KR101301512B1 - Medium composition for culture of Phellinus baumii mycelium and uses thereof - Google Patents

Abstract

The present invention is a culture medium mycelia culture medium composition containing peptone 4 ~ 6 g / L, malt extract 16 ~ 24 g / L, agar 16 ~ 24 g / L and glucose 16 ~ 24 g / L as an active ingredient, the A method of cultivating situation mushroom mycelium, characterized in that the culture medium by inoculating the situation mushroom mycelium, inoculating the situation mushroom mycelium on the medium composition to incubate the situation mushroom mycelium; And a method for producing a situation mushroom culture grain, the method comprising inoculating the cultured situation mushroom mycelium after inoculation into grains, and an antioxidant or anticancer activity enhancement comprising an extract of the situation mushroom culture grain prepared by the method as an active ingredient. It relates to a composition and a health functional food.

Description

Medium composition for culture of Phellinus baumii mycelium and uses carrot}

The present invention is inoculated with a mushroom mushroom mycelia culture medium composition containing peptone 4 ~ 6 g / L, malt extract 16 ~ 24 g / L and agar 16 ~ 24 g / L as an active ingredient, the medium composition A method of culturing situation mushroom mycelium, characterized by culturing by inoculating the situation mushroom mycelium to the culture medium composition culturing the situation mushroom mycelium; And a method for producing a situation mushroom culture grain, the method comprising inoculating the cultured situation mushroom mycelium after inoculation into grains, and an antioxidant or anticancer activity enhancement comprising an extract of the situation mushroom culture grain prepared by the method as an active ingredient. It relates to a composition and a health functional food.

Situation mushroom is a perennial mushroom belonging to the Democratic Fungi, Pine Scales (Hymenochaetaceae) and Mud Mushrooms, and grows on old trees such as mulberry. Situation mushrooms have been used for antiperspirant or gynecological diseases as a valuable herbal medicine since ancient times, but nowadays, it is confirmed that situation mushrooms have a very strong anti-cancer effect, and many studies on artificial cultivation of situation mushrooms have.

Conventional situation mushroom artificial cultivation is usually grown in the order of securing a strain and cultivating the mycelium from the strain in the culture medium, and then growing the situation mushroom in which fruiting bodies are formed from the mycelium in the cultivator. There is a method to obtain strains from wild situation mushrooms or to obtain strains from microbial deposit research institutes, but wild situation mushrooms are very rare, so it is not easy to get a seed from them. Buy and use.

Situation mushroom is not known to the general public because it grows only in the old tree of mulberry (桑 木: mulberry), and little has been studied about its cultivation method, and it has not attracted much attention from the general public. For this reason, it is very difficult to obtain the mycelium of the situation mushroom, moreover, the situation mushroom itself is not well cultured in the commonly used sawdust medium, peptone medium, pepper medium, myer's medium, malt extract medium, etc. Not specifically known. However, in recent years, situation mushrooms have been introduced to the general public as having anti-cancer activity, and the situation mushroom has become interested in artificial mass cultivation as it is known that the anti-cancer effect is far superior to any known mushrooms. It did not reach a satisfactory level.

Korean Patent Registration No. 0262632 discloses a method of cultivating situation mushrooms, but it is different from the method of cultivating a situation mushroom mycelium culture medium composition and situation mushroom mycelium.

The present invention is derived from the above requirements, the object of the present invention is to improve the antioxidant and anti-cancer activity, and economically by establishing the optimum conditions such as medium composition and culture method for producing a large amount of situation mushroom mycelium To provide a stable mass production of situation mushroom mycelium, it is to provide a situation mushroom mycelium and a situation mushroom culture grain using the same.

In order to solve the above problems, the present invention provides a medium mushroom mycelium culture medium composition containing peptone 4 ~ 6 g / L, malt extract 16 ~ 24 g / L and agar 16 ~ 24 g / L as an active ingredient .

