KR101305146B1 - Granule tea comprising extracts of glycosides derived from flammulina velutipes and method for preparing the same - Google Patents

Abstract

PURPOSE: A production method of functional and medicinal Collybia velutipes granule tea is provided to remove bad smells and tastes of Collybia velutipes, and to offer excellent immunity improvement and anti-tumor effects. CONSTITUTION: Glycoside is separated from a Collybia velutipes extract obtained from Collybia velutipes cultivated in a medium containing 5-15 wt% of waste medical herb sludge, and the glycoside is concentrated to obtain Collybia velutipes glycoside concentrate. The Collybia velutipes glycoside concentrate is mixed with a sweetener. The mixture of the Collybia velutipes glycoside concentrate and the sweetener is molded into granules. The molded granules are dried, and sieved into a size of 15-20 meshes. The sieved granules are packed. The waste medical herb sludge has the particle size of 50-150 meshes, pH of 6.5-6.8, and the water content of 64-68%. The waste medical herb sludge is residues obtained by producing medical herb extracts of more than one medical herbs selected from Scutellariae radix, Acanthopanax senticosus, Eucommia ulmoides, Angelica acutiloba Kitagawa, Cnidii rhizoma, milk vetch root, Paeoniae radix, Atractylodis rhizoma alba, tuckahoe, Rehmanniae radix preparata, cinnamon, Glycyrrhizae radix, or Puerariae radix. [Reference numerals] (AA) Cultivate Collybia velutipes in a medium containing waste medical herb sludge; (BB) Produce the extract of Collybia velutipes; (CC) Separate and concentrate glycoside; (DD) Mix the glycoside of Collybia velutipes and a sweetener; (EE) Produce granules; (FF) Dry; (GG) Sieve into the size of 15-20 meshes; (HH) Pack

Description

Granule tea comprising extracts of glycosides derived from Flammulina velutipes and method for preparing the same}

The present invention relates to a granule tea for drinking the enoki mushroom having an immune enhancing and anti-tumor effect by culturing in a medium containing waste herbal sludge and a method for producing the same.

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.

Therefore, the inventors of the present invention, when used in the medium for the cultivation of mushrooms by the use of these discarded lung herbal sludge not only increases the growth rate of the mushrooms by the nutrients contained in the waste herbal sludge, but also in particular the immune enhancing activity and It was found that the tumor suppressor effect was further increased, which was filed in patent application No. 10-2011-0015264.

On the other hand, the inventors of the present invention cultivated using the technology for growing mushrooms using the above-described waste herbal sludge has been made diligently to develop such an easily ingestible formulation of the mushrooms that have been further enhanced immune activity and drug suppression ability As a result, the present invention has been completed.

One object of the present invention is characterized by containing 4 to 12% by weight of the glycoside concentrate of the enoki mushroom cultured in a culture medium containing 5 to 15% by weight of the spent herbal sludge based on the dry weight of the mushroom fungus culture medium It is to provide an enoki mushroom granule tea.

In addition, another object of the present invention is to provide a method for producing the enoki mushroom granular tea.

In one embodiment, the present invention is that the waste herbal sludge containing 4 to 12% by weight of the glycoside concentrate of the mushrooms cultured in the medium containing 5 to 15% by weight based on the dry weight of the mushroom fungus culture medium It provides an enoki mushroom granular tea characterized by.

In the present invention, the waste herbal sludge 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 preferably has a particle size of 50 to 150 mesh, a pH of 6.5 to 6.8, and a water content of 64 to 68%. In the case of the waste herbal sludge under such conditions, the mixing is easy during the preparation of the medium, the water supply is uniform, and the mushroom hyphae culture is uniform, and the growth of the mushroom is good.

In the present invention, the mushroom fungus culture medium includes a conventional medium component selected from corn cob, rice bran, bran, calcite, rice pine sawdust, beet pulp, and cotton husk in addition to the above-described waste herbal sludge. 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.

In the present invention, the mushrooms cultivated by the culture medium containing the spent herbal sludge is superior to the growth rate and mycelial density of the mycelia compared to the mushrooms cultivated by the normal mycelial culture medium, the average yield is superior, and also deformed Mushroom production rate, the second-class mushroom production and mushroom disease incidence is significantly reduced (see Example 1).

In particular, the mushrooms cultivated by the medium has at least two-fold enhanced effects on immune function and tumor suppression effect known as the 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. . Such immunopotentiating and antitumor activity is described in detail in patent application No. 10-2011-0015264 filed by the inventor. More specifically, the mushrooms grown by the medium of the present invention induced proliferation of splenocytes compared to the mushrooms grown by the normal fungal culture medium, IL-6 is about 27.3 times, TNF- about 33 times, IFN -Induces secretion by about 15.5 times, induces B cell proliferation more than 2 times, increases the production of immunoglobulins by B cells, induces nitric oxide separation of macrophages and releases of cytokines secreted by macrophages. Production was increased, and in the measurement of tumor suppression effect on ascites cancer, the mushrooms of the present invention had an increased inhibitory effect of about 16.11% by conventional mushrooms.

