KR102183814B1 - Preparation method of liquid culture of Lentinus edode mycelia and the liquid culture of Lentinus edode mycelia - Google Patents

Abstract

The present invention relates to a method for manufacturing a culture solution composition of Lentinula edodes mycelium having excellent enzyme activity, and to the culture solution composition thereof. According to the present invention, provided is the method for manufacturing the culture solution composition of Lentinula edodes mycelium comprising the following steps: (A) extracting a fruit body of Lentinula edodes to manufacture a liquid culture medium; (B) inoculating Lentinula edodes mycelium; (C) culturing the same for 7 to 21 days in 20 to 30°C, and a shaking culture (100 to 150 rpm) condition; (D) crushing a cell wall by treating the entire culture solution with a blender after completion of the culture; and (E) culturing the culture solution treated with the blender at 45 to 55°C for 24 to 72 hours, and conducting autodigestion thereof. The culture solution composition of Lentinula edodes mycelium manufactured according to the manufacturing method is greatly increased with enzyme activity, thereby being able to be usefully used as an extraction adjuvant of natural products.

Description

Preparation method of liquid culture of Lentinus edode mycelia and the liquid culture of Lentinus edode mycelia with excellent enzyme activity

The present invention relates to a method for preparing a shiitake mushroom mycelium culture solution composition having excellent enzyme activity, and to a culture solution composition thereof, and specifically, a shiitake mushroom mycelium culture solution composition having high enzyme activity is prepared by using the shiitake mushroom fruiting body as a culture medium. It relates to a technology used in the manufacture of.

There are various extraction methods such as high temperature extraction, high pressure extraction, ultrasonic extraction, supercritical extraction, and enzyme reaction extraction as methods for increasing the extraction efficiency of useful components and physiologically active substances from natural products. Among them, Enzymatic Extraction (Enzyme-assisted Extraction) is an extraction method that improves extraction efficiency using enzymes. Enzymes are proteins produced by living organisms and are catalysts that accelerate chemical reactions. Chemical catalysts require the use of organic solvents and high temperature and pressure conditions, whereas enzymes have the advantage of having high catalytic power and substrate specificity under moderate reaction conditions and less generation of by-products. In addition, it is environmentally friendly to react in an aqueous solution without using an organic solvent.

Mushrooms are organisms that form fruiting bodies among fungi, and biologically belong to fungi. Mushrooms are composed of mycelium, which is a nutritional organ, and a fruit body with spores, which is a breeding organ, and the mycelium corresponds to the roots, stems, and leaves of plants, and the fruiting body corresponds to flowers. Mushrooms have no distinction between roots, stems, and leaves, and do not have chlorophyll, so they use mycelium to absorb nutrients generated by other organisms. Mushrooms can be divided into by-products, wood decay bacteria, mycorrhizal fungi, and parasites according to the nutrient absorption method.

Shiitake (Lentinus edodes) belongs to the family Fungi, Basidiomyces, Pleurotus oysteraceae, oyster family, and grows on dry trees such as broad-leaf trees such as chestnut, Japanese oak, and oak, by natural forestry and artificial cultivation. All can be produced. Shiitake mushrooms are wood decay bacteria and grow by ingesting necessary nutrients by decaying and decomposing the medium using enzymes of mycelium. The mycelium of shiitake mushrooms decomposes indigestible polysaccharides such as cellulose, hemicellulose, and lignin into components that can be used for metabolism by their own enzymes.

On the other hand, the fruiting body of shiitake mushrooms contains a lot of minerals such as protein, lipid, carbohydrate, ash, calcium, phosphorus, iron, sodium, potassium, and vitamins (B1, B2), and is rich in nutrients.

The inventors of the present invention were studying a method to increase the production efficiency of enzymes derived from shiitake mushroom mycelium, and the shiitake mushroom mycelium culture solution composition prepared by a specific method using the shiitake fruiting body extract as a culture medium is a natural product used as a raw material for cosmetics, etc. The present invention was completed by discovering that it can be usefully used as a catalyst for extraction of natural products by showing an effect of increasing extraction efficiency and increasing activity.

Republic of Korea Patent Publication No. 10-2012-0078327 (2012.07.10.) Republic of Korea Patent Publication No. 10-2017-0078900 (2017.07.10.)

An object of the present invention is to provide a method for preparing a shiitake mushroom mycelium culture solution composition having high enzyme activity by using shiitake mushrooms as a culture medium.

In addition, another object of the present invention is to provide a shiitake mushroom mycelium culture solution composition that can be used as an extraction catalyst or extraction solvent of natural products by the above production method.