The present invention also provides a culture method of the situation mushroom mycelium, characterized in that the culture by inoculating the situation mushroom mycelium.

In addition, the present invention comprises inoculating the situation mushroom mycelium in the medium composition culturing the situation mushroom mycelium; And it provides a method for producing a situation mushroom culture grains characterized in that the culture after inoculating the cultured situation mushroom mycelium.

The present invention also provides an antioxidant or anticancer activity enhancing composition containing the extract of the situation mushroom culture grains prepared by the method as an active ingredient.

In another aspect, the present invention provides an antioxidant or anti-cancer activity enhancing health functional food containing the extract of the situation mushroom culture grains prepared by the method as an active ingredient.

By cultivating the situation mushroom mycelium which had to be cultured using conventional wood or sawdust, etc., it is not difficult to secure a culture raw material and can be produced in large quantities, and the situation mushroom mycelium cultured with the medium composition is antioxidant and As anticancer activity is enhanced, it can greatly contribute to health promotion of consumers.

Figure 1 compares the growth rate of the mycelia of the collection strain.
Figure 2 compares the growth rate of the situation mushroom mycelium according to the type of medium.
Figure 3 compares the growth rate of the situation mushroom mycelium according to the culture temperature.
Figure 4 compares the growth rate of the situation mushroom mycelium according to the pH of the medium.
Figure 5 compares the growth rate of the situation mushroom mycelium according to the culture temperature of grain medium.
Figure 6 compares the growth rate of the situation mushroom mycelium according to the pH of the distilled water immersed grain, the raw material of the grain medium.
Figure 7 compares the growth rate of the situation mushroom mycelium according to the immersion time of grains as a raw material of grain medium.
Figure 8 compares the growth rate of the situation mushroom mycelium according to the carbon source added to the distilled water immersed grain, the raw material of the grain medium.
Figure 9 compares the growth rate of the situation mushroom mycelium according to the nitrogen source added to the distilled water immersed grain, the raw material of the grain medium.
Figure 10 compares the growth rate of the situation mushroom mycelium according to the type of soybean medium and culture temperature.
Figure 11 compares the DPPH radical scavenging ability of the ethanol extract of the situation mushroom culture grains at different culture temperatures.
Figure 12 is a comparison of colon cancer cell viability according to the water extract treatment of the situation mushroom red Tommy with different incubation temperature.
Figure 13 is a comparison of kidney cancer cell viability according to the extract treatment of the situation mushroom culture soybeans different from the soybeans and the extraction solvent.

In order to achieve the object of the present invention, the present invention is a medium for mycelial mushroom culture medium composition containing peptone 4 ~ 6 g / L, malt extract 16 ~ 24 g / L and agar 16 ~ 24 g / L as an active ingredient to provide.

The medium composition of the present invention may preferably contain 5 g / L peptone, 20 g / L malt extract and 20 g / L agar as an active ingredient, the medium composition composed of the above composition compared to other compositions the situation mushroom mycelium Was able to improve the growth.

In the medium composition of the present invention, the situation mushroom mycelium may be preferably Phellinus baumii , but is not limited thereto.

The present invention also provides a culture method of the situation mushroom mycelium, characterized in that the culture by inoculating the situation mushroom mycelium.

The culture method of the situation mushroom mycelium of the present invention specifically

(a) adjusting the pH of the medium containing peptone 4-6 g / L, malt extract 16-24 g / L, and agar 16-24 g / L to 5-6 and sterilizing; And

(b) after inoculating the situation mushroom mycelium in the sterilized medium of step (a) may include the step of culturing at 29 ~ 31 ℃,

More specifically,

(a) adjusting the pH of the medium containing 5 g / L peptone, 20 g / L malt extract and 20 g / L agar to 5-6, and then sterilizing; And

(b) inoculating the situation mushroom mycelium on the sterilized medium of step (a) may comprise the step of culturing at 30 ℃.

