional method and the results of the investigation of the immuno-enhancing activity and anti-tumor activity of the extract, the mushroom of the present invention is significantly more than the conventional mushroom It could be confirmed that it exhibits high immune enhancing activity and antitumor activity.

Therefore, the mushrooms cultivated by the medium of the present invention are useful as raw materials of medicines, cosmetics and functional foods for the purpose of immunosuppression and / or tumor suppression due to their excellent quality and significantly increased immunopotentiating and antitumor activity. Could be used. In addition, the glycoside extract for the mushroom of the present invention is also the same as the mushroom of the present invention, due to the significantly increased immunopotentiating and antitumor activity, the raw material of medicines, cosmetics and functional foods for the purpose of immunosuppression and / or tumor suppression It can be useful as.

Mushroom cultivated by the medium of the present invention has excellent growth rate and density of mushroom hyphae compared to mushrooms grown in a medium that does not contain conventional Chinese herbal sludge, mushroom disease incidence rate, malformed mushroom incidence and second-class mushroom production This can be drastically reduced, increasing the average yield and contributing to the increase in farm income. In addition, the mushrooms cultivated by the medium of the present invention exhibits a significantly increased effect compared to the known immune and antitumor effects in conventional mushrooms, so that the functionality of medicines, cosmetics and foods for the purpose of enhancing immunity and suppressing antitumor. It may be usefully used as a raw material.

Figure 1 is a figure observing the growth of the enoki mushroom mycelia according to the concentration of the spent herbal sludge.
Figure 2 is a diagram showing the mycelial growth of the enoki mushroom on day 5 in the plate medium to which the waste herbal sludge is added.
Figure 3 is a figure observing the degree of mycelial growth of mushrooms, green mushrooms and shiitake mushrooms in the medium to which 10% by weight of the spent herbal sludge was added.
Figure 4 is a result of examining the reducing sugar content of the culture medium components and waste herbal sludge of the cultivated mushrooms.
5 is a result of examining the pH content of the culture medium components and waste herbal sludge of the cultivated mushrooms.
6 is a view showing the result of cultivating and cultivating the enoki mushroom using a medium containing the waste herbal sludge of the present invention.
Figure 7 shows the results of measuring the proliferation response of splenocytes after the addition of the fungus glycoside extract cultivated using the culture medium containing the lung herbal sludge of the present invention to the splenocytes isolated from the mice by incubation for 72 hours.
Figure 8 is a result of measuring the type and secretion of cytokines secreted when the fungi glycoside extract cultivated using the culture medium containing the waste herbal sludge of the present invention induces the proliferation of splenocytes.
9 is added to the B cells of splenocytes isolated from mice by the concentration of the fungus glycosides cultivated using the culture medium containing the lung herbal sludge of the present invention by measuring the growth of B cells after 72 hours of incubation The result is.
Figure 10 is added to the B cells of splenocytes isolated from the mouse cultivated by using the culture medium containing the lung herbal sludge of the present invention to B cells of splenocytes isolated from the mouse and cultured for 48 hours, IgM, IgG1, IgG2a contained in the supernatant It is the result of measuring the quantity of, IgG2b, and IgG3.
Figure 11 is added to the macrophage isolated from the mice to the macrophage isolated cultivated by using the culture medium containing the waste herbal sludge according to the concentration of the NO ₂ ^- concentration produced by the macrophages in culture after 48 hours of incubation Is the result of measurement.
12 is a result of measuring the secretion amount of cytokines contained in the supernatant after addition of the fungus glycoside extract cultivated using the culture medium containing the waste herbal sludge of the present invention to macrophages isolated from mice and incubated for 24 hours to be.
Figure 13 is a result of measuring the tumor suppression effect on ascites cancer by injecting mice with ethanol extract of the fungus glycosides cultivated using the culture medium containing the lung herbal sludge of the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples. However, embodiments according to the present invention can be modified in many different forms, the scope of the present invention should not be construed as limited to the embodiments described below. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Example 1 Comparative Experimental Materials and Methods for the Production of Mushrooms in a Medium Containing Waste Herbal Sludge

1-1. Test strain and test material

Enoki mushroom strains were pre-packed with Flammulina velutipes Fv1-5 stored in the laboratory of Greenpis Enoki Mushroom Farm in Cheongdo-gun, Gyeongbuk, Korea Used. Waste herbal sludges were collected by using Chinese medicine in the city's oriental medicine clinic, and then used for disposal. The media materials such as corncope and rice bran were used as the materials used in Greenpeace Enoki Mushroom Farm.

