KR20210027875A - Culture method for enhancing cordycepin content of Paecilomyces javanicus Mycelium using Light-Emitting Diode - Google Patents

Culture method for enhancing cordycepin content of Paecilomyces javanicus Mycelium using Light-Emitting Diode Download PDF

Info

Publication number
KR20210027875A
KR20210027875A KR1020190108888A KR20190108888A KR20210027875A KR 20210027875 A KR20210027875 A KR 20210027875A KR 1020190108888 A KR1020190108888 A KR 1020190108888A KR 20190108888 A KR20190108888 A KR 20190108888A KR 20210027875 A KR20210027875 A KR 20210027875A
Authority
KR
South Korea
Prior art keywords
content
mycelium
light
cordycepin
cordyceps sinensis
Prior art date
Application number
KR1020190108888A
Other languages
Korean (ko)
Other versions
KR102356058B1 (en
Inventor
양재경
정지영
하시영
Original Assignee
경상국립대학교산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 경상국립대학교산학협력단 filed Critical 경상국립대학교산학협력단
Priority to KR1020190108888A priority Critical patent/KR102356058B1/en
Publication of KR20210027875A publication Critical patent/KR20210027875A/en
Application granted granted Critical
Publication of KR102356058B1 publication Critical patent/KR102356058B1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/40—Cultivation of spawn
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/20—Culture media, e.g. compost
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/50—Inoculation of spawn
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/60—Cultivation rooms; Equipment therefor
    • A01G18/69—Arrangements for managing the environment, e.g. sprinklers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/04—Electric or magnetic or acoustic treatment of plants for promoting growth
    • A01G7/045—Electric or magnetic or acoustic treatment of plants for promoting growth with electric lighting

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Environmental Sciences (AREA)
  • Mycology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Botany (AREA)
  • Ecology (AREA)
  • Forests & Forestry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Mushroom Cultivation (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

The present invention relates to a culturing method for increasing the content of cordycepin in Cordyceps tenuipes mycelia using an LED. The culturing method comprises: an inoculation step of inoculating Cordyceps tenuipes mycelia to a culture medium; and culturing the inoculated culture medium under light conditions to obtain cordycepin from the cultured Cordyceps tenuipes mycelia and a culture liquid. It has been confirmed that Cordyceps tenuipes mycelia have maximum cordycepin contents according to a culture medium and a light condition. An optimal light quantity, a culture medium glucose content, and incubation time conditions can be derived through a reaction surface analysis. By using the same as culturing conditions, it is possible to efficiently obtain the maximum amount of cordycepin from the Cordyceps tenuipes mycelia.

Description

LED를 이용한 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법{Culture method for enhancing cordycepin content of Paecilomyces javanicus Mycelium using Light-Emitting Diode}Culture method for enhancing cordycepin content of Paecilomyces javanicus Mycelium using Light-Emitting Diode} using LED

본 발명은 LED를 이용한 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법에 관한 것이다.The present invention relates to a culture method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis using LED.

동충하초(冬蟲夏草)는 겨울에는 벌레 여름에는 버섯으로 나온다는 뜻에서 이름이 붙여졌다. 대부분이 곤충병원성진균(entomopathogenic fungi)으로 약 800여종이 알려져 있으며, 기주 곤충에 감염되어 내부에서 균사 생장을 하여 자실체를 생성하는 것이 큰 특징이다. 이러한 동충하초는 아시아(중국, 한국, 일본 등)에서 예전부터 약초로 쓰여 왔다. 그 효능으로는 면역력 증강, 성인병 예방, 천식, 빈혈에 좋은 약재로 사용되어져 왔다. Cordyceps hacho (冬蟲夏草) was named because it comes out as a bug in winter and a mushroom in summer. About 800 species are known as most of them as entopathogenic fungi, and it is characterized by being infected with host insects and producing a fruiting body by growing mycelium inside. These cordyceps have been used as medicinal herbs in Asia (China, Korea, Japan, etc.) from a long time ago. For its efficacy, it has been used as a good medicine for enhancing immunity, preventing adult diseases, asthma, and anemia.

또한, 동충하초는 작물들 중 단백질 함량이 가장 높은 (건조함량의 28%이상) 군에 속하고, 면역력을 강화시키는 물질이 다량 함유되어 있다. 특히, 동충하초의 면역활성물질 중에는 키닉산(quinic acid)의 이성체로 밝혀진 '코디세핀(Cordycepin :3'-deoxy-adenosine)' 이 포함되어 있다. 코디세핀은 핵산 물질로서 세포의 유전정보에 관여하면서 저하된 면역기능을 활성화하여 정상세포가 암세포로 전환되는 것을 방지하는 작용을 한다. 동충하초를 투입한 임상실험에서 암세포를 죽이는 면역세포인 NK세포(natural killer cell/자연살해세포)와 면역세포에서 분비되는 사이토카인의 함량이 18%~25% 증가되는 것으로 확인되어 코디세핀이 혈액암치료용으로 활용되고, 건강기능식품으로서도 많은 관심을 받고 있다. In addition, 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. In particular, cordycepin (3'-deoxy-adenosine), which is found to be an isomer of quinic acid, is included among the immunologically active substances of Cordyceps sinensis. Cordycepin is a nucleic acid substance that is involved in the genetic information of cells and activates a reduced immune function to prevent 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), which are immune cells that kill cancer cells, and cytokines secreted from immune cells, increased by 18% to 25%. It is used for treatment and is receiving a lot of attention as a health functional food.

이러한 결과를 토대로 국내에서는 코디세핀이 함유된 동충하초가 건강기능식품으로 식약처 인증을 받았으며, 코디세핀의 면역증강활성, 항암활성 외 항바이러스 효과와 항염증 효과 등 다양한 기능 등이 발표되고 있다.Based on these results, cordycepin-containing cordyceps has been certified by the Ministry of Food and Drug Safety as a health functional food in Korea, and various functions such as antiviral and anti-inflammatory effects in addition to cordycepin's immunity enhancing activity and anticancer activity have been announced.

상기와 같은 코디세핀의 기능으로 인해 최근에는 동충하초로부터 코디세핀을 추출하는 연구가 많이 진행되고 있는데, 한국공개특허 제2001-0054264호에 동충하초로부터 분리 추출된 항암제용 코디세핀과 그 제조방법이 개시되어 있으나, 상기 한국공개특허 제2001-0054264호에 개시된 분리방법은 HPLC를 이용한 정제방법이기 때문에 소량으로밖에 활성물질을 분리하지 못해 상업적으로 적당하지 않은 문제점이 있어 왔으며, 현재까지 동충하초로부터 코디세핀 함량 증대에 적합한 최적 조건에 관한 연구는 미비한 실정이다.In recent years, due to the function of cordycepin as described above, research on extracting cordycepin from Cordyceps sinensis has been conducted a lot, and Korean Patent Laid-Open Patent No. 2001-0054264 discloses cordycepine for anticancer drugs extracted from Cordyceps plant and a method of manufacturing the same. However, since the separation method disclosed in Korean Patent Laid-Open No. 2001-0054264 is a purification method using HPLC, there has been a problem that it is not commercially suitable because it cannot separate the active substance in a small amount. Research on the optimal conditions suitable for the situation is insufficient.

따라서 눈꽃동충하초 균사체의 코디세핀 함량을 증대시켜 코디세핀 수득량을 증가시키기 위한 최적 방법에 대한 연구가 필요한 실정이다.Therefore, there is a need for research on an optimal method for increasing the amount of cordycepine by increasing the content of cordycepin in the mycelium of Cordyceps sinensis.

1. 대한민국공개특허 제10-2001-0054264호(2001.07.02.)1. Korean Patent Application Publication No. 10-2001-0054264 (2001.07.02.)

본 발명의 목적은 눈꽃동충하초의 최대 코디세핀 함량을 위한 최적의 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법을 제공하는 데에 있다.It is an object of the present invention to provide a cultivation method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis for the maximum content of cordycepin in Cordyceps sinensis.

또한, 본 발명의 다른 목적은 상기 방법에 따른 눈꽃동충하초로부터 수득된 코디세핀 조성물을 제공하는 데에 있다.In addition, another object of the present invention is to provide a cordycepin composition obtained from the Snow Cordyceps sinensis according to the above method.

또한, 본 발명의 다른 목적은 눈꽃동충하초 균사체의 최대 코디세핀 함량 예측방법을 제공하는 데에 있다.In addition, another object of the present invention is to provide a method for predicting the maximum content of cordycepin in the mycelium of Cordyceps sinensis.

상기 목적을 달성하기 위하여, 본 발명은 눈꽃동충하초 균사를 배지에 접종하는 접종단계; 및 접종된 배지를 광조건에서 배양하여 배양된 눈꽃동충하초 균사체 및 배양액에서 코디세핀을 얻는 단계; 를 포함하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법을 제공한다.In order to achieve the above object, the present invention is an inoculation step of inoculating the mycelium of Cordyceps sinensis into a medium; And culturing the inoculated medium under light conditions to obtain cordycepin from the cultured mycelium of Cordyceps sinensis and the culture medium. It provides a cultivation method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis comprising a.

또한, 본 발명은 상기 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법에 따라 수득된 코디세핀 조성물을 제공한다.In addition, the present invention provides a cordycepin composition obtained according to the culture method for increasing the cordycepin content of the mycelium of Cordyceps sinensis.

또한, 본 발명은 상기 눈꽃동충하초 균사체의 코디세핀 함량을 예측하는 방법에 있어서, (a) 박스 벤켄 계획법(Box-Behnken design)으로 광량(X1), 배지의 글루코스(glucose) 함량(X2), 배양시간(X3)에 대하여, -1, 0 및 1로 코드화하여 실험범위를 설계하는 단계; (b) 상기 단계 (a)의 설계된 실험범위로, 상기 광량, 배지의 글루코스 함량 및 배양시간에 대한 실험값을 얻는 단계; (c) 상기 단계 (b)의 실험값을 이용하여 하기 수학식 1로 표시되는 이차 회귀식 모델을 도출하는 단계; 및 (d) 상기 단계 (c)에서 도출된 수학식 1로 표시되는 이차 회귀식 모델을 변량분석(ANOVA)하여 상기 눈꽃동충하초 균사체의 코디세핀 함량을 예측하는 단계; 를 포함하는 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량 예측방법을 제공한다.In addition, the present invention in the method for predicting the content of cordycepin in the mycelium of Cordyceps sinensis, (a) the amount of light (X 1 ) by Box-Behnken design, and the amount of glucose (glucose) of the medium (X 2 ) , For incubation time (X 3 ), designing an experimental range by coding as -1, 0 and 1; (b) obtaining experimental values for the amount of light, the glucose content of the medium, and the incubation time in the experimental range designed in step (a); (c) deriving a quadratic regression model represented by Equation 1 below using the experimental value of step (b); And (d) predicting the content of cordycepin in the mycelium of Cordyceps sinensis by variate analysis (ANOVA) of the quadratic regression model represented by Equation 1 derived in step (c); It provides a method for predicting the content of cordycepin in the mycelium of Cordyceps sinensis, comprising a.