In the culture method of the present invention, there is a problem that the growth rate of the mycelium decreases when culturing the situation mushroom mycelium outside the temperature range of the step (a) and (b).

In addition, in the culture method of the present invention, the sterilization of the step (a) can be carried out for 10 to 20 minutes at 110 ~ 130 ℃ and 1 ~ 1.4 atm, preferably can be carried out for 15 minutes at 121 ℃ and 1.2 atm. However, the present invention is not limited thereto.

The present invention also relates to

Inoculating the mushroom mushroom mycelium on the medium composition of the present invention to incubate the mushroom mushroom mycelium; And

It provides a method for producing a situation mushroom culture grains comprising the step of inoculating the cultured situation mushroom mycelium and then incubating the grain.

In the manufacturing method of the present invention, the grains may be white rice, red rice, black rice, kidney beans, black beans, green beans, soybeans or red beans, but is not limited thereto.

In addition, in the production method of the present invention, when the grain is white rice, red soil or black rice, the production method of the situation mushroom culture grain is preferably

(a) inoculating the situation mushroom mycelium on the medium composition of the present invention to incubate the situation mushroom mycelium;

(b) adding 1 to 3% glucose to the distilled water by weight to distilled water and then adjusting the pH to 7.5 to 8.5;

(c) washing with white rice, red rice or black rice, immersing in distilled water for 3 to 5 hours, distilling water for 20 to 40 minutes, and sterilizing it in a culture bag to sterilize grain medium; And

(d) after inoculating the cultured mushroom mycelium of step (a) in the prepared grain medium may include the step of culturing at 33 ~ 35 ℃,

More preferably,

(a) inoculating the situation mushroom mycelium on the medium composition of the present invention to incubate the situation mushroom mycelium;

(b) adjusting the pH to 8 after adding 2% glucose to distilled water by weight;

(c) washing with white rice, red rice or black rice, immersing in distilled water adjusted for pH for 4 hours, distilling water for 30 minutes, and sterilizing it in a culture bag to prepare a grain medium; And

(D) it may comprise the step of inoculating the cultured mushroom mycelium cultured in the step (a) to the prepared grain medium at 34 ℃.

In the production method of the present invention, by adding glucose as a carbon source to the distilled water of step (b) it was possible to increase the growth rate of the mycelia. In addition, a nitrogen source may be additionally added to the distilled water of step (b), wherein the nitrogen source may be added to the malt extract in the case of white rice medium, glutamic acid in the case of red rice medium, 1 to 3% by weight of distilled water. , Preferably 2% can be added, by adding a nitrogen source was able to increase the growth rate of the situation mushroom mycelium in the culture using white and red rice medium, the growth rate of the mycelium is not added in the case of black rice It was the fastest.

In addition, in the production method of the present invention, when the grains are kidney beans, black beans, mung beans, soybeans or red beans, the method of producing a situation mushroom culture grains is preferably

(a) inoculating the situation mushroom mycelium on the medium composition of the present invention to incubate the situation mushroom mycelium;

(b) Washing kidney beans, black beans, mung beans, soybeans, or red beans, soak in tap water, drain water for 10 to 14 hours, and sterilize at 110 ~ 130 ℃ for 2 ~ 3 hours in 500 ~ 600 parts by weight in culture bags. To prepare a grain medium; And

(C) after inoculating the cultured mushroom mycelium cultured in the step (a) to the prepared grain medium may include the step of culturing at 23 ~ 27 ℃,

More preferably,

(a) inoculating the situation mushroom mycelium on the medium composition of the present invention to incubate the situation mushroom mycelium;

(b) Washing kidney beans, black beans, green beans, soybeans, red beans or red beans, soak in tap water, drain water for 12 hours, sterilize grain medium by sterilization at 121 ℃ for 2 hours 30 minutes in 500-600 parts by weight in a culture bag. Manufacturing; And

(C) the inoculated cultured mushroom mycelium of step (a) to the prepared grain medium may include the step of culturing at 25 ℃.