1-2. Pretreatment and Addition Concentration of Waste Herb Sludge

The waste herbal sludges collected from oriental medicine clinics in the city were naturally dried and stored in a low temperature warehouse while being crushed into 80 mesh.

First, in order to investigate the effect of the concentration of waste herbal sludge addition, 0, 5, 10 and 15% (w / w) of the pulverized waste herbal sludge were added to the corn cope medium used in the mushroom cultivation farm, respectively, and 120 ° C. After sterilizing for 15 minutes to make a plate medium, inoculate it in the center of the plate medium prepared with the block-shaped Enoki mushroom spawn, and then in a thermostat at 28 ° C (the result of the experiment was described as 25 ° C. During 9 days of incubation, the mycelial growth of the enoki mushroom was observed. As a control, a test group without PDA and waste herbal sludge was used. The results are shown in FIGS. 1 and 2.

As can be seen in Figures 1 and 2, the total fungus mycelial growth in all the plate medium as the number of culture days 3, 5 and 9 days in the corn-coated medium and lung herbal sludge added as a whole In the case of vigorous PDA, mycelial growth was slightly slower than in other media. Although the effect of the concentration was hardly observed in the test section in which the waste herbal sludge was added by concentration, the mycelial growth density was slightly fast and dense in the 10% addition test group (FIG. 2 d). As a result, it was judged that there is a possibility of developing the mushroom mushroom medium using waste herbal sludge discharged from oriental medicine clinics in the city, and the concentration was determined to be 10%.

1-3. Investigation of mycelial growth in mushroom cultivation medium containing waste herbal sludge

In order to investigate the mycelial growth in the culture medium containing waste herbal sludges of the present invention, 10% of each of the waste herbal sludges pulverized was added to the beet pulp medium used in the actual mushroom cultivation farms and mixed well, followed by a test tube having a volume of 100 ml ( 200 mm ㅧ 30 mm), sterilized at 121 ° C. for 30 minutes, and then inoculated 5 ml of each liquid spawn, such as oyster mushrooms, rust mushrooms, and shiitake mushrooms, previously incubated at 25 ° C. The growth length of the enoki mushroom mycelium was observed while incubating for 9 days in a thermostat. The results are shown in Figure 3, the control was used as a medium without the addition of the lung herbal sludge (A, B-a of Figure 3).

As shown in FIG. 3, the fungus grew about 2.2 ~ 3.4 cm in the control mushrooms on the 6th day of cultivation, but the hyphae growth was very strong in the test bowl (Fig. 3A, Bb) to which the lung herbal sludge was added. ~ 6.4 ㎝ mycelia were grown, the density of mycelium was also found to grow very dense. As a result of observing the mycelial growth of green and shiitake mushrooms, mycelial growth of green rust mushrooms was 8.8 ~ 9.7 ㎝ and mycelial growth of shiitake mushrooms was 4 ~ 4.5 ㎝. As a result, it was judged that the development of mushroom medium using waste herbal sludge was possible not only in mushrooms but also in cultivation of mainland such as shiitake mushrooms.

1-4. Analysis of General Components of Mushroom Cultivation Medium

General components such as corncobs, which are used as waste herbal sludge and mushroom cultivation medium, especially enoki mushroom medium, were analyzed. Specifically, the general composition analysis of the lung herbal sludge and corn cope, etc. were analyzed by AOAC method, the ash content was 550 ℃ direct liquefaction method, crude fat soxhlet fat extraction method, crude protein micro-Kjeldahl method (micro-Kjeldahl method) Measured by. The crude fiber was boiled with an acid detergent solution for 60 minutes, and then measured ADF (acid detergent fiber) which is a component that does not dissolve in this solution. The results are shown in Table 1 below.

