KR102350979B1 - Medium Composition for Culturing Mushroom Mycelium, Culturing Media using it and the Manufacturing Method Thereof - Google Patents

Abstract

The present invention relates to a garlic concentrate for culturing mushroom mycelium and a mushroom mycelium culture using the same. It is possible to provide a mushroom mycelium culture medium having a high content of phenolic compounds and flavonoids and excellent antioxidant activity.

Description

Medium Composition for Culturing Mushroom Mycelium, Mushroom Mycelium Culture Using Same, and Method for Manufacturing the Same

The present invention relates to a medium composition for culturing mushroom mycelium, a mushroom mycelium culture solution using the same, and a method for producing the same, and more particularly, a medium composition for culturing a mushroom mycelium having a high beta-glucan content using only a garlic concentrate without additional materials, and It relates to a mushroom mycelium culture solution using the same, and a method for manufacturing the same.

Mushrooms have been traditionally eaten because of their unique taste and flavor as a high-quality food containing nutrients including proteins, lipids, carbohydrates, minerals, amino acids and vitamins evenly. As this is identified, interest in it is growing.

Recently, it is emerging as a new functional food material that can greatly contribute to the prevention of various adult diseases such as cancer, circulatory disease, and diabetes using various functional substances contained in mushrooms as well as useful ingredients in terms of food.

Recently, interest in mushrooms is increasing in beta-glucan (β-D-glucan). Beta-glucan is a type of polysaccharide that exists in large amounts in the cell wall of mushrooms, yeast, grains, etc., and forms a major structural component of the cell wall. .

Beta-glucan does not directly attack cancer cells, but activates the immune function of normal human cells through a non-specific immune response, inhibits the proliferation and recurrence of cancer cells, has excellent blood sugar lowering and blood cholesterol reduction effects, and improves lipid metabolism to promote body fat formation and It is reported to have an anti-obesity effect by inhibiting the accumulation.

On the other hand, garlic ( Allium sativum L.) is one of the major spice vegetables consumed the most by Koreans. It contains not only simple seasoning but also various physiologically functional ingredients, which play an important role in protecting the health of the people.

As important physiological activities of garlic, antibacterial, anticancer, antiviral, antioxidant, immune enhancement, blood clotting inhibition, liver function recovery, blood sugar reduction effect, hyperlipidemia and arteriosclerosis improvement, brain function improvement, etc. are known.

Mushrooms and garlic are currently one of the most popular functional materials worldwide. However, each of these products has a commercialization limit as a single product, and commercialization in consideration of the synergistic effect has hardly been made.

Application No. 10-2003-0042746 utilizes garlic for mushroom cultivation by disclosing a two-step method for culturing Heurica oleracea using a garlic extract and a brain function improving agent containing the culture product. According to this, the first step discloses a method of culturing the mycelium using an optimal mycelium culture medium and continuing culturing by adding a garlic extract to the second step.

According to the patent, the growth of mycelium is fast by liquid culture of the mycelium using the optimal medium in the first step culture, but the garlic extract added in the second step culture is extracted by adding 50 times water to 200 g garlic, There are disadvantages in that many components are required, the culture process is complicated, and when garlic extract is added to the mycelium culture medium by 20% or more, the mycelium size is rather reduced.

Therefore, when preparing an extract using raw garlic, it is expected that the mycelium growth will be inhibited due to various sulfur compounds such as allin and allicin remaining in the garlic extract. There is a need for research and development on a medium composition for culturing mushroom mycelium with excellent growth of mushroom mycelium and a mushroom mycelium culture solution using the same.

Therefore, the present invention provides a medium composition for culturing mushroom mycelium having excellent mushroom growth and high free sugar content by removing volatile substances that inhibit the growth of mushroom mycelium while still being able to utilize the active ingredient of the garlic concentrate that has undergone the extraction and concentration process and An object of the present invention is to provide a method for manufacturing the same.

In addition, the present invention makes it possible to culture mushroom mycelium only with a simplified garlic concentrate without a separate culture medium, and utilizes a culture medium containing garlic as a main component as a medium for mushroom mycelium to mix the two materials of garlic and mushroom. An object of the present invention is to provide a mushroom mycelium culture solution that can be used in the material industry and a method for manufacturing the same.

