KR102517994B1 - Culture medium composition for culturing cordyceps mycelia comprising damaged wood by pine wilt disease - Google Patents

Culture medium composition for culturing cordyceps mycelia comprising damaged wood by pine wilt disease Download PDF

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KR102517994B1
KR102517994B1 KR1020200155419A KR20200155419A KR102517994B1 KR 102517994 B1 KR102517994 B1 KR 102517994B1 KR 1020200155419 A KR1020200155419 A KR 1020200155419A KR 20200155419 A KR20200155419 A KR 20200155419A KR 102517994 B1 KR102517994 B1 KR 102517994B1
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medium
cordyceps
mycelium
medium composition
monochamus alternatus
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KR20220068536A (en
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양재경
하시영
정지영
이동환
최지원
박재현
유찬열
박준호
박성웅
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경상국립대학교산학협력단
경상남도
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    • 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
    • A01G18/40—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
    • A01G18/50—Inoculation of spawn
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

본 발명은 소나무재선충피해목을 포함하는 동충하초 배양용 배지조성물에 관한 것으로, 보다 상세하게는 소나무재선충피해목 분말 및 탄화된 소나무재선출피해목 분말이 포함된 배지조성물에 배양된 동충하초 균사체는 종래 액체배지에서 배양된 동충하초 균사체와 비교하여 우수한 균사체의 생장량 및 코디세핀 함량 증가 효과를 나타내는 것이 확인됨에 따라, 상기 배지조성물은 솔수염하늘소 동충하초 균사체의 대량생산 및 유용한 생리활성 물질인 코디세핀 생산 증가를 위한 동충하초 배양용 배지조성물로 제공될 수 있다.The present invention relates to a medium composition for culturing Cordyceps sinensis containing pine wilt nematode damaged tree, and more particularly, Cordyceps mycelium cultured in a medium composition containing pine ash nematode damaged tree powder and carbonized pine ash nematode damaged tree powder. As it was confirmed that the mycelium growth rate and cordycepin content increase effect were confirmed compared to cordyceps mycelium cultured in the medium, the medium composition was used for mass production of Monochamus alternatus cordyceps mycelium and for increasing the production of cordycepin, a useful physiologically active substance It can be provided as a medium composition for culturing Cordyceps sinensis.

Description

소나무재선충피해목을 포함하는 동충하초 균사체 배양용 배지조성물{Culture medium composition for culturing cordyceps mycelia comprising damaged wood by pine wilt disease}Medium composition for culturing cordyceps mycelia containing pine wood nematode damaged trees {Culture medium composition for culturing cordyceps mycelia damaged comprising wood by pine wilt disease}

본 발명은 소나무재선충피해목을 포함하는 솔수염하늘소 동충하초의 균사체 생장 및 코디세핀 함량 증가를 위한 배지조성물에 관한 것이다.The present invention relates to a medium composition for growing mycelium and increasing cordycepin content of Monochamus alternatus cordyceps, including trees damaged by pine wood nematodes.

동충하초는 겨울에는 곤충의 몸에 있다가 여름에는 풀처럼 나타난다는 데서 이름지어진 일종의 버섯으로, 곰팡이의 일종인 동충하초균이 살아있는 곤충의 몸속으로 들어가 발생하는 곤충기생성 약용버섯이다. 동충하초로는 시네시스 동충하초 (Cordyceps sinensis), 밀리타리스 동충하초(Codyceps militaris, 번데기 동충하초), 눈꽃 동충하초(Paecilomyces japonica) 등이 잘 알려져 있는데, 장수하늘소 유충을 기주로 하는 장수하늘소 동충하초(Cordyceps staphylinidicola)는 가장 많이 이용되는 번데기 동충하초(Cordyceps miliatris)에 비해서도 높은 코디세핀을 함유하는 균주들이 자연에 존재한다.Cordyceps sinensis is a type of mushroom named for the fact that it resides in the body of an insect in winter and appears like grass in summer. Cordyceps sinensis, Codyceps militaris, Paecilomyces japonica, etc. are well known. Strains containing higher levels of cordycepin exist in nature even compared to the most used pupal cordyceps (Cordyceps miliatris).

동충하초는 동의보감을 통해서 오랫동안 많은 효능이 인정되어 각종 성인병 질환의 치료 및 건강유지를 위하여 사용되어 왔다. 중국에서는 육상선수들이 동충하초를 복용하고 기적의 기록들을 나타낸다고 하여 동충하초를 3대 천연식품 건강 명약으로 인정하고 있다.Cordyceps sinensis has long been recognized for its efficacy through Donguibogam, and has been used for the treatment of various adult diseases and maintenance of health. In China, cordyceps is recognized as one of the three major natural food health medicines because track and field athletes take it and show miraculous records.

동충하초는 작물들 중 단백질 함량이 가장 높은 (건조함량의 28%이상) 군에 속하고, 면역력을 강화시키는 물질이 다량 함유되어 있다. 동충하초의 면역활성물질 중에는 키닉산(quinic acid)의 이성체로 밝혀진 '코디세핀 (Cordycepin :3'-deoxy-adenosine)' 이 포함되어 있으며, 동충하초 중에서도 밀리타리스 동충하초가 다른 동충하초에 비하여 코디세핀의 함량이 월등히 높다. 코디세핀은 핵산 물질로서 세포의 유전정보에 관여하면서 저하된 면역기능을 활성화하여 정상세포가 암세포로 전환되는 것을 방지하는 작용을 한다. 동충하초를 투입한 임상 실험에서 암세포를 죽이는 면역세포인 NK세포(natural killer cell/자연살해세포)와 면역세포에서 분비되는 사이토카인의 함량이 18%~25% 증가되는 것으로 확인되어 혈액암치료용으로 활용되고, 건강기능식품으로서도 많은관심을 받고 있다. 이러한 결과를 토대로 국내에서는 코디세핀이 함유된 동충하초가 버섯으로는 유일하게 건강 기능식품으로 식약처인증을 받았다. 또한 동충하초는 혈압과 당뇨를 조절하는 효능과 폐와 간 기능 등에도 많은 도움을 주는 것으로 임상연구결과가 발표되고 있다.Cordyceps 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. Among the immunoactive substances of Cordyceps sinensis, 'Cordycepin (3'-deoxy-adenosine)', which was found to be an isomer of quinic acid, is included. this is extremely high Cordycepin is a nucleic acid substance that acts to prevent conversion of normal cells into cancer cells by activating the reduced immune function while being involved in the genetic information of cells. In a clinical experiment 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 and is receiving a lot of attention as a health functional food. Based on these results, Cordyceps sinensis, which contains Cordycepin, is the only mushroom in Korea that has been certified as a health functional food by the Ministry of Food and Drug Safety. In addition, clinical research results have been announced that Cordyceps sinensis is helpful in controlling blood pressure and diabetes, as well as lung and liver functions.

동충하초는 버섯류 중에서도 많은 소비가 이루어지고 있지만 천연에서 채집되는 양은 매우 적어서 수요를 충당할 수 없으므로 전 세계의 많은 농가들이 인공재배법으로 재배사를 만들어서 대량 재배하고 있으나 동충하초는 성장속도가 느리고, 외부환경에 민감하여 농약을 살포할 수 없는 식물이어서 재배가 매우 까다로와 오랫동안 재배를 해온 농가에서도 동충하초 재배의 수확률이 저조한 문제점이 있다.Cordyceps is consumed a lot among mushrooms, but the amount collected in nature is very small and cannot meet the demand, so many farmers around the world have created cultivation houses through artificial cultivation and are cultivating in large quantities. However, Cordyceps has a slow growth rate and is sensitive to the external environment. Because it is a plant that cannot be sprayed with pesticides, cultivation is very difficult, and even in farmhouses that have been cultivated for a long time, there is a problem in that the yield of cordyceps cultivation is low.

