KR100366033B1 - A process for cultivation of Paecilomyces japonica in pupae of the Antheraea pernyi - Google Patents

A process for cultivation of Paecilomyces japonica in pupae of the Antheraea pernyi Download PDF

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KR100366033B1
KR100366033B1 KR10-1999-0011661A KR19990011661A KR100366033B1 KR 100366033 B1 KR100366033 B1 KR 100366033B1 KR 19990011661 A KR19990011661 A KR 19990011661A KR 100366033 B1 KR100366033 B1 KR 100366033B1
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pupa
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cordyceps
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박남숙
이호웅
박상봉
장창식
이상몽
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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
    • A01G18/40—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
    • C12N1/04—Preserving or maintaining viable microorganisms

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Abstract

본 발명은 동충하초 종균을 작잠 번데기에 직접 주사하여 감염시킨 후 자실체 형성을 유도하는 작잠 번데기 동충하초 재배방법에 관한 것으로 본 발명은 동충하초 종균액을 작잠 번데기에 직접 주사하는 안정한 방법으로 접종한 후 온도 22 ~ 26℃, 습도 80 ~ 95%를 유지시키면서 5 ~ 9 일간 보호하여 100% 감염시키고, 다시 온도 18 ~ 22℃, 습도 85 ~ 98%를 유지시켜 중, 상급 이상의 자실체를 97% 형성하는 뛰어난 효과가 있다.The present invention relates to a method of cultivating a nasal chrysalis cordyceps to induce fruiting bodies after directly injecting the cordyceps seedlings into a nasal chrysalis. It protects for 5-9 days while maintaining 26 ℃ and humidity 80 ~ 95% and infects 100%, and maintains temperature 18 ~ 22 ℃ and humidity 85 ~ 98% to form 97% of medium and higher fruiting bodies. have.

Description

작잠 번데기 동충하초 재배방법{A process for cultivation of Paecilomyces japonica in pupae of the Antheraea pernyi}A process for cultivation of Paecilomyces japonica in pupae of the Antheraea pernyi}

본 발명은 작잠 번데기 동충하초 재배방법에 관한 것이다. 더욱 상세하게는, 본 발명은 동충하초 종균을 작잠 번데기에 직접 주사하여 감염시킨 후 자실체 형성을 유도한 작잠 번데기 동충하초 재배방법에 관한 것이다.The present invention relates to a method for cultivating napa pupa cordyceps. More specifically, the present invention relates to a method of cultivating nacre chrysalis Cordyceps sinensis induced by the fruiting body formation after infection by injecting Cordyceps fungi directly into the nasal chrysalis.

동충하초는 동충하초균이 곤충체내에 침입하여 충체를 기주로 하여 내생 균사핵을 형성한 후 충체의 피부를 뚫고 충체의 체표면에 자실체를 형성하거나 충체위에 포자과를 형성하는 곤충+자실체(이하, '자실체'로 약칭함) 그 자체를 말하는 것으로 처음에는 곤충기생균(Entomopathogenic fungi)의 일종인 코디셉스 시넨시스(Cordyceps sinensis)에 의해 감염된 중국산 자실체를 지칭하였으나 현재는 곤충, 거미 및 일부 균류에 기생하여 발생한 자실체를 총칭하고 있다(이 등, 1996). 농업에 있어 해충방제를 위해 과다한 화학살충제를 사용함으로 인해 발생하는 여러 가지의 문제를 환경 친화적으로 해결하기 위한 방안의 하나로 동충하초균을 이용한 해충방제 연구가 국내의 학자들에 의해 시도되어졌다(성 등, 1993; 추 등, 1994). 이와같이 해충방제를 위한 생물농약으로서 개발가능성이 높았던 동충하초균은 최근 그 자실체 및 균사체에 항암성분 등 유용 약리성분이 함유되어 있는 것으로 밝혀지고 있으며 그 결과 누에를 이용한 누에번데기 동충하초의 대량 생산기술개발을 통한 동충하초의 산업화가 모색되고 있다. 그리하여 1998년 춘잠기부터 양잠농가는 대한잠사회로부터 해당 종균을 구입하여 누에 번데기 동충하초를 상당량 생산하였다. 그러나 산누에나방과에서는 자생 동충하초가 생성된다는 문헌적 근거는 있으나 아직 인공생산방법에 대한 연구는 극히 적은 것으로 알려져 있다(청수, 1997).Cordyceps sinensis is an insect + fruiting body that enters into the insect body and forms endogenous mycelium nuclei based on the worms, then penetrates the skin of the worms, forms fruiting bodies on the surface of the worms, or forms spores on the worms. By itself, it refers to the Chinese fruiting body infected by Cordyceps sinensis , a type of Entomopathogenic fungi , but is now a fruiting body caused by insects, spiders and some fungi. Are collectively (Lee et al., 1996). In order to solve environmentally friendly problems caused by the use of excessive chemical pesticides for pest control in agriculture, research on pest control using cordyceps fungi has been attempted by domestic scholars (Sung et al. , 1993; Chu et al., 1994). As such, Cordyceps fungi, which has been highly developed as a biopesticide for pest control, have recently been found to contain useful pharmacological components such as anticancer components in fruiting bodies and mycelium. As a result, it has been developed through mass production technology of silkworm pupa cordyceps using silkworms. Industrialization of Cordyceps sinensis is being sought. Therefore, since the early spring and early 1998, sheep farmers purchased the spawn from the Korean subsidiaries and produced a significant amount of silkworm pupa. However, although there are some literatures on the production of native Cordyceps in the wild silkworm moth, there are very few studies on artificial production methods (Chungsu, 1997).