According to one aspect of the present invention, the present invention provides a step of preparing a garlic extract by adding 2.5 to 4 times the weight of raw garlic to raw garlic and then extracting it at 110 to 120° C. for 7 to 9 hours, and the garlic extract 100 It provides a method for preparing a medium composition for culturing mushroom mycelium, comprising the step of obtaining a garlic concentrate by vacuum concentration at ~120° C. for 7 to 9 hours, and a medium composition for culturing mushroom mycelium prepared by this method.

Preferably, the concentration of sugar in the garlic concentrate is 20.0-25.0 brix.

According to another aspect of the present invention, a step of preparing a garlic extract by adding 2.5 to 4 times the weight of raw garlic to raw garlic and extracting it at 110 to 120° C. for 7 to 9 hours, wherein the garlic extract is prepared at 100 to 120° C. A method for preparing a mushroom mycelium culture solution comprising the steps of obtaining a garlic concentrate by vacuum concentration for 7 to 9 hours in A mushroom mycelium culture medium is provided.

It is possible to provide a medium composition for culturing mushroom mycelium having a high free sugar content and excellent mushroom growth while utilizing the active ingredient of garlic in the present invention.

In addition, the present invention makes it possible to culture mushroom mycelium only with a simplified garlic concentrate without a separate culture medium, and utilizes a culture medium containing garlic as a main component as a medium for mushroom mycelium to mix the two materials of garlic and mushroom. It is possible to provide a mushroom mycelium culture solution that can be used in the material industry.

According to the present invention, there is provided a medium composition for culturing mushroom mycelium, a mushroom mycelium culture solution using the same, and a method for producing the same.

Hereinafter, a medium composition for culturing mushroom mycelium according to the present invention and a method for producing a mushroom mycelium culture solution using the same will be described.

After adding water to raw garlic, a high-speed vacuum low temperature concentration extractor (Cosmos 660, Kyungseo E&P, Korea) is used to prepare garlic extract. At this time, the amount of water is 1 to 4 times, preferably 2.5 to 4 times, more preferably 3 times the amount of water compared to the weight of garlic is added to the raw garlic.

After putting garlic and water in an ultra-high-speed vacuum low-temperature concentration extractor, extraction is performed at 110 to 120° C., preferably at 115° C. for 7 to 9 hours, preferably for 8 hours.

Thereafter, the concentrate is cooled at room temperature, centrifuged to remove the residue, and filtered using a 0.45 μm membrane filter.

The sugar concentration of the garlic concentrate thus prepared is 20.0 to 25.0 brix, preferably 23.0 brix.

Since the garlic concentrate for mushroom mycelium prepared according to the present invention can be prepared without a residue removal process, the garlic concentrate for mushroom mycelium can be prepared very easily, and the active ingredient can be used as it is, and the content of free sugar is high In addition, volatile components inhibiting the growth of mushroom mycelium are also removed, thereby having the advantage of promoting the growth of mushroom mycelium.

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A medium composition for culturing mushroom mycelium is prepared by diluting the garlic concentrate for culturing mushroom mycelium prepared in the same manner as above. At this time, the garlic concentrate is diluted so that the sugar concentration is 5 to 6 birx, preferably 5.5 brix.

Next, a mushroom mycelium culture solution according to the present invention is prepared using the mushroom mycelium culture medium composition. That is, the mycelium of king oyster mushroom or mycelium of the Sangha mushroom is inoculated in the dilution solution, and the culture is shaken for 35 to 45 days.
After homogenizing the culture solution cultured with shaking in this way using a homogenizer, it may be used by centrifugation.
As a result of measuring the beta-glucan content of the culture medium containing the mushroom mycelium after shaking culture, it was 0.20-0.31%.

Hereinafter, the present invention will be described in more detail with reference to specific examples, but the scope of the present invention is not limited to these examples.

Mushroom mycelium used in the following comparative examples and examples is shown in Table 1 below, and was used after receiving the sale from the Jangheung Mushroom Industry Research Institute. Mushroom mycelium was cultured for 14 days at each optimum culture temperature using PDA (potato dextrose agar, difco, USA) medium.