Furtherance(%) Waste Herb Sludge Konkov Rice bran bran moisture 9.7 9.8 9.3 9.8 Crude fat 5.1 0.3 19.7 3.6 Crude protein 8.8 3.6 5.3 8.6 Views min 4.6 4.7 7.9 4.9 Crude fiber 32.2 40.8 9.8 12.4

As can be seen in Table 1, the water content was less than 10% in all media components, including lung herbal sludge, crude fat content was the highest in rice bran 17.9%, followed by lung herbal sludge and bran 5.1% respectively. And 3.6%. Crude protein content was 8.8%, 8.6%, 5.3% and 3.6% in lung herbal sludge, bran, rice bran and corn cope, respectively. The ash content of rice bran was slightly higher (approximately 7.9%), but in other herbal sludges, corn cobs and bran, the content was similar to 4.6 ~ 4.9%. As a result of measuring crude fiber content, it was 32 ~ 41% in corncob and lung herbal sludge, but 12.4 ~ 9.8% in bran and rice bran.

As a result, waste herbal sludge has a higher content of crude fat and crude protein than corn cope which is used as a main ingredient in the preparation of the enoki mushroom medium, and shows little difference with rice bran and bran used as auxiliary additives. It was judged that it could be used as an additive.

1-5. Reducing Sugar Content in Enoki Mushroom Medium

Reducing sugar content of corn cob, beet pulp and waste herbal sludge, etc., which are used as a mushroom mushroom medium material, are shown in FIG. 4.

As can be seen in Figure 4, cotton silk and rice bran showed a similar content of 5.9 ~ 6.2 mg / g, lung herbal sludge showed 4.8 mg / g. Miso sawdust, bran, corncope and beet pulp showed a low reducing sugar content of 1.1 ~ 2.4 mg / g.

1-6. PH Measurement of Enoki Mushroom Medium

10 g of the pulverized herbal sludge crushed with 80 mesh was mixed in 90 mL of sterile distilled water, and then shaken vigorously at room temperature for 90 minutes, and then the pH of the supernatant obtained by centrifugation for 12,000, g and 15 minutes was measured. In the same manner, the pH of corncob, beet pulp and the like were measured, and the results are shown in FIG. 5.

As can be seen in Figure 5, the pH of the lung herbal sludge, rice chopped sawdust, corn cob and beet pulp showed a weak acidity of about 4.9 ~ 5.4, but the pH of rice bran, cottonseed and bran, etc. is about 6.59 ~ 7.1 It was.

1-7. Field Application Test of Waste Herb Sludge Medium

The cultivation test of the top mushrooms using the medium containing waste herbal sludge was directly performed in the cultivation of the top mushroom of Greenpeace Co., Ltd. Enoki mushrooms were grown using a 1,100 mL mushroom cultivation bottle, and the control components were corncope 49% (w / w), rice bran 39% (w / w), bran 7% (w / w) and calcite 5% ( w / w) was mixed well to adjust the water content to 66.7%, pH 6.68. Waste herbal sludge added medium, a test group, was added with 10% (w / w) waste herbal sludge instead of 10% (w / w) of corn cop in the control medium. The other medium composition and conditions were prepared in the same manner. . In addition, the quality of the mushrooms produced was observed by visual observation of the size, thickness, and color of the mushrooms. Special features include mushrooms and commodities with a uniform diameter and length of 12 cm or more, with a diameter of 10 mm or less. (Secondary) is a mushroom shade with a diameter of 10 mm or more, but more than 30% pin mushrooms and deformed mushrooms have two stems, or the shades are connected. A mushroom with no commodity value.

The results are shown in Figure 6 and Table 2. First, as can be seen in Figure 6, the mycelial culture of the enoki mushroom (see Fig. 6A) and the formation of fruiting bodies (see Fig. 6 B, C) in the test zone to which the lung herbal sludge was added compared to the control There was no difference at all, and the growth was observed. In addition, the results of the observation of the average yield of mushrooms, the amount of malformed mushrooms, second-class mushrooms, inferior growth and mushroom disease incidence (see Table 2), and the occurrence of white mold and bacterial brown pattern disease in the control and test It could not be observed. The yields of diseased top mushrooms in the experimental groups A, B and C were about 5% lower than those of the control group, but the incidence of malformed mushrooms and the production of the second-class mushrooms were significantly lower in the experimental group containing lung herbal sludge. As a result, it is considered that there is sufficient possibility of producing mushrooms with the enoki mushroom medium containing waste herbal sludge, which will contribute to reducing the production cost of mushrooms in the future.