The method and the effect of cultivating the mushrooms in the medium containing the waste herbal sludge as described above are described in more detail in Patent Application No. 10-2011-0015264 filed by the present inventors. Accordingly, the present invention includes patent application No. 10-2011-0015264 filed by the inventors as a reference and content of the present invention.

In the present invention, the glycoside concentrate of the enoki mushroom is separated only from the extract of the enoki mushroom cultured in the medium containing 5 to 15% by weight of the above-mentioned waste herbal sludge based on the dry weight of the mushroom fungus culture medium, and concentrated Say that.

In one specific embodiment, an extract of the enoki mushroom was prepared, centrifuged, and then concentrated twice. Then, the glycoside was separated using a column and concentrated. Here, as an example, the column is an open column, and an ion exchange resin is used as the stationary phase, and water and spirits are used as the mobile phase.

Concentrates of such glycosides can be used as they are in liquid form or dried. The drying method includes, but is not limited to, methods such as freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying, infrared drying and the like.

On the other hand, the extract of the enoki mushroom can be obtained using various extraction methods known in the art. Preferably (a) water, (b) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) a mixed solvent of the lower alcohol with water, (d) acetone, (e) ethyl acetate, (f) chloroform, (g) 1,3-butylene glycol or mixtures thereof can be obtained as an extraction solvent. More preferably, it can extract using methanol and ethanol aqueous solution, Most preferably, it can extract using ethanol aqueous solution. On the other hand, it is apparent to those skilled in the art that the mushroom extract of the present invention can obtain an extract having substantially the same effect using not only the aforementioned extraction solvent but also other extraction solvents. The specific extraction conditions (temperature, time, etc.) required in addition to the extraction solvent can be selected and adjusted by those skilled in the art depending on the properties of the extraction solvent.

The enoki mushroom granular tea of the present invention contains the enoki mushroom glycoside concentrate in an amount of 4 to 12% by weight, preferably 5 to 12% by weight of the total granular tea weight. When the content of the enoki mushroom glycoside concentrate is less than 4% by weight, it may not have sufficient immunity and antitumor effects according to the drinking of the final granular tea, and when it exceeds 12% by weight, the granular tea may be due to high viscosity during the preparation of the final granular tea. There are many difficulties in manufacturing, and sensory evaluation has a very bad problem.

In addition to the above-mentioned mushroom mushroom glycoside concentrate, the enoki mushroom granule tea of the present invention further comprises glucose, vitamin C, and polydextrose or skim milk powder in order to enhance the taste and aroma of the final product enoki mushroom granule tea. The glucose is contained in 70 to 80 parts by weight, preferably 72 to 79 parts by weight based on the weight part of the total granular tea. The vitamin C is contained 0.6 to 0.8 parts by weight, preferably 0.7 to 0.75 parts by weight based on the weight part of the total granular tea. The polytextrose or skim milk powder is contained in an amount of 14 to 16 parts by weight, preferably 15 to 15.5 parts by weight based on the weight part of the total granular tea. When the content of glucose, vitamin C, and polydextrose or skim milk powder is within the above range, it is easy to mold into a granule formulation in preparation of the enoki mushroom granule tea according to the present invention and has the best taste and flavor.

In a more specific embodiment, the enoki mushroom granular tea according to the present invention is 15.3 wt% skim milk powder or polydextrin, 78.99 wt% glucose, and 0.71 wt% vitamin C when the enoki mushroom glycoside concentrate is 5.0 wt%. It is contained.

In another specific embodiment, the enoki mushroom granular tea according to the present invention comprises 15.3 wt% of skim milk powder or polydextrin, 72.01 wt% of glucose, and 0.71 wt% of vitamin C when the enoki mushroom glycoside concentrate is 11.98 wt%. It is contained as.

Enoki mushroom granule tea according to the present invention was easy to prepare granules, was excellent in solubility when prepared as granules, was evaluated as the best taste and flavor for drinking. In particular, the enoki mushroom granular tea according to the present invention increases the immunity in vivo without toxicity, and the anticancer effect is very excellent compared to the conventional mushroom tea and enoki mushroom tea. In particular, according to an embodiment of the present invention, the effect of inhibiting the size and weight of tumor cells from mice orally administered the enoki mushroom granular tea of the present invention, cytokines IL-2, IL-6, IFN-γ, TNF- observe the effect of increasing the secretion of α, the number of natural killer cells (NK1.1 +), and the cytotoxic activity of natural killer cells and antigen-specific cytotoxic T cells Could. Therefore, the enoki mushroom granular tea according to the present invention may be provided as a functional food having an immune enhancing and antitumor inhibitory effect upon drinking.