In the preparation method of the present invention, the soaking of the step (b) may be performed for 2 hours 30 minutes to 3 hours 30 minutes in case of kidney beans or black beans, preferably for 3 hours, and in case of meso beans 1 The time can be carried out for 30 minutes to 2 hours 30 minutes, preferably for 2 hours, mung bean or red beans can be carried out for 7 to 9 hours, preferably for 8 hours.

The present invention also provides an antioxidant or anticancer activity enhancing composition containing the extract of the situation mushroom culture grains prepared by the method as an active ingredient.

In the composition of the present invention, the extract may be water or ethanol extract, but is not limited thereto.

The present invention also provides an antioxidant or anti-cancer activity enhancing health functional food containing the extract of the situation mushroom culture grain produced by the method as an active ingredient.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Experimental Example  1: Culture Conditions for Mass Production of Situation Mushroom Mycelium

In order to determine the culture conditions 5 strains of situation mushrooms were collected and the following experiment was carried out.

A) Measurement of growth rate of collected strain

In order to determine the growth rate of mycelia according to the collected strain, PDA (potato dextrose agar) medium generally used for cultivating mushroom mycelia was used. After sterilizing the medium for 15 minutes at 121 ° C. and 1.2 atm, it is dispensed into a sterile Petri plate in a clean bench and allowed to cool completely. Then, the culture medium is inoculated in the center of the medium using a cork puncturer with a diameter of 8 mm, The cells were cultured in an incubator adjusted to 26 ° C. The average growth rate of the mycelial growth was determined by measuring the length and length of the radius of the mycelia, and the mycelial density was visually observed.

B) Measurement of mycelial growth according to medium

In order to determine the growth rate of mycelia according to the medium, experiments were carried out using 9 kinds of media such as PDA, Cz, ME1, ME2, YE, MCM, MMM, YM, and HU, which are commonly used to culture mushroom mycelia with situational strains. The medium composition is shown in Table 1 below. Cultures were inoculated in the center of the medium using a cork perforator having a diameter of 5 mm and cultured in an incubator adjusted to 26 ° C. Mycelial growth was measured in the same manner as the above-described collection strain growth rate measurement.

C) Measurement of mycelial growth by temperature

In order to determine the growth rate of mycelia according to the culture temperature, PDA (potato dextrose agar) medium which is generally used for mushroom mycelial culture was used. Temperatures were adjusted to 15, 17, 20, 22, 24, 26, 28, 30 and 32 ° C., respectively, and cultured in an incubator. Cultures were inoculated in the center of the medium using a cork perforator having a diameter of 5 mm, and the mycelial growth was measured in the same manner as in the above-described collection growth rate measurement.

Experimental Example  2: Grain medium  Culture Condition of Situation Mushroom Mycelia

A) Measurement of mycelial growth in grain medium by temperature

After washing each grain (white rice, red rice, black rice), soak for 4 hours and drain for 30 minutes, and then put 50 g of each grain in a transparent cylindrical test tube of 24 mm diameter and 200 mm height, and then 121 ° C. After sterilization and cooling for 15 minutes at S. mushroom mycelium was inoculated on the surface portion of the grain and incubated in the incubator at 24, 26, 28, 30, 32, 34 and 38 ℃, respectively. The growth of mycelium was measured from the length of the mycelium which grew downward from the inoculation surface.

B) Measurement of mycelial growth according to pH in grain medium

After adjusting the pH to 4.0-8.0 using 1N HCl and 1N NaOH in distilled water, washing white rice, red rice, and black rice, respectively, immersing in distilled water for 4 hours and performing draining for 30 minutes, 50 g of each grain was placed in a transparent cylindrical test tube having a diameter of 24 mm and a height of 200 mm, sterilized by cooling at 121 ° C. for 15 minutes, and then inoculated with the surface mushroom mycelium and incubated in an incubator controlled at 30 ° C. . The growth of mycelium was measured from the length of the mycelium which grew downward from the inoculation surface.