Example 2 Experimental Method for Investigating the Immune Function Effect of Mushrooms Cultivated in Mushroom Cultivation Medium Containing Oriental Herbal Sludge

2-1. Experimental animal

The experimental animals were supplied with specific pathogen free C57BL / 6 and ICR mice from the Korean Experimental Animal Center, maintaining 6 densities per polycarbonate breeding box (18 × 20cm) in the experimental animal breeding room. The females were used 8-12 weeks of age, with sufficient water and feed, and 12-hour day and night cycles to raise mice as little stress as possible at room temperature for two weeks.

2-2. Experimental material

Three types of top mushrooms A, B, and C are top mushrooms with different cultivation methods. A is a normal farming method and is a top mushroom grown in basic medium + rice bran and B is a cultivation method using waste sludge. Enoki mushroom is grown in 10% Chinese medicine. C, a combination of waste sludge and general farming methods, is an enoki mushroom grown in basic medium + 10% rice bran + 10% Chinese herbal medicine.

 To extract the three kinds of mushrooms (3Kg) was extracted twice with a reflux condenser at 80 ℃ using 18L of 70% ethanol for 3 hours. Then, the extract was concentrated under reduced pressure to 2L, and the glycosides were separated through the water extract using an open column (HP-20). Second, the remaining filtrate was extracted using 95% ethanol. The final extract was concentrated under reduced pressure again to pretreat the general and sugar components. The extract of the mushrooms was used after suspended in phosphate (PBS) and filtered with a 0.2 ㎛ filter, and the ethanol extract of the mushrooms was suspended in DMSO (dimethyl sulfoxide).

All extracts were dried sufficiently at 110 ° C. and used by measuring dry weight.

2-3. Reagent

Antibiotic-antimycotic (FCC) and fetal calf serum (FCS) were used as PAA in addition to the medium RPMI 1640 required for cell culture, sodium bicarbonate (NaHCO ₃ ), 2-ME (2-mercaptoethanol), sulfanilamide, N-1-naphthyl-ethylen-diamine was used as Sigma (St. Louis, USA). In addition, the reagent used to measure cell proliferation (Cell Kit-8) was manufactured by DOJINDO (Japan), and the antibody required for cytokine measurement by Pharmingen (San Diego, USA).

2-4. Isolation of Splenocytes

Splenocytes of mice were used to measure the effect on the proliferation of immune cells. The mice were sacrificed with cervical vertebra and the spleen was aseptically separated to form a single cell suspension using tweezers or a mesh. Single cell suspensions were washed by centrifugation with RPMI 1640 cultures three times, and then diluted to 10x FCS-RPMI 1640 medium to a concentration of 5 × 10 ⁶ cells / mL, and then 100 μL per well was added to 96well plates. At this time, the test materials were added by concentration and incubated for 72 hours at 37 ℃, 5% CO ₂ incubator, and then measured cell proliferation using the Cell Counting Kit-8 (DOJINDO, Japan).

2-5. B cell separation method

B cells were diluted in 10 mL of 10% FCS-RPMI 1640 in splenocyte suspension, and 40 μl of anti-Thy1.2 mAb ascites were incubated at 4 ° C. for 60 minutes. After that, rabbit complement was added and incubated for 45 minutes in a 37 ° C. water bath to remove T cells. Then, B cells were purified by removing the remaining adherent cells using Sephadex G-10 column. Proliferation was measured using Cell Counting Kit-8 (DOJINDO, Japan).

2-6. Cell proliferation assay

Cell proliferation was measured by inserting the isolated splenocytes and B cells into 96well plate, and then adding the glycoside extract to each concentration, and measuring the proliferation. 10 μl of Cell Counting Kit-8 (DOJINDO, Japan) was added to the cell culture wells and incubated in 37 ° C., 5% CO ₂ incubator for 4-8 hours, followed by microplate reader (Titertek Multiscan Plus, Finland) at 450 nm. Absorbance was measured.