[수학식 1][Equation 1]

Y1=3224.64+974.20X1+571.52X2-307.48X3+250.54X1X2-187.12X1X3-95.55X2X3-1651.51X1 2-272.72X2 2-663.51X3 2 Y 1 =3224.64+974.20X 1 +571.52X 2 -307.48X 3 +250.54X 1 X 2 -187.12X 1 X 3 -95.55X 2 X 3 -1651.51X 1 2 -272.72X 2 2 -663.51X 3 2

(상기 수학식 1에서 Y1은 눈꽃동충하초 균사체의 코디세핀 함량(mg/L), X1은 광량, X2는 배지의 글루코스 함량 및 X3는 배양시간을 의미함.)(In Equation 1, Y 1 is the cordycepine content (mg/L) of the mycelium of Cordyceps sinensis, X 1 is the amount of light, X 2 is the glucose content of the medium, and X 3 is the culture time.)

본 발명에 따라 눈꽃동충하초 균사체는 SDB(Sabouraud dextrose broth) 액체배지에서 파란색 및 적색 혼합 LED 광을 조사하여 배양한 경우 최대 코디세핀 함량을 나타내었으며, 반응 표면 분석을 통하여 최적의 광량, 배지의 글루코스 함량 및 배양시간 조건을 도출하고, 이를 배양조건으로 이용함으로써 눈꽃동충하초 균사체로부터 최대 함량의 코디세핀을 효율적으로 수득할 수 있는 효과가 있다.According to the present invention, when the mycelium of Cordyceps sinensis was cultured by irradiation with blue and red mixed LED light in SDB (Sabouraud dextrose broth) liquid medium, the maximum amount of cordycepine was shown, and the optimal amount of light and glucose content of the medium were obtained through reaction surface analysis. And by deriving the culture time conditions, and using this as a culture condition, there is an effect of efficiently obtaining the maximum amount of cordycepine from the mycelium of Cordyceps sinensis.

또한, 본 발명에 따른 배양방법에 따라 수득된 눈꽃동충하초 균사체 및 배양액에는 면역증강활성, 항암활성, 항바이러스 및 항염증 효과가 뛰어난 코디세핀 함량이 높아 건강기능식품으로도 유용하게 사용될 수 있다.In addition, the mycelium of Cordyceps sinensis and the culture solution obtained according to the cultivation method according to the present invention have high content of cordycepin, which has excellent immunity enhancing activity, anticancer activity, antiviral and anti-inflammatory effect, and thus can be usefully used as a health functional food.

도 1은 눈꽃동충하초(Paecilomyces javanicus) 균사체를 나타낸 도면이다.
도 2는 눈꽃동충하초의 배양과정 및 코디세핀 함량 측정 과정을 나타낸 도면이다.
도 3은 눈꽃동충하초의 액체배지 종류에 따른 균사체의 코디세핀 함량을 나타낸 도면이다(데이터는 3번의 실험 결과를 평균±표준편차로 나타냈다).
도 4는 눈꽃동충하초의 배양기간에 따른 균사체의 코디세핀 함량을 나타낸 도면이다(데이터는 3번의 실험 결과를 평균±표준편차로 나타냈다).
도 5는 형광등 및 UV-A가 설치된 shaking incubator를 나타낸 도면이다.
도 6은 형광등 및 UV-A 광량 및 on/off 조절을 위한 컨트롤 박스를 나타낸 도면이다.
도 7은 형광등 및 UV-A의 조사에 따른 눈꽃동충하초 균사체의 코디세핀 함량 변화를 나타낸 도면이다(데이터는 3번의 실험 결과를 평균±표준편차로 나타냈다).
도 8은 LED 단일광(red, green, blue)이 설치된 shaking incubator를 나타낸 도면이다.
도 9는 LED 단일광 광량 및 on/off 조절을 위한 컨트롤 박스를 나타낸 도면이다.
도 10은 눈꽃동충하초의 LED 단일광(red, green, blue)조사에 따른 균사체의 코디세핀 함량 변화를 나타낸 도면이다(데이터는 3번의 실험 결과를 평균±표준편차로 나타냈다).
도 11은 LED 단일광의 조사시간에 따른 눈꽃동충하초 균사체의 코디세핀 함량 변화를 나타낸 도면이다(데이터는 3번의 실험 결과를 평균±표준편차로 나타냈다).
도 12는 눈꽃동충하초의 LED 단일광 및 혼합광 종류에 따른 균사체의 코디세핀 함량 변화를 나타낸 도면이다(데이터는 3번의 실험 결과를 평균±표준편차로 나타냈다).
도 13은 눈꽃동충하초 균사체에서 혼합광 red*blue의 혼합비율에 따른 코디세핀 함량 변화를 나타낸 도면이다(데이터는 3번의 실험 결과를 평균±표준편차로 나타냈다).
도 14는 눈꽃동충하초 균사체에서 혼합광 red*blue(5:5)의 조사시간에 따른 균사체 코디세핀 함량 변화를 나타낸 도면이다(데이터는 3번의 실험 결과를 평균±표준편차로 나타냈다).
도 15는 눈꽃동충하초 균사체 배양 조건과 균사체 코디세핀 함량간의 상관관계를 나타낸 도면이다.
도 16은 LED 제어조건과 눈꽃동충하초 균사체 코디세핀 함량간의 fit model을 나타낸 도면이다.
도 17은 눈꽃동충하초 균사체의 최대 코디세핀 함량을 위한 최적 LED 제어조건을 나타낸 도면이다.
도 18은 눈꽃동충하초 균사체의 최대 코디세핀 함량을 위한 최적 LED 제어조건 반응 모델의 적합도를 나타낸 도면이다.1 is a snowflake cordyceps ( Paecilomyces) javanicus ) is a diagram showing the mycelium.
Figure 2 is a view showing the cultivation process and cordycepin content measurement process of the Snow Cordyceps sinensis.
Figure 3 is a view showing the content of cordycepine in mycelium according to the type of liquid medium of Cordyceps sinensis (data shows the results of three experiments as mean ± standard deviation).
Figure 4 is a diagram showing the content of cordycepin in mycelium according to the cultivation period of Cordyceps sinensis (data shows the results of three experiments as mean ± standard deviation).
5 is a diagram showing a shaking incubator in which a fluorescent lamp and UV-A are installed.
6 is a view showing a fluorescent lamp and a control box for adjusting the amount of UV-A light and on/off.
7 is a view showing the change in the content of cordycepin in the mycelium of Cordyceps sinensis according to irradiation with a fluorescent lamp and UV-A (data are shown as mean ± standard deviation of the results of the three experiments).
8 is a diagram showing a shaking incubator in which single LED lights (red, green, blue) are installed.
9 is a view showing a control box for adjusting the amount of light and on/off of a single LED light.
10 is a view showing the change in the content of cordycepin in the mycelium according to the LED single light (red, green, blue) irradiation of the Snow Cordyceps sinensis (data shows the results of three experiments as mean ± standard deviation).
11 is a view showing the change in the content of cordycepin in the mycelium of Cordyceps sinensis according to the irradiation time of a single LED light (data are shown as mean ± standard deviation of the results of three experiments).
12 is a view showing the change in the content of cordycepin in mycelium according to the type of LED single light and mixed light of the Snow Cordyceps sinensis (data shows the results of three experiments as mean ± standard deviation).
13 is a view showing the change in the content of cordycepin according to the mixing ratio of mixed light red*blue in the mycelium of Cordyceps sinensis (data shows the results of three experiments as mean ± standard deviation).
14 is a view showing the change in mycelium cordicepin content according to the irradiation time of mixed light red*blue (5:5) in the mycelium of Cordyceps sinensis (data shows the results of three experiments as mean ± standard deviation).
15 is a view showing the correlation between the mycelium culture conditions of Cordyceps sinensis and the content of mycelium cordicepin.
16 is a view showing a fit model between the LED control conditions and the content of the mycelium cordycepine of Cordyceps sinensis.
17 is a view showing the optimal LED control conditions for the maximum cordycepine content of the mycelium of Snow Cordyceps sinensis.
18 is a view showing the suitability of the optimal LED control condition response model for the maximum cordycepine content of the mycelium of Snow Cordyceps sinensis.

이하에서는 본 발명을 구체적으로 설명한다.Hereinafter, the present invention will be described in detail.

본 발명자들은 SDB(Sabouraud dextrose broth) 액체배지에서 파란색 및 적색 혼합 LED 광을 1일 12시간으로 2일 동안 조사하여 배양한 경우, 눈꽃동충하초 균사체의 최대 코디세핀 함량을 나타냄을 확인하였으며, 반응 표면 분석을 통하여 최적의 광량, 배지의 글루코스 함량 및 배양시간 조건을 도출하고, 이를 배양조건으로 이용함으로써 눈꽃동충하초 균사체로부터 최대 함량의 코디세핀을 효율적으로 수득할 수 있음을 밝혀내어 본 발명을 완성하였다.The present inventors confirmed that when the blue and red mixed LED light was irradiated for 2 days for 12 hours per day in a liquid medium of SDB (Sabouraud dextrose broth) and cultured, the maximum cordycepine content of the mycelium of Cordyceps sinensis was shown, and the reaction surface analysis The present invention was completed by finding that the maximum amount of cordycepine can be efficiently obtained from the mycelium of Cordyceps sinensis by deriving the optimal amount of light, the glucose content of the medium, and the culture time conditions, and using this as a culture condition.

본 발명은 눈꽃동충하초 균사를 배지에 접종하는 접종단계; 및 접종된 배지를 광조건에서 배양하여 배양된 눈꽃동충하초 균사체 및 배양액에서 코디세핀을 얻는 단계; 를 포함하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법을 제공한다.The present invention is an inoculation step of inoculating the mycelium of Cordyceps sinensis into the medium; And culturing the inoculated medium under light conditions to obtain cordycepin from the cultured mycelium of Cordyceps sinensis and the culture medium. It provides a cultivation method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis comprising a.

이때, 상기 배지는 사브로드 덱스트로스 브로스(Sabouraud dextrose broth) 배지일 수 있으며, 상기 배양은 1일 내지 4일 동안 수행하는 것을 특징으로 하나, 바람직하게는 2일일 수 있다.In this case, the medium may be a sabouraud dextrose broth medium, and the culture may be performed for 1 to 4 days, preferably 2 days.