대한민국 공개특허 제10-2020-0048792호 (2020.05.08. 공개)Republic of Korea Patent Publication No. 10-2020-0048792 (2020.05.08. Publication)

본 발명은 솔수염하늘소 동충하초의 균사체 생장 향상 및 코디세핀 함량을 증가시키기 위해 소나무재선충피해목을 포함하는 동충하초 배양용 배지조성물을 제공하고자 한다.The present invention is to provide a medium composition for culturing Cordyceps sinensis containing Pine wilt nematode damaged tree to improve mycelial growth and increase cordycepin content of Monochamus alternatus Cordyceps.

본 발명은 기본배지 및 소나무재선충피해목을 포함하는 솔수염하늘소 동충하초 배양용 배지조성물을 제공한다.The present invention provides a medium composition for culturing Monochamus alternatus Cordyceps sinensis, including a basic medium and pine wood nematode-damaged trees.

또한, 본 발명은 상기 배지조성물에 동충하초 균사체를 접종하는 단계; 및 상기 배지조성물에 접종된 동충하초 균사체를 배양하는 단계를 포함하는 솔수염하늘소 동충하초 균사체 배양방법을 제공한다.In addition, the present invention comprises the steps of inoculating the mycelium of Cordyceps sinensis in the medium composition; and culturing the mycelium of Cordyceps sinensis inoculated in the medium composition.

본 발명에 따르면, SDB 1.5 중량%, 대두 2.5 중량% 및 솔수염하늘소 4.1 중량%를 포함하는 액체배지에 소나무재선충피해목 분말 및 탄화된 소나무재선출피해목 분말이 포함된 배지조성물에 배양된 동충하초 균사체는 종래 액체배지에서 배양된 동충하초 균사체와 비교하여 우수한 균사체의 생장량 및 코디세핀 함량 증가 효과를 나타내는 것이 확인됨에 따라, 상기 배지조성물은 솔수염하늘소 동충하초 균사체의 대량생산 및 유용한 생리활성 물질인 코디세핀 생산 증가를 위한 동충하초 배양용 배지조성물로 제공될 수 있다.According to the present invention, Cordyceps sinensis cultured in a medium composition containing pine ash nematode damaged tree powder and carbonized pine ash ash damaged tree powder in a liquid medium containing 1.5% by weight of SDB, 2.5% by weight of soybean, and 4.1% by weight of Monochamus alternatus. As it was confirmed that the mycelium exhibits an effect of increasing the growth amount and cordycepin content of the mycelium compared to the mycelium of cordyceps mycelium cultured in a conventional liquid medium, the medium composition is used for mass production of Monochamus alternatus cordyceps mycelium and cordycepin, a useful physiologically active substance It can be provided as a medium composition for culturing Cordyceps sinensis for increased production.