According to the present invention to achieve the above object, (A) preparing a liquid culture medium by extracting the shiitake fruiting body; (B) inoculating shiitake mushroom mycelium; (C) 20 ~ 30 ℃, in the shaking culture (100 ~ 150rpm) conditions, culturing for 7 ~ 21 days; (D) crushing the cell wall by processing the entire culture medium after completion of the culture with a blender; And (E) there is provided a method for producing a shiitake mushroom mycelium culture solution composition comprising the step of self-digesting and incubating the culture solution treated with a blender at 45 to 55°C for 24 to 72 hours.

The medium in step (A) is characterized in that it is prepared by mixing the shiitake fruiting bodies with an extraction solvent and extracting them at 90 to 100°C for 2 hours. In the step (D), the blender treatment is characterized in that it is performed for 3 to 5 minutes at a speed of 10,000 to 20,000 rpm.

In order to achieve the above other object, according to the present invention there is provided a shiitake mushroom mycelium culture solution composition prepared by the above manufacturing method. The mycelium culture liquid composition is characterized in that it is used as an extraction catalyst or extraction solvent of natural products.

The shiitake mushroom mycelium culture solution composition prepared according to the present invention has excellent enzyme activity and can be usefully used as an extraction catalyst or extraction solvent for natural products used as raw materials for cosmetics.

Hereinafter, the present invention will be described in more detail.

Conventionally, shiitake mushroom mycelium has been used as a natural fermentation strain. Republic of Korea Patent Laid-Open Patent No. 10-2017-0078900 "Cosmetic composition containing fermented parrot tree fermentation extract as an active ingredient" discloses the use of shiitake mushroom mycelium in the manufacture of fermented parrot tree.

In addition, Korean Patent Application Publication No. 10-2012-0078327 "Moisturizing and/or whitening cosmetic composition containing polysaccharides extracted from shiitake mushroom mycelium cultures as an active ingredient" includes the shiitake mushroom mycelium culture itself to be used as a whitening or moisturizing cosmetic. It is disclosed that you can.

The present invention is a technical feature that the shiitake mushroom mycelium culture solution composition prepared by using the shiitake fruiting body as a culture medium and undergoing a two-step enzyme production enhancement process exhibits excellent enzyme activity.

According to the present invention, (A) preparing a liquid culture medium by extracting the fruiting body of shiitake mushrooms; (B) inoculating shiitake mushroom mycelium; (C) 20 ~ 30 ℃, in the shaking culture (100 ~ 150rpm) conditions, culturing for 7 ~ 21 days; (D) crushing the cell wall by processing the entire culture medium after completion of the culture with a blender; And (E) there is provided a method for producing a shiitake mushroom mycelium culture solution composition comprising the step of self-digesting and incubating the culture solution treated with a blender at 45 to 55°C for 24 to 72 hours.

The medium in the step (A) is prepared by mixing the shiitake fruiting bodies with an extraction solvent and extracting them at 90 to 100°C for 2 hours. Water may be used as the extraction solvent, and a solvent capable of usefully extracting the fruiting components of shiitake mushrooms, such as ethanol, methanol, and acetone, may be used.

The shiitake mushroom fruit body is preferably mixed in a ratio of 1 to 10% by weight based on the total weight of the mixture.

In the step (D), the blender treatment is preferably performed for 3 to 5 minutes at a speed of 10,000 to 20,000 rpm.

It was confirmed that the enzyme activity of the shiitake mushroom mycelium culture solution composition of the present invention prepared through a two-step process of cell wall disruption and induction of self-digestion through a blender treatment, using a nutrient-rich shiitake fruiting body as a medium ( Test example).

Therefore, the shiitake mushroom mycelium culture solution composition of the present invention can be usefully used as an extraction catalyst or extraction solvent of natural products used as raw materials for cosmetics, pharmaceuticals, and foods.

[Example]

Hereinafter, the present invention will be described in more detail based on the following Examples and Test Examples. However, the following examples are for illustrative purposes only, and the present invention is not limited by the following examples.

Examples 1 to 5: Preparation of shiitake mushroom mycelium culture solution composition

Shiitake mushroom mycelium was mixed with purified water as a solvent in the ratio of Table 1, extracted for 2 hours at 90 to 100°C, and filtered to obtain a filtrate. The obtained filtrate was sterilized at 121° C. for 20 minutes to prepare a liquid culture medium.