또한, 상기 배지에는 80 내지 120 mesh 크기의 볏짚을 5 내지 20 g/L 첨가할 수 있으며, 바람직하게는 80 mesh 크기의 볏짚을 15g/L 첨가할 수 있으나, 이에 제한되는 것은 아니다.In addition, 5 to 20 g/L of rice straw having a size of 80 to 120 mesh may be added to the medium, and preferably 15 g/L of rice straw having a size of 80 mesh may be added, but the present invention is not limited thereto.

또한, 상기 광조건은 LED 단일광 또는 혼합광을 접종된 배지에 조사하여 균사체를 배양하며, LED 단일광은 400nm 내지 500nm의 파장을 갖는 파란색광이며, LED 혼합광은 400nm 내지 500nm의 파장을 갖는 파란색광과 620nm 내지 780nm의 파장을 갖는 적색광을 혼합한 광인 것을 특징으로 하나, 바람직하게는 400nm 내지 500nm의 파장을 갖는 파란색광과 620nm 내지 780nm의 파장을 갖는 적색광을 혼합한 광을 이용할 수 있으며, 상기 LED 파란색 및 적색 혼합광의 혼합비율은 각각 7 : 3 내지 3 : 7인 것을 특징으로 하나, 바람직하게는 5 : 5일 수 있다. 이때 상기의 바람직한 광조건에서 눈꽃동충하초 균사체의 최대 코디세핀 함량을 나타낼 수 있다.In addition, the light condition is to cultivate the mycelium by irradiating LED single light or mixed light onto the inoculated medium, and the LED single light is blue light having a wavelength of 400 nm to 500 nm, and the LED mixed light is blue light having a wavelength of 400 nm to 500 nm. It is characterized in that the light is a mixture of light and red light having a wavelength of 620 nm to 780 nm, but preferably, light obtained by mixing blue light having a wavelength of 400 nm to 500 nm and red light having a wavelength of 620 nm to 780 nm may be used. The mixing ratio of the blue and red mixed light of LED is characterized in that each of 7:3 to 3:7, and preferably may be 5:5. At this time, it may represent the maximum content of cordycepin in the mycelium of Cordyceps sinensis under the above preferred light conditions.

또한, 상기 LED 단일광 또는 혼합광을 1일 5 내지 15시간 동안 접종된 배지에 조사하여 균사체를 배양하는 것을 특징으로 하며, 바람직하게는 1일 12시간 동안 조사할 수 있으나, 이에 제한되는 것은 아니다.In addition, it is characterized in that the mycelium is cultured by irradiating the LED single light or the mixed light on the inoculated medium for 5 to 15 hours per day, and preferably, it may be irradiated for 12 hours per day, but is not limited thereto. .

이때, 상기와 같은 배양조건 및 광조건을 벗어나면 본 발명에 따른 눈꽃동충하초 균사체의 코디세핀 함량이 증대되지 않아 코디세핀의 수득 수율이 현저히 낮아져 경제적이지 못한 문제가 야기될 수 있다.At this time, if the above cultivation conditions and light conditions are deviated, the content of cordycepin in the mycelium of Cordyceps sinensis according to the present invention is not increased, so that the yield of cordycepine is significantly lowered, which may cause an economic problem.

또한, 상기 눈꽃동충하초 균사체의 코디세핀 함량은 광량, 배지의 글루코스 함량 및 배양시간과 하기의 수학식 1과 같은 상관관계를 가지는 것을 특징으로 할 수 있다.In addition, the cordycepin content of the mycelium of Cordyceps sinensis may be characterized by having a correlation with the amount of light, the glucose content of the medium, and the culture time as shown in Equation 1 below.

[수학식 1][Equation 1]

Y1=3224.64+974.20X1+571.52X2-307.48X3+250.54X1X2-187.12X1X3-95.55X2X3-1651.51X1 2-272.72X2 2-663.51X3 2 Y 1 =3224.64+974.20X 1 +571.52X 2 -307.48X 3 +250.54X 1 X 2 -187.12X 1 X 3 -95.55X 2 X 3 -1651.51X 1 2 -272.72X 2 2 -663.51X 3 2

(상기 수학식 1에서 Y1은 눈꽃동충하초 균사체의 코디세핀 함량(mg/L), X1은 광량, X2는 배지의 글루코스 함량 및 X3는 배양시간을 의미함.)(In Equation 1, Y 1 is the cordycepine content (mg/L) of the mycelium of Cordyceps sinensis, X 1 is the amount of light, X 2 is the glucose content of the medium, and X 3 is the culture time.)

이때, 눈꽃동충하초 균사체의 최대 코디세핀 함량을 얻기 위한 상기 광량, 배지의 글루코스 함량 및 배양시간의 배양조건은 500 내지 1500 lux, 5 내지 15 g/50mL, 30 내지 100 시간인 것을 특징으로 하며, 바람직하게 상기 광량, 배지의 글루코스 함량 및 배양시간의 배양조건은 각각 990.723 lux, 9.65274 g/50mL, 61.2026 시간일 수 있으나, 이에 제한되는 것은 아니다.At this time, the cultivation conditions of the amount of light, the glucose content of the medium, and the cultivation time for obtaining the maximum cordycepin content of the mycelium of Cordyceps sinensis are 500 to 1500 lux, 5 to 15 g/50 mL, and 30 to 100 hours, preferably Thus, the amount of light, the glucose content of the medium, and the culture conditions of the culture time may be 990.723 lux, 9.65274 g/50mL, and 61.2026 hours, respectively, but are not limited thereto.

또한, 본 발명은 상기의 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법에 따라 수득된 코디세핀 조성물을 제공한다.In addition, the present invention provides a cordycepin composition obtained according to the culture method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis.

상기와 같이 배양된 눈꽃동충하초 균사체 및 배양액에는 코디세핀 함량이 높아 면역증강활성, 항암활성을 위한 건강기능식품으로도 유용하게 이용될 수 있을 것이다.Since the content of cordycepin is high in the mycelium and culture medium of Snow Cordyceps cultured as described above, it may be usefully used as a health functional food for immunity enhancing activity and anticancer activity.

또한, 본 발명은 상기 눈꽃동충하초 균사체의 코디세핀 함량을 예측하는 방법에 있어서, (a) 박스 벤켄 계획법(Box-Behnken design)으로 광량(X1), 배지의 글루코스(glucose) 함량(X2), 배양시간(X3)에 대하여, -1, 0 및 1로 코드화하여 실험범위를 설계하는 단계; (b) 상기 단계 (a)의 설계된 실험범위로, 상기 광량, 배지의 글루코스 함량 및 배양시간에 대한 실험값을 얻는 단계; (c) 상기 단계 (b)의 실험값을 이용하여 하기 수학식 1로 표시되는 이차 회귀식 모델을 도출하는 단계; 및 (d) 상기 단계 (c)에서 도출된 수학식 1로 표시되는 이차 회귀식 모델을 변량분석(ANOVA)하여 상기 눈꽃동충하초 균사체의 코디세핀 함량을 예측하는 단계; 를 포함하는 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량 예측방법을 제공한다.In addition, the present invention in the method for predicting the content of cordycepin in the mycelium of Cordyceps sinensis, (a) the amount of light (X 1 ) by Box-Behnken design, and the amount of glucose (glucose) of the medium (X 2 ) , For incubation time (X 3 ), designing an experimental range by coding as -1, 0 and 1; (b) obtaining experimental values for the amount of light, the glucose content of the medium, and the incubation time in the experimental range designed in step (a); (c) deriving a quadratic regression model represented by Equation 1 below using the experimental value of step (b); And (d) predicting the content of cordycepin in the mycelium of Cordyceps sinensis by variate analysis (ANOVA) of the quadratic regression model represented by Equation 1 derived in step (c); It provides a method for predicting the content of cordycepin in the mycelium of Cordyceps sinensis, comprising a.

[수학식 1][Equation 1]

Y1=3224.64+974.20X1+571.52X2-307.48X3+250.54X1X2-187.12X1X3-95.55X2X3-1651.51X1 2-272.72X2 2-663.51X3 2 Y 1 =3224.64+974.20X 1 +571.52X 2 -307.48X 3 +250.54X 1 X 2 -187.12X 1 X 3 -95.55X 2 X 3 -1651.51X 1 2 -272.72X 2 2 -663.51X 3 2

(상기 수학식 1에서 Y1은 눈꽃동충하초 균사체의 코디세핀 함량(mg/L), X1은 광량, X2는 배지의 글루코스 함량 및 X3는 배양시간을 의미함.)(In Equation 1, Y 1 is the cordycepine content (mg/L) of the mycelium of Cordyceps sinensis, X 1 is the amount of light, X 2 is the glucose content of the medium, and X 3 is the culture time.)

이때, 눈꽃동충하초 균사체의 최대 코디세핀 함량을 얻기 위한 상기 광량, 배지의 글루코스 함량 및 배양시간의 배양조건은 500 내지 1500 lux, 5 내지 15 g/50mL, 30 내지 100 시간인 것을 특징으로 하며, 바람직하게 상기 광량, 배지의 글루코스 함량 및 배양시간의 배양조건은 각각 990.723 lux, 9.65274 g/50mL, 61.2026 시간일 수 있으나, 이에 제한되는 것은 아니다.At this time, the cultivation conditions of the amount of light, the glucose content of the medium, and the cultivation time for obtaining the maximum cordycepin content of the mycelium of Cordyceps sinensis are 500 to 1500 lux, 5 to 15 g/50 mL, and 30 to 100 hours, preferably Thus, the amount of light, the glucose content of the medium, and the culture conditions of the culture time may be 990.723 lux, 9.65274 g/50mL, and 61.2026 hours, respectively, but are not limited thereto.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention, to those of ordinary skill in the art to which the present invention pertains. It will be self-evident.

<< 실시예Example 1> 동충하초 균사체의 액체배양기술 확립 1> Establishment of liquid culture technology for cordyceps mycelium

1. 공시재료1. Disclosure material

동충하초 균사체의 코디세핀(cordycepin) 함량을 평가하기 위하여 눈꽃동충하초(Paecilomyces javanicus)(KCCM 60299)를 한국미생물보존센터(KCCM)로부터 분양받았다(도 1).In order to evaluate the cordycepin content of the cordyceps mycelium, the snowflake cordyceps ( Paecilomyces javanicus ) (KCCM 60299) was sold from the Korea Microbiological Conservation Center (KCCM) (Fig. 1).