따라서 본 발명자들은 야생견사곤충의 일종이면서 산업화 가치가 비교적 큰 작잠 번데기에 동충하초 종균을 주사하여 작잠 번데기 동충하초 자실체를 유도하므로써 본 발명을 완성하였다.Therefore, the present inventors have completed the present invention by inducing the nasal chrysalis Cordyceps Sinensis fruiting by injecting the species of Cordyceps sinensis into the nap chrysalis, which is a kind of wild dog insect and has a relatively high industrial value.

본 발명의 목적은 작잠 번데기에 동충하초 종균을 주사하여 감염시킨 후 자실체를 유도하는 작잠 번데기 동충하초 재배방법을 제공함에 있다.It is an object of the present invention to provide a method for cultivating a nasal chrysalis cordyceps to induce fruiting bodies after inoculating the nasal chrysalis by injecting the cordyceps fungi.

본 발명의 상기 목적은 98년 춘기에 인공적으로 조성한 상수리나무 사료 포장에 직접 야외 방사하여 생산한 작잠(Antheraea pernyi) 번데기의 충체에 동충하초 균액을 직접주사하여 번데기를 동충하초 균으로 감염시킨 후 온도와 습도를 적정수준으로 유지시켜 자실체를 형성하므로써 달성하였다.The object of the present invention is to directly inject the cordyceps fungus into the fungus of the Nathe ( Antheraea pernyi ) chrysalis produced by direct outdoor spinning on the artificial oak feed package prepared in the spring of 1998 to infect the chrysalis with cordyceps fungus It was achieved by forming the fruiting body by maintaining the proper level.

이하, 본 발명의 구성 및 작용을 설명한다.Hereinafter, the configuration and operation of the present invention.

도 1은 눈꽃 동충하초(Paecilomyces japonica) 균액을 작잠(Antheraea pernyi) 번데기에 주사한 후 번데기 몸체로부터 균사가 발생하여 자실체를 형성하기 까지의 과정을 나타낸 사진도이다.FIG. 1 is a photograph showing the process up to the formation of fruiting bodies from the pupa body after injecting Paecilomyces japonica mycelia into the chrysalis ( Antheraea pernyi ) pupa.

도 2는 눈꽃 동충하초 자실체가 형성된 작잠 번데기의 종단면과 횡단면을 나타낸 사진도이다.Figure 2 is a photograph showing a longitudinal section and a cross-section of the nasal chrysalis formed snowflake Cordyceps sinensis fruiting body.

도 3은 작잠 번데기에 눈꽃 동충하초 균액을 주사한 결과, 번데기의 감염율 100%와 자실체 형성율 97%를 나타낸 사진도이다.Figure 3 is a photograph showing the infection rate of pupa chrysalis 100% and fruiting body formation rate as a result of injecting snow fungus Cordyceps fungus in the nap pupa.