C) Measurement of mycelial growth by immersion time

After washing with white, red and black rice, they were soaked in distilled water for 2, 4 and 8 hours and drained for 30 minutes, and then 50 g of each grain was put in a transparent cylindrical test tube having a diameter of 24 mm and a height of 200 mm. After sterilization and cooling at 121 ° C. for 15 minutes, the situation mushroom mycelium was inoculated on the surface portion of the grain and incubated in an incubator adjusted to 30 ° C. The growth of mycelium was measured from the length of the mycelium which grew downward from the inoculation surface.

D) Measurement of mycelial growth according to carbon source in grain medium

Glucose, lactose, maltose, starch and sucrose are selected as carbon sources, and each carbon source is dissolved in distilled water at 2% concentration to prepare a carbon source solution. It was. After washing with white rice, red rice and black rice, they were immersed in the respective carbon source solution for 4 hours and drained for 30 minutes, and then 50 g of each grain was put in a transparent cylindrical test tube having a diameter of 24 mm and a height of 200 mm. After sterilization and cooling at 121 ° C. for 15 minutes, the situation mushroom mycelium was inoculated on the surface portion of the grain and incubated in an incubator adjusted to 30 ° C. The growth of mycelium was measured from the length of the mycelium which grew downward from the inoculation surface.

E) Measurement of mycelial growth according to nitrogen source in grain medium

Ammonium tartrate, glutamic acid, malt extract, potassium nitrate, and urea were selected as nitrogen sources, and each nitrogen source was dissolved in distilled water at a concentration of 2%. A nitrogen source solution was prepared and prepared. After washing with white, red and black rice, each of the prepared nitrogen source solution was soaked for 4 hours and drained for 30 minutes, and then 50 g of each grain was placed in a transparent cylindrical test tube having a diameter of 24 mm and a height of 200 mm. Then, after sterilization for 15 minutes at 121 ℃ cooled to inoculate the surface mushrooms mycelium and incubated in an incubator adjusted to 30 ℃. The growth of mycelium was measured from the length of the mycelium which grew downward from the inoculation surface.

F) Measurement of mycelial growth in legume medium

Experiments were conducted using soybeans, soybeans, kidney beans, black beans, green beans, and red beans. After washing the beans, the soybeans, kidney beans, and black beans were soaked in tap water for 4 hours, and the green beans and red beans were soaked for 8 hours, drained for 1 hour, and then placed in a transparent cylindrical test tube 24 mm in diameter and 200 mm high. 50 g of soybeans were added, sterilized for 40 minutes at 121 ° C., and then cooled, followed by inoculation of the surface mushroom mycelium on the surface of the grains and incubated in an incubator controlled at 30 ° C. The growth of mycelium was measured from the length of the mycelium which grew downward from the inoculation surface.

G) Mass cultivation in legume medium

The soybeans were soaked in tap water for 2 hours, the kidney beans and black beans were soaked for 3 hours, and the green beans and red beans were soaked for 8 hours, followed by draining for at least 12 hours. After sterilization and cooling for minutes, the situation mushroom mycelium was inoculated.

Experimental Example  3: Assay of bioactive substance and anticancer effect according to culture condition

A) material

(1) Experimental material

Situation cultured and cultured mycelium according to the culture temperature was cultured on soybeans (Red beans, red beans, black beans, soybeans, green beans) at Jangheung-gun Mushroom Research Institute.

(2) Reagent

Reagents used in the bioactivity assay were used as the first-class and the special reagent.

(3) sample extraction

Each of the test materials was ground and extracted with stirring with water or 80% ethanol. It was then concentrated and used as a sample while storing frozen in 25 ml.