In another embodiment, the present invention provides a method for producing the above-mentioned mushroom mushroom granite tea.

As a specific aspect, the manufacturing method of the enoki mushroom granule tea of the present invention,

Preparing glycoside glycoside concentrate by separating the glycoside from the extract of the enoki mushroom cultured in the medium containing 5 to 15% by weight based on the dry weight of the mushroom fungus culture medium for waste herbal sludge;

Mixing a sweetener to the glycoside concentrate of the prepared mushrooms;

Shaping the mixture of glycoside concentrate and sweetener of the enoki mushroom into granules;

Drying the molded granules and then sieving them to a size of 15 to 20 mesh; And

Packing the sifted granules.

Hereinafter, with reference to FIG. 13, the manufacturing method of the pine mushroom granular tea of this invention is demonstrated in detail.

As a first step, the isolated herbal glycosides are prepared by separating the glycoside from the extract of the enoki mushroom cultured in the medium containing 5 to 15% by weight based on the dry weight of the mushroom fungus culture medium.

Here, the lung herbal sludge refers to the residue left after extracting the extract from the herbal medicines such as golden, thorny goose, worms, herds, Cheongung, Astragalus, Peony, Baekryong, Sugijihwang, cinnamon, licorice, brown root as described above. In the following, the waste herbal sludge is omitted as it is as described above.

In addition, the extract of the enoki mushroom and the enoki mushroom glycoside concentrate obtained by separating and concentrating the glycoside therefrom are also referred to above.

As a second step, a sweetener is mixed with the glycoside concentrate of the prepared mushroom.

The sweetener is to improve or enhance the taste and aroma of the enoki mushroom granulated tea produced by the present invention, examples of which include glucose, vitamin C, polydextrose or skim milk powder. Preferably, the sweetener is used by mixing glucose, vitamin C, and polydextrose or skim milk powder. These sweeteners contain 88 to 96% by weight based on the total weight of the granular tea. More specifically, the glucose is contained in 70 to 80 parts by weight, preferably 72 to 79 parts by weight based on the weight part of the total granular tea. The vitamin C is contained 0.6 to 0.8 parts by weight, preferably 0.7 to 0.75 parts by weight based on the weight part of the total granular tea. The polytextrose or skim milk powder is contained in an amount of 14 to 16 parts by weight, preferably 15 to 15.5 parts by weight based on the weight part of the total granular tea. More preferred composition of the granular tea according to the present invention is 15.3% by weight of skim milk powder or polydextrin, 78.99% by weight of glucose, and 0.71% by weight of vitamin C, or encapsulated glycoside glycoside when the concentration of pine mushroom glycoside is 5.0% by weight At 11.98 wt% water, 15.3 wt% skim milk powder or polydextrin, 72.01 wt% glucose, and 0.71 wt% vitamin C. Most preferably, the composition of the granular tea according to the present invention is 11.98% by weight of the mushroom mushroom glycoside concentrate, 15.3% by weight of skim milk powder or polydextrin, 72.01% by weight of glucose, and 0.71% by weight of vitamin C.

In a third step, the mixture of the glycoside concentrate and the sweetener of the enoki mushroom is molded into granules.

The step is to put the mixture of the glycoside concentrate and the sweetening agent of the enoki mushroom into a conventional granulator, for example, a counter-rotating granules and to form a granule formulation.

As a fourth step, the molded granules are dried and then separated into a size of 15 to 20 mesh.

In the step of drying the granules, the granules formed from the mixture of the glycoside concentrate and the sweetening agent of the enoki mushroom have a moisture content of 5% or less using a conventional dryer, for example, a dryer having a method such as hot air drying or natural drying. Dry as much as possible.

In addition, since drinking a granule tea is required for a certain size of granules in order to dissolve in water quickly and easily, it is a step of sifting only granules having a size of 15 to 20 mesh.

As a fifth step, the step of packaging the sifted granules.

The packaging here is preferably packaged using a wrapping paper that is easy to store granule tea, for example, can be packaged using an aluminum foil tea bag wrapping paper.

Enoki mushroom granule tea according to the present invention is excellent in the sensory and the smell of the fungus is removed and is easy to drink because it is in the form of granules. In particular, the enoki mushroom glycosides included in the granule tea of the present invention may effectively provide these effects through the granule tea according to the present invention because it is superior in immunity improvement and antitumor effect compared to any conventional enoki mushroom glycoside.

