KR20210006158A - Method for culture of Cordyceps militaris containing high concentration cordycepin - Google Patents

Abstract

The present invention relates to a method for the culture of Cordyceps militaris containing a high concentration of cordycepin. More particularly, the present invention relates to a method for the culture of Cordyceps militaris containing a high concentration of cordycepin by inoculating a mycelium in solids remained after the extraction of fruits or medicinal plants and exposing the product to a red wavelength for mycelium development and a blue wavelength that promotes the secretion of cordycepin.

Description

Method for culture of Cordyceps militaris containing high concentration cordycepin}

The present invention relates to a method for culturing cordyceps sinensis containing high concentration cordycepin. More specifically, by inoculating the cultivated cordyceps mycelium on the remaining solids after extracting fruits or medicinal plants, and exposing them to a red wavelength for mycelium growth and a blue wavelength that promotes the synthesis of cordycepine, cordyceps containing a high concentration of cordycepine is prepared. It relates to how to cultivate.

Cordyceps is a type of mushroom named because it resides in the body of insects in winter and appears like grass in summer. It is an insect parasitic medicinal mushroom that occurs when cordyceps fungi, a type of fungus, enters the body of a living insect. Cordyceps sinensis , Cordyceps sinensis , Codyceps militaris , and Paecilomyces japonica are well known as cordyceps.

Cordyceps sinensis has long been recognized for its many efficacy through Donguibogam, and has been used to treat various adult diseases and maintain health. In China, athletes take cordyceps and show miracle records, so cordyceps are recognized as one of the top three natural health foods.

Cordyceps sinensis belongs to the group with the highest protein content (more than 28% of dry content) among crops, and contains a large amount of substances that enhance immunity. Cordycepin (3'-deoxy-adenosine) is included among the immunologically active substances of cordyceps, and among cordyceps, Militaris cordyceps has a significantly higher content of cordycepin than other cordyceps. Cordycepin is a nucleic acid substance that activates the reduced immune function of cells and prevents normal cells from being converted into cancer cells. In clinical trials with cordyceps sinensis, it was confirmed that the content of NK cells (natural killer cells) and cytokines secreted from immune cells, which are immune cells that kill cancer cells, increased by 18% to 25%. It is utilized and is receiving a lot of attention as a health functional food. Based on these results, in Korea, cordyceps containing cordyceps was the only mushroom that was certified by the Ministry of Food and Drug Safety as a health functional food. In addition, the results of clinical studies have been published that cordyceps sinensis helps control blood pressure and diabetes, as well as lung and liver functions.

Cordyceps are consumed a lot among mushrooms, but the amount that is collected from nature is very small and cannot meet the demand, so many farmers around the world have made cultivation houses using artificial cultivation methods and cultivated them in large quantities. However, cordyceps grows slower than plants, and cultivation is very difficult because it is a fungus that cannot spray pesticides because it is sensitive to the external environment. Although successful in mass cultivation, it is known that the harvest rate of cordyceps cultivation is low due to pollution even in farms that have been cultivated for a long time. That is, when 10 cells are cultured and harvested, about 1 to 2 cells are contaminated or abnormal strains grow, resulting in a lower yield.

Currently, there are many studies on the cultivation method of cordyceps in order to increase the content of cordycepine, such as Korean Patent No. 10-1446001 and Korean Patent Publication No. 10-2009-0054114, but compared to the existing content of cordycepine. There is no significant increase in the effect, so there is an urgent need for a technology to produce high-concentration cordycepine.

Accordingly, the inventors of the present invention confirmed that the content of cordycepine increased when cordyceps were cultivated by irradiating different wavelengths of light while studying a method of culturing Cordyceps sinensis in fruits or medicinal plants to cultivate cordyceps containing high concentration cordycepine. Completed the method.

