KR20200095208A - Co-culture method of mushroom mycelium - Google Patents

Abstract

The present invention relates to a method for culturing mushroom mycelium comprising a step of mixing a composition containing a mushroom extract with mycelium of Sparassis crispa, Grifola frondosa, and Phellinus linteus and culturing the same thereafter. Using the culture method of mushroom mycelium, the mycelium mass and polysaccharide recovery rate can be significantly increased.

Description

Mixture culture method of mushroom mycelium {Co-culture method of mushroom mycelium}

The present invention relates to a method for mixing mushroom mycelium.

Basidiomycetes are mushrooms that are attracting the most attention as mushrooms that produce natural products that are effective in treating and preventing various diseases. Basidiomycete mushroom absorbs substances from organic substances containing various nutrients to produce various nutrients important to the human body such as proteins, amino acids, enzymes, vitamins, inorganic salts, fats and sugars, and controls a wide range of pharmacological actions. It has been widely used as a raw material.

In order to use effective pharmacological ingredients from basidiomycete as food or medicine, the method of collecting or cultivating fruiting bodies and using the fruiting bodies as materials, and the method of separating the mycelium from the fruiting bodies and culturing the mycelium to extract the active ingredient from the cultured mycelium are used. do. However, when wild fruiting bodies are collected and used, there is an environmental problem that biological resources are depleted and the ecosystem is destroyed, and there is a problem that it is difficult to obtain the necessary amount of fruiting bodies in the case of rare basidiomycete. In addition, when obtaining fruiting bodies by cultivation methods using sawdust, special cultivation facilities are required, and due to high cost, fruiting bodies can be produced only for a certain period of time, and the cultivation period also takes 3 to 6 months. There is this. On the other hand, the method of separating and culturing the mycelium from the fruiting body can solve the above problems at once, but it is difficult to purely separate the mycelium, the recovery rate of the polysaccharide is low, and the culture conditions of the isolated mycelium are difficult compared to bacteria, etc. Since the growth of the isolated mycelium was slow, it was difficult to establish facilities and culture conditions for cultivation in a fermenter without contamination of various bacteria, and thus it was not generalized.

Recently, a culture method capable of mass-producing basidiomycete mycelium has been actively studied. Korean Patent Registration No. 10-1301512 discloses a medium composition for culturing Pseudomonas mushroom mycelium containing peptone, malt extract, agar and glucose as active ingredients, and a method for culturing Pseudomonas mushroom mycelium using the same. In addition, Republic of Korea Patent No. 10-0706132 is the cultivation of Pellinus linteus mycelium containing GLM (glucose monohydrate and mannose), CSP (corn leach powder) or CSL (corn leach solution), yeast extract, soy peptone, malt extract and distilled water. Disclosed are a medium composition for use and a method of culturing a Pellitus linteus strain using the same. In addition, Republic of Korea Patent Registration No. 10-1865292 shows that the amount of mycelium and polysaccharide of Pellinus linteus cultivated in a medium containing a Pellitus linteus extract, a Maitake mushroom extract, or a flower mushroom extract was significantly increased compared to the case of culturing in a conventional medium. Disclosing the information.

On the other hand, Phellinus Linteus (Phellinus Linteus) or Pseudomonas Mushroom is a mushroom belonging to the Aphyllphorales and Pineapples family (Hymenochaetaceae), and is a basidiomycete mushroom that grows naturally in old trees such as mountain mulberry, oak, chestnut or oak. to be. Pelinus linteus has compounds showing various physiological activities such as antibiotics, antimutagenic substances, and anticholesterol substances in mushrooms, so it is widely studied not only for food but also as a development material for pharmaceuticals. Pellitus linteus or Pseudomonas mushrooms (Mulberry serrata) are known to contain the most protein polysaccharides with immune enhancing functions. Since 1968, the National Cancer Institute of Japan announced that the antitumor activity of Pseudomonas mushrooms reached 96.7% and showed the highest activity among edible mushrooms and medicinal mushrooms, it has been the subject of much interest, and as various physiologically active functions were revealed, fruiting bodies and mycelium Research on a method that can be mass cultured is being actively conducted.