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.
Figure 6 is a diagram showing the results of cultivating and cultivating the top mushrooms using a medium containing waste herbal sludge.
Figure 7 is a result of examining the proliferation of splenocytes of mice (C57BL / 6) orally administered the preparation examples C and D samples of the enoki mushroom granular tea according to an embodiment of the present invention.
8 is a result of observing the proliferative response of T cells and B cells after separating the spleen cells of mice (C57BL / 6) orally administered the preparation examples C and D samples of the fungus granule tea according to an embodiment of the present invention .
Figure 9 shows the size of the cancer cells over time after injection subcutaneously injected with cancer cell line B16F0 (melanoma) in the mice (C57BL / 6) orally administered the sample C and D samples of the fungus granule tea according to an embodiment of the present invention Observed.
10 is a sacrifice of cancer cells after 21 days of injection of cancer cell line B16F0 (melanoma) subcutaneously in the mice (C57BL / 6) orally administered mice C and D samples prepared according to an embodiment of the present invention The figure shows the result of comparing size by measuring the size and weight.
Figure 11 shows the spleen cells three weeks after the subcutaneous injection of cancer cell line B16F0 (melanoma) to the mice (C57BL / 6) orally administered the preparation example C and D samples of the enoki mushroom granular tea according to an embodiment of the present invention The results of the isolation and observation of the proliferative response of T cells and B cells.
Figure 12 shows the spleen cells after 3 weeks after the injection of cancer cell line B16F0 (melanoma) in the mice (C57BL / 6) orally administered the preparation example C and D samples of the enoki mushroom granular tea according to an embodiment of the present invention This figure shows the result of measuring the concentration of cytokine after separation.
Figure 13 illustrates an example of a method for producing a fungus granule tea according to an embodiment 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 strain was obtained by preserving Flammulina velutipes Fv1-5 stored in the laboratory of Greenpeace Enoki Mushroom Farm in Cheongdo-gun, Gyeongbuk, and incubated in PDA (potato dextrose agar) plate medium and refrigerated for 4 ℃. Used. Waste herbal sludges were extracted by using Chinese medicine from the oriental medicine clinic in the city 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 the sterilization for 15 minutes to prepare a plate medium, the inoculated in the center of the plate medium prepared in the block-shaped mushrooms, and then incubated for 9 days at 25 ℃ incubator to observe the mycelial growth of the mushrooms. 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 It was very active, but the PDA flat medium showed slightly slower mycelial growth than 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 real mushroom cultivation farms, and then mixed well. 200 mm × 30 mm), sterilized at 121 ° C. for 30 minutes, and then uniformly inoculated 5 ml of each liquid spawn, such as the mushroom, green mushroom, and shiitake mushroom, prepared in advance and incubated in the upper portion of the medium 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, in the case of the enoki mushroom on the 6th day of culture, the hyphae grew about 2.2 ~ 3.4 cm in the control, but the hyphae growth was very strong in the test sphere (Fig. 3A and Bb) to which the lung herbal sludge was added. The mycelium grew to 6.4 cm, and the density of the mycelium was also grown very densely. As a result of observing the mycelial growth of rust mushrooms and shiitake mushrooms, the mycelial growth of rust mushrooms was 8.8 ~ 9.7 cm and mycelial growth of shiitake mushrooms was about 4 ~ 4.5 cm. 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, 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, corncope and beet pulp showed a weak acidity of about 4.9 ~ 5.4 but the pH of rice bran, cottonseed and bran, etc. showed a neutral degree of 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 Tea Preparation of Enoki Mushrooms Cultivated Using Waste Herb Sludge

2-1. Preparation of Concentrated Glycoside Glycosides

In order to preserve the intrinsic taste of the cultivated mushrooms using the waste herbal sludge prepared in Example 1, and to use as a main raw material, 70% (10v / v) ethanol was repeated three times at 80 ℃ Extracted by reflux for 5 hours, centrifuged (8,000rpm, 20 minutes), concentrated to twice the total capacity, and using the open column ion exchange resin (Daion HP-20) as a fixed phase, and using only water and alcohol as the mobile phase. Glycosides were isolated from the mushrooms, and the extract was concentrated and used as the main raw material.

2-2. Preparation and mixing of subsidiary materials

In consideration of the flavor and color of the final product, the enoki mushroom granular tea, and the injection aptitude of the granular form, first, the ingredients as shown in Table 3 were selected, prepared for each content, mixed, and then ground to 80 mesh sizes. The ground submaterial was mixed with the enoki mushroom glycoside.