B) Experimental method

(1) antioxidant activity

(A) DPPH scavenging capacity measurement

Each sample was dispensed in 70 μl into a 96well microplate. As a positive control, 70 μl of BHT and vitamin C were diluted to a concentration of 0.1 mg / ml, and 140 μl of 0.075 mM DPPH prepared immediately before use was added and reacted at 25 ° C. for 30 minutes. Blank for color correction was added 210 μl of ethanol. Thereafter, the absorbance at the wavelength of 517 nm was measured with an ELISA reader and calculated by substituting the following equation.

% Clearance = [(AO-A1) / AO] × 100

AO: Absorbance of the control (sampled with ethanol)

A1: absorbance of the experimental group

(2) anticancer activity

(A) Cell line and cell culture

Caki and HT-29 cells were distributed from Korea Cell Line Bank and cultured with RPMI by adding 10% fetal bovine serum (FBS, Gibco) and 1% Antibiotic. The cells were cultured in a humidified incubator at 37 ° C. and 5% CO ₂ , and when cells grew about 80% of the dishes, washed with PBS, treated with Trypsin-EDTA (Gibco), and passaged every 2 to 3 days.

(B) MTS cytotoxicity test (anticancer activity)

Toxicity of the cell lines to the extract was analyzed by 5- (3-caroboxymeth-oxyphenyl) -2H-tetra-zolium inner salt (MTS) assay. This measures the conversion of MTS to formazan by mitochondrial dehydrogenases. 1 × 10 ⁴ cells / well were dispensed into 96 well plates and the extracts were treated for 24 hours. After adding 20 μl of MTS solution per well and reacting for 4 hours at 37 ° C., 5% CO ₂ incubator, the absorbance was measured at 450 nm using a microplate reader (DYNEX, Opsys MR, USA). Cell viability was expressed as a percentage. The absorbance of each sample was corrected by comparing the absorbance of the control group and the experimental group by culturing the medium without the cells.

Example  1: of strain Culture Investigate physiological characteristics

end) Collection strain  Growth Rate and Mycelial Density according to Mycelial Growth

The results of comparing the growth rate of the hyphae for each collection strain are shown in FIG. 1. Growth rate is JMI-P2 ( Phellinus baumii )> JMI-P1 ( Phellinus linteus ), JMI-P3 ( Phellinus robustus )> JMI-P5 ( Phellinus baumii )> JMI-P4 ( Phellinus linteus ). Among the strains, the growth of the JMI-P2 strain was confirmed to be the best. Mycelial density was also found to be the best in the JMI-P2 strain. As a result, the JMI-P2 strain is a strain that can be used as a food and is superior to other strains in growth, and is cultivated a lot in Jangheung. The scientific name of this strain was Phellinus It was baumii (situation mushroom).

B) Selection of optimal medium

In order to select the optimal mycelial growth medium of the JMI-P2 strain, the results of the mycelial growth were examined by disclosing 9 kinds of media as shown in FIG. 2. The mycelial growth was the best in ME2 medium and the mycelial growth was good in HU, PDA, YM, and ME1 medium, but the mycelial growth was poor in YE and Cz medium. The mycelial density was good in MCM, ME1, ME2, PDA, and YM media, but poor in HU, MMM, YE, and CZ media. As a result of this experiment, it was considered that the difference in the degree of mycelial growth was large depending on the composition of the medium, and ME2 medium among the 9 medium was the most suitable for mycelial growth.

C) Selection of optimum temperature

In order to investigate the optimum strain culture temperature of the JMI-P2 strain, the results of the mycelial growth at different culture temperatures are shown in FIG. 3. The growth rate of the JMI-P2 strain was the fastest at 30 ℃, it was confirmed that the growth of mycelia at 32 ℃ rather. In addition, the mycelial growth was low even at the conditions of 17 ~ 24 ℃. The density of mycelia was best at 28 ~ 30 ℃. This confirms that the mycelial culture of the JMI-P2 strain is suitable for 30 ℃.