본 발명은 동충하초 균주를 PDA 고체배지에서 배양한 후 PD 배지에서 액체종균배양하여 에탄올로 소독한 작잠 번데기에 상기 배양한 종균액을 직접 주사하여 접종한 다음 충체내에서 내생균핵을 형성시키고 온도와 습도를 적절히 유지시켜 자실체를 형성하는 것으로 구성된다.The present invention is cultured in Cordyceps sinensis strain in PDA solid medium and inoculated by direct injection of the cultured spawn solution in the nasal pupa sterilized with ethanol by liquid spawn culture in PD medium to form the endogenous nucleus in the body and temperature and humidity It is composed of maintaining the appropriately to form the fruiting body.

본 발명에서 작잠 번데기에 주사하는 동충하초 액체 종균의 포자농도는 107~ 109spores/mL개이며 가장 바람직하게는 108spores/mL개이다.In the present invention, the spore concentration of Cordyceps sinensis spawn injected into the nasal pupa is 10 7 to 10 9 spores / mL and most preferably 10 8 spores / mL.

본 발명에서 작잠 번데기에 주사하는 동충하초 액체 종균의 양은 0.4 ~ 0.7mL이며, 가장 바람직하게는 0.5mL이다.In the present invention, the amount of Cordyceps sinensis spawn injected into the nasal pupa is 0.4-0.7 mL, most preferably 0.5 mL.

본 발명에서 작잠 번데기에 동충하초 종균 주사 후 내생균핵 형성 조건은 온도 22 ~ 26℃, 습도 80 ~ 95%에서 5 ~ 9일간 보호하는 것이며 가장 바람직하게는 온도 24℃, 습도 90%에서 7일간 보호하는 것이다In the present invention, the endogenous nucleus formation conditions after the injection of Cordyceps spp. In the nasal pupa are protected for 5 to 9 days at a temperature of 22 to 26 ° C and a humidity of 80 to 95%, and most preferably for 7 days at a temperature of 24 ° C and a humidity of 90%. will be

본 발명에서 작잠 번데기에 동충하초 자실체를 유도하는 조건은 온도 18 ~ 22℃, 습도 85 ~ 98%이며 가장 바람직하게는 온도 20℃, 습도 95%이다.In the present invention, the conditions for inducing Cordyceps sinensis to the nasal pupa are at a temperature of 18 to 22 ° C., a humidity of 85 to 98%, and most preferably at a temperature of 20 ° C. and a humidity of 95%.

이하, 본 발명의 구체적인 방법을 실시예를 들어 상세히 설명하고자 하지만 본 발명의 권리범위는 이들 실시예에만 한정되는 것은 아니다.Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