Grifola frondosa is a mushroom that belongs to the Democratic Mushroom Order and Polyporaceae. It occurs in clusters around the branches and roots of oaks, broadleaf trees and dead trees in summer and autumn. , It is a basidiomycete mushroom distributed in Europe and the United States. Maitake mushroom is a functional mushroom that is edible and has excellent pharmacological action. It is known to be effective in inhibiting cancer by increasing the immunity of the human body, excellent tumor suppression rate, and increasing the activity of natural killer cells and macrophages.

Sparassis crispa (Sparassis crispa) is a fungus belonging to the order of Mushrooms, and the family of Sparassis mushrooms. It is a basidiomycete mushroom that grows naturally by gathering on the stems or stumps near the roots of living trees from summer to autumn. Blossom mushrooms are known to have effects such as enhancing immunity, suppressing hypertension, suppressing rise in blood sugar, improving blood circulation, and hematopoietic action.

Accordingly, in the present invention, while searching for a method for efficiently separating useful components from basidiomycete, when the mushroom mycelium is extracted from the mushroom mycelium culture mixture obtained through mixing culture of the three basidiomycete as described above, mushrooms The present invention was completed by confirming that the recovery rate of mycelium and polysaccharide can be significantly increased.

It is an object of the present invention to provide a method for culturing mushroom mycelium comprising the step of culturing by mixing and cultivating a composition containing a mushroom extract with Pellitus linteus, Maitake mushroom and Flower mushroom mycelium.

In order to achieve the above object, the present invention provides a method for culturing a mushroom mycelium comprising the step of culturing by mixing and culturing the mycelium of Pelinus linteus, Maitake mushroom and Flower mushroom in a composition containing a mushroom extract.

The present invention relates to a method for culturing a mushroom mycelium comprising the step of culturing a composition containing a mushroom extract by mixing and culturing the mycelium of Pelinus linteus, Maitake mushroom, and Mushroom mushroom. When using the method of culturing the mushroom mycelium of the present invention The mycelium mass and polysaccharide recovery rate can be significantly increased.

1 is a schematic diagram showing the process of mixing mushroom mycelium and extracting polysaccharides.
Figure 2 is a schematic view showing the manufacturing process of a complex mixture of mushroom and wild ginseng cultured root extract (Amex 7).
3 shows the effect of a complex mixture of mushroom and wild ginseng cultured root extracts on the size (A) and weight (B) of a tumor formed by subcutaneously inoculating human colon cancer cells into Balb/c nude mice.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for culturing a mushroom mycelium comprising the step of culturing by mixing and cultivating a composition containing a mushroom extract of Pellinus linteus, Maitake mushroom, and Flower mushroom mycelium.

The mushroom extract may be an extract of any one or more mushrooms selected from the group consisting of Pelinus linteus, Maitake mushroom, Flower mushroom, Chaga mushroom, Cordyceps sinensis, Cloud mushroom, Ganoderma lucidum mushroom, and Yellow oyster mushroom.

Pelinus linteus used to prepare the mushroom extract and Pelinus linteus used in cultivation may be those deposited under accession number KCTC0399BP. The strain is made of a fruiting body without a stipe and a horseshoe-shaped wood, and the pores may be formed in multiple layers. In addition, the cap is semicircular, flat, about 10 cm wide, and the surface may be dark brown and the back side may be yellowish brown to dark brown. Spores are spore-shaped, pale yellow, can be about 5 um in size, setae multiple, and can be thick-membranous.

Specifically, the mushroom extract may be a mycelium extract of mushrooms. The extract may be extracted using water, C1 to C4 lower alcohol or a mixture thereof as a solvent. In one embodiment of the present invention, the solvent may be water. The extraction solvent may be extracted by adding 0.5 to 10 times the weight of the culture.