2. 눈꽃동충하초(2. Snowflake Cordyceps ( PaecilomycesPaecilomyces javanicusjavanicus )의 액체배양 및 코디세핀 함량 분석) In liquid culture and analysis of cordycepine content

눈꽃동충하초(Paecilomyces javanicus) 균사체의 코디세핀 함량 증대를 위한 배양환경 조건을 확립하기 위해 하기 표 1의 배양조성을 갖는 3종(YMG(yeast extract, malt extract, glucose 배지), PDB(potato dextrose broth), SDB(Sabouraud dextrose broth)의 액체 배양배지를 이용하였다.Snowflake Cordyceps ( Paecilomyces javanicus ) 3 species (YMG (yeast extract, malt extract, glucose medium), PDB (potato dextrose broth), SDB (Sabouraud dextrose)) having the culture composition shown in Table 1 below to establish the culture environment conditions for increasing the content of cordycepine in mycelium. broth)'s liquid culture medium was used.

배지 명Badge name 배지 pHMedium pH 배지 조성(g/L)Medium composition (g/L) Yeast extractYeast extract Malt extractMalt extract GlucoseGlucose Potato extractPotato extract PeptonePeptone YMGYMG 6.06.0 44 1010 44 -- -- PDBPDB 5.25.2 -- -- 2020 44 -- SDBSDB 4.54.5 -- -- 2020 -- 1010

상기 KCCM으로부터 분양받은 눈꽃동충하초(Paecilomyces javanicus) 균사를 PDA(Potato Dextrose Agar) 평판배지를 이용하여 항온 배양기에서 25℃로 7일간 배양하였다. 이후, 배양된 균사의 가장자리 부위를 직경 5 mm 코르크 보러를 사용하여 3core씩 액체배지(YMG, PDB, SDB)에 접종하여 25℃에서 7일간 진탕배양(100 rpm)하여 액체 종균을 생산하였으며, 하루간격으로 배양된 액체 종균의 균사체 코디세핀 함량을 측정하였다. Snow flower cordyceps (Paecilomyces) sold from the KCCM javanicus ) mycelium was cultured for 7 days at 25°C in an incubator using a PDA (Potato Dextrose Agar) plate medium. Thereafter, the edge of the cultured mycelium was inoculated into a liquid medium (YMG, PDB, SDB) by 3 cores using a cork borer with a diameter of 5 mm, and cultured with shaking (100 rpm) at 25°C for 7 days to produce a liquid seed. The content of the mycelium cordycepine of the liquid seed cultured at intervals was measured.

상기 YMG, PDB 또는 SDB 배지는 100mL 삼각플라스크에 50mL 씩 각각 투입하고 121℃에서 15분간 살균한 다음 냉각시켜 사용하였다.The YMG, PDB, or SDB medium was added to each of 50 mL in a 100 mL Erlenmeyer flask, sterilized at 121° C. for 15 minutes, and then cooled and used.

또한, 배양된 눈꽃동충하초 균사체 및 배양액의 코디세핀 함량은 Wang et al. (2005)의 비색법에 의해 하기의 방법으로 측정되었으며, 눈꽃동충하초 균사체의 배양과정 및 코디세핀 함량 측정 과정을 도 2에 나타내었다.In addition, the contents of cordycepin in the cultured mycelium Cordyceps sinensis and the culture medium were determined by Wang et al . It was measured by the following method by the colorimetric method of (2005), and the process of culturing the mycelium of Cordyceps sinensis and measuring the content of cordycepine is shown in FIG. 2.

① 배양이 끝난 균사체 배양액을 펠콘 튜브(falcon tube)에 옮김.① Transfer the cultured mycelium culture solution to a falcon tube.

② 원심분리 (4000 rpm, 10 min, 2회, Union 32R Plus, HANIL, Korea)를 실시함.② Perform centrifugation (4000 rpm, 10 min, 2 times, Union 32R Plus, HANIL, Korea).

③ 배양액과 균사체 분리 후 배양액은 냉장보관, 균사체는 60 ℃에서 24시간 건조시킴.③ After separating the culture medium from the mycelium, keep the culture medium in a refrigerator and dry the mycelium at 60 ℃ for 24 hours.

④ 60 ℃에서 24시간 건조된 균사체에 50% 에탄올 10 mL 를 투입함.④ Add 10 mL of 50% ethanol to the mycelium dried for 24 hours at 60 ℃.

⑤ 1시간동안 50 ℃에서 초음파 처리(ultrasonication) (40 kHz 250 W, JINWOO 2010, Korea)를 실시함.⑤ Conduct ultrasonication (40 kHz 250 W, JINWOO 2010, Korea) at 50 ℃ for 1 hour.

⑥ 1000 rpm으로 20분간 원심분리 (Union 32R Plus, HANIL, Korea)한 후 상등액을 코디세핀(cordycepin) 측정에 이용함.⑥ After centrifugation at 1000 rpm for 20 minutes (Union 32R Plus, HANIL, Korea), the supernatant is used to measure cordycepin.

⑦ 안트론(Anthron) 0.2g을 90% 황산 100 mL에 녹여 준비함 (Solvent A).⑦ Prepare by dissolving 0.2 g of Anthron in 100 mL of 90% sulfuric acid (Solvent A).

⑧ 냉장보관된 배양액과 균사체 에탄올 추출물 1mL에 Solvent A 5mL를 투입함.⑧ Add 5mL of Solvent A to 1mL of ethanol extract of mycelium and culture medium stored in the refrigerator.

⑨ 100 ℃ 워터 베스(water bath)에서 10분 동안 반응시킨 후 UV-spectrophotometer (7205, ZENWAY, UK)를 이용하여 460 nm에서 흡광도 측정.⑨ After reacting in a 100 ℃ water bath for 10 minutes, the absorbance was measured at 460 nm using a UV-spectrophotometer (7205, ZENWAY, UK).

⑩ 표준 시약 (cordycepin, sigma) 검량선 y=0.0075x+0.0378 (r2=0.9913)에 대입하여 코디세핀(cordycepin) 함량 도출.⑩ Standard reagents (cordycepin, sigma) by substituting the calibration curve y=0.0075x+0.0378 (r 2 =0.9913) to derive the content of cordycepin.

3. 눈꽃동충하초(3. Snowflake Cordyceps ( PaecilomycesPaecilomyces javanicusjavanicus )의 액체배양 배지에 따른 코디세핀 함량) Of cordycepine according to the liquid culture medium

눈꽃동충하초 균사체의 액체배지 종류에 따른 균사체 및 배양액의 코디세핀 함량은 도 3에 나타냈다.The contents of cordycepin in the mycelium and the culture medium according to the type of the liquid medium of the mycelium cordyceps sinensis are shown in FIG. 3.

결과적으로, 눈꽃동충하초 균사체를 SDB 배지에서 배양했을 때, 배양액(extra-mycelium)은 124.8 μg/mL, 균사체(intra-mycelium)는 55.9 μg/mL으로 가장 높은 코디세핀(cordycepin) 함량을 나타냈으며, 눈꽃동충하초 균사체 보다 배양액에서 더 높은 코디세핀 함량을 나타냄을 확인하였다.As a result, when the mycelium of Cordyceps sinensis was cultured in SDB medium, the culture medium (extra-mycelium) was 124.8 μg/mL, and the mycelium (intra-mycelium) was 55.9 μg/mL, showing the highest cordycepin content. It was confirmed that the content of cordycepine was higher in the culture medium than the mycelium of Cordyceps sinensis.

또한, 배양기간에 따른 눈꽃동충하초의 코디세핀 함량을 분석한 결과, 도 4와 같이 2일 경과 후 유의성 있게 높아짐을 나타내어 3일 이후 코디세핀 함량의 감소를 나타내어 눈꽃동충하초 균사체의 코디세핀 함량 측정을 위한 배양기간을 2일로 고정하였다.In addition, as a result of analyzing the content of cordycepine of Cordyceps sinensis according to the culture period, as shown in FIG. 4, it was significantly increased after 2 days, indicating a decrease in the content of cordicepine after 3 days. The incubation period was fixed to 2 days.

<< 실시예Example 2> 형광등 및 UV-A가 동충하초 균사체의 코디세핀 함량에 미치는 영향 평가 2> Evaluation of the effect of fluorescent lamps and UV-A on cordycepin content in Cordyceps mycelium

1. 공시재료 및 눈꽃동충하초 균사체의 코디세핀 함량 평가1. Evaluation of Codycepin Content in Disclosure Materials and Mycelium of Cordyceps sinensis

형광등 및 UV-A의 조사에 따른 상기 <실시예 1-1>에서 분양받은 눈꽃동충하초 균사체의 코디세핀 함량 변화를 평가하기 위하여 도 5와 같이 shaking incubator에 형광등과 UV-A (356 nm)를 설치하였다. 상기 광원은 배양액의 약 30cm 높이에 설치하였으며, 광도는 100pmol·m-2·s-1 로 설정하였다. A fluorescent lamp and UV-A (356 nm) were installed in a shaking incubator as shown in FIG. 5 in order to evaluate the change in the content of cordycepine in the mycelium of Snow Cordyceps in <Example 1-1> according to irradiation with a fluorescent lamp and UV-A. I did. The light source was installed at a height of about 30cm of the culture solution, and the luminous intensity was set to 100pmol·m -2 ·s -1.

또한, 상기 광원의 on/off를 위한 컨트롤 박스는 도 6과 같이 (주)이에스레즈의 식물생장용 LED 조명 브랜드인 빛솔 LED로부터 구매하여 설치하였으며, 배양은 상기 <실시예 1-2>와 같은 조건으로 SDB 배지에서 25℃로 7일간 진탕배양(100 rpm)하여 액체 종균을 생산하였고, 형광등 및 UV-A는 12 h/day로 2일 동안 조사하여 비색법에 의해 배양된 동충하초 균사체 및 배양액의 코디세핀 함량을 측정하였다.In addition, the control box for on/off of the light source was purchased and installed from Bitsol LED, an LED lighting brand for plant growth of ESRED, Inc., as shown in FIG. 6, and the cultivation was the same as in <Example 1-2>. As conditions, a liquid seed was produced by shaking culture (100 rpm) at 25°C for 7 days in SDB medium, and a fluorescent lamp and UV-A were irradiated for 2 days at 12 h/day, and the coordination of the cordyceps mycelium and culture medium cultured by colorimetric method. Sepine content was measured.

대조군으로는 암배양 조건으로 배양된 눈꽃동충하초 균사체를 이용하였다. As a control, the mycelium of Cordyceps sinensis cultivated under dark culture conditions was used.

2. 눈꽃동충하초의 형광등 및 UV-A 광원에 따른 코디세핀 함량2. Cordycepine content according to fluorescent lamp and UV-A light source of Snowflake Cordyceps sinensis

도 7은 눈꽃동충하초의 형광등 및 UV-A 조사에 따른 코디세핀 함량을 나타낸 것으로, 눈꽃동충하초는 암배양이나 형광등 광원보다 UV-A에서 배양했을 때 가장 많은 코디세핀 함량을 나타냄을 확인하였으며, 암배양보다 약 8배 더 많은 코디세핀 함량을 나타내는 것으로 확인되었다.7 shows the content of cordycepine according to the fluorescent lamp and UV-A irradiation of the Snow Cordyceps sinensis, and it was confirmed that the Snow Cordyceps showed the highest content of cordycepine when cultured in UV-A than the dark culture or fluorescent light source, and the dark culture It was found to exhibit about 8 times more cordycepin content than.