D) optimal pH selection

The results of mycelial growth according to pH are shown in FIG. 4. Mycelial growth tended to decrease with increasing acidity above pH 5 ~ 6, and mycelial growth was very low at pH 4 condition. These results confirmed that the optimal pH value of the JMI-P2 strain was pH 5-6.

hemp) Carbon source , Mycelial growth according to nitrogen source

The carbon source is an essential nutrient source for the growth of strains as a source of synthesis and energy for carbohydrates, proteins, lipids and nucleic acids in fungi. Experimental results for selecting the optimal carbon source for mycelial growth of JMI-P2 showed that the growth was accelerated in all treatments compared to the control, and was the best in the glucose treatment. Experimental results for the selection of the optimal nitrogen source showed that the growth was slower than that of the control in the treatments of ammonium tartrate and glutamic acid, and that the growth was faster in the malt extract, potassium nitrate and urea treatments than in the control. It was good.

Example  2: Grain medium  Investigation of Culture Characteristics of Situation Mushroom Mycelia

end) In grain medium  Check the optimum temperature for mycelial growth

In order to confirm the optimum culture temperature of the situation mushroom mycelium using grains, the growth of the mycelium was measured according to the culture temperature, and the experimental results are shown in FIG. 5. The growth of mushroom mycelium was the most prominent among white grains. When grown in white rice, 20 days after the culture showed the most vigorous growth at 34 ℃ condition, growth was inhibited at 36 ℃ rather, the mycelia did not grow at 38 ℃ was confirmed to die. The same result was confirmed in the conditions of red and black rice, and it was confirmed that the optimum culture temperature of the situation mushroom mycelium was 34 ° C. in cultured rice.

I) In grain medium  Optimal Mycelial Growth pH  Confirm

In order to confirm the optimum pH of the situation mushroom mycelium using grains, the growth of mycelium was measured according to pH, and the experimental results are shown in FIG. 6. White, red and black rice all showed the best growth under the condition of pH 8.

C) Optimal mycelial growth Immersion time  Confirm

As a result of comparing the mycelial growth according to the immersion time of the situation mushroom mycelium using grains is shown in FIG. If the moisture content is too low, there is a problem that the growth of the mycelium is delayed because the water supply to the mushroom mycelium is not sufficient.On the contrary, if the water content of the medium is large, the starch contained in the grain is increased by increasing each grain medium. As a result, the mushroom mycelium becomes cohesive in the medium, and the supply of oxygen to the medium is insufficient, as well as the penetration of the mushroom mycelium is slowed down. For this reason, moisture content is important for mycelial growth.

As a result of comparing the mycelial growth with soaking time, the soaking time did not significantly affect the mycelial growth. In case of mass cultivation in a bag, the soaked in 4 hours soaked rice is no different from the soaked rice for 8 hours.

la) In grain medium On carbon  Mycelial growth according to

In order to determine the optimal carbon source of the situation mushroom mycelium using grains, the growth of the mycelium according to the carbon source was measured and shown in FIG. 8. In the case of white rice, the best growth was obtained under the condition of adding sucrose and glucose among carbon sources, whereas the growth was slower than the condition of adding distilled water in the condition of adding lactose, maltose and starch. In the case of red rice, the best growth was observed under the conditions in which glucose was added, whereas in the rest conditions, growth was lower than the conditions in which distilled water was added. In the case of black rice, the best growth was observed under the conditions of adding glucose, and the growth rate was similar to that under the addition of distilled water. As a result, it was confirmed that the proper carbon source is glucose when cultivating the situation mycelia in grain medium.

hemp) In grain medium  Mycelial growth according to nitrogen source

In order to confirm the optimum nitrogen source of the situation mushroom mycelium using grains, the growth of mycelium according to the nitrogen source was measured and shown in FIG. 9. In the case of white rice, the best growth was observed in the test group containing malt extract. In the case of red rice, the best growth was observed in the glutamic acid-added test, while the rest was lower than the distilled water. In the case of black rice, it was confirmed that the growth rate of the situation mycelium was the best under the condition of distilled water rather than the nitrogen source. In addition, urea suppressed the growth of situational mycelium under the condition that urea was added.