As a method for extracting the extract, all conventional methods known in the art, such as reduced pressure high temperature extraction, hot water extraction, filtration method, hot water extraction, immersion extraction, reflux extraction, cold needle extraction, steam extraction, room temperature extraction, or ultrasonic extraction, can be used. I can. In one embodiment of the present invention, the extraction method may be hot water extraction. The extraction may be performed at a temperature of 80°C to 110°C, specifically 85°C to 105°C, and more specifically 90°C to 100°C. Meanwhile, the extraction may be performed for 1 to 8 hours, specifically 2 to 7 hours, more specifically 3 to 6 hours.

The extract extracted by the above method may be filtered and concentrated, and may be powdered using spray drying, freeze drying or hot air drying. The mushroom extract may be contained in an amount of 1 to 50% (w/v), specifically 3 to 40% (w/v), and more specifically 5 to 30% (w/v) with respect to the total medium composition. .

The composition may further include any one or more selected from the group consisting of a carbon source and a nitrogen source.

The carbon source is arabinose, fructose, galactose, glucose, mannose, rhamnose, xylose, lactose, maltose, raffinose, cellulose, cottonseed flour, dextrin, maltodextrin, starch, glycerol, sorbitol, mannitol and It may be any one or more selected from the group consisting of inositol. In one embodiment of the present invention, the carbon source may be glucose. The glucose may be included in an amount of 0.5 to 20% (w/v), specifically 1 to 15% (w/v), and more specifically 2 to 10% (w/v) with respect to the total medium composition.

The nitrogen sources are soypeptone, soybean meal, yeast extract, beef extract, tryptone, peptone, fish meal, urea, casamino acid, asparagine, corn steep liquor, N-Zamine, NH4NO3, NH4Cl, KNO3, NH4H2PO4, NaNO2, NaNO3, (NH4)2SO4, (NH4)2CO3, Ca(NO3)2, and may be any one or more selected from the group consisting of CH3COONH4. In one embodiment of the present invention, the nitrogen source may be any one or more selected from the group consisting of yeast extract and soypeptone. The yeast extract may be included in an amount of 0.05 to 5.0% (w/v), specifically 1.0 to 4.0% (w/v), and more specifically 2.5 to 3.5% (w/v) with respect to the total medium composition. The soypeptone may be included in an amount of 0.01 to 3.0% (w/v), specifically 0.05 to 2.0% (w/v), and more specifically 0.1 to 1.0% (w/v).

The method of culturing the mushroom mycelium may further include pre-culturing the mycelium of the mushroom before the step of culturing by mixing and culturing the composition containing the mushroom extract with the mycelium of Pelinus linteus, the Maitake mushroom, and the Mushroom mushroom. At this time, in the pre-cultivation step, Pelinus linteus, Maitake mushrooms, or flowering mushrooms are YMG (yeast malt dextrose) medium, YM (yeast malt peptone dextrose) medium, PIM (yeast soypeptone dextrose) medium, PSA (potato sucrose agar) medium, PDA (potato dextrose agar) medium, MEA (malt extract agar) medium, QEA (quercus extract agar) medium, PEA (poplar extract agar) medium, RBA (rice bran extract agar) medium, WBEA (wheat bran extract agar) medium, MCM (mushroom complete medium) medium, CDA (Czapedox agar) medium, SCA (Spawn complex agar) medium and OMA (oatmeal agar) medium can be cultured in any one or more medium selected from the group consisting of.

In a specific embodiment of the present invention, the strain of Pelinus linteus was pre-cultured in YMG medium, Maitake mushrooms were pre-cultured in YMG medium, and flower mushrooms were pre-cultured in PIM medium.

The mycelium is a mixture ratio of 30 to 70 parts by weight of Maitake mushrooms, 15 to 50 parts by weight of Pellitus linteus, and 15 to 50 parts by weight of flower mushrooms, 40 to 60 parts by weight of Maitake mushrooms, 20 to 40 parts by weight of Pellius linteus, and blossoms It may be characterized in that the mixing ratio of 20 to 40 parts by weight of mushrooms.

In a specific embodiment of the present invention, the present inventors prepared a medium in which a mixture of Pellinus linteus mycelium, Maitake mushroom mycelium, and flower mushroom mycelium culture medium was prepared, and hot water extraction was performed to prepare a mushroom mycelium extract (see Examples 1 to 3).