도 1은 동충하초 균사체의 코디세핀 함량 증대에 효과적인 대두 (Soybean) 첨가 액체배지 개발을 위한 실험 모식도이다.
도 2는 대두 첨가량에 따라 제조된 PDB 및 SDB 액체배지이다.
도 3은 PDB 액체배지에서 대두 첨가량에 따른 균사체 생장량 변화를 확인하기 위해 균사체의 건조 무게를 확인한 결과로, PDBC: PDB (Potato Dextrose Broth) 단독; PDBS1: PDB+2.5 g/L soybean; PDBS2: PDB+5.0 g/L soybean; PDBS3: PDB+10.0 g/L soybean; PDBS4: PDB+20 g/L soybean을 의미한다. 각 결과 값은 평균 ± SE (n = 5)로 나타내었다.
도 4는 SDB 액체배지에서 대두 첨가량에 따른 균사체 변화를 확인하기 위해 균사체의 건조 무게를 확인한 결과로, SDBC: SDB (Sabouraud Dextrose Broth) 단독; SDBS1: SDB+2.5 g/L soybean; SDBS2: SDB+5.0 g/L soybean; SDBS3: SDB+10.0 g/L soybean; SDBS4: SDB+20 g/L soybean을 의미한다. 각 결과 값은 평균 ± SE (n = 5)로 나타내었다.
도 5는 PDB 액체배지에서 대두 첨가량에 따른 균사체량을 확인을 위해 600 nm에서 흡광도를 확인한 결과로, PDBC: PDB (Potato Dextrose Broth); PDBS1: PDB+2.5 g/L soybean; PDBS2: PDB+5.0 g/L soybean; PDBS3: PDB+10.0 g/L soybean; PDBS4: PDB+20 g/L soybean을 의미한다. 각 결과 값은 평균 ± SE (n = 5)로 나타내었다.
도 6은 SDB 액체배지에서 대두 첨가량에 따른 균사체량을 확인을 위해 600 nm에서 흡광도를 확인한 결과로, SDBC: SDB (Sabouraud Dextrose Broth) 단독; SDBS1: SDB+2.5 g/L soybean; SDBS2: SDB+5.0 g/L soybean; SDBS3: SDB+10.0 g/L soybean; SDBS4: SDB+20 g/L soybean을 의미한다. 각 결과 값은 평균 ± SE (n = 5)로 나타내었다.
도 7은 PDB 및 SDB 액체배지에서 대두 첨가량에 따른 균사체 생장을 확인한 결과이다.
도 8은 Cordycepin standard의 calibration curve (R2=0.9999189)이다.
도 9는 PDB 액체배지에서 생장한 동충하초 균사체 배양액의 코디세핀 성분 피크를 확인한 결과이다.
도 10은 SDB 액체배지에서 생장한 동충하초 균사체 배양액의 코디세핀 성분 피크를 확인한 결과이다.
도 11은 PDB 액체배양액과 SDB 액체배양액의 코디세핀 성분 피크를 비교한 결과이다.
도 12는 대두 2.5 g/L이 첨가된 SDB 액체배지(SDBS1)에서 생장한 동충하초 균사체와 배양액의 코디세핀 성분 피크를 확인한 결과이다.
도 13은 PDB 및 SDB 배지에서 대두 첨가량에 따른 동충하초 균사체 배양액의 코디세핀 함량을 확인한 결과로, PDBC: PDB (Potato Dextrose Broth) 단독; PDBS1: PDB+2.5 g/L soybean; PDBS2: PDB+5.0 g/L soybean; PDBS3: PDB+10.0 g/L soybean; PDBS4: PDB+20 g/L soybean. SDBC: SDB (Sabouraud Dextrose Broth) 단독; SDBS1: SDB+2.5 g/L soybean; SDBS2: SDB+5.0 g/L soybean; SDBS3: SDB+10.0 g/L soybean; SDBS4: SDB+20 g/L soybean을 의미한다. 각 결과 값은 평균 ± SE (n = 5)로 나타내었다.
도 14는 pH 조건에 따른 액체배지이다.
도 15는 액체배지에서 pH 조건에 따른 균사체 생장량을 확인한 결과이다. 각 결과 값은 평균 ± SE (n = 5)로 나타내었다.
도 16은 흡광도를 이용한 액체배지에서 pH 조건에 따른 균사체 생장량 변화를 확인한 결과이다. 각 결과 값은 평균 ± SE (n = 5)로 나타내었다.
도 17은 액체배지에서 pH 조건에 따른 균사체 배양액의 코디세핀 함량을 확인한 결과이다.
도 18은 솔수염하늘소 투입 형태에 따른 액체배지이다.
도 19는 2.5 g/L이 첨가된 SDB 액체배지(SDBS1)에서 솔수염하늘소 첨가 형태에 따른 균사체 생장량을 확인한 결과이다.
도 20은 2.5 g/L이 첨가된 SDB 액체배지(SDBS1)에서 솔수염하늘소 첨가 형태에 따른 코디세핀 함량을 확인한 결과이다.
도 21은 2.5 g/L이 첨가된 SDB 액체배지(SDBS1)에서 솔수염하늘소 첨가량에 따른 균사체 생장량을 확인한 결과이다.
도 22는 2.5 g/L이 첨가된 SDB 액체배지(SDBS1)에서 솔수염하늘소 첨가량에 따른 코디세핀 함량을 확인한 결과이다.
도 23은 솔수염하늘소 분말도에 따른 액체배지이다.
도 24는 2.5 g/L이 첨가된 SDB 액체배지(SDBS1)에서 솔수염하늘소 분말도에 따른 균사체 생장량을 확인한 결과이다.
도 25는 2.5 g/L이 첨가된 SDB 액체배지(SDBS1)에서 솔수염하늘소 분말도에 따른 코디세핀 함량을 확인한 결과이다.
도 26은 동충하초 균사체의 코디세핀 함량 증대를 위한 솔수염하늘소 최적 투입 조건을 확인한 결과이다.
도 27은 솔수염하늘소 첨가배지에서 재선충피해목 분말과 탄화된 재선충피해목 분말 혼합에 따른 동충하초 균사체의 코디세핀 함량을 확인한 결과이다.1 is a schematic diagram of an experiment for the development of a soybean-added liquid medium effective for increasing the cordycepin content of cordyceps mycelium.
Figure 2 is a PDB and SDB liquid medium prepared according to the amount of soybeans added.
Figure 3 is a result of checking the dry weight of the mycelium to confirm the change in mycelial growth according to the amount of soybean addition in the PDB liquid medium, PDBC: PDB (Potato Dextrose Broth) alone; PDBS1: PDB+2.5 g/L soybean; PDBS2: PDB+5.0 g/L soybean; PDBS3: PDB+10.0 g/L soybean; PDBS4: Means PDB+20 g/L soybean. Each resultant value is expressed as the mean ± SE (n = 5).
4 is a result of checking the dry weight of the mycelium to confirm the change in mycelium according to the amount of soybeans added in the SDB liquid medium, SDBC: SDB (Sabouraud Dextrose Broth) alone; SDBS1: SDB+2.5 g/L soybean; SDBS2: SDB+5.0 g/L soybean; SDBS3: SDB+10.0 g/L soybean; SDBS4: means SDB+20 g/L soybean. Each resultant value is expressed as the mean ± SE (n = 5).
Figure 5 is a result of confirming the absorbance at 600 nm to confirm the amount of mycelium according to the amount of soybeans added in the PDB liquid medium, PDBC: PDB (Potato Dextrose Broth); PDBS1: PDB+2.5 g/L soybean; PDBS2: PDB+5.0 g/L soybean; PDBS3: PDB+10.0 g/L soybean; PDBS4: Means PDB+20 g/L soybean. Each resultant value is expressed as the mean ± SE (n = 5).
Figure 6 is a result of confirming the absorbance at 600 nm to confirm the amount of mycelium according to the amount of soybeans added in SDB liquid medium, SDBC: SDB (Sabouraud Dextrose Broth) alone; SDBS1: SDB+2.5 g/L soybean; SDBS2: SDB+5.0 g/L soybean; SDBS3: SDB+10.0 g/L soybean; SDBS4: means SDB+20 g/L soybean. Each resultant value is expressed as the mean ± SE (n = 5).
Figure 7 is the result of confirming the growth of mycelium according to the amount of soybean addition in PDB and SDB liquid medium.
8 is a calibration curve (R2 = 0.9999189) of the Cordycepin standard.
9 is a result of confirming the cordycepin component peak of the cordyceps mycelium culture medium grown in PDB liquid medium.
10 is a result of confirming the cordycepin component peak of Cordyceps sinensis mycelium culture medium grown in SDB liquid medium.
11 is a result of comparing the cordycepin component peaks of the PDB liquid culture medium and the SDB liquid culture medium.
12 is a result of confirming the cordycepin component peak of cordyceps mycelium and culture medium grown in SDB liquid medium (SDBS1) to which 2.5 g/L of soybean was added.
13 is a result of confirming the cordycepin content of the cordyceps mycelium culture medium according to the amount of soybeans added in PDB and SDB medium, PDBC: PDB (Potato Dextrose Broth) alone; PDBS1: PDB+2.5 g/L soybean; PDBS2: PDB+5.0 g/L soybean; PDBS3: PDB+10.0 g/L soybean; PDBS4: PDB+20 g/L soybean. SDBC: SDB (Sabouraud Dextrose Broth) alone; SDBS1: SDB+2.5 g/L soybean; SDBS2: SDB+5.0 g/L soybean; SDBS3: SDB+10.0 g/L soybean; SDBS4: means SDB+20 g/L soybean. Each resultant value is expressed as the mean ± SE (n = 5).
14 is a liquid medium according to pH conditions.
15 is a result of confirming the mycelium growth according to the pH conditions in the liquid medium. Each resultant value is expressed as the mean ± SE (n = 5).
16 is a result of confirming the change in mycelial growth according to the pH condition in a liquid medium using absorbance. Each resultant value is expressed as the mean ± SE (n = 5).
17 is a result of confirming the cordycepin content of the mycelium culture medium according to the pH conditions in the liquid medium.
18 is a liquid medium according to Monochamus alternatus input type.
19 is a result of confirming the growth of mycelium according to the type of Monochamus alternatus added in SDB liquid medium (SDBS1) to which 2.5 g/L was added.
20 is a result of confirming the content of cordycepin according to the added form of Monochamus alternatus in SDB liquid medium (SDBS1) to which 2.5 g/L was added.
21 is a result of confirming the mycelium growth according to the amount of Monochamus alternatus added in SDB liquid medium (SDBS1) to which 2.5 g / L was added.
22 is a result of confirming the cordycepin content according to the amount of Monochamus alternatus added in SDB liquid medium (SDBS1) to which 2.5 g/L was added.
23 is a liquid medium according to Monochamus alternatus powder.
24 is a result of confirming the mycelium growth according to the powder degree of Monochamus alternatus in SDB liquid medium (SDBS1) to which 2.5 g/L was added.
25 is a result of confirming the cordycepin content according to Monochamus alternatus powder in SDB liquid medium (SDBS1) to which 2.5 g/L was added.
26 is a result of confirming the optimal input conditions for Monochamus alternatus for increasing the cordycepin content of Cordyceps sinensis mycelium.
27 is a result of confirming the content of cordycepin in Cordyceps sinensis mycelium according to the mixture of P. nematode damaged tree powder and carbonized W. elegans damaged tree powder in Monochamus alternatus supplemented medium.

이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.

동충하초는 기주의 대상이 되는 곤충의 몸에 침입하여 기주의 양분을 통해 자실체를 형성시키는 약용버섯으로, 동물성 단백질을 영양성분으로 하기 때문에 일반 버섯류에서는 찾아볼 수 없는 유용한 물질, 즉 인간의 각종 질병치료에 이용되어질 수 있는 유용한 성분을 포함하여 기능성 식품으로서의 고부가가치가 있으나, 대량 재배가 어렵고 외부환경에 민감하여 재배가 매우 까다로운 문제점이 있는 바, 상기 문제점을 해결하기 위하여 본 발명을 완성하였다.Cordyceps sinensis is a medicinal mushroom that invades the body of a target insect and forms a fruiting body through the nutrients of the host. Because it uses animal protein as a nutritional component, it is a useful substance that cannot be found in general mushrooms, that is, it treats various human diseases. Although it has high added value as a functional food, including useful ingredients that can be used for, there is a problem in that it is difficult to grow in large quantities and sensitive to the external environment, so cultivation is very difficult. In order to solve the above problem, the present invention was completed.

