CN105379562A - Method for cultivating sterile silkworm insect grass - Google Patents
Method for cultivating sterile silkworm insect grass Download PDFInfo
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- CN105379562A CN105379562A CN201510882691.7A CN201510882691A CN105379562A CN 105379562 A CN105379562 A CN 105379562A CN 201510882691 A CN201510882691 A CN 201510882691A CN 105379562 A CN105379562 A CN 105379562A
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- silkworm
- aseptic
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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
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Abstract
The invention provides a method for cultivating sterile silkworm insect grass. The method comprises the steps that silkworm eggs in the head pigmentation stage or before the head pigmentation stage are disinfected and sterilized; the disinfected and sterilized silkworm eggs are placed in a sterile isolator to be hatched; sterile newly-hatched silkworms which are hatched in the sterile isolator are fed with sterile artificial feed; cordyceps militaris bacterium liquid is prepared; the cordyceps militaris bacterium liquid is injected into abdominal cavities of the sterile silkworms of the third to fifth day of five ages; died and stiff sterile silkworms which grow a small amount of hyphae are taken out from a raising plate and then transferred into a sterile environment alternating illumination at 25 DEG C in the daytime with dark at 20 DEG C at night to be cultivated for 38-42 days, the environment humidity is kept 95% or above in the cultivating process, and then the sterile silkworm insect grass of which the whole body completely grows the hyphae and sporophores of different lengths is obtained.
Description
Technical field
The present invention relates to the cultivation of silkworm Chinese caterpillar fungus, particularly relate to aseptic culture silkworm Chinese caterpillar fungus.
Background technology
Cordyceps sinensis has another name called Chinese caterpillar fungus, is me
nationalbetween the usual famous and precious tonic herb of one, its nutrient component, higher than ginseng, can be used as medicine, also edible, is superior delicacies, has very high nutritive value.Cordyceps sinensis, due to wherein contained cordycepin, can strengthen the immunity of body, nourishing lung and kidney, have obvious inhibitory action to lung cancer, liver cancer etc.Clinically to chronic cough of deficiency lung, asthma, hemoptysis of pulmonary tuberculosis, night sweat, soreness of waist and knee joint of suffering from a deficiency of the kidney, impotence and seminal emission, the red blood cell decreased after neurasthenia and chemotherapy, radiotherapy is all effective in cure.Cordycepin content number adult grass quality good or not important mark refer to.
But Cordyceps sinensis is due to its scarcity, and price is very expensive, develops inexpensive high-quality silkworm Chinese caterpillar fungus and substitute rare famous and precious Cordyceps sinensis, become the hot subject that silkworm Chinese caterpillar fungus is researched and developed.Silkworm Chinese caterpillar fungus also claims silkworm Chinese caterpillar fungus or silkworm Chinese caterpillar fungus, is the entomogenous fungi combination with artificial culture on cordyceps militaris link bacterial strain spore inoculating to silkworm.
The distribution of cordycepin is followed successively by Mycelium culture liquid, mycelium, stroma increase gradually.Improve the quality of Chinese caterpillar fungus, have only and make mycelial growth vigorous, expand growth area; Promote stroma growth population and length.But in existing silkworm Chinese caterpillar fungus culture technique, the cordycepin content obtained is lower, cannot reach the mean level of Cordyceps sinensis.Therefore, there are the needs improved silkworm Chinese caterpillar fungus cultural method in this area always, to improve the content of cordycepin in silkworm Chinese caterpillar fungus further.
Summary of the invention
The invention provides a kind of method of cultivating aseptic silkworm Chinese caterpillar fungus, it raises by specific condition and feed the host that aseptic silkworm grows as cordyceps, Development and Production third generation Chinese caterpillar fungus, its cordycepin content be the decades of times of wild Cordyceps sinensis to hundreds of times, other has active bio matter generation apparently higher than Cordyceps sinensis.