In a specific example of the present invention, it was confirmed that the final pH value of the composition containing the mixture of each mushroom mycelium extract was lower than the composition containing the single cultured mushroom mycelium extract (see Table 2).

In addition, it was confirmed that the mycelium amount was significantly increased in the composition containing the mixture of each mushroom mycelium extract than the composition containing the single cultured mushroom mycelium extract, and contained a mixture of each mushroom mycelium extract than the composition containing the single cultured mushroom mycelium extract. It was confirmed that the polysaccharide recovery rate was significantly increased in the composition, and the α-methyl-D(+)-glucoside content significantly increased in the composition containing the mixture of each mushroom mycelium extract than the composition containing the single cultured mushroom mycelium extract. Was confirmed (see Tables 3 to 5).

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited thereto.

Example 1. Preparation of mycelium culture medium of Pellinus linteus, Maitake mushroom and Flower mushroom

1-1. Preparation of the culture medium of Pellinus linteus mycelium

Pelinus linteus (accession number KCTC 0399BP) strain isolated from wild mushrooms was inoculated in YMG solid medium (Yeast extract 4, Malt extract 10, Dextrose 4, Agar 20g/L), and then incubated at a temperature of 29±1℃. Cultured in. A week later, a block was made in YMG solid medium (Yeast extract 4, Malt extract 10, Dextrose 4, Agar 20g/L) in which Pellinus linteus was cultured using a round cork borer (Korea Ace), and 200 mL of 10 to 20 blocks were aseptically inoculated into a 1 L baffle flask containing YMG liquid medium, and cultured for 24 hours in a shaking incubator at 29±1°C. 10% (w/v) glucose (Samyang's hydrated crystalline glucose, D2010), 1.6% (w/v) of corn leachate (Samyang's corn thickened immersion liquid, CLS) and 0.3% (w/v) of yeast extract (Bio Springer SA, NuCel 751MG) 3 L liquid medium containing pH 5.0 in a 5 L fermenter and sterilized, and then inoculated with Pellinus linteus strain cultured for 24 hours in the shaking incubator, and inoculated with 29±1° C. A culture solution was prepared by pre-culturing for 3 days (72 hours) under conditions of temperature, rotation speed of 250 rpm and air volume of 2.0 L/min.

1-2. Preparation of Maitake Mushroom Mycelium Culture Solution

The Maitake mushroom seed was inoculated on YMG solid medium and then cultured in an incubator at a temperature of 29±1°C. A week later, a block was made in YMG solid medium (Yeast extract 4, Malt extract 10, Dextrose 4, Agar 20g/L) in which the Maitake mushroom seed was cultured using a round cork borer (Korea Ace), and 200 ml of 10 to 20 blocks were aseptically inoculated into a 1 L baffle flask containing YMG liquid medium, and cultured for 3 days (72 hours) in a shaking incubator under a temperature condition of 29±1°C. 10% (w/v) glucose (Samyangsa hydrated crystalline glucose, D2010), 3.3% (w/v) yeast extract (Bio Springer SA, NuCel 751MG) and 1.0% (w/v) soypeptone (The Tatua Co-Operative Dairy Co. Ltd., HSP-349) pH 5.0 liquid medium containing 3 L of pH 5.0 in a 5 L fermenter and sterilized, and then cultured for 3 days (72 hours) in the shaking incubator. The seed was inoculated, and pre-cultured for 3 days (72 hours) at a temperature of 29±1° C., a rotation speed of 250 rpm, and an air volume of 2.0 L/min to prepare a culture solution.