본 발명은 기본배지 및 소나무재선충피해목을 포함하는 솔수염하늘소 동충하초 배양용 배지조성물을 제공할 수 있다.The present invention can provide a medium composition for culturing Monochamus alternatus Cordyceps, including a basic medium and pine wood nematode-damaged trees.

상기 소나무재선충피해목은 소나무재선충피해목 분말, 탄화된 소나무재선충피해목 분말 또는 이들의 혼합물인 것일 수 있다.The pine wood nematode damaged tree may be pine wood nematode damaged wood powder, carbonized pine wood nematode damaged wood powder, or a mixture thereof.

상기 배지조성물은 소나무재선충피해목 10 내지 40 중량부 및 기본배지 60 내지 90 중량부로 포함되는 것일 수 있다. The medium composition may be included in 10 to 40 parts by weight of pine wood nematode-damaged trees and 60 to 90 parts by weight of the basal medium.

상기 기본배지는 조성물 100 중량부에 대하여, 배지 1 내지 5 중량부, 대두 1 내지 10 중량부, 솔수염하늘소 5 내지 15 중량부 및 물 70 내지 90 중량부를 포함하는 것일 수 있다.The basal medium may include 1 to 5 parts by weight of medium, 1 to 10 parts by weight of soybeans, 5 to 15 parts by weight of Monochamus alternatus, and 70 to 90 parts by weight of water, based on 100 parts by weight of the composition.

보다 상세하게는 상기 배지조성물은 물 100 중량%에 대하여, SDB 3 중량%, 대두 5 중량% 및 솔수염하늘소 8.2 중량%를 포함하는 것일 수 있으나, 이에 제한되는 것은 아니다.More specifically, the medium composition may include 3% by weight of SDB, 5% by weight of soybeans, and 8.2% by weight of Monochamus alternatus based on 100% by weight of water, but is not limited thereto.

상기 배지는 PDB (Potato Dextrose Broth) 및 SDB (Sabouraud Dextrose Broth)으로 이루어진 군에서 선택되는 것일 수 있다.The medium may be selected from the group consisting of Potato Dextrose Broth (PDB) and Sabouraud Dextrose Broth (SDB).

상기 솔수염하늘소는 건조된 원형 또는 분말로 첨가되는 것일 수 있다.The monochamus alternatus may be added as a dried form or powder.

상기 배지조성물은 pH 4 내지 pH 6인 것일 수 있으며, 보다 바람직하게는 pH 4.5 내지 5.6일 수 있으나, 이에 제한되는 것은 아니다.The medium composition may be pH 4 to pH 6, more preferably pH 4.5 to 5.6, but is not limited thereto.

상기 배지조성물은 솔수염하늘소 동충하초 균사체의 생장 및 코디세핀 함량을 증가시키는 것일 수 있다.The medium composition may increase the growth and cordycepin content of Monochamus alternatus cordyceps mycelium.

본 발명은 상기 배지조성물에 동충하초 균사체를 접종하는 단계; 및 상기 배지조성물에 접종된 동충하초 균사체를 배양하는 단계를 포함하는 솔수염하늘소 동충하초 균사체 배양방법을 제공할 수 있다.The present invention comprises the steps of inoculating the mycelium of Cordyceps sinensis in the medium composition; and culturing the Cordyceps mycelium inoculated in the medium composition.

보다 상세하게는 배지조성물에 접종된 동충하초 균사체를 배양하는 단계는 24℃에서 5 내지 10일간 배양하는 것일 수 있으나, 이에 제한되는 것을 아니다.More specifically, the step of culturing the mycelium of Cordyceps sinensis inoculated into the medium composition may be culturing at 24 ° C. for 5 to 10 days, but is not limited thereto.

상기 배양방법은 솔수염하늘소 동충하초 균사체의 생장량 및 코디세핀 함량을 증가시키는 것일 수 있다.The culturing method may be to increase the growth amount and cordycepin content of Monochamus alternatus cordyceps mycelium.

상기 동충하초 균사체의 코디세핀 함량은 솔수염하늘소의 투입량, 투입크기 및 배양시간과 하기의 수학식 1과 같은 상관관계를 가지는 것일 수 있다.The cordycepin content of the Cordyceps sinensis mycelium may have a correlation as shown in Equation 1 below with the input amount, input size, and incubation time of Monochamus alternatus.

[수학식 1][Equation 1]

Y1=-3624.37595+161.74173A+649.65098B+1062.81758C-17.98434AB-23.56214AC-2.33472BC-4.62463A2-76.22227B2-60.03783C2 Y1=-3624.37595+161.74173A+649.65098B+1062.81758C-17.98434AB-23.56214AC-2.33472BC-4.62463A 2 -76.22227B 2 -60.03783C 2

상기 수학식 1에서 Y1은 밀리타리스 동충하초 균사체의 코디세핀 함량 (mg/L), A는 분말도 (mm), B는 투입량 (g/50mL) 및 C는 배양기간 (day)을 나타낸다.In Equation 1, Y1 represents the cordycepin content (mg/L) of the mycelium of Militaris Cordyceps sinensis, A is the fineness (mm), B is the input amount (g/50mL), and C is the incubation period (day).

이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<실시예 1> 동충하초 균사체 (<Example 1> Cordyceps mycelium ( Cordyceps militarisCordyceps militaris ) 생장 및 코디세핀 함량 증가를 위한 액체 배지 선별) liquid medium screening for growth and increased cordycepin content

1. 대두 (soybean) 첨가량에 따른 동충하초 균사체 생장량 확인1. Check the growth of cordyceps mycelium according to the amount of soybean added

동충하초 균사체 생장량 향상을 위한 액체 배지 개발을 위해, Potato Dextrose Broth (PDB) 및 Sabouraud Dextrose Broth (SDB)를 사용하였으며, 표 1과 같은 함량으로 대두 분말을 액체배지에 첨가하고 교반기를 이용하여 혼합하여 도 2와 같이 다양한 함량의 대두가 포함된 액체배지를 준비하였다.To develop a liquid medium for improving the growth of Cordyceps mycelium, Potato Dextrose Broth (PDB) and Sabouraud Dextrose Broth (SDB) were used, and soybean powder was added to the liquid medium with the contents shown in Table 1 and mixed using an agitator. As in 2, a liquid medium containing soybeans of various contents was prepared.

배지badge Potato extract (g)Potato extract (g) Dextrose (g)Dextrose (g) Peptone (g)Peptone (g) Soybean (g)Soybeans (g) Agar Agar
(g)(g) PDBPDB 44 2020 PDBS1PDBS1 44 2020 2.52.5 PDBS2PDBS2 44 2020 55 PDBS3PDBS3 44 2020 1010 PDBS4PDBS4 44 2020 2020 SDBSDB 2020 1010 SDBS1SDBS1 2020 1010 2.52.5 SDBS2SDBS2 2020 1010 55 SDBS3SDBS3 2020 1010 1010 SDBS4SDBS4 2020

상기 대두가 첨가된 액체배지를 고압멸균기 (autoclave)를 이용하여 121℃에서 15분간 고압멸균하고, 고압멸균된 액체배지에 동충하초 균사체 (Cordyceps militaris, 5 mm)를 3 core 투입한 다음 24℃, 100 rpm 진탕배양기 (shaking incubator)에서 7일간 배양하였다.The liquid medium to which the soybeans were added was sterilized at 121 ° C. for 15 minutes using an autoclave, and 3 cores of Cordyceps militaris ( Cordyceps militaris , 5 mm) were added to the high-pressure sterilized liquid medium, followed by 24 ° C., 100 It was cultured for 7 days in an rpm shaking incubator.