bar) In the soy sauce  Mycelial growth during mass cultivation

Conditioned mycelial culture conditions were confirmed for mass culture in the pulse media. 2 hours of soybeans, 3 hours of black beans and kidney beans, and 8 hours of green beans were appropriate. However, in the case of red beans, the same time of immersion as green beans seems to be appropriate, but in the case of hardened red beans, it was not called even after increasing the time. Therefore, it is considered proper to incubate with red bean soaking time of 8 hours. When the water draining time is shortened after soaking, the water content during the mycelial culture is a factor that hinders the growth of the mycelia, so the water draining time is appropriate for more than 12 hours. Sterilization time was 1 hour and 30 minutes was contaminated and mycelia did not grow, 2 hours 30 minutes was suitable at 121 ℃. In addition, as a result of culturing at different temperature of 25 ℃ and 30 ℃, mycelia were similarly grown. However, when incubated at 30 ℃ the grains of the lower part showed a tangled phenomenon appeared to be excessive moisture content (Fig. 10). It is considered that the culture is suitable at 25 ° C. during the mass culture of soybeans, and the possibility of mass culture of the situation mushroom mycelium using soybeans was confirmed.

Example  3: Bioactive substance and anticancer effect assay

1) different culture conditions Situation Cultivated  Antioxidant activity

DPHT scavenging activity was tested for antioxidant effects, BHT, 0.1 mg / ml, 89.97 ± 0.02% and Vit C, 94.82 ± 0.01%. In the case of ethanol extracts of circumferential white rice, circumferential red rice and circumferential black rice, most of the cultured rice cultured at 34 ° C showed high DPPH scavenging ability, indicating that the antioxidant activity was high.

2) anticancer activity

When HT-29 cells (colon cancer) were treated with the contextual Tommy water extract, the survival rate of the cells was confirmed that the extract of the situational Tommy cultured at 34 ° C. showed the lowest survival rate (FIG. 12). In addition, when the samples were treated with Caki-1 cells (renal cancer), the viability of the cells was lower in the survival rate of kidney cancer cells than in the bean and the ethanol extracts inoculated with the mycelium. This enhancement was confirmed (FIG. 13).

Claims (9)

delete delete delete delete delete (a) adjusting the pH of the medium containing peptone 4-6 g / L, malt extract 16-24 g / L, and agar 16-24 g / L to 5-6 and sterilizing;
(b) inoculating the situation mushroom mycelium on the sterilized medium of step (a) and culturing at 29-31 ° C .;
(c) adding 1 to 3% glucose to the distilled water by weight to distilled water and then adjusting the pH to 7.5 to 8.5;
(d) after washing white rice, red rice or black rice, immersed in distilled water adjusted to pH of step (c) for 3 to 5 hours, deducting water for 20 to 40 minutes, sterilized by placing in a culture bag and sterilizing Manufacturing step; And
(e) inoculating the cultured mushroom mycelium cultured in the step (b) in the prepared grain medium and then culturing at 33 ~ 35 ℃ manufacturing method of the situation mushroom culture grains comprising the step of manufacturing. (a) adjusting the pH of the medium containing peptone 4-6 g / L, malt extract 16-24 g / L, and agar 16-24 g / L to 5-6 and sterilizing;
(b) inoculating the situation mushroom mycelium on the sterilized medium of step (a) and culturing at 29-31 ° C .;
(c) After washing the kidney beans, black beans, mung beans, soybeans or red beans, immerse in tap water and withdraw water for 10 to 14 hours, sterilize for 2 to 3 hours at 110 ~ 130 ℃ in 500 ~ 600 parts by weight in a culture bag To prepare a grain medium; And
(d) inoculating the cultured mushroom mycelium of step (b) on the prepared grain medium, and then culturing at 23-27 ° C. delete delete

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