1-3. Preparation of flower mushroom mycelium culture solution

Blossom mushroom seeds were inoculated in PIM solid medium (Yeast extract 15, Soy peptone 4.5, Dextrose 30, Agar 20g/L) and cultured in an incubator at a temperature of 29±1°C. A week later, a block was made in PIM solid medium (Yeast extract 15, Soy peptone 4.5, Dextrose 30, Agar 20g/L) in which the blossom mushroom spawn was cultured using a round cork borer (Korea Ace), and 200 mL of 10 to 20 blocks were aseptically inoculated into a 1 L baffle flask containing PIM liquid medium, and cultured for one day (24 hours) in a shaking incubator at a temperature of 29±1°C. Sterilized by putting 3 L of pH 5.0 liquid medium containing 10% (w/v) glucose, 3.3% (w/v) yeast extract and 1.0% (w/v) soypeptone in a 5 L fermentor After that, inoculate the blossom mushroom seeds cultured for one day (24 hours) in the shaking incubator, and before for 3 days (72 hours) under conditions of a temperature of 29±1℃, a rotation speed of 250 rpm, and an air volume of 2.0 L/min. A culture solution was prepared by culturing.

Example 2. Preparation of culture medium mixed with mushroom mycelium culture medium

Prepare a 30 L liquid medium of pH 5.0 containing 10% (w/v) glucose, 3.3% (w/v) yeast extract and 1.0% (w/v) soypeptone, and the liquid medium was 50 After sterilization by putting it in an L fermentor, the culture solutions prepared in Examples 1-1, 1-2, and 1-3 were mixed and inoculated in the following 6 ratios, and a temperature of 29±1° C. and rotation speed of 250 rpm And cultured for 3 days (72 hours) under the condition of an air volume of 2.0 L/min to prepare a mixed culture solution (mixed culture solution of mushroom mycelium culture solution).

A: Pelinus Linteus 3 L + Maitake 1 L + Flower Mushroom 1 L

B: Pelinus linteus 1 L + Maitake mushroom 3 L + Blossom mushroom 1 L

C: Pelinus linteus 1 L + Maitake mushroom 1 L + Flower mushroom 3 L

D: Pelinus linteus 2 L + Maitake mushroom 2 L + Flower mushroom 1 L

E: Pelinus linteus 2 L + Maitake mushroom 1 L + Blossom mushroom 2 L

F: Pelinus Linteus 1 L + Maitake 2 L + Flower Mushroom 2 L

Example 3. Preparation of mushroom mycelium extract

10 L of mushroom mycelium mixed and cultured in a 50 L fermenter by the method of Example 2 was filtered and washed 2 or 3 times with water, and 1 L of water was added to the filtered mycelium. The mycelium to which water was added was put in a flask, and hot water was extracted for 4 hours at 100°C in a water bath connected to a cooling tube. After cooling the mycelium hot water extract at room temperature, it is filtered twice using a filter paper and a 10-fold (w/v) concentrate obtained by evaporating purified water as a solvent using a rotary vacuum concentrator.Pellinus linteus mycelium, Maitake mushroom mycelium, and flower mushrooms A mixture of mycelium extract was prepared.

Example 4. Preparation of complex mixture of mushroom extract and wild ginseng cultured root extract

Cultured wild ginseng root (Hans Biotech, Lot. No. G8060461) was extracted with purified water at 90°C for 3 hours, and the resulting product was filtered with a pore size of 150 μm to obtain a wild ginseng cultured root extract. The wild ginseng cultured root extract was used as phytosanitary mushroom (Hans Biotech, Lot.No.M7050952), shiitake (HK Biotech, 30180322LEP), fingering mushroom (Hans Biotech, Lot.No.H7121952), cordyceps (Hans Biotech, Lot.No.C7061352). ), flower mushroom (Hans Biotech, Lot.No.K8052451), roe deer mushroom (Hans Biotech, Lot.No.L6110252) and Maitake mushroom (Hans Biotech, Lot.No.B7080252) extract powder and 200 rpm for 20 minutes. It was mixed at a rotational speed of to obtain a mixture. The mixture was filtered with a pore size of 5 μm and sterilized at 98° C. for 10 minutes to prepare a complex mixture of mushroom extract and wild ginseng cultured root extract (Amex 7, Korea New Drug).