이후 액체배지에서 배양된 균사체 생장량을 측정하기 위해 배양 종료된 액체배지를 원심분리하여 상등액을 제거하고 분리된 균사체를 멸균수로 세척한 후 동결건조기를 이용하여 72시간 건조 후 무게를 측정하였다.Then, in order to measure the growth of the mycelium cultured in the liquid medium, the culture medium was centrifuged to remove the supernatant, the separated mycelium was washed with sterile water, and the weight was measured after drying for 72 hours using a freeze dryer.

그 결과, 도 3과 같이 PDB 액체배지에 대두 10.0 g/L가 첨가된 PDBS3이 가장 높은 균사체 생장량을 나타내었으며, 대두가 첨가되지 않은 PDB 배지와 비교하여 PDBS3에서는 1.8배의 균사체 생장량이 확인되었다. 대두 10.0 g/L 이상 첨가된 PDB 배지에서는 대두 첨가량이 균사체에 유의적인 영향을 나타내지 않는 것으로 확인됨에 따라, 대두 10.0 g/L이 첨가된 PDBS3 배지가 동충하초 균사체 생장에 가장 효과적인 것으로 확인되었다.As a result, as shown in FIG. 3, PDBS3 in which 10.0 g / L of soybean was added to the PDB liquid medium showed the highest mycelial growth, and compared to the PDB medium without soybean, the mycelium growth was 1.8 times higher in PDBS3. As it was confirmed that the addition of soybeans did not have a significant effect on mycelium in the PDB medium containing 10.0 g/L or more of soybeans, PDBS3 medium containing 10.0 g/L of soybeans was found to be most effective for the growth of Cordyceps mycelium.

또한, 도 4와 같이 SDB 액체배지에서는 대두 2.5 g/L가 첨가된 SDBS1에서 가장 높은 균사체량이 확인되었으며, 대두가 첨가되지 않은 SDBC 배지와 비교하여 SDBS1에서 1.2배 이상의 균사체량이 확인되었다.In addition, as shown in FIG. 4, in the SDB liquid medium, the highest mycelial mass was confirmed in SDBS1 with 2.5 g / L of soybeans added, and more than 1.2 times more mycelial mass was confirmed in SDBS1 compared to SDBC medium without soybeans.

상기 결과로부터 PDB와 SDB 배지 모두 대두가 첨가되었을 때 균사체량이 증가하는 것으로 확인되었으며, 특히 SDB 배지보다 PDB 배지에 10 g/L 대두를 첨가하였을 때 가장 높은 균사체량 (0.22 g/50mL)을 나타내는 것을 확인할 수 있었다.From the above results, it was confirmed that mycelial mass increased when soybeans were added to both PDB and SDB media, and in particular, the highest mycelial mass (0.22 g/50mL) was shown when 10 g/L soybean was added to PDB medium than SDB medium. I was able to confirm.

또한, 균사체 생장량을 흡광도로 확인하기 위해, 배양 기간 동안 1일 1회 균질화된 배양액 1 mL를 수집하였으며, 수집된 배양액은 UV-spectrophotometer를 이용하여 600 nm에서 흡광도를 측정하였다.In addition, in order to confirm the mycelium growth by absorbance, 1 mL of the homogenized culture medium was collected once a day during the culture period, and the absorbance of the collected culture medium was measured at 600 nm using a UV-spectrophotometer.

그 결과, 도 5 내지 도 6과 같이 대두가 첨가된 PDB와 SDB 배지 모두 6일차에서 급격히 균사체량이 증가하는 것으로 확인되었으며, 특히 PDBS3이 가장 높은 균사체량 증가 효과를 나타내었다.As a result, as shown in FIGS. 5 to 6, it was confirmed that both PDB and SDB medium to which soybeans were added rapidly increased mycelial mass on the 6th day, and in particular, PDBS3 showed the highest mycelial mass increasing effect.

상기 결과들로부터 동충하초 균사체의 높은 생장량 증대를 나타낸 PDBS3이 동충하초 균사체 생장에 가장 효과적인 것이 확인되었다.From the above results, it was confirmed that PDBS3, which showed a high growth increase of Cordyceps mycelium, was most effective for the growth of Cordyceps mycelium.

한편, 대두 투입량에 따른 동충하초 균사체의 코디세핀 (cordycepin) 함량을 HPLC를 이용하여 확인하였다.On the other hand, the cordycepin content of cordyceps mycelium according to the amount of soybean input was confirmed using HPLC.

건조된 균사체 1 mg을 HPLC 등급의 메탄올 1 mL와 혼합하고 HPLC 분석기기를 이용하여 코디세핀을 정성·정량 분석하였다.1 mg of the dried mycelium was mixed with 1 mL of HPLC-grade methanol, and cordycepin was analyzed qualitatively and quantitatively using an HPLC analyzer.

HPLC 분석 컬럼은 Agilent Eclipse Pluse C18 (4.6×250 mm, 5 μm)을 사용하였으며, 이동상으로 HPLC 등급의 메탄올과 물을 15 : 85 (w:w)로 혼합하여 사용하였다. 유속 1 mL/min, 컬럼 온도 30℃, PDA 260 nm detector, injection volume 10 μL 조건으로 분석을 수행하였다.An HPLC analysis column was Agilent Eclipse Pluse C18 (4.6 × 250 mm, 5 μm), and HPLC-grade methanol and water were mixed at a ratio of 15:85 (w:w) as a mobile phase. Analysis was performed under conditions of a flow rate of 1 mL/min, column temperature of 30°C, PDA 260 nm detector, and injection volume of 10 μL.

정성분석은 cordycepin standard의 retention time을 이용하여 검출을 확인하였으며, 정량분석은 농도별 cordycepin standard의 HPLC 정량 분석 결과로부터 작성된 도 8의 calibration curve에 이용하여 도출하였다. Qualitative analysis confirmed the detection using the retention time of the cordycepin standard, and quantitative analysis was derived using the calibration curve of FIG. 8 prepared from the results of HPLC quantitative analysis of the cordycepin standard for each concentration.

그 결과, 도 9 내지 도 13을 참고하면 대두 2.5 g/L가 첨가된 SDB 배지(SDBC1)에서 생장한 동충하초 균사체의 코디세핀 함량이 가장 높은 것으로 확인되었으며, 대두가 첨가되지 않은 SDB 배지와 비교하여 대두 첨가는 동충하초 균사체 배양액 내 코디세핀 함량을 증가시키는 효과적임이 확인되었다.As a result, referring to FIGS. 9 to 13, it was confirmed that the cordycepin mycelium of cordyceps mycelium grown in SDB medium (SDBC1) with 2.5 g / L of soybean had the highest cordycepin content, compared to SDB medium without soybean. It was confirmed that the addition of soybean was effective in increasing the cordycepin content in the cordyceps mycelium culture medium.

또한, PDB 배지에서 생장한 동충하초 균사체 배양액보다 SDB 배지에서 생장한 동충하초 균사체 배양액에서 높은 코디세핀 함량이 확인되었다.In addition, a higher cordycepin content was confirmed in the Cordyceps mycelium culture medium grown in the SDB medium than in the Cordyceps mycelium culture medium grown in the PDB medium.