Preparation Example A Preparation Example B Compounding ingredient content(%) Compounding ingredient content(%) Enoki Mushroom Glycoside 14.35 Enoki Mushroom Glycoside 11.98 oligosaccharide 56.71 Polytextloss 30.6 glucose 28.23 glucose 56.71 Vitamin C 0.71 Vitamin C 0.71 Sum 100 Sum 100 Preparation Example C Preparation Example D Compounding ingredient content(%) Compounding ingredient content(%) Enoki Mushroom Glycoside 5.0 Enoki Mushroom Glycoside 11.98 Skim milk powder 15.3 Polytextloss 15.3 glucose 78.99 glucose 72.01 Vitamin C 0.71 Vitamin C 0.71 Sum 100 Sum 100

2-3. Preparation of Enoki Mushroom Granulated Tea with Enoki Mushroom Glycoside Concentrate

After the granules were prepared by mixing the fungus glycosides concentrate and the component material prepared in Example 2-2, the granules were formed by using a reverse-rotating granule, dried to 5% or less (60 ° C., 5 days), and then separated into 15 20 mesh sizes. Packed in aluminum foil in the form of tea bags. Determination of the composition and ratio of the mushroom polysaccharide concentrate and the ingredient of the enoki mushroom was determined the final titer concentration through the mixing aptitude, the reverse granulation injection aptitude, the sensory evaluation.

Example  3: enoki mushroom Granular  Sensory test

A sensory test of five out of ten was performed on ten expert sensory panels for the fungus granule tea prepared in Example 2, and the results are shown in Table 4 below.

color incense Bad taste sweetness Overall flavor Preparation Example A 3.4 1.8 2.7 4.5 3.1 Preparation Example B 3.9 2.6 3.2 4.6 3.6 Preparation Example C 4.3 3.8 4.2 4.8 4.3 Preparation Example D 3.9 2.6 3.2 4.6 3.6

As can be seen in Table 4, the enoki mushroom granule tea (Preparation Example C) having a content of the enoki mushroom glycoside (5.0 wt%) was found to have the best taste, aroma and color, and then the content of the enoki mushroom glycoside was 11.98% by weight Enoki mushroom granule tea (Preparation Examples C and D) was found to have good taste, aroma and color, and the most common result was Enoki mushroom granule tea (Preparation Example A) which contains 14.35% wt. Indicated.

On the other hand, although not specifically described in the present embodiment, in the case of Preparation A was very difficult to formulate into granules due to the high viscosity in the granule manufacturing process. Therefore, the content and proportion of the enoki mushroom granule tea according to the present invention were excluded from Preparation Example A.

Example  4: prepared mushrooms Granular  Quality characteristic and stability investigation

As a result of the sensory test of Example 3 for the oyster mushroom granules prepared in Example 2, the quality characteristics of the oyster mushroom granules of Preparation Examples B to D except Preparation Example A were investigated. The results are shown in Table 5 below.

division Characteristics Flavor
Enoki mushroom content
Solubility
How to take
Packing unit Harmony of the unique taste and sweetness of the top mushroom
Preparation Examples B and D: 11.98 wt%, Preparation Example C: 5.0 wt%
60 ℃ within 10 seconds
3g / 1 day
3.0 g / bag

Example  5: top mushroom according to the present invention Granule tea  Immune Enhancement and Anticancer Activity

5-1. Materials and methods

1) Experimental animals

The experimental animals were supplied with specific pathogen free C57BL / 6 mice from Orient Bio Co., Ltd., and maintained at 5 densities per polysulfone cage (365 × 207 × 130mm) in the laboratory animal laboratory. These mice were used for experiments with 8-10-week-old mice, fed enough water and feed at room temperature for 2 weeks, and kept as stress-free as possible by adjusting the day and night cycles for 12 hours.

2) Test Sample Administration

In experiments in which the mushrooms were observed orally by administering an enoki mushroom granular tea (hereinafter referred to as a sample), the sample was diluted in 200 μl of PBS at 150 mg / kg / day, then orally administered once daily for 4 weeks, and the mice were sacrificed. In experiments that observed tumor suppression, the samples were diluted in 200 μl of PBS at 150 mg / kg / day, then orally administered once daily for 4 weeks, followed by subcutaneous injection of 3 × 10 ⁵ cells / mouse of B16F0 cells. Samples were continued at 150 mg / kg / day for 3 weeks.

3) Reagent

RPMI-1640, antibiotics (antibiotic-antimycotic), Fetal Bovine Serum (FBS) for cell culture, Hyclone products, 2-mecaptoethanol (2ME), sodium bicarbonate (NaHCO ₃ ), Lipopolysaccharide (LPS) Concanavalin A (Con A) used Sigma Chemical Co. (St. Louis, USA).