Korean Patent Registration No. 10-1446001 (Name of the invention: Cordyceps soybean with improved content of cordyceps and its manufacturing method, Applicant: Hwanghwaseok, registration date: 2014.09.23.) Korean Patent Laid-Open Patent No. 10-2009-0054114 (Title of invention: Method for stably producing high content of cordycepine during artificial cultivation of pupa chinensis mushroom, Applicant: Yesan County Agricultural Technology Center, Publication date: 2009.05.29) Korean Patent Laid-Open Patent No. 10-2017-0121866 (Name of the invention: Militaris Cordyceps sinensis extract, its manufacturing method, and a composition for preventing or treating liver cancer comprising as an active ingredient, cordycepine isolated therefrom, Applicant: Mustech, Inc. Publication date: 2017.11.03.)

An object of the present invention is to provide a method for culturing Cordyceps sinensis containing high concentration cordycepin. More specifically, it is intended to provide a method for culturing Cordyceps sinensis containing high concentration of cordycepine by inoculating the cordyceps mycelium on the remaining solids after juiced fruits or medicinal plants and irradiating the red and blue wavelengths during cultivation.

The present invention comprises the steps of (first step) juicing a fruit or medicinal plant and preparing a juiced solid;

(Second step) inoculating the strain of Cordyceps sinensis to the juiced solid of the first step;

(3rd step) The step of culturing the solid inoculated with the cordyceps strain of the second step and irradiating the red wavelength; And

It relates to a cordyceps cultivation method comprising a high-concentration cordycepin comprising; (4th step) bright culture after the third step of cancer culture and irradiating a blue wavelength.

The fruits or medicinal plants of the first step may be apples, bokbunja, deodeok ( Codonopsis lanceolata ) or aronia ( Aronia melanocarpa ).

The cordyceps militaris strain of the second step may be Cordyceps militaris .

The red wavelength of the third step may be irradiated with a wavelength of 640 to 700 nm for 2 to 5 days.

The blue wavelength of the fourth step may be irradiated with a wavelength of 400 to 500 nm for 20 to 40 days.

Hereinafter, the present invention will be described in detail.

The cordyceps strain of the second step may be obtained by first culturing the mycelium in a solid medium and then pulverizing the cultured mycelium, and then culturing the pulverized mycelium in YM liquid medium. At this time, the solid medium may contain 2.0 to 4.0 g of Yeast extract, 2.0 to 4.0 g of Malt extract, 3.0 to 7.0 g of Peptone, 5.0 to 15.0 g of Glucose, and 15.0 to 25.0 g of Agar based on 1 L of solvent. In addition, the YM liquid medium may contain 2.0 to 4.0 g of Yeast extract, 2.0 to 4.0 g of Malt extract, 3.0 to 7.0 g of Peptone, and 5.0 to 15.0 g of Glucose based on 1 L of solvent.

Inoculating the cordyceps fungus strain to the juiced solid of the second step is to prepare a medium by mixing the juiced solid of the first step and YM liquid medium to which aspartate is added, and then the Codyceps militaris strain can be cultured. The aspartate may be added 1.0 to 10.0g to the YM liquid medium. If the aspartate is less than 1.0 g, the cordyceps strain may not be sufficiently cultured or cordycepine may not be sufficiently secreted, and if it exceeds 10.0 g, there is no significant difference in the content of cordycepine, which is not economically desirable.

It is preferable to culture by irradiating the red wavelength at 620 to 720 nm for 1 to 5 days during the dark culture of the third step. More preferably, it is good to irradiate at 640 to 700 nm for 2 to 3 days. At this time, the dark culture may be cultured by irradiating a red wavelength in the absence of light.

In the light culture of the fourth step, the blue wavelength is preferably cultivated by irradiation at 380 to 520 nm for 20 to 40 days. More preferably, it is good to irradiate at 400 to 500 nm for 25 to 30 days. If the blue wavelength irradiation period is less than 20 days, cordycepine may not be sufficiently secreted, and if it exceeds 40 days, it is not economically desirable.

In addition, the light culture may be cultured at 10 to 30°C under conditions of light irradiation of 800 to 1300 Lux.

The culture cultured after the fourth step may be juiced to prepare a juice culture, and a health functional food may be prepared by mixing a fruit or medicinal plant with a juice juice juiced in the first step. It can be prepared by mixing 1 to 10 parts by weight of the juice culture based on 100 parts by weight of the juice.