Figure 2 is a schematic diagram showing the manufacturing process of the complex mixture of mushroom and wild ginseng cultured root extract (Amex 7), Table 1 shows the constituents and ratios of the complex mixture of the mushroom extract and wild ginseng cultured root extract.

compound Mixing ratio (%) Volume (ml) Mushroom 1.340 1.005 Shiitake mushrooms 0.400 0.300 Fingering mushroom 0.400 0.300 Cordyceps 0.400 0.300 Flower mushroom 0.400 0.300 Roe deer mushroom 0.400 0.300 Maitake Mushroom 0.400 0.300 Wild Ginseng Cultured Root 0.130 0.098 Purified water 81.464 61.098 etc 14.666 10.999 gun 100.000 75.000

Experimental Example 1. Analysis of pH of the medium according to the mixing ratio of mushroom mycelium

In the process of mixing and culturing the mycelium of Pelinus linteus, Maitake mushroom, and Flower mushroom by the method of Example 2, the pH change of the culture medium according to the mixing ratio of the mycelium of Pelinus linteus, Maitake mushroom, and Flower mushroom was measured using a pH meter.

Badge condition Initial pH Final pH Pelinus linteus control 5.0 5.21 Maitake control 5.0 5.35 Flower mushroom control 5.0 5.33 A (3 Fell + 1 Leaf + 1 Flower) 5.0 4.60 B (1 Pel + 3 Leaf + 1 Flower) 5.0 5.14 C (1 Pel + 1 Leaf + 3 Flower) 5.0 5.19 D (Pel 2 + Leaf 2 + Flower 1) 5.0 4.72 E (2 Fell + 1 Leaf + 2 Flower) 5.0 4.81 F (Pel 1 + Leaf 2 + Flower 2) 5.0 5.20

As a result, as shown in Table 2, the final pH value of the composition containing the mixture of each mushroom mycelium extract was lower than the composition containing the single-cultured mushroom mycelium extract. In particular, the pH value of the culture medium in the A and D conditions where the mixing ratio of Pellinus linteus was high was significantly lower than that of other conditions.

Experimental Example 2. Analysis of mycelium mass according to the mixing ratio of mushroom mycelium

The amount of mycelium according to the mixing ratio of Pelinus linteus, Maitake mushroom and flower mushroom mycelium when mixed and cultured with the method of Example 2 was measured using the following mycelial dry weight method.

Specifically, 1 L was obtained after uniformly mixing the mixed culture solution of the Pellitus linteus, Maitake mushroom, and flower mushroom mycelium culture solution prepared by the method of Example 2, filtered and washed three times with water, and then at 60° C. for 12 hours. After drying, the weight was measured and expressed as the hyphae dry weight per 1 L.

Badge condition Mycelial dry weight (g/L) Pelinus linteus control 9.7 Maitake control 10.5 Flower mushroom control 8.9 A (3 Fell + 1 Leaf + 1 Flower) 16.1 B (1 Pel + 3 Leaf + 1 Flower) 17.5 C (1 Pel + 1 Leaf + 3 Flower) 14.9 D (Pel 2 + Leaf 2 + Flower 1) 17.0 E (2 Fell + 1 Leaf + 2 Flower) 15.4 F (Pel 1 + Leaf 2 + Flower 2) 16.2

As a result, as shown in Table 3, the mycelium amount was significantly increased in the composition containing the mixture of each mushroom mycelium extract than the composition containing the single-cultured mushroom mycelium extract. In particular, when cultured under the condition of B, which had the highest mix ratio of Maitake mushrooms, the mycelium amount was highest, and then, even in the conditions of D and F, where the mixing ratio of Maitake mushrooms was high, the amount of mycelium was higher than when cultured under other conditions.

Experimental Example 3. Analysis of polysaccharide recovery rate according to the mixing ratio of mushroom mycelium

The polysaccharide recovery rate was analyzed according to the mixing ratio of Pelinus linteus, Maitake mushroom, and Flower mushroom mycelium in the mixture culture of Pellitus linteus, Maitake mushroom, and Flower mushroom mycelium by the method of Example 2.