4) hematological analysis

Before sacrifice of mice, blood was collected by orbital bleeding and placed in an EDTA (Etylene diamineteraacetic acid) tube for anticoagulant treatment. Within 5 hours, the following items were measured with a hemocytometer (HemaVet, Drew, USA). White blood cell (WBC) and white blood cell composition (Neutrophil, NE, Lymphocyte, LY, Monocyte, MO, Eosinophil, EO, Basophil, BA), Red blood cell Red Blood Cell (RBC), Hemoglobin (Hb), Hematocits (Hct), Mean Red Blood Cells (Mean Corpuscular Volume, MCV) Mean Red Blood Cells (Mean Corpuscular Hemoglobin (MCH), Average Red Blood Cell Pigment Concentrations (Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet (PLT), Mean Platelet Volume (Mean Plasma Volume, MPV)) were measured.

5) Spleen Cell Proliferation Measurement

Spleens of mice (C57BL / 6) were isolated and single cell suspensions were prepared using tweezers. Single cell suspensions were washed three times with RPMI 1640 medium, diluted to a concentration of 5 × 10 ⁶ cells / ml, and then 100 μl per 96 wells were added. At this time, 1, 3, 10 ㎍ / ㎖ LPS or 0.1, 0.3, 1.0 ㎍ / ㎖ Con A was incubated for 48 hours in 37 ℃, 5% CO ₂ incubator. 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. That is, 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 to 8 hours, followed by 450 nm with a Microplate reader (Titertek Multiscan Plus, Finland). Absorbance was measured at.

6) Splenocyte cytokine assay

Splenocytes (5 × 10 ⁶ ) were treated with LPS (10 μg / ml) and Con A (1 μg / ml), and then incubated in 37 ° C. and 5% CO ₂ incubator for 24 hours. After incubation, the supernatant was collected and the amount of IL-2, IL-6, IFN-γ, and TNF-α contained in the supernatant was measured using an enzyme-linked immunosorbent assay (ELISA). In other words, dilute the primary antibody (2 ㎍ / ㎖) in the coating buffer (0.1 M NaHCO ₃ , pH8.2) in a plate-bottom micro-well and dispense 50 ㎕ / well, and overnight at 4 ℃, washing solution Washed with (0.05% Tween 20 / PBS). The washed micro-wells were blocked with PBS added with 10% FBS, and the culture supernatants collected in the experiment were diluted in an appropriate ratio and then dispensed into each well to react at room temperature. Then, 100 μl / well of the secondary antibody (1 μg / ml) to which biotin was attached was reacted at room temperature for a predetermined time, and then 100 μl / well of avidin-peroxidase (2.5 μg / ml) was added. Finally, the substrate (2, 2-azino-bis, 0.1 M citritic acid, H ₂ O ₂ ) was added and developed, and then the absorbance was measured at 405 nm using a micro-plate reader. The measured concentrations of IL-2, IL-6, IFN-γ and TNF-α were converted using a standard curve and the limit of the measured concentration was 10 pg / ml.

7) Cell surface molecule analysis

Thymic and splenocytes (5 × 10 ⁵ ) of mice were collected and blocked with anti-CD16 / CD32 (FcγIII / II Receptor) mAb at 4 ° C. for 30 minutes and washed with washing solution (1% FBS0.1% NaN ₃ / PBS). PE-conjugated anti-CD8 mAb, FITC-conjurated anti-CD4, and CD-19 mAb were stained at 4 ° C. for 30 minutes. The stained cells were washed again with washing solution, suspended in phosphate buffer solution, and analyzed by flow cytometry.

8) Cytotoxicity of Natural Killer Cells and Cytotoxic T Cells

Cultured YAC-1 cells and B16F0 cells were harvested and washed twice with washing medium. The washed two cells were suspended in 4 × 10 ⁵ cells / ml in 5% FBS RPMI medium and dispensed at 50 μl / well in a 96-well U bottom plate. 41-2 was added to 37 ℃, 5% CO ₂ incubator such that: the cells are suspended in the RBC is removed, the dispensing well splenocytes in 5% FBS RPMI medium effect cells and the ratio of target cells to 50: 1 or 100 After incubation for 24 hours, 50 g of the supernatant was collected by spin down at 250 g, 25 ° C for 4 minutes. The collected supernatant was measured by the cytotoxic ability of the cancer cells in ^® Non-Radioactive Cytotoxicity Assay nature CytoTox96 killed in the Kit by splenocytes measuring LDH using (promega, USA) cells and cytotoxic T cells.

[Experimental formula]

9) B16F0 Tumor Size and Weight Measurements

Tumor size was measured once daily using Digimatic caliper (Mitutoyo, Japan) from the 11th day after tumor formation after B16F0 cell administration. Photographed.

5-2. Immunopotentiation Activity Assay

1) Hematological analysis

Hematological analysis after oral administration once daily for 4 weeks is shown in Table 6 below. As can be seen in Table 6, there was no significant change in most items, but the number of neutrophils and lymphocytes (lymphocytes) in the experimental group to which the oyster mushroom granule tea (Preparation C and D) samples of the present invention were administered compared to the control group. There was a slight increase, but there was no statistical significance.