실시예 1: 작잠 번데기 동충하초 생산Example 1: Nap pupa cordyceps sinensis production

본 실시예에서 사용한 곤충은 공시 곤충으로서는 농업과학기술원 잠사곤충부의 방사(放飼)포장에서 98춘잠기에 상수리나무로 구성된 인공 작수림에서 야외방사하여 수확한 작잠 번데기를 사용하였으며 공시 동충하초균주는 강원대학교 성재모 교수로부터 분양받은 눈꽃 동충하초(Paecilomyces japonica)를 사용하였다. 균주의 고체배지배양은 PDA배지에서 행하였으며 주사형태의 종균은 PD 배지에서 배양한 액체 종균으로 포자농도는 108spores/mL개 였고 작잠 번데기당 주사량은 0.5mL로 하였다. 주사직전의 작잠 번데기는 70% 에탄올로 약 5분간 번데기의 몸체를 침지하여 체표면을 소독한 후 꺼내어 크린벤치에서 건조시켰다. 작잠 번데기에 종균을 번데기의 두부로부터 꼬리 쪽 방향으로 체내에 1회용 1mL 주사기로 직접 주사한 후 충체내에 내생균핵이 형성될 때까지(충체가 딱딱해지는 시기) 온도 24℃, 습도 90%에서 1주일간 보호하였으며 그후 자실체 형성시까지 온도 20℃, 습도 95%에서 보호하였다. 실험결과, 도 1에 종균액을 작잠 번데기의 두부에 직접 주사한 후 번데기 몸체로부터 균사가 발생하여 자실체를 형성하기까지의 과정을 나타낸 바와 같이 번데기의 충체 내부가 균사핵덩어리로 가득차서 만지면 딱딱한 느낌을 줄 정도로 변하였다. 이는 균액주사 후 약 7일 정도 지났을 때이며 그 이후 균사체가 번데기 피부를 뚫고 자실체를 생성할 때 까지는 20℃에서 약 7일 정도 소요되었다. 즉, 균액을 번데기에 주사한 후 자실체 길이가 3cm이상 되어 작잠 동충하초로서 수확이 가능하기 까지는 약 2주가 소요되었다. 도 2에는 자실체가 형성된 작잠 번데기 내부의 균사핵 형성정도를 알기 위해 번데기의 종단면과 횡단면을 관찰한 결과를 나타낸 것으로 피부 및 내부 각 조직의 경계막을 제외한 대부분의 조직 및 세포들은 균사로 가득차 있음이 확인되었다. 표 1에는 작잠 번데기에 동충하초균을 주사하여 2주간의 생체 배양 후 작잠 번데기 동충하초를 수확한 결과를 정리한 것으로 주사한 33개의 번데기 모두 감염되어 감염률은 100%이었으며 자실체 형성율도 33개 번데기중 중·상위급 이상의 양호한 자실체 형성개체가 32개였으므로 약 97%였다. 또 번데기 무게는 종균주사전에 ♀·♂ 평균 9.38g정도 였으나 자실체 형성 후에는 7.87g으로 약 16%정도 체중감소 현상이 나타났다. 이와같은 체중감소 현상은 생체 배양중 내생균핵 생성과 자실체 형성에 따른 수분감소와 영양분 소모의 결과로 사료되었다. 이상과 같이 작잠 번데기에 종균을 주사하여 작잠 번데기 동충하초를 생산하는 방법은 종균의 번데기감염율 100%로서 매우 좋은 결과를 보였으며 도 3에 100% 감염율과 97% 자실체 형성율을 나타냈다.Insects used in the present example were used as a public insect, and the napa pupa harvested by outdoor radiation in artificial arbors composed of oak trees in the spring and autumn seasons at the radial packing of the Nasa insects in the Agricultural Science and Technology Institute. Paecilomyces japonica , which was sold by Professor Sung Jae Mo , was used. Solid cultures of the strains were carried out in PDA medium, and the injection type spawn was a liquid seed cultured in PD medium. The spore concentration was 10 8 spores / mL and the injection volume per nap pupa was 0.5 mL. The nap pupa just before injection was sterilized by the body of the pupa for about 5 minutes with 70% ethanol to disinfect the surface of the pupa and then dried in a clean bench. In the nasal pupa, the spawn is injected directly into the body from the pupa to the tail with a disposable 1 mL syringe in the body, until the endogenous nucleus is formed in the umbilical cord (when the body becomes hard). It was then protected at a temperature of 20 ° C. and a humidity of 95% until the formation of fruiting bodies. As a result of the experiment, the seed solution was injected directly to the head of the nap pupa, and then the hyphae generated from the pupa body to form the fruiting body. As shown in FIG. Changed to give. This was about 7 days after the injection of the fungal solution, and it took about 7 days at 20 ° C until the mycelium penetrated the pupa skin and produced fruiting bodies. In other words, after injecting the fungus into the pupa, the fruiting body length was more than 3 cm, and it took about 2 weeks to be harvested as a nasal cordyceps. Figure 2 shows the results of observing the longitudinal section and cross section of the pupa to determine the degree of mycelial formation in the nasal pupa inside the fruiting body is formed is confirmed that most tissues and cells except the boundary membrane of the skin and each tissue is filled with the hyphae It became. Table 1 summarizes the results of harvesting nasal chrysalis after two weeks of incubation with nasal chrysalis in nasal chrysalis. All 33 chrysalis were infected with 100% infection rate and the rate of fruiting body formation among 33 chrysalis. Since there were 32 good fruit-body forming objects of higher grade or more, it was about 97%. In addition, the weight of pupa was 9.38g on average after the spawn injection, but it was 7.87g after fruiting. This weight loss phenomenon is thought to be the result of the decrease of water and nutrient consumption by endogenous nucleus formation and fruiting body formation during incubation. As described above, the method of producing a nap pupa cordyceps by injecting the seed to the nap pupa showed a very good result as the pupal infection rate of 100% of the spawn and showed 100% infection rate and 97% fruiting body formation rate in FIG. 3.