The present invention relates to a cordyceps cultivation method containing a high concentration of cordycepin, and by inoculating a cordyceps strain and exposing them to red and blue wavelengths, the development of mycelium and the secretion of cordicepin are promoted to obtain cordyceps containing a high concentration of cordycepin. can do.

Figure 1 shows a flow chart of a cordyceps cultivation method according to the present invention.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to sufficiently convey the spirit of the present invention to those skilled in the art so that the contents introduced herein are thorough and complete.

Example 1. Juice of fruit or medicinal plant

In this example, apples, bokbunja, deodeok ( Codonopsis lanceolata ) or Aronia ( Aronia melanocarpa ) were used, and each was juiced. The juiced solids of apples, bokbunja, deodeok, or aronia remaining after juice was used in the present invention.

Example 2. Cordyceps mycelium culture

Example 2-1. Solid medium culture of cordyceps mycelium

In the present invention, a cordyceps militaris strain isolated from the cordyceps militaris was collected from near Byeongji valley in Hoengseong-gun, Gangwon-do, and was cultured in a solid medium.

The solid medium was YM (Yeast Mold) agar medium. 3.0g of Yeast extract, 3.0g of Malt extract, 5.0g of Peptone, 10.0g of Dextrose and 20.0g of Agar were mixed with 1 liter of distilled water, and sterilized using an autoclave (autoclave, 121°C, 1.2 atm, 20 minutes). The sterilized medium was cooled and solidified to prepare a solid medium.

Cordyceps militaris strain, which is a cordyceps militaris strain, was plated on the solid medium and cultured at 25° C. for 7 days.

Example 2-2. Crushing of cordyceps mycelium

The cordyceps mycelium cultured in Example 2-1 was pulverized through a homogenizer to crush the tissue of the mycelium colony.

Example 2-3. Liquid medium culture of cordyceps mycelium

The mycelial tissue crushed in Example 2-2 was cultured in a YM (Yeast Mold) liquid medium.

In the YM liquid medium, 3.0g of Yeast extract, 3.0g of Malt extract, 5.0g of Peptone, 10.0g of Dextrose and 0.15g of vegetable oil were mixed in 1L of distilled water, and the autoclave was used (autoclave, 121℃, 1.2 atm, 20 minutes). And sterilized to prepare a liquid medium.

The mycelial tissue (diameter 10 mm) pulverized in Example 2-2 was inoculated into the liquid medium and cultured at 25°C for 3 days.

Example 3. Cultivation of Cordyceps Sinensis of Juiced Solid

The cordyceps mycelia cultured in Example 2 was inoculated and cultured in the juice solid prepared in Example 1.

First, the culture was prepared by mixing 100 g of the juice solid of Example 1 and the liquid medium prepared by adding 1, 5, and 10 g of Aspartate to the YM liquid medium of Example 2, respectively, and the 15 ml of cordyceps mycelium suspension liquid cultured in Example 2-3 was mixed and cultured.

Culture conditions were divided into dark culture conditions and light culture conditions. First, the dark culture conditions were cultured by irradiating a red wavelength of 640 to 700 ㎚ for 3 days in a dark condition at 25 ℃ without light. Afterwards, bright culture was performed and cultured by irradiating a blue wavelength of 400 to 500 nm for 30 days under conditions of 1,000 Lux of light irradiation.

division Contents Example 3-1 Cordyceps mycelia cultured in a medium containing 1 g of aspartate Example 3-2 Cordyceps mycelia cultured in a medium containing 5 g of aspartate Example 3-3 Cordyceps mycelium cultured in a medium containing 10 g of aspartate

Comparative Example 1. Cordyceps mycelium cultured with different culture conditions

Cordyceps sinensis was cultured as in Example 3, but cultured under different culture conditions. In more detail, the culture was conducted with different red wavelength and blue wavelength irradiation, and the blue wavelength was irradiated during dark culture, and the red wavelength was irradiated when light culture was cultured, which are shown in Table 2 below.

division Culture conditions Cancer culture Culture Comparative Example 1-1 Red wavelength White wavelength Comparative Example 1-2 No wavelength irradiation White wavelength + blue wavelength Comparative Example 1-3 No wavelength irradiation No wavelength irradiation Comparative Example 1-4 Blue wavelength White wavelength + red wavelength Comparative Example 1-5 Red wavelength White wavelength + red wavelength Comparative Example 1-6 Blue wavelength White wavelength + blue wavelength