2) Splenocyte proliferation

After oral administration once daily for 4 weeks, splenocytes were isolated and stimulated with Con A and B cell proliferation inducers, LPS, for 48 hours, followed by incubation for 48 hours. And FIG. 7 (3 repetitions, mean ± standard deviation). As can be seen in Table 7 and FIG. 7, the proliferative response was found to be slightly lower in the experimental group to which the inventive mushroom fungus granule tea (preparation examples C and D) of the present invention was administered compared to the control group, but there was no statistical significance.

3) Spleen Cell Cytokine Measurement

After oral administration once daily for 4 weeks, splenocytes were isolated and stimulated with Con A and B cell proliferation inducer, LPS, for 24 hours, followed by culture supernatant to collect IL-2, The concentrations of IL-6, IFN-γ, and TNF-α were measured (3 replicates, mean standard deviation). The results are shown in Table 8 below. As can be seen in Table 8, the concentration of IFN-γ was slightly lower in the experimental group to which the present invention was administered the oyster mushroom granule tea (Preparation C and D) compared to the control group, but there was no statistical significance.

4) Surface molecule analysis of splenocytes

After oral administration once daily for 4 weeks, splenocytes were isolated to helper T cells (CD4 ⁺ ), cytotoxic T cells (CD8 ⁺ ), B cells (B cells, CD19 ⁺ ), In order to determine the number of natural killer cells (NK1.1 +), as a result of staining with an antibody to the specific protein molecules of each cell, as shown in the present invention, compared to the control group, the enoki mushroom granular tea (Preparation C And D) there was no significant statistical difference in the number of cells in the experimental group administered the sample (see Table 9).

5) Thymic Cell Proliferation

After oral administration once daily for 4 weeks, the number of thymus cells was determined by separating the thymus, and the number of thymus cells in the experimental group administered with the sample of the present invention was obtained. Significantly increased (see Table 10).

6) Analysis of surface molecules of thymic cells

Four weeks once daily after oral administration, a double speech by removing the thymocytes in the thymus, T-cell undifferentiated (CD4 ^- CD8 ^-) or a double positive (CD4 ⁺ CD8 ⁺⁾ T cells and helper T cells are differentiated over ( helper T cell, CD4 ⁺ CD8 ^-) or cytotoxic T cells (cytotoxic T cell, CD4 ^- in order to examine the percentage of CD8 ^+), and stained with antibodies as measured results for specific protein molecules in each cell, the control group In comparison, there was no significant statistical difference in the ratio of each cell in the experimental group to which the present invention was administered the enoki mushroom granule tea (Preparation Examples C and D) (see Table 11).

7) Cytotoxicity of Natural Killer Cells and Cytotoxic T Cells

After oral administration once daily for 4 weeks, to determine the cytotoxic activity of the natural killer cells (NK1.1 +) contained in the splenocytes and to the cancer cells of the spleen cells, cancer cell line YAC- As a result of measuring the cytotoxicity against 1, there was no significant statistical difference in the cytotoxic activity of natural killer cells in the experimental group to which the present invention obtained the present invention mushroom granule tea (Preparation C and D) compared to the control group (Table 12 Reference).

8) Differentiation ability into dendritic cells

After oral administration once daily for 4 weeks, in order to determine the differentiation ability of the bone marrow cells into dendritic cells, IL-4 and GM-CSF were added to the isolated bone marrow cells to induce differentiation into dendritic cells. As a result of measuring the number of colonies of the cells, there was no significant statistical difference in the differentiation ability of dendritic cells into cells in the experimental group to which the present invention was obtained. ).

9) Comprehensive Results

As can be seen from the above experimental results, the results of the administration of the enoki mushroom granular tea sample of the present invention to mice once daily for 4 weeks showed no statistically significant change in various experimental items, thereby activating the immune function. Significant effects could not be confirmed, but there was no significant toxic effect from long-term administration.

5-3. Anticancer Effect Analysis

As a result of experiments by subcutaneous injection of cancer cells after the administration of the granulated oyster mushroom tea (Preparation Examples C and D) according to the present invention, the effect of inhibiting the size and weight of tumor cells, cytokines IL-2, IL-6, IFN -γ, increase the secretion of TNF-α, increase the number of natural killer cells (NK1.1 +), and cytotoxic activity of natural killer cells and antigen-specific cytotoxic T cells The increasing effect was observed to be statistically significant. More specific results are as follows.

1) Results of tumor suppression experiment

After the injection of cancer cell line B16F0 (melanoma) subcutaneously, the size of the cancer cells was measured and compared daily. As a result, the control group significantly increased the size of the cancer cells over time. C and D) in the experimental group to which the sample was administered, the size increase rate of the cancer cells was significantly decreased compared to the control group (see Table 14 and FIG. 8).