[Table 1]

[Comparative Example 1] Ground garlic control and preparation of culture solution using the same

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After purchasing peeled garlic sold in the market, the garlic was steamed at 100° C. for 30 minutes and cooled by leaving it at room temperature. Equal amounts (w:w) of steamed garlic and distilled water were homogenized in a mixer for 3 minutes to prepare ground garlic. As a result of measuring the sugar concentration of ground garlic, it was found to be 20.2 brix.

The ground garlic was diluted to have a sugar concentration of 5.5 brix (corresponding to the ground garlic control in Tables 2 to 4).
After autoclaving 100 mL of the diluted solution, 5 pieces of the oyster mushroom mycelium (8 mm in diameter) cultured in PDA were inoculated and cultured with shaking.

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[Comparative Example 2]

A culture medium was prepared in the same manner as in Comparative Example 1, except that the Sanghwang mushroom mycelium was used instead of the king oyster mushroom mycelium.

[Comparative Example 3] Liver garlic extract control and preparation of culture solution using the same

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After purchasing peeled garlic sold in the market, the garlic was steamed at 100° C. for 30 minutes and cooled by leaving it at room temperature. Equal amounts (w:w) of steamed garlic and distilled water were homogenized in a mixer for 3 minutes to prepare ground garlic. Then, in order to increase the extraction efficiency, 1% each of celluclast and alcalase (Novozymes, Denmark) was added, followed by enzymatic reaction at 50° C. for 24 hours. After the enzymatic reaction, the residue and the filtrate were separated from the liver garlic extract, and the filtrate was concentrated at 105° C. for 10 hours. As a result of measuring the concentration of sugar in the concentrate, 22.0 brix was obtained.

The liver garlic extract was diluted to 5.5 brix (corresponding to the garlic extract control in Tables 2 to 4).
After autoclaving 100 mL of the dilution, 5 pieces of the oyster mushroom mycelium (8 mm in diameter) cultured in PDA were inoculated and cultured with shaking.

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[Comparative Example 4]

A culture medium was prepared in the same manner as in Comparative Example 3, except that the Sanghwang mushroom mycelium was used instead of the king oyster mushroom mycelium.

[Example 1] Garlic concentrate control and preparation of culture solution using the same

Raw garlic and water were mixed in a ratio of 1:3, extracted at 115°C for 8 hours, and vacuum concentrated at 110°C for 8 hours. As a result of concentrating the garlic extract, the concentration of sugar in the garlic extract was 23.0 brix.

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The garlic concentrate was diluted to 5.5 brix (corresponding to the garlic concentrate control in Tables 2 to 4).
After autoclaving 100 mL of the dilution, 5 pieces of the oyster mushroom mycelium (8 mm in diameter) cultured in PDA were inoculated and cultured with shaking.

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[Example 2]

A culture medium was prepared in the same manner as in Example 1, except that the Sanghwang mushroom mycelium was used instead of the king oyster mushroom mycelium.

Beta-glucan, antioxidant activity, total phenolic compound content and flavonoid content of the liver garlic control, garlic extract control, garlic concentrate control, and mushroom mycelium cultures prepared in Comparative Examples 1 to 4 and Examples 1 and 2 were measured. For use in this measurement, the garlic extract and garlic concentrate were cooled at room temperature, and the garlic concentrate was centrifuged to remove residues (residues), filtered using a 0.45 μm membrane filter, and stored frozen.

Then, the culture medium inoculated with each mushroom strain was cultured with shaking for 40 days, homogenized for 5 minutes using a homogenizer (D-500, Wiggenhauser, Germany), centrifuged at 4000 rpm for 20 minutes, and the supernatant was taken and the experiment was performed. proceeded.

Both mycelium of King oyster mushroom and Sanghwang mushroom mycelium were visually observed in both the liver garlic culture medium and the garlic extract culture medium, and the culture was not smooth. This is thought to be because the growth of mushroom mycelium was inhibited due to the relatively low nutritional components required for the maintenance of antibacterial activity of garlic or the growth of mycelium under the conditions of the liver garlic culture medium and the garlic extract culture medium.

1. Beta glucan content

Beta-glucan content of garlic mushroom culture was measured using Megazyme kit (Mushroom and Yeast beta-glucan Assay Procedure K-YBGL).