상기 결과로부터 대두가 첨가된 SDB 배지는 동충하초 균사체의 생장 및 코디세핀 함량을 모두 증가시킬 수 있음이 확인되었으며, 특히 대두 2.5 g/L가 첨가된 SDB 배지가 균사체 생장 및 코디세핀 함량 증가를 위한 최적의 배지 조건임이 확인되었다.From the above results, it was confirmed that the SDB medium to which soybeans were added can increase both the growth of cordyceps mycelium and the cordycepin content. It was confirmed that the medium condition of

2. 액체 배지 pH에 따른 균사체 생장량 및 코디세핀 함량 확인2. Confirmation of mycelium growth and cordycepin content according to liquid medium pH

도 14와 같이 1N HCl과 1M NaOH를 사용하여 대두 2.5 g/L가 첨가된 SDB 배지의 pH를 pH 4.5, pH 5.0, pH 5.5 및 pH 6.0 조건으로 조절한 후 pH에 따른 균사체 생장량 및 코디세핀 함량을 확인하였다.As shown in FIG. 14, after adjusting the pH of the SDB medium to which 2.5 g/L of soybeans was added using 1N HCl and 1M NaOH to pH 4.5, pH 5.0, pH 5.5 and pH 6.0 conditions, mycelial growth and cordycepin content according to pH confirmed.

상기 액체 배지에 앞선 실험과 동일한 방법으로 동충하초 균사체 (5 mm)를 3 core 투입한 다음 24℃, 100 rpm 진탕배양기에서 7일간 배양하였다.3 cores of cordyceps mycelium (5 mm) were added to the liquid medium in the same manner as in the previous experiment, and then cultured for 7 days in a shaker at 24 ° C and 100 rpm.

배양 종료 후 액체 배지를 원심분리하여 상등액을 제거하고 균사체를 멸균수로 세척한 후 동결건조기를 이용하여 72시간 건조 후 무게를 측정하였으며, 앞선 실험과 동일한 방법으로 흡광도를 측정하여 배양된 균사체의 생장량을 확인하였다.After the completion of the culture, the liquid medium was centrifuged to remove the supernatant, and the mycelia were washed with sterile water, dried for 72 hours using a freeze dryer, and then the weight was measured. confirmed.

그 결과, 도 15 및 도 16과 같이 pH 5.5에서 가장 높은 동충하초 균사체 생장량이 확인되었다. As a result, as shown in FIGS. 15 and 16, the highest growth rate of cordyceps mycelium was confirmed at pH 5.5.

상기 결과로부터 동충하초 균사체 생장을 위한 액체 배지의 pH 조건은 pH 5.5인 것으로 확인되었으나, pH 조절 전, SDB의 pH가 5.5~5.6인 점을 고려했을 때, 동충하초 균사체 생장을 위해서 pH 조절이 필수적이지 않은 것으로 확인되었다.From the above results, it was confirmed that the pH condition of the liquid medium for the growth of Cordyceps mycelium was pH 5.5. confirmed to be

한편, 배지의 pH 조건에 따른 동충하초 균사체의 코디세핀 함량을 HPLC를 이용하여 앞선 실험과 동일한 방법으로 분석하였다.On the other hand, the cordycepin content of cordyceps mycelium according to the pH conditions of the medium was analyzed using HPLC in the same manner as in the previous experiment.

그 결과, 도 17과 같이 동충하초 균사체 또는 배양액의 pH가 낮을수록 코디세핀 함량이 증가하는 것이 확인되었으며, 상기 결과는 종래 보고된 연구(Chung 등, 2019)와 동일한 경향을 나타내는 것으로 확인되었다. As a result, as shown in FIG. 17, it was confirmed that the cordyceps mycelium or the culture solution had a lower pH, the cordycepin content increased, and the results were confirmed to show the same trend as previously reported studies (Chung et al., 2019).

상기 결과로부터 동충하초 균사체 또는 배양액의 코디세핀 함량 증가를 위해서는 대두 2.5 g/L가 첨가된 SDB 배지의 pH는 4.5가 적합한 것으로 확인되었다.From the above results, it was confirmed that pH 4.5 of the SDB medium supplemented with 2.5 g/L of soybeans was suitable for increasing the cordycepin content of Cordyceps sinensis mycelium or culture medium.

<실시예 2> 동충하초 균사체 생장에 적합한 솔수염하늘소 첨가 배지 개발<Example 2> Development of a medium containing Monochamus alternatus suitable for the growth of Cordyceps sinensis mycelium

1. 솔수염하늘소 첨가 형태에 따른 동충하초 균사체 생장량 및 코디세핀 함량 확인1. Confirmation of cordyceps mycelium growth and cordycepin content according to Monochamus alternatus addition type

액체배지에 동충하초 균사체 생장을 위해 솔수염하늘소 투입형태 조건을 확인하였다. 솔수염하늘소를 경남산림환경연구원에서 제공받았으며, 실험에서 사용한 액체배지는 각각 대두 2.5 g/L가 투입된 SDB 배지를 이용하였다. 액체배지에 투입된 솔수염하늘소의 투입형태는 원형 및 분말 형태로 전건 무게로 표 2와 같은 조건으로 첨가하였다. For the growth of Cordyceps sinensis mycelium in liquid medium, Monochamus alternatus input type conditions were confirmed. Monochamus alternatus was provided by the Gyeongnam Forest Environment Research Institute, and the liquid medium used in the experiment was SDB medium containing 2.5 g/L of soybeans, respectively. The input form of Monochamus alternatus introduced into the liquid medium was added under the conditions shown in Table 2 in dry weight in round and powder form.

솔수염하늘소 분말은 원형상태를 60℃에서 3일간 건조한 후 분쇄하였으며, 분쇄된 분말을 체를 이용하여 40 mesh를 통과시켜 사용하였다.Monochamus alternatus powder was dried in its original form at 60 ° C for 3 days and then pulverized, and the pulverized powder was used by passing through 40 mesh using a sieve.

상기 조건과 같이 솔수염하늘소가 첨가된 액체배지에 앞선 실험과 동일한 방법으로 동충하초 균사체를 첨가하여 배양한 후 솔수염하늘소 첨가 형태에 따른 균사체 생장량 및 코디세핀 함량 변화를 확인하였다.As in the above conditions, Cordyceps mycelium was added and cultured in the liquid medium to which Monochamus alternatus was added in the same manner as in the previous experiment, and then changes in mycelium growth and cordycepin content were confirmed according to the type of Monochamus alternatus added.

그 결과, 도 19 및 도 20과 같이 솔수염하늘소가 원형으로 첨가되었을 때, 가장 높은 균사체량과 코디세핀 함량이 확인되었다.As a result, when Monochamus alternatus was added in a circular shape as shown in FIGS. 19 and 20, the highest mycelial mass and cordycepin content were confirmed.

항목item 미처리untreated 원형circle 분말powder 분말도fineness -- -- 40 mesh40 mesh 투입량input -- 20 g/L20g/L 20 g/L20g/L 배양 기간incubation period 7일7 days 7일7 days 7일7 days 배양 온도incubation temperature 24 ℃24℃ 24 ℃24℃ 24 ℃24℃ RPMRPM 100100 100100 100100

2. 솔수염하늘소 첨가량에 따른 동충하초 균사체 생장량 확인2. Confirmation of the growth of Cordyceps sinensis mycelium according to the amount of Monochamus alternatus added

표 3과 같은 조건으로 액체배지에 솔수염하늘소를 첨가하였다.Monochamus alternatus was added to the liquid medium under the same conditions as in Table 3.

솔수염하늘소의 첨가량은 전건무게 기준으로 배지 1L 당 2.5-30 g/L를 첨가하였으며, 솔수염하늘소가 첨가된 액체배지를 121℃ 15분 동안 고압멸균기를 이용하여 고압멸균하여 제조하였다. 제조된 솔수염하늘소 액체 배지에 동충하초 균사체 (5 mm) 3 core를 투입하였으며, 24℃, 100 rpm의 진탕배양기에서 7일간 배양하였다. 배양 후 앞선 실험과 동일한 방법으로 균사체 생장량 및 코디세핀 함량 변화를 확인하였다.The amount of Monochamus alternatus added was 2.5-30 g/L per 1 L of medium based on the dry weight, and the liquid medium to which Monochamus alternatus was added was sterilized at 121 ° C. for 15 minutes using a high-pressure sterilizer. 3 cores of cordyceps mycelium (5 mm) were added to the prepared Monochamus alternatus liquid medium, and cultured for 7 days in a shaker at 24 ° C and 100 rpm. After culturing, changes in mycelial growth and cordycepin content were confirmed in the same manner as in the previous experiment.