<< 실시예Example 3> LED 3> LED 단일광의Monolithic 제어조건(파장 종류, 조사 시간)에 따른 동충하초 균사체의 코디세핀 함량 평가 Evaluation of cordycepine content of cordyceps mycelium according to control conditions (wavelength type, irradiation time)

1. 공시재료 및 눈꽃동충하초 균사체의 코디세핀 함량 평가1. Evaluation of Codycepin Content in Disclosure Materials and Mycelium of Cordyceps sinensis

LED 단일광의 조사에 따른 눈꽃동충하초 균사체 코디세핀 함량 변화를 평가하기 위하여 도 8과 같이 shaking incubator에 LED 단일광을 설치하였다. 단일광은 red, green, blue로 총 3종의 색을 설치했으며, 각 색상 당 120개의 수량으로 설치하였다(표 2). 상기 광원은 배양액의 약 30cm 높이에 설치하였으며, 광도는 64.9~108.0 pmol·m-2·s-1 로 설정하였다.In order to evaluate the change in the content of cordycepin in the mycelium Cordyceps sinensis according to the irradiation of the LED single light, a single LED light was installed in the shaking incubator as shown in FIG. 8. For single light, a total of 3 colors were installed: red, green, and blue, and 120 pieces were installed for each color (Table 2). The light source was installed at a height of about 30cm of the culture solution, and the luminous intensity was set to 64.9-108.0 pmol·m -2 ·s -1 .

또한, 상기 LED 및 컨트롤 박스는 도 9와 같이 (주)이에스레즈의 식물생장용 LED 조명 브랜드인 빛솔 LED를 구매하여 설치하였으며, <실시예 1-2>와 같은 조건으로 PDB 배지에서 25℃에서 7일간 진탕배양(100 rpm)하여 액체 종균을 생산하였고, 단일광 3종은 12 h/day로 2일 동안 조사하여 비색법에 의해 배양된 동충하초 균사체 및 배양액의 코디세핀 함량을 측정하였다. In addition, the LED and the control box were installed by purchasing the LED lighting brand for plant growth of ESrez Co., Ltd., as shown in Fig. 9, Bissol LED, in the same conditions as in <Example 1-2> in the PDB medium at 25 ℃ A liquid seed was produced by shaking culture for 7 days (100 rpm), and three single light species were irradiated for 2 days at 12 h/day to measure the content of cordycepin in the cultured cordyceps mycelium and the culture medium by colorimetric method.

대조군으로는 암배양, 형광등 및 UV-A 조건(실시예 2)으로 배양된 동충하초 균사체를 이용하였다.As a control, cordyceps mycelium cultured under dark culture, fluorescent lamp and UV-A conditions (Example 2) was used.

색상color 파장(nm)Wavelength(nm) LED 수량LED quantity PCB 수량PCB quantity PCB 한 개당 LED 수량Number of LEDs per PCB RedRed 650650 120120 1010 1212 GreenGreen 525525 120120 1010 1212 BlueBlue 450450 120120 1010 1212

2. 눈꽃동충하초의 LED 2. LED of Snowflake Cordyceps 단일광Single light 조건에 따른 코디세핀 함량 Cordycepine content depending on conditions

도 10은 눈꽃동충하초의 LED 단일광 조사에 따른 균사체의 코디세핀 함량을 나타낸 것으로, 암배양, 형광등 배양, UV-A 배양, red광 배양, green광 배양, blue광 배양 각각 124.8 mg/L, 301.6 mg/L, 492.2 mg/L, 1186.9 mg/L, 1132.2 mg/L, 3437.6 mg/L의 코디세핀 함량을 나타내어, 눈꽃동충하초는 blue 광 조사 시, 암배양보다 27배, 형광등보다 11배, UV-A보다 7배 높은 코디세핀 함량을 나타내는 것을 확인하였다.Figure 10 shows the content of cordycepin in mycelium according to the LED single light irradiation of the Snow Cordyceps sinensis, dark culture, fluorescent lamp culture, UV-A culture, red light culture, green light culture, and blue light culture, respectively, 124.8 mg/L, 301.6 Cordycepine content of mg/L, 492.2 mg/L, 1186.9 mg/L, 1132.2 mg/L, and 3437.6 mg/L is shown. It was confirmed that the content of cordycepine was 7 times higher than that of -A.

따라서, LED blue 광은 눈꽃동충하초 균사체의 코디세핀 함량 증대에 효과적인 것으로 판단되며, 도 11은 LED 단일광 중 blue 광의 조사시간에 코디세핀 함량 변화를 나타낸 것으로, 하루에 12시간 blue 광을 조사했을 때 가장 높은 코디세핀 함량을 나타냄을 확인하였다.Therefore, LED blue light is judged to be effective in increasing the content of cordycepin in the mycelium of Cordyceps sinensis, and FIG. 11 shows the change in the content of cordycepine during the irradiation time of blue light among LED single lights, when blue light is irradiated for 12 hours a day. It was confirmed that it showed the highest cordycepin content.

<< 실시예Example 4> LED 4> LED 혼합광의Mixed mania 제어조건( Control condition ( 혼합광Mixed light 종류, 혼합비율, 조사 시간)에 따른 동충하초 균사체의 코디세핀 함량 평가 Evaluation of cordycepine content of cordyceps mycelium according to type, mixing ratio, and irradiation time)

1. 공시재료 및 눈꽃동충하초 균사체의 코디세핀 함량 평가1. Evaluation of Codycepin Content in Disclosure Materials and Mycelium of Cordyceps sinensis

LED 혼합광의 조사에 따른 눈꽃동충하초 균사체의 코디세핀 함량 변화를 평가하기 위하여 혼합광은 red*green (650+525nm), red*blue (650+450nm), green*blue (525+450nm)를 이용하였으며, 상기 광원의 광도는 64.9~108.0 pmol·m-2·s-1 로 설정하였다.Red*green (650+525nm), red*blue (650+450nm), and green*blue (525+450nm) were used to evaluate the change of cordycepin content in the mycelium of Cordyceps sinensis according to the irradiation of LED mixed light. , The luminous intensity of the light source was set to 64.9-108.0 pmol·m -2 ·s -1.

또한, 배양은 <실시예 1-2>와 같은 조건으로 SDB 배지에서 25℃에서 7일간 진탕배양(100 rpm)하여 액체 종균을 생산하였으며, 혼합광 3종은 12 h/day로 2일 동안 조사하여 비색법에 의해 배양된 동충하초 균사체 및 배양액의 코디세핀 함량을 측정하였다.In addition, culture was carried out in SDB medium under the same conditions as in <Example 1-2> by shaking culture (100 rpm) at 25° C. for 7 days to produce liquid seed, and 3 types of mixed light were irradiated for 2 days at 12 h/day. Thus, the contents of cordycepin in the cordyceps mycelium and the culture medium cultured by the colorimetric method were measured.

2. 눈꽃동충하초의 LED 2. LED of Snowflake Cordyceps 혼합광Mixed light 조건에 따른 코디세핀 함량 Cordycepine content depending on conditions

도 12는 눈꽃동충하초의 LED 혼합광 조사에 따른 균사체의 코디세핀 함량을 나타낸 것으로, red*blue 조사 시, 가장 높은 코디세핀 함량인 3654.8 mg/L, red*green 혼합광 조사 시 가장 낮은 코디세핀 함량인 1397.5 mg/L를 나타냈으며, 단일광 blue를 적용했을 때 (3437.0 mg/L) 보다 혼합광 red*blue를 적용했을 때 (3654.8 mg/L) 더 높은 코디세핀 함량을 나타냈다.Figure 12 shows the content of cordycepine in mycelium according to the LED mixed light irradiation of Snow Cordyceps sinensis, when red*blue irradiation, the highest cordicepin content 3654.8 mg/L, the lowest cordicepine content when irradiated with red*green mixed light Phosphorus showed 1397.5 mg/L, and when mixed light red*blue was applied (3654.8 mg/L) than when single light blue was applied (3437.0 mg/L), the content of cordycepine was higher.

따라서 혼합광 중 red*blue를 동충하초 균사체에 조사했을 때, 균사체의 코디세핀 함량 증대에 가장 효과적인 것으로 확인되었다. Therefore, when red*blue was irradiated to the mycelium of Cordyceps sinensis, it was found to be most effective in increasing the content of cordycepine in the mycelium.

3. 눈꽃동충하초의 LED 혼합비율 조건에 따른 코디세핀 함량3. Cordycepine content according to LED mixing ratio condition of Snowflake Cordyceps sinensis

상기와 같이 눈꽃동충하초 균사체의 코디세핀 함량 증대에 가장 효과적이었던 red*blue 혼합광의 혼합비율을 red:blue를 7:3, 5:5, 3:7로 설정하고, 상기 <실시예 1-2>와 같은 방법으로 배양된 동충하초 균사체 및 배양액의 코디세핀 함량을 측정하였다.As described above, the mixing ratio of the red*blue mixed light, which was most effective in increasing the cordycepin content of the mycelium Cordyceps sinensis, was set to red:blue as 7:3, 5:5, and 3:7, and the <Example 1-2> Cordycepin content of the cordyceps mycelium and the culture medium cultured in the same manner as described above was measured.

그 결과, 도 13과 같이 red:blue가 5:5일 때 가장 높은 코디세핀 함량을 나타냄을 확인하였다.As a result, as shown in FIG. 13, when red:blue is 5:5, it was confirmed that the highest cordicepine content was displayed.

4. 눈꽃동충하초의 LED 조사시간 조건에 따른 코디세핀 함량4. Cordycepin content according to LED irradiation time condition of Snowflake Cordyceps sinensis

LED 혼합광 red*blue(5:5)의 조사시간에 따른 코디세핀 함량 평가를 위하여 6 h/day, 12 h/day, 24 h/day 조건으로 조사하였으며, 상기 <실시예 1-2>와 같은 방법으로 배양된 동충하초 균사체 및 배양액의 코디세핀 함량을 측정하였다.In order to evaluate the cordycepin content according to the irradiation time of the LED mixed light red*blue (5:5), irradiation was conducted under the conditions of 6 h/day, 12 h/day, and 24 h/day, and Cordycepin content of the cordyceps mycelium and the culture medium cultured in the same way was measured.