First, in order to measure total glucan, a tube was prepared, 12M H ₂ SO ₄ 2 mL was added to 90 mg of the sample, and left on ice for 2 hours, and vortexed frequently for complete dissolution of beta-glucan. 10 mL of tertiary distilled water was added to each tube and reacted again for 2 hours in a water bath at 100°C.

After cooling the reaction solution at room temperature, an appropriate amount of 0.2 M sodium acetate buffer (pH 5.0) was added, and 6 mL of 10M KOH was added and mixed.

0.2 M sodium acetate buffer (pH 5.0) was added to this mixture, adjusted to 100 mL, and centrifuged (1,500×g, 10 min) to obtain a supernatant. 0.1 mL of exo-1,3-β-glucanase (20 U/mL)+β-glucosidase (4 U/mL) solution was added to 0.1 mL of the supernatant, and reacted in a water bath at 40°C for 60 minutes. To this reaction solution, 3 mL of GOPOD (glucose oxidase/peroxidase, Megazyme) reagent was added and reacted at 40° C. for 20 minutes, and then absorbance was measured at a wavelength of 510 nm.

In order to measure alpha glucan (a-glucan), 2 mL of 2M KOH was added to 100 mg of the sample and reacted with stirring on ice for 20 minutes. To this reaction solution, add 8 mL of 1.2M sodium acetate buffer (pH 3.8), mix, add 0.2 mL of amyloglucosidase (1630 U/mL)+invertase (500 U/mL) solution, and stir intermittently for 30 minutes in a 40℃ water bath. After centrifugation (1,500 × g, 10 min) to obtain a supernatant.

To 0.1 mL of this supernatant, 0.1 mL of 0.2M sodium acetate buffer (pH 5.0) and 3 mL of GOPOD reagent were added, reacted at 40°C for 20 minutes, and absorbance was measured at a wavelength of 510 nm and used to calculate the alpha-glucan content.

The measured absorbances of total glucan and alpha-glucan were measured using the absorbance of a reaction solution obtained by reacting a standard substance, glucose solution (1 mg/mL) and a control, 0.2 M sodium acetate buffer (pH 5.0) with GOPOD reagent, to determine the respective content (%). , w/w) was obtained.

The program used for the calculation was used by downloading K-YBGL CALC from Mega-Calc provided from the Megazyme website (www.megazyme.com), and the beta-glucan content was calculated by subtracting the alpha-glucan content from the total glucan content.

Beta-glucan values for the control and Comparative Examples 1-4, Examples 1-2 are shown in Table 2 below.

[Table 2]

From Table 2, the beta-glucan content of the liver garlic control and the garlic extract control that were not inoculated with the mushroom mycelium was 0.054%, and the beta-glucan content of the garlic concentrate control was 0.194%, which was about 3.5 times higher than that of the liver garlic and garlic extract. It was high content.

Looking at group B inoculated with king oyster mushroom mycelium, when the liver garlic and garlic extracts were inoculated with king oyster mushroom, it was as low as 0.058% and 0.050%, respectively, whereas Example 1 using the garlic concentrate according to the present invention as a medium was 0.301%. It was about 5.1 times and 6.0 times higher than that of ground garlic and garlic extract medium.

In addition, looking at group C inoculated with the mycelium of Sanghwang mushroom, known as medicinal mushroom, in Example 2 using the garlic concentrate according to the present invention as a medium, the beta-glucan content was about 4.24 times higher than that of ground garlic and garlic extract.

The beta-glucan content of the mycelium cultured in ground garlic and garlic extract did not show almost the same or no difference, because both extracts were not formulated in an optimal state for the growth of mushroom mycelium, sulfur compounds and nutrients, which are strong antibacterial substances in garlic. is considered to be due to

Although the garlic concentrate processing method according to the present invention is a simple process, active ingredients that were not easily eluted in the extraction process were extracted during the concentration process while going through all the extraction and concentration processes without removing the residue in advance. It is judged that the mycelium was more smoothly used as it was further decomposed. After the garlic extract preparation process, the sugar concentration was 22 brix (Comparative Example 3), but after the garlic concentrate process, it was 23 brix (Example 1).