그 결과, 도 21 및 도 22와 같이 솔수염하늘소 원형 20.0 g/L이 첨가 군의 코디세핀 함량이 가장 높은 것을 확인할 수 있었다.As a result, as shown in FIGS. 21 and 22, it was confirmed that the original 20.0 g/L of Monochamus alternatus had the highest cordycepin content in the added group.

항목item 미처리untreated 원형circle 분말powder 분말도fineness -- -- 40 mesh40 mesh 투입량input -- 2.5 g/L
5 g/L
10 g/L
20 g/L
30 g/L2.5g/L
5g/L
10 g/L
20g/L
30g/L 2.5 g/L
5 g/L
10 g/L
20 g/L
30 g/L2.5g/L
5g/L
10 g/L
20g/L
30g/L 배양 기간incubation period 7일7 days 7일7 days 7일7 days 배양 온도incubation temperature 24 ℃24℃ 24 ℃24℃ 24 ℃24℃ RPMRPM 100100 100100 100100

3. 배지의 솔수염하늘소 분말도에 따른 동충하초 균사체 생장량 및 코디세핀 수준 확인3. Confirmation of cordyceps mycelium growth and cordycepin level according to Monochamus alternatus fineness of medium

솔수염하늘소 분말도에 따른 동충하초 균사체의 생장량 및 코디세핀 수준 변화를 확인하였다.Changes in the growth amount and cordycepin level of Cordyceps sinensis mycelium were confirmed according to the powder level of Monochamus alternatus.

솔수염하늘소를 60℃ 건조기에서 3일간 건조 후 분쇄하고, 체(sieve)를 이용하여 40 mesh, 80 mesh, 100 mesh를 통과한 분말을 각각 제조하였다.Monochamus alternatus was dried in a dryer at 60 ° C for 3 days, then pulverized, and powders passing through 40 mesh, 80 mesh, and 100 mesh were prepared using a sieve, respectively.

표 4와 같은 조건으로 액체배지에 제조된 솔수염하늘소 분말을 첨가하고 121℃, 15분간 autoclave를 이용하여 고압멸균하였다. 고압멸균된 솔수염하늘소 액체배지에 앞선 실험과 동일한 방법으로 동충하초 균사체를 접종한 후 배양하여 균사체 생장량 및 코디세핀 함량 변화를 확인하였다.Monochamus alternatus powder prepared in a liquid medium under the conditions shown in Table 4 was added and sterilized under high pressure using an autoclave at 121 ° C. for 15 minutes. The mycelium of Cordyceps sinensis was inoculated into the high-pressure sterilized Monochamus alternatus liquid medium in the same manner as in the previous experiment, and then cultured to confirm changes in mycelial growth and cordycepin content.

그 결과, 도 24 및 도 25와 같이 원형의 솔수염하늘소가 첨가된 실험군에서 높은 동충하초 균사체 생장량이 확인되었으며, 특히 코디세핀 함량이 매우 증가된 것을 확인할 수 있었다.As a result, as shown in FIGS. 24 and 25, in the experimental group to which monochamus alternatus was added, a high growth rate of Cordyceps sinensis mycelium was confirmed, and in particular, it was confirmed that the cordycepin content was greatly increased.

항목item 미처리untreated 원형circle 분말powder 분말도fineness -- -- 원형
(natural type)
0.952mm
0.0952mmcircle
(natural type)
0.952mm
0.0952mm 투입량input -- 20 g/L20g/L 20 g/L20g/L 배양 기간incubation period 7일7 days 7일7 days 7일7 days 배양 온도incubation temperature 24 ℃24℃ 24 ℃24℃ 24 ℃24℃ RPMRPM 100100 100100 100100

4. 반응표면분석(RSM)을 이용한 코디세핀 함량 증대를 위한 솔수염하늘소 첨가배지의 최적 조건 확인4. Confirmation of Optimal Conditions for Monochamus alternatus-added Medium to Increase Cordycepin Content Using Response Surface Analysis (RSM)

투입량, 분말도 및 배양 기간을 변수로 하고, Design-expert version 12를 이용하여 표 5와 같이 코디세핀 최대 함량을 위한 최적의 솔수염하늘소 첨가배지 코드 및 실측값을 얻었으며, 하기 수학식 1을 이용하여 코디세핀 함량을 확인하였다.Using Design-expert version 12, using the input amount, fineness and culture period as variables, the optimal Monochamus alternatus supplemented medium code and measured values for the maximum cordycepin content were obtained as shown in Table 5, and Equation 1 below Cordycepin content was confirmed using

[수학식 1][Equation 1]

Y1=-3624.37595+161.74173A+649.65098B+1062.81758C-17.98434AB-23.56214AC-2.33472BC-4.62463A2-76.22227B2-60.03783C2 Y1=-3624.37595+161.74173A+649.65098B+1062.81758C-17.98434AB-23.56214AC-2.33472BC-4.62463A 2 -76.22227B 2 -60.03783C 2

상기 수학식 1에서 Y1은 밀리타리스 동충하초 균사체의 코디세핀 함량 (mg/L), A는 분말도 (mm), B는 투입량 (g/50mL) 및 C는 배양기간 (day)을 나타낸다.In Equation 1, Y1 represents the cordycepin content (mg/L) of the mycelium of Militaris Cordyceps sinensis, A is the fineness (mm), B is the input amount (g/50mL), and C is the incubation period (day).

그 결과, 도 26과 같이 SDB 배지 1.5 g/mL, 대두 (Soybean) 2.5 g/mL, 솔수염하늘소 원형 4.1 g/mL 및 증류수 50 mL의 배지 조건으로 7일간 배양할 때, 코디세핀 최대 함량이 2171.25 mg/L (R2=0.9720)인 것으로 확인되었다.As a result, as shown in FIG. 26, when cultured for 7 days under the medium conditions of 1.5 g/mL of SDB medium, 2.5 g/mL of soybean, 4.1 g/mL of Monochamus alternatus prototype, and 50 mL of distilled water, the maximum content of cordycepin was 2171.25 mg/L (R 2 =0.9720).

RunRun CodedCoded ActualActual Cordycepin contentCordycepin content X1X1 X2X2 X3X3 X1X1 X2X2 X3X3 Y1Y1 1One -1-One 00 -1-One 0.09520.0952 33 1One 124.43124.43 22 00 00 00 0.9520.952 33 77 1730.921730.92 33 -1-One 00 1One 0.09520.0952 33 1414 2119.282119.28 44 1One 1One 00 9.529.52 66 77 308.74308.74 55 00 00 00 0.9520.952 33 77 1765.121765.12 66 00 -1-One -1-One 0.9520.952 1.51.5 1One 112.81112.81 77 1One 00 -1-One 9.529.52 33 1One 104.18104.18 88 -1-One -1-One 00 0.09520.0952 1.51.5 77 1793.161793.16 99 1One 00 1One 9.529.52 33 1414 320.56320.56 1010 00 -1-One 1One 0.9520.952 1.51.5 1414 655.23655.23 1111 -1-One 1One 00 0.09520.0952 66 77 1952.111952.11 1212 00 1One 1One 0.9520.952 66 1414 613.48613.48 1313 00 00 00 0.9520.952 33 77 1731.831731.83 1414 00 00 00 0.9520.952 33 77 1712.591712.59 1515 1One -1-One 00 9.529.52 1.51.5 77 913.36913.36 1616 00 1One -1-One 0.9520.952 66 1One 155.11155.11 1717 00 00 00 0.9520.952 33 77 1712.731712.73 Independent variablesIndependent variables LevelsLevels -1-One 00 1One X1: Particle size, mmX 1 : Particle size, mm 0.09520.0952 0.9520.952 9.529.52 X2: Input weight, gX 2 : Input weight, g 1.51.5 33 66 X3: Incubation time, dayX 3 : Incubation time, day 1One 77 1414