대조군으로는 암배양된 동충하초 균사체를 이용하였다.As a control, a cancer-cultured cordyceps mycelium was used.

그 결과, 도 14와 같이 혼합광 red*blue(5:5)을 하루에 12시간 조사했을 때, 3657.5 mg/L로 가장 높은 코디세핀 함량을 나타냈으며, 이는 암배양 보다 약 29배 높은 코디세핀 함량을 나타냄을 확인하였다.As a result, when the mixed light red*blue (5:5) was irradiated for 12 hours a day, as shown in FIG. 14, the highest cordicepine content was shown at 3657.5 mg/L, which was about 29 times higher than in cancer culture. It was confirmed that the content was indicated.

<실시예 5> LED 혼합광과 볏짚이 포함된 배지에 따른 동충하초 균사체의 코디세핀 함량 평가<Example 5> Evaluation of cordycepin content of cordyceps mycelium according to a medium containing LED mixed light and rice straw

1. 공시재료 및 눈꽃동충하초 균사체의 코디세핀 함량 평가1. Evaluation of Codycepin Content in Disclosure Materials and Mycelium of Cordyceps sinensis

LED 혼합광 red*blue(5:5)의 12시간 조사에서 SDB 배지에 볏짚을 포함했을 때, 눈꽃동충하초 균사체의 코디세핀 함량 변화를 평가하기 위하여 볏짚은 분쇄 후 ASTM 규격 mesh No. 80, No. 100, No. 120로 분급하여 5 ~ 20 g/L 첨가하였으며, 상기 <실시예 1-2>와 같은 방법으로 배양된 동충하초 균사체 및 배양액의 코디세핀 함량을 측정하였다.When rice straw was included in the SDB medium in 12 hours of irradiation with LED mixed light red*blue (5:5), in order to evaluate the change in the content of cordycepin in the mycelium of Cordyceps sinensis, rice straw was pulverized and then pulverized according to ASTM standard mesh No. 80, No. 100, No. It was classified at 120 and added 5 to 20 g/L, and the contents of cordycepin in the cultured cordyceps mycelium and the culture medium were measured in the same manner as in <Example 1-2>.

대조군으로는 암배양된 동충하초 균사체를 이용하였으며, LED 혼합광은 red*blue (5:5), 조사시간은 12시간으로 고정하여 평가하였다.As a control, dark-cultured cordyceps mycelium was used, and the LED mixed light was fixed at red*blue (5:5) and the irradiation time was fixed at 12 hours.

2. 눈꽃동충하초의 LED 혼합광과 볏짚이 포함된 배지 조건에 따른 코디세핀 함량2. The content of cordycepine according to the conditions of the culture medium containing LED mixed light and rice straw of Snowflake Cordyceps sinensis

LED 혼합광 red*blue(5:5) 조사와 볏짚의 크기에 따른 코디세핀 함량 변화를 표 3에 나타냈으며, 이때의 볏짚 첨가량은 15 g/L로 고정하였다.Table 3 shows the change of cordycepin content according to the red*blue (5:5) irradiation of LED mixed light and the size of the rice straw, and the amount of rice straw added at this time was fixed at 15 g/L.

결과적으로, 볏짚을 포함하지 않는 SDB 배지에서 혼합광 red*blue (5:5)를 적용했을 때, 3657.5 mg/L, Mesh No. 80 pass의 볏짚을 포함한 SDB 배지에서 혼합광 red*blue (5:5)를 적용했을 때, 3813.1 mg/L의 코디세핀 함량을 나타냈다.As a result, when mixed light red*blue (5:5) was applied in SDB medium containing no rice straw, 3657.5 mg/L, Mesh No. When the mixed light red*blue (5:5) was applied in the SDB medium containing 80 pass of rice straw, the content of cordycepine was 3813.1 mg/L.

따라서, 동충하초 균사체에서 LED 혼합광 red*blue (5:5)를 볏짚이 포함된 SDB 배지에 적용했을 때, 코디세핀 함량 증대에 대한 시너지 효과가 있는 것으로 판단되며, 볏짚의 크기는 No. 100 mesh 및 No. 120 mesh 보다 No. 80 mesh가 코디세핀 증대에 가장 효과적인 것으로 확인하였다.Therefore, when the LED mixed light red*blue (5:5) in the cordyceps mycelium was applied to the SDB medium containing rice straw, it was judged that there was a synergistic effect on increasing the content of cordycepine, and the size of the rice straw was No. 100 mesh and No. No. than 120 mesh It was confirmed that 80 mesh was the most effective in increasing cordycepin.

또한, 표 4는 배지에서 볏짚의 첨가량 (5 ~ 20 g/L)에 따른 눈꽃동충하초의 코디세핀 함량을 나타낸 것으로, 볏짚 크기는 mesh No. 80 pass로 고정하였다. In addition, Table 4 shows the content of cordycepin in Snow Cordyceps according to the amount of rice straw added (5 ~ 20 g/L) in the medium, and the size of the rice straw is mesh No. It was fixed at 80 pass.

그 결과, 15 g/L의 볏짚을 배지에 첨가했을 때, 가장 높은 코디세핀 함량(3915.5 mg/L)을 나타내었으며, 이로써 80 mesh 크기의 볏짚을 15 g/L로 SDB 배지에 포함시켰을 때 눈꽃동충하초의 코디세핀 함량 증대가 높은 것으로 확인하였다.As a result, when 15 g/L of rice straw was added to the medium, the highest cordycepine content (3915.5 mg/L) was exhibited. Accordingly, when 80 mesh-sized rice straw was included in the SDB medium at 15 g/L, the snow flower It was confirmed that the increase in the content of cordycepin in Cordyceps sinensis was high.

조건Condition 코디세핀 함량(㎍/mL)Cordycepine content (㎍/mL) Red:Blue (5:5)Red:Blue (5:5) 3657.5 ±0.0d3657.5 ±0.0d Red:Blue (5:5) + media based 80mesh pass rice strawRed:Blue (5:5) + media based 80mesh pass rice straw 3813.1 ±0.1a3813.1 ±0.1a Red:Blue (5:5) + media based 100mesh pass rice strawRed:Blue (5:5) + media based 100mesh pass rice straw 3696.8 ±0.0b3696.8 ±0.0b Red:Blue (5:5) + media based 120mesh pass rice strawRed:Blue (5:5) + media based 120mesh pass rice straw 3681.3 ±0.0c3681.3 ±0.0c

조건Condition 코디세핀 함량(㎍/mL)Cordycepine content (㎍/mL) Red:Blue (5:5)Red:Blue (5:5) 3657.5 ±0.0e3657.5 ±0.0e Red:Blue (5:5) + media based 5g rice straw/LRed:Blue (5:5) + media based 5g rice straw/L 3662.4 ±0.0d3662.4 ±0.0d Red:Blue (5:5) + media based 10g rice straw/LRed:Blue (5:5) + media based 10g rice straw/L 3768.8 ±0.0c3768.8 ±0.0c Red:Blue (5:5) + media based 15g rice straw/LRed:Blue (5:5) + media based 15g rice straw/L 3813.1 ±0.1a3813.1 ±0.1a Red:Blue (5:5) + media based 20g rice straw/LRed:Blue (5:5) + media based 20g rice straw/L 3800.5 ±0.1b3800.5 ±0.1b

<< 실시예Example 6> 균사체의 최대 코디세핀 함량을 위한 LED의 최적 제어 조건 도출 6> Derivation of optimal control conditions of LED for maximum cordycepine content of mycelium

1. LED 제어조건과 균사체 코디세핀 함량1. LED control conditions and mycelium cordycepine content 간의Liver 상관관계 도출 Derivation of correlation

LED 제어조건을 포함한 동충하초 균사체 배양 조건과 균사체 코디세핀 함량간의 상관관계 도출은 피어슨 상관계수(Hauke and Kossowski, 2001)와 히트맵(Wilf et al., 2016)을 이용하였으며, 통계 프로그램은 R program (version 3.4.3)을 이용하였다.Pearson correlation coefficient (Hauke and Kossowski, 2001) and heat map (Wilf et al., 2016) were used to derive the correlation between cordyceps mycelium culture conditions including LED control conditions and mycelium cordycepine content, and the statistical program was R program ( version 3.4.3) was used.

눈꽃동충하초 균사체 배양조건 인자는 LED 파장, LED 광량, LED 조사 시간, 배지의 글루코스(glucose) 함량, 배지의 pH로 설정하였다.The parameters of the culture conditions for mycelium of Cordyceps sinensis were set as LED wavelength, LED light intensity, LED irradiation time, glucose content of the medium, and pH of the medium.

상기 피어슨 상관계수에서 계수가 높은 수치를 나타낼수록 배양 조건 인자와 코디세핀 함량간의 상관관계가 높다고 평가하였으며, LED 제어조건과 동충하초 균사체의 코디세핀 함량간의 회귀식을 도출하기 위하여 CurveExpert program (version 3.4.3)의 fit model을 이용하였다.In the Pearson correlation coefficient, the higher the coefficient was, the higher the correlation between the culture condition factor and the cordycepine content was evaluated, and to derive a regression equation between the LED control condition and the cordycepine content of Cordyceps mycelium, the CurveExpert program (version 3.4. The fit model of 3) was used.

결과적으로, 눈꽃동충하초 균사체 배양 조건과 균사체 코디세핀 함량간의 상관관계를 도 15에 나타내었으며, 눈꽃동충하초 균사체 코디세핀 함량에 가장 큰 영향을 미치는 배양 조건은 LED 파장으로, 이때 LED 파장은 균사체 코디세핀 함량과 음의 상관관계(-0.50)를 나타내는 것으로 확인되었다.As a result, the correlation between the mycelium culture conditions of Cordyceps sinensis and mycelium cordycepine content is shown in FIG. It was found to show a negative correlation (-0.50) with.

또한, LED 제어조건과 동충하초 균사체의 코디세핀 함량간의 fit model은 도 16에 나타내었으며, LED 제어 조건과 균사체 코디세핀 함량간의 가장 상관관계가 높았던 LED 파장은 ‘Ratkowsky’ model인 것으로 확인되었다. 이를 이용한 회귀식은 표 5에 나타내었으며, 도출된 LED 제어조건과 균사체 코디세핀 함량간의 회귀식을 통해 LED 제어조건(파장, 광량, 조사시간)만을 이용하여 코디세핀 함량을 예측할 수 있을 것으로 판단된다.In addition, the fit model between the LED control condition and the cordycepin content of the cordyceps mycelium is shown in FIG. 16, and the LED wavelength, which had the highest correlation between the LED control condition and the mycelium cordycepine content, was confirmed to be the'Ratkowsky' model. The regression equation using this is shown in Table 5, and through the regression equation between the derived LED control conditions and the mycelial cordycepine content, it is judged that the cordycepine content can be predicted using only the LED control conditions (wavelength, light quantity, irradiation time).