Specifically, cultures of Pellinus linteus, Maitake mushrooms, and zinnia mushrooms were obtained by the method of Example 2, mixed uniformly, and an extract was obtained by the method of Example 3. To the concentrated mycelium hot water extract, 2 to 4 times the amount of ethanol compared to the extract was slowly added and precipitated while stirring, and left at 4°C for 12 hours. Thereafter, the precipitate was separated by centrifugation for 20 minutes at a rotation speed of 10,000 rpm. The separated precipitate was recovered and dissolved in water, and then lyophilized to recover the polysaccharide, and the amount was measured to analyze the recovery rate. The polysaccharide recovery rate was expressed as a ratio of the amount of recovered polysaccharide to the weight of 1 L of mycelium.

Badge condition Polysaccharide recovery rate (%) Pelinus linteus control 6.4 Maitake control 7.0 Flower mushroom control 5.5 A (3 Fell + 1 Leaf + 1 Flower) 8.3 B (1 Pel + 3 Leaf + 1 Flower) 10.2 C (1 Pel + 1 Leaf + 3 Flower) 7.2 D (Pel 2 + Leaf 2 + Flower 1) 9.5 E (2 Fell + 1 Leaf + 2 Flower) 8.1 F (Pel 1 + Leaf 2 + Flower 2) 8.5

As a result, as shown in Table 4, the polysaccharide recovery rate was significantly increased in the composition containing the mixture of each mushroom mycelium extract than the composition containing the single-cultured mushroom mycelium extract. In particular, when cultured under the condition of B, which has the highest mixing ratio of Maitake mushrooms, the polysaccharide recovery rate was maximized, and even under the conditions of D and F, where the mixing ratio of Maitake mushrooms was high, the polysaccharide recovery rate was higher than when cultured under other conditions. It was high.

Experimental Example 4. Analysis of α-methyl-D(+)-glucoside content according to mixing ratio of mushroom mycelium

The content of α-methyl-D(+)-glucoside among the components of the polysaccharide recovered by the method of Experimental Example 3 was measured by gas chromatography (Shimadzu GC-9A).

Specifically, 100 mg of the polysaccharide recovered by the method of Experimental Example 3 was put into a 20 mL flask, and then 2 mL of methanolic 0.75 N hydrochloric acid solution (SAMCHUN, 072318) was additionally added. The flask was placed in a water bath at a temperature of 80±5° C. equipped with a cooler and reacted for 24 hours. The reaction solution was cooled and filtered at room temperature, and the filtrate was concentrated under reduced pressure at 60°C. 2 mL of methanol was added to the residue concentrated under reduced pressure to completely dissolve, and then concentrated under reduced pressure at 60°C. 1 mL of anhydrous pyridine (Wako, 166-05316) was added to the residue concentrated under reduced pressure to dissolve, and 0.1 mL of TMCS (trimethylsilyl chloride) (Sigma, 386529) and 0.2 mL of HMDS (hexamethyldisilazane) (Sigma, 52619) were added. After vigorous shaking for 3 minutes, 1 mL of n-hexane (Sigma, 296090) was added to prepare a sample solution. In addition, 1 mL of anhydrous pyridine (Wako, 166-05316) was added to 20 mg of α-methyl-D(+)-glucoside (Wako, 139-02542) to dissolve, and 0.1 mL of TMCS (Sigma, 386529) and After adding 0.2 mL of HMDS (Sigma, 52619) and shaking vigorously for 3 minutes, 1 mL of n-hexane (Sigma, 296090) was added thereto to prepare a standard solution. Gas chromatography was performed using the sample solution and standard solution prepared by the above method. The sample solution and standard solution were injected in an amount of 5 uL, and a DB-17 Capillary Column (Agilent Technologies, 122-1732) and a hydrogen flame ionization detector (SHIMADZU, GC-2010) were used. The temperature of the column was increased by 2.5°C/min from 160°C to 200°C, and the final temperature was 220°C, the injection temperature was 200°C, and the detector temperature was 220°C. Helium was used as the carrier gas, and it was flowed at a flow rate of 30 ml/min. When analyzed according to the test conditions, using the peak area between RT 13.5 to 13.6 and the amount of standard α-methyl-D(+)-glucoside, Pellinus linteus, Maitake mushroom and The content of α-methyl-D(+)-glucoside, a polysaccharide component contained in flower mushrooms, was analyzed.