5. 소나무재선충피해목 분말 및 탄화된 소나무재선충피해목 분말 첨가에 따른 동충하초 균사체의 코디세핀 함량 확인5. Confirmation of Cordycepin Content in Cordyceps Sinensis Mycelium According to Addition of Pine Ash Nematode Damaged Tree Powder and Carbonized Pine Ash Nematode Damaged Tree Powder

최적의 솔수염하늘소 첨가배지에서 소나무재선충피해목 분말 20 g/L와 탄화된 소나무재선충피해목 분말 20 g/L를 각각 투입했을 때와 소나무재선충피해목 분말 및 탄화된 소나무재선충피해목 분말을 각각 10 g/L 씩 동시에 투입했을 때의 코디세핀 함량을 확인하였다.When 20 g/L of pine ash nematode damaged tree powder and 20 g/L of carbonized pine ash nematode damaged tree powder were added in the optimal Monochamus alternatus supplemented medium, respectively, and pine ash nematode damaged tree powder and carbonized pine ash nematode damaged tree powder, respectively The content of cordycepin when 10 g/L was simultaneously added was confirmed.

소나무재선충피해목을 분쇄 후 100 mesh를 통과시켜 분말로 제조하였으며, 탄화된 소나무재선충피해목 분말은 소나무재선충피해목을 500℃, 1시간 동안 탄화로에서 탄화시킨 후 상기 방법으로 제조하였다.The pine nematode damaged wood was ground and passed through 100 mesh to make powder, and the carbonized pine wood nematode damaged wood powder was prepared by the above method after carbonizing the pine wood nematode damaged wood at 500 ° C. for 1 hour in a carbonization furnace.

제조된 소나무재선충피해목 분말 및 탄화된 소나무재선충피해목 분말을 최적의 솔수염하늘소 액체배지에 첨가한 후 앞선 실험들과 동일한 방법으로 동충하초 균사체를 접종한 후 배양하여 코디세틴 함량을 확인하였다.After adding the prepared pine wilt nematode damaged tree powder and carbonized pine ash nematode damaged tree powder to the optimal Monochamus alternatus liquid medium, Cordyceps sinensis mycelium was inoculated and cultured to confirm the content of cordycetin.

그 결과, 도 27과 같이 최적 솔수염하늘소 액체배지에 소나무재선충피해목 분말이 첨가된 실험군에서 코디세틴 함량이 약 1.2배 증가된 것을 확인할 수 있었으며, 특히 소나무재선충피해목 분말과 탄화된 소나무재선충피해목 분말이 함께 첨가된 실험군에서 가장 높은 코디세핀 함량이 확인되었다. As a result, as shown in FIG. 27, it was confirmed that the cordycetin content increased by about 1.2 times in the experimental group in which the powder of pine wilt nematodes was added to the optimal Monochamus alternatus liquid medium. The highest cordycepin content was confirmed in the experimental group in which throat powder was added together.

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

기본배지 및 소나무재선충피해목을 포함하는 솔수염하늘소 동충하초 배양용 배지조성물로서,
상기 기본배지는 조성물 100 중량부에 대하여, 배지 1 내지 5 중량부, 대두 1 내지 10 중량부, 솔수염하늘소 5 내지 15 중량부 및 물 70 내지 90 중량부를 포함하는 것을 특징으로 하는 솔수염하늘소 동충하초 배양용 배지조성물.As a medium composition for culturing Monochamus alternatus cordyceps, including a basic medium and pine wood nematode damaged trees,
The basic medium comprises 1 to 5 parts by weight of medium, 1 to 10 parts by weight of soybeans, 5 to 15 parts by weight of Monochamus alternatus, and 70 to 90 parts by weight of water, based on 100 parts by weight of the composition. Medium composition for culture. 청구항 1에 있어서, 상기 소나무재선충피해목은 소나무재선충피해목 분말, 탄화된 소나무재선충피해목 분말 또는 이들의 혼합물인 것을 특징으로 하는 솔수염하늘소 동충하초 배양용 배지조성물.The medium composition for culturing Monochamus alternatus Cordyceps sinensis according to claim 1, wherein the pine wood nematode damaged tree is pine wood nematode damaged wood powder, carbonized pine wood nematode damaged wood powder, or a mixture thereof. 청구항 1에 있어서, 상기 배지조성물은 소나무재선충피해목 10 내지 40 중량부 및 기본배지 60 내지 90 중량부로 포함되는 것을 특징으로 하는 솔수염하늘소 동충하초 배양용 배지조성물.The medium composition for culturing Monochamus alternatus Cordyceps sinensis according to claim 1, wherein the medium composition comprises 10 to 40 parts by weight of pine wood nematode-damaged trees and 60 to 90 parts by weight of the basic medium. 삭제delete 청구항 1에 있어서, 상기 배지는 PDB (Potato Dextrose Broth) 및 SDB (Sabouraud Dextrose Broth)으로 이루어진 군에서 선택되는 것을 특징으로 하는 솔수염하늘소 동충하초 배양용 배지조성물.The medium composition for culturing Monochamus alternatus Cordyceps sinensis according to claim 1, wherein the medium is selected from the group consisting of PDB (Potato Dextrose Broth) and SDB (Sabouraud Dextrose Broth). 청구항 1에 있어서, 상기 솔수염하늘소는 건조된 원형 또는 분말로 첨가되는 것을 특징으로 하는 솔수염하늘소 동충하초 배양용 배지조성물.The medium composition for culturing Monochamus alternatus Cordyceps sinensis according to claim 1, characterized in that the monochamus alternatus is added in a dried form or powder. 청구항 1에 있어서, 상기 배지조성물은 pH 4 내지 pH 6인 것을 특징으로 하는 솔수염하늘소 동충하초 배양용 배지조성물.The medium composition for culturing Monochamus alternatus Cordyceps sinensis according to claim 1, wherein the medium composition is pH 4 to pH 6. 청구항 1에 있어서, 상기 배지조성물은 솔수염하늘소 동충하초 균사체의 생장 및 코디세핀 함량을 증가시키는 것을 특징으로 하는 솔수염하늘소 동충하초 배양용 배지조성물.The medium composition for culturing Monochamus alternatus Cordyceps according to claim 1, characterized in that the medium composition increases the growth and cordycepin content of Monochamus alternatus Cordyceps mycelium. 청구항 1 내지 청구항 3 및 청구항 5 내지 청구항 8 중 어느 한 항에 따른 배지조성물에 동충하초 균사체를 접종하는 단계; 및
상기 배지조성물에 접종된 동충하초 균사체를 배양하는 단계를 포함하는 솔수염하늘소 동충하초 균사체 배양방법.Inoculating the mycelium of Cordyceps sinensis in the medium composition according to any one of claims 1 to 3 and 5 to 8; and
A method for culturing Cordyceps sinensis mycelium comprising the step of culturing the Cordyceps mycelium inoculated in the medium composition. 청구항 9에 있어서, 상기 배양방법은 솔수염하늘소 동충하초 균사체의 생장량 및 코디세핀 함량을 증가시키는 것을 특징으로 하는 솔수염하늘소 동충하초 균사체 배양방법.The method according to claim 9, wherein the culturing method increases the growth amount and cordycepin content of Monochamus alternatus cordyceps mycelium.
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