No.No. χχ yy 회귀식Regression 상수a constant 1One 파장wavelength 코디세핀 함량Cordycepine content y=a/(1+eb-cχ)y=a/(1+eb -cχ ) a=0.98; b=3.86; c=0.03a=0.98; b=3.86; c=0.03 22 광량Light intensity 코디세핀 함량Cordycepine content y=a/(1+eb-cχ)y=a/(1+eb -cχ ) a=3.79; b=162.80; c=0.12a=3.79; b=162.80; c=0.12 33 조사시간Investigation time 코디세핀 함량Cordycepine content y=(a+bχ)/(1+cχ+dχ2)y=(a+bχ)/(1+cχ+dχ 2 ) a=27.07; b=-0.29; c=-3.05; d=2.04a=27.07; b=-0.29; c=-3.05; d=2.04

2. 균사체 코디세핀 함량 증대를 위한 최적 LED 조건 확립2. Establishment of optimal LED conditions for increasing mycelium cordycepine content

눈꽃동충하초 균사체 코디세핀 함량 증대를 위한 최적 LED 제어 조건은 반응 표면 모델(Response Surface Model;RSM)의 박스 벤켄 계획법(Box-Benkhen design;BBD) 실험설계를 이용하였으며, 프로그램은 Design-Expert Software Version 10을 이용하였다. The optimal LED control conditions for increasing the content of cordycepin in the mycelium cordyceps sinensis were used for the Box-Benkhen design (BBD) experimental design of the Response Surface Model (RSM), and the program was Design-Expert Software Version 10. Was used.

고정 인자는 눈꽃동충하초(P. javanicus) 균사체이며, 변수는 광량 조건, 배지의 글루코스 함량, 배양시간으로 설정하였다. 이때, 변수의 범위는 예비 실험을 통하여 설정하였으며, BBD 실험 설계는 하기 표 6에서와 같이 총 17조건으로 설정하였다. The fixation factor was the mycelium of Snow Cordyceps (P. javanicus ), and the variables were set as the light quantity condition, the glucose content of the medium, and the cultivation time. At this time, the range of the variable was set through a preliminary experiment, and the BBD experimental design was set to a total of 17 conditions as shown in Table 6 below.

RunRun 독립변수(Independent variables)Independent variables
(coded)(coded) 독립변수(Independent variables)Independent variables
(actual)(actual) 코디세핀 함량(CordycepinCordycepin content (Cordycepin
content), mg/Lcontent), mg/L XX 1One XX 22 XX 33 XX 1One XX 22 XX 33 YY 1One 1One 1One 00 -1-One 13381338 7.57.5 2424 2074.182074.18 22 00 00 00 669669 7.57.5 7272 3100.923100.92 33 1One 00 1One 13381338 7.57.5 120120 1320.561320.56 44 -1-One 1One 00 00 1010 7272 186.37186.37 55 00 00 00 669669 7.57.5 7272 3165.123165.12 66 00 -1-One -1-One 669669 55 2424 1772.231772.23 77 -1-One 00 -1-One 00 7.57.5 2424 124.43124.43 88 1One -1-One 00 13381338 55 7272 1913.361913.36 99 -1-One 00 1One 00 7.57.5 120120 119.28119.28 1010 00 -1-One 1One 669669 55 120120 1112.811112.81 1111 1One 1One 00 13381338 1010 7272 3008.743008.74 1212 00 1One 1One 669669 1010 120120 2613.482613.48 1313 00 00 00 669669 7.57.5 7272 3031.833031.83 1414 00 00 00 669669 7.57.5 7272 3112.593112.59 1515 -1-One -1-One 00 00 55 7272 93.1693.16 1616 00 1One -1-One 669669 1010 2424 3655.113655.11 1717 00 00 00 669669 7.57.5 7272 3712.733712.73 Independent variablesIndependent variables LevelsLevels -1-One 00 1One X1: 광량, luxX 1 : Light quantity, lux 00 669669 13381338 X2: 글루코스 함량, g/50 mLX 2 : glucose content, g/50 mL 55 7.57.5 1010 X3: 배양시간, h X 3 : Incubation time, h 2424 7272 120120

도 17은 각 인자간의 상호관계를 반응 표면으로 나타낸 3차원 반응 표면 그래프와 유도된 2차 다항방정식 회귀식 모델을 나타낸 것으로, 결과적으로 눈꽃동충하초 균사체의 최대 코디세핀 함량을 위한 최적 배양 조건이 광량 990.723 lux, 배지의 glucose 함량 9.65274 g/50mL, 배양기간 61.2026시간 일 때, 3779.2 mg/L까지 코디세핀 함량이 증대되는 것으로 확인되었다.FIG. 17 shows a three-dimensional response surface graph showing the relationship between each factor as a response surface and an induced quadratic polynomial regression model. When the lux, the glucose content of the medium was 9.65274 g/50mL, and the culture period was 61.2026 hours, it was confirmed that the content of cordycepine increased to 3779.2 mg/L.

또한, 상기 2차 다항방정식 회귀식 모델을 변량분석(ANOVA)하여 도 18에서와 같이 반응 모델의 적합도를 나타내는 R2 (결정계수)를 얻었다. 그 결과 R2 (결정계수)는 0.8910으로 높은 신뢰성을 나타내었으며, 이에 따라 상기 도 17에서 유도된 2차 다항방정식은 눈꽃동충하초의 최대 코디세핀 함량을 위한 최적 배양 조건의 반응값 예측에 적합한 것으로 판단된다.In addition, the second-order polynomial regression model was subjected to variate analysis (ANOVA ) to obtain R 2 (coefficient of determination) indicating the fitness of the reaction model as shown in FIG. 18. As a result, R 2 (coefficient of determination) showed high reliability as 0.8910, and accordingly, the second-order polynomial equation derived in FIG. 17 was judged to be suitable for predicting the response value of the optimal culture conditions for the maximum cordycepine content of Cordyceps sinensis. do.

Claims (16)