Badge condition α-methyl-D(+)-glucoside content (mg/g) Pelinus linteus control 38.9 Maitake control 43.5 Flower mushroom control 36.7 A (3 Fell + 1 Leaf + 1 Flower) 57.3 B (1 Pel + 3 Leaf + 1 Flower) 68.2 C (1 Pel + 1 Leaf + 3 Flower) 51.8 D (Pel 2 + Leaf 2 + Flower 1) 62.5 E (2 Fell + 1 Leaf + 2 Flower) 53.5 F (Pel 1 + Leaf 2 + Flower 2) 58.4

As a result, as shown in Table 5, α-methyl-D(+)-glucoside content was significantly increased in the composition containing the mixture of each mushroom mycelium extract than the composition containing the single cultured mushroom mycelium extract. In particular, when cultured under the condition of B, where the mixing ratio of Maitake mushrooms was the highest, the α-methyl-D(+)-glucoside content was highest. The content of α-methyl-D(+)-glucoside was higher than that of cultured under conditions.

Experimental Example 5. Anticancer efficacy of complex mixture of mushroom and wild ginseng cultured root extract

The anticancer efficacy of the complex mixture of mushroom and wild ginseng cultured root extract prepared by the method of Example 4 was confirmed by the following method.

Specifically, human colon cancer cells HT-29 (Korea Cell Bank) were used in 10% FBS (Biowest, S1520-500), 100 u/mL penicillin (WELGENE, LS 202-02), and 100 ug/mL streptomycin (WELGENE, LS 202-02) in RPMI-1640 medium (WELGENE, LM 011-01) supplemented with 37° C., 5% CO ₂ conditions, and cultured HT-29 cells 3×10 ⁶ Balb/c of 5-week-old male The nude mice (Nara Biotech) were inoculated subcutaneously on the right hind limb. A complex mixture of the mushroom extract and wild ginseng cultured root extract prepared by the method of Example 4 from when the tumor reached a size of about 180-200 mm ³ until the average tumor size of the control group reached about 2,000 mm ³ (control: Distilled water, 1.25 mL/kg, 6.25 mL/kg, or 12.5 mL/kg) was administered twice a day, and then the size of the tumor and the weight of the tumor were measured.

As a result, as shown in Figure 3, the complex mixture of mushroom and wild ginseng cultured root extract significantly reduced the growth of tumors formed by xenograft colon cancer cells.

Claims (10)

A method of culturing mushroom mycelium comprising the step of culturing by mixing and culturing a composition containing a mushroom extract with Felinus linteus, Maitake mushroom and Flower mushroom mycelium.
The mushroom mycelium of claim 1, wherein the mushroom extract is an extract of any one or more mushrooms selected from the group consisting of Pelinus linteus, Maitake mushrooms, Flower mushrooms, Chaga mushrooms, Cordyceps sinensis, Cloud mushrooms, Ganoderma lucidum mushrooms and Yellow oyster mushrooms. Cultivation method.
The method of claim 1, wherein the mushroom extract is a mushroom mycelium extract.
The method of claim 3, wherein the extract is extracted with water, a lower alcohol of C1 to C4, or a mixture thereof.
The method according to claim 2, wherein the Pellitus linteus is deposited with accession number KCTC0399BP.
The method of claim 1, wherein the composition further comprises any one or more selected from the group consisting of a carbon source and a nitrogen source.
The method of claim 6, wherein the carbon source is glucose.
The method of claim 6, wherein the nitrogen source is any one or more selected from the group consisting of corn steep liquor, soy peptone and yeast extract.
The method of claim 1, further comprising the step of pre-culturing the mushroom mycelium prior to the step of culturing by mixing and culturing the composition containing the mushroom extract with Pellitus linteus, Maitake mushroom and flowering mushroom mycelium, cultivation of mushroom mycelium Way.
The method of claim 1, wherein the mycelium is a mixture ratio of 30 to 70 parts by weight of Maitake Mushrooms, 15 to 50 parts by weight of Pelinus Linteus, and 15 to 50 parts by weight of Flower Mushrooms.

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