The shiitake mushroom mycelium for inoculation prepared by subculturing on PDA (Potato Dextrose Agar) plate medium was cut into a size of 1cm X 1cm and inoculated in the prepared liquid culture medium, and cultured with shaking (100rpm) for 14 days at a temperature of 25°C. . Thereafter, the mycelium was removed by filtration and centrifugation to prepare a shiitake mushroom mycelium culture solution composition.

division Shiitake mushroom fruiting body weight (%) Example 1 One Example 2 2 Example 3 4 Example 4 8 Example 5 10

Examples 6 to 10: Preparation of Shiitake Mushroom Mycelium Culture Solution Composition

After preparing a liquid culture medium in the same manner as in Examples 1 to 5 with the composition of Table 2, cut the shiitake mushroom mycelium for inoculation prepared by subculturing on a PDA (Potato Dextrose Agar) plate medium to a size of 1 cm X 1 cm. It was inoculated into the prepared liquid culture medium and cultured with shaking (100 rpm) for 14 days at a temperature of 25°C.

The culture medium was treated with a waring blender for 3 minutes to disrupt the cell walls of the mycelium. Thereafter, the mycelium was removed by filtration and centrifugation to prepare a shiitake mushroom mycelium culture solution composition.

division Shiitake mushroom fruiting body weight (%) Example 6 One Example 7 2 Example 8 4 Example 9 8 Example 10 10

Examples 11 to 15: Preparation of Shiitake Mushroom Mycelium Culture Solution Composition

After preparing a liquid culture medium in the same manner as in Examples 1 to 5 with the composition of Table 3, cut the shiitake mushroom mycelium for inoculation prepared by subculturing on a PDA (Potato Dextrose Agar) plate medium to a size of 1 cm X 1 cm. It was inoculated into the prepared liquid culture medium and cultured with shaking (100 rpm) for 14 days at a temperature of 25°C. The culture medium was treated with a waring blender for 3 minutes to disrupt the cell walls of the mycelium.

Subsequently, the culture medium having the cell wall crushed was cultured at 45° C. for 60 hours to induce self-digestion. Thereafter, the mycelium was removed by filtration and centrifugation to prepare a shiitake mushroom mycelium culture solution composition.

division Shiitake mushroom fruiting body weight (%) Example 11 One Example 12 2 Example 13 4 Example 14 8 Example 15 10

Comparative Example 1: Preparation of Shiitake Mushroom Mycelium Culture Solution Composition

A shiitake mushroom mycelium culture solution composition was prepared in the same manner as in Example 12, except that 2.4% of a general liquid culture medium, PDB (Potato Dextrose Broth) medium was used.

Test Example 1: Quantification of protein in shiitake mushroom mycelium culture solution composition

The Bradford method was performed to check the protein content contained in the shiitake mushroom mycelium culture composition. An enzyme is a type of protein, meaning that the higher the protein content, the higher the amount of enzyme produced. The results are shown in Table 4.

Sample name Protein content (%) Example 1 0.10 Example 2 0.13 Example 3 0.12 Example 4 0.13 Example 5 0.12 Example 6 0.15 Example 7 0.19 Example 8 0.17 Example 9 0.18 Example 10 0.18 Example 11 0.25 Example 12 0.33 Example 13 0.31 Example 14 0.32 Example 15 0.33 Comparative Example 1 0.12

Test Example 2: Enzyme Activity Confirmation of Shiitake Mushroom Mycelium Culture Solution Composition

In order to confirm the enzyme activity of the shiitake mushroom mycelium culture solution compositions of the above Examples and Comparative Examples, the API ZYM kit was used to analyze. API ZYM kit is an enzyme assay kit containing 19 kinds of hydrolase substrates. After dispensing 65 µl of a sample into each cuple of API ZYM kit, incubating at 37°C for 4 hours, add a drop of reaction reagent to each cuple, and then measure the enzyme activity by checking the degree of change in reaction color after 5 minutes. I did. Depending on the degree of color change of each cuple, it can be displayed as a value from 0 to 5, where 0 is the negative response, 5 is the maximum intensity response, and 1-4 is the intermediate response value. The higher the degree of change of the reaction color is, the higher the enzyme activity is, and the results are shown in Table 5 below. As a control group, a sample was placed in a Qple without an enzyme substrate and a reaction reagent was added.