눈꽃동충하초 균사를 배지에 접종하는 접종단계; 및
접종된 배지를 광조건에서 배양하여 배양된 눈꽃동충하초 균사체 및 배양액에서 코디세핀을 얻는 단계; 를 포함하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법.Inoculation step of inoculating the mycelium of Cordyceps sinensis into the medium; And
Culturing the inoculated medium under light conditions to obtain cordycepin from the cultured mycelium of Cordyceps sinensis and the culture medium; Cultivation method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis comprising a. 제 1항에 있어서,
배지는 사브로드 덱스트로스 브로스(Sabouraud dextrose broth) 배지인 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법.The method of claim 1,
The culture method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis, characterized in that the medium is a sabouraud dextrose broth medium. 제 1항에 있어서,
배지에 80 내지 120 mesh 크기의 볏짚을 5 내지 20 g/L 첨가하는 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법.The method of claim 1,
A culture method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis, characterized in that 5 to 20 g/L of rice straw having a size of 80 to 120 mesh is added to the medium. 제 1항에 있어서,
배양은 1일 내지 4일 동안 수행하는 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법.The method of claim 1,
Culture method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis, characterized in that the cultivation is carried out for 1 to 4 days. 제 1항에 있어서,
광조건은 LED 단일광 또는 혼합광을 접종된 배지에 조사하여 균사체를 배양하는 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법.The method of claim 1,
The light condition is a culture method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis, characterized in that the mycelium is cultivated by irradiating the inoculated medium with LED single light or mixed light. 제 5항에 있어서,
LED 단일광은 400nm 내지 500nm의 파장을 갖는 파란색광이며, LED 혼합광은 400nm 내지 500nm의 파장을 갖는 파란색광과 620nm 내지 780nm의 파장을 갖는 적색광을 혼합한 광인 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법.The method of claim 5,
LED single light is a blue light having a wavelength of 400nm to 500nm, and the LED mixed light is a combination of blue light having a wavelength of 400nm to 500nm and red light having a wavelength of 620nm to 780nm. Cultivation method to increase the sepin content. 제 5항에 있어서,
LED 단일광 또는 혼합광을 1일 5 내지 15시간 동안 접종된 배지에 조사하여 균사체를 배양하는 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법.The method of claim 5,
A culture method for increasing the content of codycepin in the mycelium of Cordyceps sinensis, characterized in that the mycelium is cultured by irradiating the inoculated medium with LED single light or mixed light for 5 to 15 hours per day. 제 6항에 있어서,
400nm 내지 500nm의 파장과 620nm 내지 780nm의 파장을 갖는 LED 파란색 및 적색 혼합광이 눈꽃동충하초 균사체의 최대 코디세핀 함량을 나타내는 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법.The method of claim 6,
A cultivation method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis, characterized in that the blue and red mixed light of LED having a wavelength of 400nm to 500nm and a wavelength of 620nm to 780nm represents the maximum content of cordicepin in the cordyceps mycelium. 제 8항에 있어서,
LED 파란색 및 적색 혼합광의 혼합비율은 각각 7 : 3 내지 3 : 7인 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법.The method of claim 8,
A cultivation method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis, characterized in that the mixing ratio of the blue and red LED light is 7:3 to 3:7, respectively. 제 1항에 있어서,
눈꽃동충하초 균사체의 코디세핀 함량은,
광량, 배지의 글루코스 함량 및 배양시간과 하기의 수학식 1과 같은 상관관계를 가지는 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법:
[수학식 1]
Y1=3224.64+974.20X1+571.52X2-307.48X3+250.54X1X2-187.12X1X3-95.55X2X3-1651.51X1 2-272.72X2 2-663.51X3 2
(상기 수학식 1에서 Y1은 눈꽃동충하초 균사체의 코디세핀 함량(mg/L), X1은 광량, X2는 배지의 글루코스 함량 및 X3는 배양시간을 의미함.)The method of claim 1,
The content of cordycepine in the mycelium of Cordyceps sinensis,
A culture method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis, characterized in that it has a correlation as shown in Equation 1 below with the amount of light, the glucose content of the medium, and the culture time:
[Equation 1]
Y 1 =3224.64+974.20X 1 +571.52X 2 -307.48X 3 +250.54X 1 X 2 -187.12X 1 X 3 -95.55X 2 X 3 -1651.51X 1 2 -272.72X 2 2 -663.51X 3 2
(In Equation 1, Y 1 is the cordycepine content (mg/L) of the mycelium of Cordyceps sinensis, X 1 is the amount of light, X 2 is the glucose content of the medium, and X 3 is the culture time.) 제 10항에 있어서,
광량, 배지의 글루코스 함량 및 배양시간의 배양조건은 각각 500 내지 1500 lux, 5 내지 15 g/50mL, 30 내지 100 시간인 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법.The method of claim 10,
Culture method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis, characterized in that the amount of light, the glucose content of the medium, and the culture conditions of the culture time are 500 to 1500 lux, 5 to 15 g/50 mL, and 30 to 100 hours, respectively. 제 11항에 있어서,
광량, 배지의 글루코스 함량 및 배양시간의 배양조건은 각각 990.723 lux, 9.65274 g/50mL, 61.2026 시간인 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량을 증대하는 배양방법.The method of claim 11,
Culture method for increasing the content of cordycepin in the mycelium of Cordyceps sinensis, characterized in that the amount of light, the glucose content of the medium, and the culture conditions of the culture time are 990.723 lux, 9.65274 g/50mL, and 61.2026 hours, respectively. 제 1항 내지 제 12항 중 어느 한 항의 방법에 따라 배양된 눈꽃동충하초로부터 수득된 코디세핀 조성물.The cordycepin composition obtained from the Snow Cordyceps cultivated according to the method of any one of claims 1 to 12. 제 1항에 따른 눈꽃동충하초 균사체의 코디세핀 함량을 예측하는 방법에 있어서,
(a) 박스 벤켄 계획법(Box-Behnken design)으로 광량(X1), 배지의 글루코스(glucose) 함량(X2), 배양시간(X3)에 대하여, -1, 0 및 1로 코드화하여 실험범위를 설계하는 단계;
(b) 상기 단계 (a)의 설계된 실험범위로, 상기 광량, 배지의 글루코스 함량 및 배양시간에 대한 실험값을 얻는 단계;
(c) 상기 단계 (b)의 실험값을 이용하여 하기 수학식 1로 표시되는 이차 회귀식 모델을 도출하는 단계; 및
(d) 상기 단계 (c)에서 도출된 수학식 1로 표시되는 이차 회귀식 모델을 변량분석(ANOVA)하여 상기 눈꽃동충하초 균사체의 코디세핀 함량을 예측하는 단계; 를 포함하는 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량 예측방법:
[수학식 1]
Y1=3224.64+974.20X1+571.52X2-307.48X3+250.54X1X2-187.12X1X3-95.55X2X3-1651.51X1 2-272.72X2 2-663.51X3 2
(상기 수학식 1에서 Y1은 눈꽃동충하초 균사체의 코디세핀 함량(mg/L), X1은 광량, X2는 배지의 글루코스 함량 및 X3는 배양시간을 의미함.)In the method for predicting the content of cordycepin in the mycelium of Cordyceps sinensis according to claim 1,
(a) Experiment by coding as -1, 0 and 1 for the amount of light (X 1 ), the glucose content of the medium (X 2 ), and the incubation time (X 3) by Box-Behnken design Designing a range;
(b) obtaining experimental values for the amount of light, the glucose content of the medium, and the incubation time in the experimental range designed in step (a);
(c) deriving a quadratic regression model represented by Equation 1 below using the experimental value of step (b); And
(d) predicting the content of cordycepine in the mycelium of Cordyceps sinensis by variate analysis (ANOVA) of the quadratic regression model represented by Equation 1 derived in step (c); Method for predicting the content of cordycepin in the mycelium of Cordyceps sinensis, characterized in that it comprises:
[Equation 1]
Y 1 =3224.64+974.20X 1 +571.52X 2 -307.48X 3 +250.54X 1 X 2 -187.12X 1 X 3 -95.55X 2 X 3 -1651.51X 1 2 -272.72X 2 2 -663.51X 3 2
(In Equation 1, Y 1 is the cordycepine content (mg/L) of the mycelium of Cordyceps sinensis, X 1 is the amount of light, X 2 is the glucose content of the medium, and X 3 is the culture time.) 제 14항에 있어서,
광량, 배지의 글루코스 함량 및 배양시간의 배양조건은 각각 500 내지 1500 lux, 5 내지 15 g/50mL, 30 내지 100 시간인 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량 예측방법.The method of claim 14,
The amount of light, the glucose content of the medium, and the culture conditions of the culture time are 500 to 1500 lux, 5 to 15 g/50 mL, and 30 to 100 hours, respectively. 제 15항에 있어서,
광량, 배지의 글루코스 함량 및 배양시간의 배양조건은 각각 990.723 lux, 9.65274 g/50mL, 61.2026 시간인 것을 특징으로 하는 눈꽃동충하초 균사체의 코디세핀 함량 예측방법.The method of claim 15,
The amount of light, the glucose content of the medium, and the culture conditions of the culture time are 990.723 lux, 9.65274 g/50mL, and 61.2026 hours respectively.
KR1020190108888A 2019-09-03 2019-09-03 Culture method for enhancing cordycepin content of Paecilomyces javanicus Mycelium using Light-Emitting Diode KR102356058B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020190108888A KR102356058B1 (en) 2019-09-03 2019-09-03 Culture method for enhancing cordycepin content of Paecilomyces javanicus Mycelium using Light-Emitting Diode

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020190108888A KR102356058B1 (en) 2019-09-03 2019-09-03 Culture method for enhancing cordycepin content of Paecilomyces javanicus Mycelium using Light-Emitting Diode

Publications (2)

Publication Number Publication Date
KR20210027875A true KR20210027875A (en) 2021-03-11
KR102356058B1 KR102356058B1 (en) 2022-01-26

Family

ID=75143288

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020190108888A KR102356058B1 (en) 2019-09-03 2019-09-03 Culture method for enhancing cordycepin content of Paecilomyces javanicus Mycelium using Light-Emitting Diode

Country Status (1)

Country Link
KR (1) KR102356058B1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220145494A (en) * 2021-04-22 2022-10-31 충북대학교 산학협력단 Cultivation method of Glehnia littoralis in plant factory

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20010054264A (en) 1999-12-04 2001-07-02 안용준 Cordycepin separated from Cordyceps spp. for an anti-cancer medicine and its production
JP2006204310A (en) * 2006-05-01 2006-08-10 Hokuto Corp Method for cultivating plant worm
KR20140073803A (en) * 2012-12-07 2014-06-17 (주)티앤아이 Method of Sparassis crispa hypa cultivating by LED light
KR20140089204A (en) * 2013-01-04 2014-07-14 안인웅 Cultivation method of ascomycota mushroom using nano-gold coloid
CN107311733A (en) * 2017-05-08 2017-11-03 李鹄鸣 A kind of production method of high content adenosine fruiting bodies of cordyceps militaris
KR101898086B1 (en) * 2015-11-30 2018-09-12 김태욱 Cultivating method of Cordyceps staphylinidicola for increasing its content of cordycepin

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20010054264A (en) 1999-12-04 2001-07-02 안용준 Cordycepin separated from Cordyceps spp. for an anti-cancer medicine and its production
JP2006204310A (en) * 2006-05-01 2006-08-10 Hokuto Corp Method for cultivating plant worm
KR20140073803A (en) * 2012-12-07 2014-06-17 (주)티앤아이 Method of Sparassis crispa hypa cultivating by LED light
KR20140089204A (en) * 2013-01-04 2014-07-14 안인웅 Cultivation method of ascomycota mushroom using nano-gold coloid
KR101898086B1 (en) * 2015-11-30 2018-09-12 김태욱 Cultivating method of Cordyceps staphylinidicola for increasing its content of cordycepin
CN107311733A (en) * 2017-05-08 2017-11-03 李鹄鸣 A kind of production method of high content adenosine fruiting bodies of cordyceps militaris

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
한국잠사곤충학회지(=Journal of sericultural and entomological science) v.52 no.1, pp.59~63, 2014년* *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220145494A (en) * 2021-04-22 2022-10-31 충북대학교 산학협력단 Cultivation method of Glehnia littoralis in plant factory

Also Published As

Publication number Publication date
KR102356058B1 (en) 2022-01-26

Similar Documents

Publication Publication Date Title
Chiang et al. Effect of light-emitting diodes on the production of cordycepin, mannitol and adenosine in solid-state fermented rice by Cordyceps militaris
Desjardin et al. Fungi bioluminescence revisited
Attaran Dowom et al. Agronomic and environmental factors affecting cultivation of the winter mushroom or Enokitake: Achievements and prospects
CN109122050A (en) A kind of cultural method of Cordyceps militaris
Zhou Cultivation of Ganoderma lucidum
KR102356058B1 (en) Culture method for enhancing cordycepin content of Paecilomyces javanicus Mycelium using Light-Emitting Diode
JP6331206B1 (en) Method for evaluating quality of fulvic acid solution and method for producing fulvic acid solution
CN104381013A (en) Method for cultivating antrodia cinnamomea by using culture medium
Shi et al. Enhanced production of cordycepic acid from Cordyceps cicadae isolated from a wild environment
KR102356057B1 (en) Culture method for enhancing cordycepin content of Cordyceps Militaris Mycelium using Light-Emitting Diode
Luangharn et al. Domestication of Ganoderma leucocontextum, G. resinaceum, and G. gibbosum Collected from Yunnan Province, China
Alcazar et al. Effect of Light-Emitting Diodes on the Mycelial Biomass Production and Antioxidant Activity of Ganoderma lucidum (W. Curt.: Fr.) P. Karst.
KR102356059B1 (en) Culture method for enhancing content of Cordyceps militaris mycelium using Light-Emitting Diode
KR100655781B1 (en) Specific primer for identification of Cordyceps militaris and identification method using the same
Tiniola et al. Light emitting diode enhances the biomass yield and antioxidant activity of Philippine wild mushroom Lentinus swartzii.
KR102356056B1 (en) Culture method for enhancing content of Paecilomyces javanicus mycelium using Light-Emitting Diode
JP2003116522A (en) Medium for culturing cordyceps sinensis sacc. and method for culturing cordyceps sinensis sacc.
Ha et al. Effect of light-emitting diodes on cordycepin production in submerged culture of Paecilomyces japonica
JP2006281010A (en) Woody waste material decomposer and woody waste material decomposing method
CHANG et al. Basidiomatal formation of Antrodia cinnamomea on artificial agar media
KR102600164B1 (en) Culture method for enhancing content of Paecilomyces javanicus mycelium using woody liquid medium and Light-Emitting Diode
KR102518524B1 (en) Culture method for enhancing content of Cordyceps militaris mycelium using woody liquid medium and Light-Emitting Diode
CN108004102A (en) A kind of bottle is planted the envirment factor control method that Cordyceps militaris prepares living Chinese caterpillar wine
Krishna et al. Conservation of Endangered Cordyceps sinensis Through Artificial Cultivation Strategies of C. militaris, an Alternate
KR102518526B1 (en) Culture method for enhancing cordycepin content of Paecilomyces javanicus mycelium using woody liquid medium and Light-Emitting Diode

Legal Events

Date Code Title Description
E902 Notification of reason for refusal
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right