Comparative Example 2. Cordyceps mycelium cultured with different aspartate content

Cordyceps sinensis was cultured in the same manner as in Example 3, but cultured with different aspartate content added to the medium. Cordyceps mycelium cultured in one medium with different aspartate content is shown in Table 3 below.

division Contents Comparative Example 2-1 Cordyceps mycelia cultured in a medium without aspartate Comparative Example 2-2 Cordyceps sinensis mycelium cultured in a medium containing 0.5 g of aspartate Comparative Example 2-3 Cordyceps mycelia cultured in a medium containing 15 g of aspartate

Experimental Example 1. Measurement of cordycepine content

In this experimental example, the cordycepin content of the cordyceps mycelium cultured in Example 3 and Comparative Examples 1 and 2 was measured.

Cordycepine content was measured using high-speed liquid chromatography (HPLC, Agilent 1100, series, USA), and the experimental conditions were Mobile phase: Acetonitrile: Water = 10: 90 Flow rate: 0.8 ㎖/min Temperature: 25°C Wavelength: 260 nm Collumn: ODS-C ₁₈ , 250x4mm Detector: SPD-10A. The measured content of cordycepin is shown in Table 4 below.

division Cordycepine content (mg/g) Example 3-1 2.4 Example 3-2 2.5 Example 3-3 2.4 Comparative Example 1-1 1.5 Comparative Example 1-2 1.7 Comparative Example 1-3 1.2 Comparative Example 1-4 1.3 Comparative Example 1-5 1.3 Comparative Example 1-6 1.5 Comparative Example 2-1 1.0 Comparative Example 2-2 1.3 Comparative Example 2-3 2.0

Referring to Table 4, it can be seen that the content of cordycepin of cordyceps cultivated in Examples 3-1 to 3-3 is 2.4 mg/g or more. On the other hand, in Comparative Examples 1-1 to 1-6 cultured with different wavelengths to be irradiated, it can be seen that the content of cordicepine is 1.7 mg/g or less, which is lower than that of Examples 3-1 to 3-3 . Therefore, it can be seen that the content of cordycepine may vary depending on the irradiated wavelength.

In addition, the cordycepine content of Comparative Example 2-1 without aspartate was the lowest at 1.0 mg/g, and Comparative Examples 2-2 and 2-3 were cultured by adding 5 g or 15 g of aspartate. It was confirmed that the content of sepin was lower than 2.0 mg/g.

Claims (6)

(First step) juice of a fruit or medicinal plant and preparing a juiced solid;
(Second step) inoculating the strain of Cordyceps sinensis to the juiced solid of the first step;
(3rd step) The step of culturing the solid inoculated with the cordyceps strain of the second step and irradiating the red wavelength; And
(Fourth step) After the dark culture of the third step, the bright culture and the step of irradiating the blue wavelength; Cordyceps cultivation method containing a high concentration cordycepin, characterized in that it comprises. The method of claim 1,
The fruit or medicinal plant of the first step is apple, bokbunja, deodeok ( Codonopsis lanceolata ) or Aronia ( Aronia melanocarpa ), characterized in that the cordyceps cultivation method containing high concentration cordycepin. The method of claim 1,
Cordyceps militaris strain of the second step is cordyceps militaris ( Cordyceps militaris ), characterized in that the cordyceps cultivation method containing a high concentration cordycepin. The method of claim 1,
The cordyceps strain of the second step is a cordyceps cultivation method containing high concentration cordycepine, characterized in that the mycelium is pulverized after culturing a solid medium and cultured in a YM liquid medium to cultivate the mycelium. The method of claim 1,
The red wavelength of the third step is a cordyceps cultivation method containing high concentration cordycepin, characterized in that irradiation with a wavelength of 640 to 700 nm for 2 to 5 days. The method of claim 1,
The blue wavelength of the fourth step is a cordyceps cultivation method containing high concentration cordycepin, characterized in that irradiation with a wavelength of 400 to 500 nm for 20 to 40 days.

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