number Type of enzyme Reaction color change Example Comparative Example 1 One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 One No enzyme substrate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 Alkaline phosphatase 0 0 0 0 0 One One One One One One One 2 One One 0 3 Esterase(C4) 0 0 0 0 0 0 0 0 0 0 0 0 One 0 0 One 4 Esterase Lipase(C8) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 Lipase(C14) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 Leucine arylamidase 0 0 0 0 0 0 One One One One One 3 4 One 3 0 7 Valine arylamidase 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 Crystine arylamidase 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9 Trypsin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 One 10 α-chymotrypsin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11 Acid phosphatase One 3 One One One One 2 2 One 2 One 4 4 3 2 0 12 Naphtol-AS-BI-phosphohydrolase One 2 One One One One One One One One One One One One One One 13 α-galactosidase 0 0 0 0 0 One 3 3 2 2 One 4 3 2 2 0 14 β-galactosidase 0 0 One 0 One One One 3 2 2 2 4 2 2 3 0 15 β-glucuronidase 0 2 One One One One 3 3 3 3 3 3 One One One 0 16 α-glucosidase 0 0 0 0 0 One 3 2 One One 2 3 2 2 2 0 17 β-glucosidase 0 0 0 0 0 One One One One One One One 3 2 One 0 18 N-acetyl-β-glucosaminidase 0 0 0 0 0 One 2 2 One One One 2 2 One 2 One 19 α-mannosidase 0 0 0 0 0 0 0 0 0 0 0 One 2 One One 0 20 α-fucosidase 0 0 0 0 0 0 0 0 0 0 0 3 3 2 One 0

As shown in Table 5 above, the shiitake mushroom mycelium culture composition of Examples 11 to 15 prepared through a two-stage treatment of blender treatment and self-digestion induction using shiitake fruit bodies as a medium was prepared using PDB medium. Enzymatic activity is very much compared to the culture solution composition (Comparative Example 1), the culture solution composition not subjected to the blender treatment and the self-digestion induction treatment (Examples 1 to 5), and the culture solution composition (Examples 6 to 10) not subjected to the autodigestion induction treatment. It can be seen that it appears high.

Test Example 3: Extraction catalyst effect test (1)

In order to test the extraction catalytic effect of the shiitake mushroom mycelium culture solution composition of the above example, natural products were extracted and extraction efficiency was confirmed. 15 kinds of natural products widely used as cosmetics were selected and used.

First, as shown in Table 6 below, 50g of each natural product was put into 1Kg of purified water, extracted for 24 hours at 50°C in an extractor equipped with a cooling condenser, and filtered to obtain a filtrate. The filtrate was prepared by evaporating the solvent with a vacuum evaporator, and the yields are shown in Table 6.

division Natural products Yield weight (g) One Ginseng 4.74 2 Gosam 4.64 3 Party drug 4.59 4 green tea 7.70 5 Centella 6.67 6 Mugwort 6.63 7 Machi Prefecture 7.02 8 Aloe 6.54 9 Rose 5.68 10 Cherry Blossom 4.65 11 Calendula flower 4.58 12 Camellia 3.68 13 Green tea seeds 3.56 14 Sunflower seeds 3.53 15 bean 4.64

In order to evaluate the extraction catalytic activity of the shiitake mushroom mycelium culture solution composition prepared in Example 12, a natural product was extracted by mixing it with an extraction solvent.

50 g of each natural product was added to 900 g of purified water, and 100 g of the culture solution composition of Example 12 was added, followed by extraction at 50° C. for 24 hours in an extractor equipped with a cooling condenser, followed by filtration to obtain a filtrate. The filtrate was prepared by evaporating the solvent with a vacuum evaporator, and the results are shown in Table 7 below.

division Natural products Yield weight (g) One Ginseng 8.11 2 Gosam 7.97 3 Party drug 7.89 4 green tea 13.15 5 Centella 11.43 6 Mugwort 11.37 7 Machi Prefecture 11.91 8 Aloe 11.22 9 Rose 9.73 10 Cherry Blossom 7.98 11 Calendula flower 7.88 12 Camellia 6.54 13 Green tea seeds 6.25 14 Sunflower seeds 6.10 15 bean 7.96

As shown in Table 7 above, a natural product was extracted by adding the shiitake mushroom mycelium culture solution composition of Example 12 prepared through a two-step process of blender treatment and self-digestion induction as a medium using shiitake fruit bodies as an extraction aid. In this case, it can be seen that the extraction yield of natural products is greatly increased. Therefore, it can be seen that the shiitake mushroom mycelium culture solution composition of the present invention can be effectively used as an extraction catalyst or extraction solvent in the extraction of natural products.

Test Example 4: Extraction catalyst effect test (2)

The content of polyphenols and flavonoids in the components of the natural extract obtained in Test Example 3 was measured to confirm the increase of useful components and the catalytic effect of extraction of the shiitake mushroom mycelium culture solution composition of the above example.