2-7. Cytokine Measurement

Glycoside extract, Con A or LPS was added to the splenocytes and incubated for 24 hours, and then the supernatant was recovered and the IL-2, 4, 6, 10, 12 IFN-γ, TNF-α, GM-CSF contained in the supernatant. The amount of was measured. That is, the primary antibody was diluted in coating buffer (0.1 M NaHCO ₃ ), added to the plate, left overnight at 4 ° C., washed with washing solution (0.05% Tween 20 / PBS), and then phosphate buffer solution containing bovine serum (10). After blocking with% FCS / PBS), the culture supernatant was appropriately diluted and added, followed by the addition of a secondary antibody bound to biotin. After a certain time, add avidin-peroxidase, and add a substrate (2.2'-azino-bis, 0.1M citritic acid, H ₂ O ₂ ) to develop a microplate using enzyme-linked immunsorbent assy (ELISA). The absorbance was measured at 405 nm with a reader. At this time, the measurement limit of each cytokine was 10 pg / mL.

2-8. Immunoglobulin Concentration

IgM, G1, G2a, G2b, and G3 concentrations in the culture medium cultured for 48 hours by adding glycoside extract or LPS to B cells isolated from splenocytes were measured at 405 nm using a microplate reader.

2-9. Nitric Oxide Measurement

The stable nitrogen monoxide oxide NO ₂ ⁻ (nitrite) was measured using the Griess reaction. After incubating the macrophage line (RAW264.7) with glycoside extract or LPS for 48 hours, 100 μL of the culture supernatant was added to a 96 well plate, and the Griess reagent (0.1% N-1-naphthyl-ethylendiamine in H ₂ O: 1% sulfanilamide in 5% H ₃ PO ₄ ) was added in the same amount for 10 minutes, and the absorbance was measured at 550 nm with a Microplate reader. Nitrite concentrations were calculated by comparing the standard curves obtained by diluting sodium nitrite from 64 μM to 0.5 μM in 2-fold.

2-10. Measurement of tumor suppression effect on ascites cancer

Float Sarcoma 180 to 5 × 10 ⁶ cells / ml and implant it into the abdominal cavity of 0.3ml (1 × 10 ⁶ cells / mouse) ICR mice to induce ascites cancer and 100mg / kg body from 24 hours after Sarcoma 180 implantation The extract of the weight concentration was dissolved in physiological saline, and the extract of Gaga was administered 0.2 ml intraperitoneally once per 7 days. In the same period, the same amount of saline was administered, and the anti-tumor effect was evaluated by the median survival time (MST) and the elongation rate (ILS).

ILS = [(TC) / C] × 100

C: Average lifespan of controls (days)

T: life expectancy in days

Example 3 Immune Function Effect of Mushrooms Cultivated in Mushroom Cultivation Medium Containing Lung Sludge

3-1. Proliferation of splenocytes

To determine the effect on the proliferation of splenocytes, the growth reaction was measured after 72 hours of incubation by adding the extract of the enoki mushroom glycoside to the splenocytes isolated from mice. As a result, it was confirmed that high proliferation reaction of splenocytes occurred by LPS (lipopolysaccharide) which specifically induces B cell proliferation and Con A (concanavalin A) which specifically induces T cell proliferation. When the glycoside extract was added, the proliferation of splenocytes could not be confirmed at low concentrations, but the splenocytes were proliferated at high concentrations of 100 and 300 ㎍ / mL compared to the untreated control (FIG. 7). In other words, the enoki mushroom glycoside extract induced proliferation of splenocytes at high concentrations, but the proliferative response was lowered at higher concentrations of 300 ㎍ / mL than 100 ㎍ / mL in ethanol A and C extracts. Is considered to have lowered proliferation. On the other hand, the maximum proliferative reaction occurred at the maximum concentration of 300 ㎍ / mL in water extract C and ethanol extract B.