In addition, 21 days after subcutaneous injection of the cancer cell line B16F0 (melanoma), the mice were sacrificed and the cancer cells were isolated and compared. As a result, compared to the control group, the enoki mushroom granule tea (Preparation C and D) was compared. In the experimental group to which the sample was administered, the size and weight of the cancer cells were statistically significantly reduced (see Table 15 and FIG. 9).

2) Induction of proliferation of T cells and B cells

The spleen cells were isolated and stimulated with Con A and B cell proliferation inducers, LPS, and cultured for 48 hours, and then observed for proliferation. C and D) There was no statistical difference in proliferative responses in the experimental group administered the samples (see Table 16 and FIG. 10). These data are the results of three replicates and mean ± standard deviation.

3) Measurement of cytokines in splenocytes

The splenocytes were isolated and stimulated with Con A, a T cell proliferation inducer, and LPS, a B cell proliferation inducer, and cultured for 24 hours, followed by harvesting the culture supernatant, IL-2, IL-6, IFN-γ, and TNF-. The concentration of α was measured. The results are shown in Table 17 and FIG.

As can be seen in Figure 11, IL-2 and IFN-γ was statistically significantly increased in the experimental group administered the sample of Preparation Example C according to the present invention compared to the control group, IL-6, TNF-α The concentration was significantly increased in the experimental group administered the samples of Preparation C and D, and the experimental group administered the sample of Preparation D to the experimental group administered the sample of Preparation C.

4) Surface molecule analysis of splenocytes

Splenocytes were isolated from helper T cells (CD4 ⁺ ), cytotoxic T cells (CD8 ⁺ ), B cells (B cells, CD19 ⁺ ), natural killer cells (NK1). In order to determine the number of 1+), the result was measured by staining with an antibody to a specific protein molecule of each cell, natural killer cells in the experimental group administered the sample of Preparation D according to the present invention compared to the control group , NK1.1 +) showed a statistically significant increase (see Table 18. * means p <0.01 compared to control).

5) Cytotoxicity

To isolate the splenocytes and determine their cytotoxic activity against cancer cells of natural killer cells (NK1.1 +) and antigen-specific cytotoxic T cells (CD8 ⁺ T cells) contained in splenocytes, As a result of measuring cytotoxicity against the cancer cell line YAC-1 or B16F0, when the ratio of the effect cell and the target cell (E: T ratio) is 50: 1, the samples of Preparation Examples C and D according to the present invention compared to the control group Cytotoxic activity of natural killer cells and antigen-specific cytotoxic T cells in the experimental group was significantly increased (see Table 19 and Figure 12, * means p <0.01 compared to the control).

Claims (9)

Enoki mushroom granulated tea characterized in that the spent herbal sludge contains 4 to 12% by weight of the glycoside concentrate of the enoki mushroom cultured in a medium containing 5 to 15% by weight, based on the dry weight of the mushroom fungus culture medium. The method of claim 1, wherein the spent herbal sludge has a particle size of 50 to 150 mesh, a pH of 6.5 to 6.8, the moisture content is 64 to 68% of the enoki mushroom granular tea. 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 Enoki mushroom granules, characterized in that the remaining residue after. The method according to claim 1, wherein the granular tea is an enoki mushroom granular tea further comprising glucose, vitamin C, and polydextrose or skim milk powder. Preparing glycoside glycoside concentrate by separating the glycoside from the extract of the enoki mushroom cultured in the medium containing 5 to 15% by weight based on the dry weight of the mushroom fungus culture medium for waste herbal sludge;
Mixing a sweetener to the glycoside concentrate of the prepared mushrooms;
Shaping the mixture of glycoside concentrate and sweetener of the enoki mushroom into granules;
Drying the molded granules and then sieving them to a size of 15 to 20 mesh; And
Packing the sifted granules; Method for producing a fungus granules tea comprising a. The method of claim 5, wherein the waste herbal sludge has a particle size of 50 to 150 mesh, a pH of 6.5 to 6.8, and a moisture content of 64 to 68%. The method of claim 5, wherein the Chinese herbal medicine sludge is a Chinese medicine extract from one or more selected from the group consisting of golden, thorny goose, tofu, Korean herb, cheongung, astragalus, peony, baekryeok, fukryeong, sagejiku, cinnamon, licorice and brown root Method for producing a top mushroom granule tea, characterized in that the remaining residue after. 6. The method of claim 5, wherein the mixture contains 4-12% by weight of the glycoside concentrate of the enoki mushroom in the total weight of the entire mixture. The method of claim 5, wherein the sweetener is glucose, vitamin C, and polydextrose or skim milk powder.

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