Total phenol content

5 mL of purified water was added to 1 mL of the extract prepared from the extracts 1 to 15 of Test Example 3 at a concentration of 1 mg/mL, and then 1 mL of Folin-Ciocalteau's phenol reagent (Sigma-Aldrich) was added and mixed, and then at room temperature for 5 minutes. Reacted. Thereafter, 1 mL of a saturated Na ₂ CO ₃ solution was added and mixed, followed by reacting at room temperature for 1 hour, and absorbance was measured at 760 nm. The total phenol content was calculated after preparing a calibration curve using tannin acid as a standard material.

division Natural products Total phenol content (mg/g) Shiitake mushroom mycelium culture medium composition-free extraction Shiitake mushroom mycelium culture medium composition added and extracted One Ginseng 2.68 5.63 2 Gosam 5.60 11.42 3 Party drug 3.48 7.09 4 green tea 26.08 53.61 5 Centella 8.31 16.82 6 Mugwort 21.07 44.21 7 Machi Prefecture 15.38 30.89 8 Aloe 8.03 16.38 9 Rose 27.28 55.46 10 Cherry Blossom 10.12 21.40 11 Calendula flower 13.82 27.96 12 Camellia 2.34 4.88 13 Green tea seeds 2.69 5.83 14 Sunflower seeds 2.43 4.98 15 bean 12.56 25.27

Total flavonoid content

The extracts 1 to 15 of Test Example 3 were mixed by adding 1.5 mL of ethanol, 0.1 mL of 10% AlCl ₃ solution, 0.1 mL of 1M Potassium acetate solution, and 2.8 mL of purified water to 0.5 mL of the extract prepared at a concentration of 1 mg/1 mL, After reacting at room temperature for 40 minutes, absorbance was measured at 415 nm. As a standard, a calibration curve was prepared using quercetin, and the total flavonoid content was calculated.

division Natural products Total flavonoid content (mg/g) Shiitake mushroom mycelium culture medium composition-free extraction Shiitake mushroom mycelium culture medium composition added and extracted One Ginseng 1.39 2.87 2 Gosam 2.01 4.20 3 Party drug 1.97 3.97 4 green tea 29.58 59.51 5 Centella 7.03 14.64 6 Mugwort 10.32 20.87 7 Machi Prefecture 9.12 18.24 8 Aloe 1.05 2.15 9 Rose 7.51 15.27 10 Cherry Blossom 3.49 7.21 11 Calendula flower 9.32 18.78 12 Camellia 1.01 2.34 13 Green tea seeds 1.24 2.83 14 Sunflower seeds 1.13 2.52 15 bean 8.51 17.30

As shown in Tables 8 and 9, a natural product by adding the shiitake mushroom mycelium culture solution composition of Example 12 prepared through a two-step process of blender treatment and self-digestion induction using shiitake fruit bodies as a medium In the case of extracting, it can be seen that the extraction yield of phenol and flavonoids, which are useful components of natural products, is greatly increased. Therefore, it can be seen that the shiitake mushroom mycelium culture solution composition of the present invention can be effectively used as an extraction catalyst or extraction solvent to increase useful components in the extraction of natural products.

Claims (6)

(A) preparing a liquid culture medium by extracting the fruiting bodies of shiitake mushrooms;
(B) inoculating shiitake mushroom mycelium;
(C) 20 ~ 30 ℃, in the shaking culture (100 ~ 150rpm) conditions, culturing for 7 ~ 21 days;
(D) crushing the cell wall by processing the entire culture medium after completion of the culture with a blender; And
(E) A method for producing a shiitake mushroom mycelium culture solution composition for an extraction catalyst or an extraction solvent comprising the step of incubating the culture solution treated with a blender at 45 to 55° C. for 24 to 72 hours and self-digesting. The method of claim 1, wherein the medium in step (A) is prepared by mixing shiitake fruiting bodies with an extraction solvent and extracting them at 90 to 100°C for 2 hours. Method of making the composition. The method of claim 2, wherein the shiitake fruiting bodies are mixed in a ratio of 1 to 10% by weight based on the total weight of the mixture. The method of claim 1, wherein the blender treatment in the step (D) is performed for 3 to 5 minutes at a speed of 10,000 to 20,000 rpm for an extraction catalyst or an extraction solvent. The shiitake mushroom mycelium culture solution composition for an extraction catalyst or an extraction solvent prepared by the method of any one of claims 1 to 4. delete