3-2. Cytokine Production in Splenocytes

When the proliferative response of splenocytes occurs, proliferating cells secrete cytokines that mediate various types of immune responses. In other words, knowing the type of cytokine secreted when the splenocyte proliferation occurs, it is possible to know the cytokine-mediated immune response and the type of proliferating cells. Therefore, we investigated what kind of cytokines secrete when the enoki mushroom glycoside extract induces the proliferation of splenocytes. LPS, which induces the proliferation of B cells among splenocytes, induces higher secretion of IL-6 and TNF-α compared to the control group, and Con A, which induces T cell proliferation, also contains large amounts of IL-2 and IFN- γ secretion was induced (FIG. 8). When stimulated with glycoside extract, it secreted a small amount of IL-6, TNF-α, and IFN-γ compared to the untreated control, but ethanol extract B had about 27.3 times IL-6 and TNF-α about 33 times IFN-γ. About 15.5 times as much secretion was induced. However, IL-2 was not induced as secreted control. IL-2 is a cytokine secreted by T cells for antigen stimulation and is known as a cytokine that induces proliferation of helper T cells and cytotoxic T cells involved in the adaptive immune response. IFN-γ is a cytokine that plays an important role in activating macrophages involved in endogenous immune responses. As a result, the encapsulated Glycoside glycoside extract did not induce T cell proliferation in splenocytes, and it was found that IL-6, TNF-α, IFN-γ secretion was induced in ethanol B extract.

3-3. Proliferation of B cells

As a result of examining the proliferation-inducing effect of the splenocytes on the glycoside extracts from Examples 3-1 and 3-2, it was confirmed that the cells induce proliferation at high concentrations (FIG. 7). At this time, the type of cytokine secreted by the proliferating splenocytes revealed that the glycoside extract did not directly induce the proliferation of T cells (FIG. 8). Therefore, since the proliferation of splenocytes is likely to be B cells, only the B cells contained in the splenocytes were isolated to measure the B cell proliferation induction effect of the glycoside extract. Glycoside extracts were added to the isolated B cells by concentration, followed by incubation for 72 hours to measure the proliferation of B cells (FIG. 9). As a result, the proliferative response was induced only to LPS, which induces the proliferative response of B cells, but not to Con A, which induces the proliferative response of T cells. In addition, the experimental group treated with glycoside extract did not appear to induce the proliferation of B cells compared to the untreated control group, but it was found that the ethanol B extract showed a two-fold proliferation effect at a high concentration of 100 ㎍ / mL. The enoki mushroom glycoside ethanol B extract was found to directly induce the proliferation of B cells, especially among splenocytes.

3-4. Immunoglobulin Production in B Cells

As a result of measuring the proliferation inducing effect of B cells from Example 3-3, it was found that glycoside ethanol B extract strongly induced proliferation of B cells (FIG. 9). B-cells that divide and proliferate produce and produce immunoglobulins. We examined whether glycoside extracts induce B-cell immunoglobulin production. First, in order to measure the amount of IgM, IgG1, IgG2a, IgG2b, and IgG3 produced by B cells, copolysaccharide extracts were added to B cells isolated from splenocytes, incubated for 48 hours, and then the supernatant was recovered and included in the supernatant. Each immunoglobulin amount was measured (FIG. 10). As a result, it was found that the amount of IgM, IgG1, and IgG3 immunoglobulin was increased in the ethanol B extract when the crude polysaccharide extract was added as compared to the untreated control. In particular, IgM produced about 8-fold and IgG3 produced about 1.8-fold. That is, ethanol B extract compared to other glycoside extracts was found to have an effect of inducing humoral immune responses by increasing the production of IgG1, IgG2a, IgG3 as well as the proliferation of B cells.

3-5. Nitric oxide production

Macrophages are cells responsible for the primary immune response when an antigen, such as bacteria, invade. The macrophages act as phagocytic cells that first eat the invading bacteria. At this time, the eaten bacteria are not immediately killed, but are killed and degraded by the bactericidal action in the macrophages. There are many known substances that macrophages secrete to kill bacteria, but one of the most recently discovered is nitric oxide. Therefore, we investigated how glycoside extracts affect nitric oxide production in macrophages. After treating the glycoside extract by concentration in macrophage line (RAW264.7) for 48 hours and incubating for 48 hours, the concentration of NO ₂ ⁻ in NO oxidized form of macrophages in culture was measured (FIG. 11). As a result, the experimental group treated with glycoside extracts induced the nitric oxide production of macrophages in a concentration-dependent manner compared to the untreated control, but the water extracts A, B, C and ethanol extracts A, C produced a small amount of nitrogen monoxide. . However, ethanol B extract produced more nitrogen monoxide than other glycoside extracts. In other words, it was found that ethanol B extract of glycoside extract induces a large amount of nitric oxide production by activating macrophages.