In addition, in the case of Examples 1 and 2, the experimental group of the garlic concentrate with well cultured mycelium, the beta-glucan content was 0.300% and 0.212%, respectively, and 1.5 times and 1.1 times increased, respectively, compared to the control group, the garlic concentrate (0.194%). . The increase rate of beta-glucan of the mycelium of Sangha mushroom is insignificant, because the increase of the mycelium of the mushroom itself is slow, so it is expected that it can be further increased through continuous culture.

Considering the above results, when preparing garlic concentrate rather than ground garlic and garlic extract, the starch of garlic itself is decomposed into monosaccharides during the extraction and concentration process, making it a good nutrient for mycelium growth. It is judged that the culture medium suitable for growth of mushroom mycelium was removed as some of the volatile sulfur compounds in garlic, which inhibit the growth of garlic, were removed.

2. Total phenolic compounds and flavonoid content

To determine the total phenolic compound content, add 0.5 mL of Foline-Ciocalteau reagent to 1 mL of the sample extraction filtrate, stir sufficiently for 3 minutes, add 0.5 mL of 10% Na ₂ CO ₃ solution, leave it in the dark at room temperature for 1 hour, and then measure the absorbance at 760 nm. measured. Using gallic acid (Sigma-Aldrich Co., St, Louis, MO, USA) as a standard material, the content of total phenolic compounds was calculated from the calibration curve obtained by analyzing the sample in the same way as gallic acid equivalent (GAE mg/L) marked with

For the total flavonoid content, 0.1 mL of 10% aluminum nitrate, 0.1 mL of 1 M potassium acetate, and 4.3 mL of 80% ethanol were sequentially added to each sample, mixed, and left at room temperature for 40 minutes, followed by a microplate reader (Perkin-Elmer Inc., Waltham). , MA, USA) was used to measure the absorbance at 415 nm. The flavonoid content was calculated from the standard calibration curve obtained using Quercetin (Sigma-Aldrich Co., St. Louis, MO, USA) as a standard material and expressed as quercetin equivalent (QE mg/L).

Table 3 below shows the measurement results of total phenolic compounds and flavonoid content for each control group, Comparative Examples 1-4, and Examples 1-2.

[Table 3]

As can be seen from Table 3, the total phenolic compound content in group A, which is the mycelium uninoculated sample, was 358.06 mg/L in the case of ground garlic, 502.68 mg/L in the case of the garlic extract prepared by enzyme treatment, and in the case of the garlic concentrate. In the case of garlic concentrate, it was 377.51 mg/L, which was the highest, and in the case of group B inoculated with king oyster mushroom mycelium, the total phenolic compounds in the garlic concentrate was 286.97~461.36 mg/L, which was lower than when the mycelium was not inoculated. The concentration was the lowest. However, in the case of group C inoculated with Sanghwang mushroom mycelium, the liver garlic and garlic extract were lower than that of the uninoculated sample, but the sample using the garlic concentrate as a culture medium was 502.68 mg/L, which was 1.3 times higher than that of the uninoculated and 1.7 times higher than that of the king oyster mushroom mycelium culture. The stomach was high.

The flavonoid content was higher in the garlic concentrate (Example 1) than in the ground garlic and garlic extract when the mycelium was not inoculated. 1.8 times higher than in the group.

From these results, it is confirmed that the content of total phenolic compounds and flavonoid components increases as the mycelium of Sanghwang mushroom is cultured, and the content of beta-glucan is low depending on the type of mycelium, but it is expected that the functionality of the culture medium may vary.

3. Measurement of antioxidant activity

DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical scavenging activity is expressed as electron donating activity to DPPH, and the method of Blosi (1958) was modified using the principle that the purple reagent becomes discolored as the antioxidant activity of the sample is high. After mixing 100 μL of DPPH solution adjusted to 1.5×10 ^-4 M concentration with ethanol and 100 μL of the sample, react at room temperature for 20 minutes, and then measure the absorbance at 525 nm using a spectrophotometer to measure the sample for the sphere without sample. It was calculated as the absorbance ratio of the addition sphere and expressed in %.

ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) radical scavenging activity was determined by dissolving potassium persulfate in 7 mM ABTS solution to 2.4 mM according to the method of Re et al. (1999), and then in the dark. After reacting for 12 to 16 hours, it was diluted with distilled water so that the absorbance at 415 nm was 1.5. 100 μL of each concentration sample solution was added to 100 μL of this solution, reacted at room temperature for 5 minutes, and absorbance was measured at 415 nm.

In order to confirm the antioxidant activity of the culture medium, DPPH and ABTS radical scavenging activity, which are often used for activity measurement, were measured, and the measurement results are shown in Table 4 below.

[Table 4]

Looking at the DPPH and ABTS radical scavenging activity with reference to Table 4, all of the mycelium culture medium (Comparative Examples 1, 2, and Example 1) had lower antioxidant activity than each control. In particular, in the case of Example 1, in which the beta-glucan content was increased due to good culture, the DPPH and ABTS radical scavenging activities were lower at 21.93% and 19.99%, respectively, and it was confirmed that the radical scavenging activity was rather decreased due to the growth of the mycelium.

On the other hand, in the case of the mycelium culture medium, in Comparative Examples 3 and 4 using liver garlic and garlic extract, the antioxidant activity was lower than that of each control, but in Example 2, the DPPH radical scavenging activity was 84.50% and ABTS radicals were well cultured. The scavenging activity was 96.07%, and the antioxidant activity was the highest.

From these results, it can be confirmed that the antioxidant activity of the mycelium culture medium of Sanghwang mushroom is 3.85 to 4.80 times higher than that of the mycelium of King oyster mushroom.

From the beta-glucan content, total phenolic compound and flavonoid content measurement and antioxidant activity measurement results, it can be seen that the mushroom mycelium exhibits a higher culturing degree in the garlic concentrate according to the present invention than in the liver garlic and garlic extract, and In this case, it is possible to obtain a culture medium with a high beta-glucan content, and when cultured using the Sangha mycelium, the total phenolic compound and flavonoid content is increased, and antioxidant activity is increased. It is expected that health-promoting materialization will be possible if the Sanghwang mushroom mycelium culture medium is used as a mixed material.

The embodiments disclosed in this specification are intended to illustrate, not to limit the present invention, and the scope of the present invention is not limited by these embodiments. The protection scope of the present invention should be construed by the following claims, and all technologies within the scope equivalent thereto should be construed as being included in the scope of the present invention.

Claims (6)

In the method for preparing a medium composition for culturing a mushroom mycelium containing a large amount of beta-glucan,
preparing a garlic extract by adding 2.5 to 4 times the weight of the raw garlic to raw garlic and then extracting it at 110 to 120° C. for 7 to 9 hours;
vacuum-concentrating the garlic extract at 100-120° C. for 7-9 hours to obtain a garlic concentrate of 20.0-25.0 brix; and
As a method for producing a medium composition for culturing mushroom mycelium comprising the step of diluting the garlic concentrate to 5.0 ~ 6.0 brix to obtain a diluted solution,
The medium composition is a method for producing a medium composition for culturing a mushroom mycelium, characterized in that it contains only the diluent as an energy source for the mushroom mycelium. A medium composition for culturing mushroom mycelium prepared by the method of claim 1. delete In the method for preparing a culture solution for culturing mushroom mycelium for culturing a mushroom mycelium containing a large amount of beta-glucan,
preparing a garlic extract by adding 2.5 to 4 times the weight of the raw garlic to raw garlic and then extracting it at 110 to 120° C. for 7 to 9 hours;
vacuum-concentrating the garlic extract at 100-120° C. for 7-9 hours to obtain a garlic concentrate of 20.0-25.0 brix;
diluting the garlic concentrate to 5.0 ~ 6.0 brix to obtain a diluted solution; and
As a method for producing a mushroom mycelium culture solution comprising the step of inoculating the mushroom mycelium in the dilution and then culturing with shaking,
The mushroom mycelium culture medium is a method for producing a mushroom mycelium culture medium, characterized in that it contains only the dilution as an energy source of the mushroom mycelium. 5. The method of claim 4,
The mushroom mycelium is a method for producing a mushroom mycelium culture medium, characterized in that the mycelium of the king oyster mushroom or the mycelium of the situation mushroom. A mushroom mycelium culture solution prepared by the method of claim 4.