작잠 번데기에 동충하초 종균을 주사한 후 자실체 형성 결과Fruiting body formation after injection of Cordyceps sinensis in nap pupa 접종된번데기수Inoculated pupa 감염된번데기수Infected chrysalis 자실체 등급Fruiting body grade 자실체를 생산한 번데기 무게Pupa weight produced by fruiting bodies 고Go 중medium 저that 접종 전Before vaccination 생체 배양 후After incubation 3333 3333 번데기30Chrysalis 30 번데기2Pupa 2 번데기1Chrysalis 1 (g)9.38(g) 9.38 (g)7.87(g) 7.87

이상, 상기 실시예를 통하여 설명한 바와 같이 본 발명은 작잠 유충에 동충하초균을 접종하여 재배하는 효과가 있으며 특히, 동충하초 종균액을 작잠 번데기에 직접 주사하여 작잠 동충하초를 생산하는 안정한 방법으로 동충하초 종균을 접종한 후 온도 22 ~ 26℃, 습도 80 ~ 95%를 유지시키면서 5 ~ 9일간 보호하여 감염율을 100%로 높히고, 다시 온도 18 ~ 22℃, 습도 85 ~ 98%를 유지시켜 중, 상급 이상의 자실체 형성율을 97%이상으로 높히는 뛰어난 효과가 있으므로 작잠 동충하초 재배 산업상 매우 유용한 발명인 것이다.As described above, the present invention has the effect of inoculating Cordyceps fungus on the nasal larvae, and in particular, inoculates Cordyceps fungi by injecting Cordyceps fungi broth directly into the nap pupa to produce a fungus Cordyceps fungus. Afterwards, the infection rate is increased to 100% by maintaining the temperature 22-26 ℃ and the humidity 80-95% for 5-9 days, and again maintaining the temperature 18-22 ℃ and the humidity 85-98% to form medium and higher fruiting bodies. Since it has an excellent effect of increasing the rate to more than 97%, it is a very useful invention for the nap cordyceps cultivation industry.

Claims (4)

액체배양한 동충하초(Paecilomyces japonica) 배양액을 곤충의 번데기에 접종하여 내생균핵을 형성시키고, 상기 형성된 내생균핵으로부터 자실체를 형성시키는 단계로 구성되는 동충하초의 인공재배 방법에 있어서,In the method of artificial cultivation of Cordyceps sinensis comprising the step of inoculating liquid cultured Paecilomyces japonica culture to the pupa of the insect to form an endogenous nucleus, and forming the fruiting body from the formed endogenous nucleus, 상기 곤충의 번데기는 작잠(Antheraea pernyi)번데기이고, 액체 배양한 동충하초 배양액을 107~108spores/mL의 농도로 희석하여 작잠 번데기의 충체에 0.4mL∼0.6mL 주사접종하는 것을 특징으로 하는 작잠 번데기 동충하초의 재배방법.The insect chrysalis is an antheraea pernyi chrysalis, and the nasal chrysalis is diluted with a concentration of 10 7 to 10 8 spores / mL in a liquid cultured Cordyceps sinensis culture, and the nasal chrysalis are inoculated with 0.4mL to 0.6mL injection. Cultivation method of pupa cordyceps. 삭제delete 삭제delete 삭제delete
KR10-1999-0011661A 1999-04-02 1999-04-02 A process for cultivation of Paecilomyces japonica in pupae of the Antheraea pernyi KR100366033B1 (en)

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CN105638244A (en) * 2016-01-21 2016-06-08 广东省农业科学院蚕业与农产品加工研究所 Silkworm cordyceps militaris culturing method

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105638244A (en) * 2016-01-21 2016-06-08 广东省农业科学院蚕业与农产品加工研究所 Silkworm cordyceps militaris culturing method

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