3-6. Cytokine Production in Macrophages

When macrophages are activated by stimulation of antigen, they secrete IL-6, TNF-α and GM-CSF to regulate immune responses. In particular, TNF-α is a cytokine that destroys tumor cells as a substance known as tumor nacrosis factor, and IL-6 induces hepatocytes to synthesize some plasma proteins such as fibrinogen and induces B cell differentiation. It acts as a B cell growth factor. And GM-CSF (granulocyte-macrophage colony stimulating factor) induces the differentiation and proliferation of myeloid progenitor cells to produce granulocytes and macrophages. Therefore, we investigated how glycoside extracts affect the secretion of these cytokines produced by macrophages. In order to determine the types of cytokines secreted, the glycoside extract was added to the macrophage line, followed by culturing for 24 hours, and the supernatant was recovered to measure the amount of cytokines contained in the supernatant (FIG. 12). The production of large amounts of IL-6, TNF-α and GM-CSF was induced by LPS, a substance that activates macrophages. In addition, when the glycoside extract was treated, it was confirmed that the secretion of IL-6 and TNF-α was increased compared to the untreated control, and in particular, the ethanol B extract showed a lot of secretion. The macrophage cytokine production was higher than the LPS that activates macrophages in TNF-α. Therefore, the ethanol B extract of the enoki mushroom glycoside extract was able to induce anti-tumor and immune activity by activating macrophages to secrete various cytokines.

3-7. Measurement of tumor suppression effect on ascites cancer

In vitro experiments with the extract of the enoki mushroom glycoside showed that the water extracts of the control and the enoki mushroom glycoside showed almost similar immune cell proliferation and cytokine secretion. On the basis of these results, the tumor suppression effect on ascites cancer was measured with the fungus ethanol extract cultivated by the cultivation method using lung medicinal herbs was measured tumor suppression effect on ascites cancer (Fig. 13).

The life expectancy of the control group was 21 days after injection of Sarcoma 180 inoculated ethanol extract of the enoki mushroom, and the mean survival time of the control group administered with 100 mg / kg body weight of ethanol extract B was 24.5 days. Life expectancy was 16.11%. Therefore, it was confirmed that the ethanol B extract in the fungus glycoside extract inhibited tumor suppression of ascites cancer by 16.11%.

Claims (8)

In the culture medium for mushroom fungus culture, containing 5 to 15% by weight of dried waste herbal sludge having a particle size of 50 to 150 mesh based on the dry weight of the medium, the pH is 6.5 to 6.8 after sterilization, the water content is 64 to 68 %, Functional mushroom fungus culture medium, characterized in that to enhance the immune-enhancing and anti-tumor activity of the cultivated mushrooms. The method according to claim 1, wherein the mushroom is a mushroom fungus cultivation medium, characterized in that the top mushroom, shiitake mushroom or rust mushroom. The method of claim 1, wherein the Chinese herbal medicine sludge is a Chinese medicine extract from one or more selected from the group consisting of golden, thorn ogapi, tofu, Korean herb, cheongung, astragalus, peony, baekryeok, bokyeong, sagejiku, cinnamon, licorice and brown root Mushroom cultivation medium, characterized in that the leftover residue. (a) mixing a medium component containing 5 to 15% by weight of dry dry herbal sludge having a particle size of 50 to 150 mesh based on the dry weight of the medium;
(b) sterilizing the mixture obtained from step (a) to prepare a medium having a pH of 6.5 to 6.8 and a water content of 64 to 68%;
(c) inoculating mushroom spawn on the medium prepared from step (b); And
(d) cultivating functional mushrooms having increased immunopotentiating and antitumor activity, comprising culturing the inoculated mushroom spawn of step (c) for 5 to 10 days at a temperature of 20 to 26 ° C. Way. The method of claim 4, wherein the mushrooms are mushrooms, shiitake mushrooms or green mushrooms. The method according to claim 4, wherein the lung herbal sludge is at least one selected from the group consisting of golden, thorn ogapi, tofu, Korean herring, cheongung, astragalus, peony, baekrye, bokyeong, sageji, cinnamon, licorice and brown root How to grow mushrooms. Functional mushrooms having increased immuno-enhancing activity and anti-tumor activity with mushrooms cultivated by the method of claims 4 to 6. Glycoside extract of the mushroom grown by the method of claims 4 to 6.

Images