Described method of the present invention comprises:
(1) by silkworm seed 75% ethanol and the 4% formalin soaking disinfection sterilizing before head pigmentation stage or head pigmentation stage;
(2) the described silkworm seed after described sterilization is placed in Sterilized isolator to hatch, what described Sterilized isolator comprised several horizontal positioned raises dish, described Sterilized isolator keeps hatching process to carry out in the dark state, described hatching process keeps the steady temperature of 25 DEG C, humidity is 70-80%, for whole described Sterilized isolator, the transfer rate through the air of aseptic process is 250-350 liter/sky, until obtain aseptic newly-hatched silkworm;
(3) the described aseptic newly-hatched silkworm sterilizing artificial feed hatched in described Sterilized isolator are fed, described sterilizing artificial feed are that artificial feed are through high pressure steam sterilization, the vitamin then supplied because of sterilizing loss obtains, wherein said sterilizing artificial feed comprise: based on the gross weight of described sterilizing artificial feed, dried mulberry leaf powder 36.5wt%, defatted soy flour 35.4wt%, mulberry green branch powder 5wt%, corn starch 11.0wt%, sucrose 4.5%, vitamin B1 .5wt%, vitamin C 1.5wt%, peaceful lemon acid 2.0wt%, crotonic acid 0.4wt%, Choline Chloride 0.2wt%, the gross weight of described sterilizing artificial feed is 100wt%, described Sterilized isolator keeps feeding process to carry out in the dark state, described feeding process keeps the temperature of 27-29 DEG C, humidity is 70-80%, for whole described Sterilized isolator, transfer rate through the air of aseptic process is 250-350 liter/sky, until obtain the aseptic silkworm in 3rd ~ 5 days 5 ages,
(4) produce cordyceps liquid, comprising by Natural C.militaris bacterial classification transferred species potato liquid nutrient medium, and described potato liquid nutrient medium is placed in the inherent 20 DEG C of lucifuge joltings of fungal culture case and cultivates 5 to 7 days;
(5) by the abdominal cavity of the aseptic silkworm in described 3rd ~ 5 days 5 ages of acquisition in described cordyceps liquid implantation step (3), injection volume is 0.2 ~ 0.3 milliliter/bar, described aseptic silkworm after injection is still placed in described Sterilized isolator raise, described Sterilized isolator keeps feeding process to carry out in the dark state, described feeding process keeps the temperature of 27-29 DEG C, humidity is 70-80%, for whole described Sterilized isolator, transfer rate through the air of aseptic process is 250-350 liter/sky, until described aseptic silkworm is dead stiff and grow a small amount of mycelia;
(6) aseptic silkworm stiff for the death growing a small amount of mycelia obtained in step (5) is taken out from described raising dish, proceed to 25 DEG C of illumination on daytime, cultivate 38-42 days in gnotobasis that night, 20 DEG C of dark replaced, keep described ambient humidity more than 95% around here, namely obtain the aseptic silkworm Chinese caterpillar fungus that whole body covers with the sporophore of mycelia and varying length.
In an embodiment of the invention, also comprise mineral salt in described sterilizing artificial feed, described mineral salt are selected from any combination of following material: calcium carbonate, sodium chloride, potassium sulfate, manganese sulphate, calcium phosphate, magnesium sulfate, hydrated copper sulfate, ferric sulfate hydrate and sodium fluoride.
In an embodiment of the invention, the method for described cultivation silkworm Chinese caterpillar fungus, wherein said raising is coiled as drawer type raises dish.
Based on the cultural method of silkworm Chinese caterpillar fungus of the present invention, the amount of the cordycepin extracted in the silkworm Chinese caterpillar fungus of the specific yield obtained significantly improves, and advantageously provides the cheap substitute products of Cordyceps sinensis.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
(1) by silkworm seed 75% ethanol and the 4% formalin soaking disinfection sterilizing before head pigmentation stage or head pigmentation stage;
(2) the described silkworm seed after described sterilization is placed in Sterilized isolator to hatch, what described Sterilized isolator comprised several horizontal positioned raises dish, described Sterilized isolator keeps hatching process to carry out in the dark state, described hatching process keeps the steady temperature of 25 DEG C, humidity is 70-80%, for whole described Sterilized isolator, the transfer rate through the air of aseptic process is 250-350 liter/sky, until obtain aseptic newly-hatched silkworm;
(3) the described aseptic newly-hatched silkworm sterilizing artificial feed hatched in described Sterilized isolator are fed, described sterilizing artificial feed are that artificial feed are through high pressure steam sterilization, the vitamin then supplied because of sterilizing loss obtains, wherein said sterilizing artificial feed comprise: based on the gross weight of described sterilizing artificial feed, dried mulberry leaf powder 36.5wt%, defatted soy flour 35.4wt%, mulberry green branch powder 5wt%, corn starch 11.0wt%, sucrose 4.5%, vitamin B1 .5wt%, vitamin C 1.5wt%, peaceful lemon acid 2.0wt%, crotonic acid 0.4wt%, Choline Chloride 0.2wt%, the gross weight of described sterilizing artificial feed is 100wt%, described Sterilized isolator keeps feeding process to carry out in the dark state, described feeding process keeps the temperature of 27-29 DEG C, humidity is 70-80%, for whole described Sterilized isolator, transfer rate through the air of aseptic process is 250-350 liter/sky, until obtain the aseptic silkworm in 3rd ~ 5 days 5 ages,
(4) produce cordyceps liquid, comprising by Natural C.militaris bacterial classification transferred species potato liquid nutrient medium, and described potato liquid nutrient medium is placed in the inherent 20 DEG C of lucifuge joltings of fungal culture case and cultivates 5 to 7 days;
(5) by the abdominal cavity of the aseptic silkworm in described 3rd ~ 5 days 5 ages of acquisition in described cordyceps liquid implantation step (3), injection volume is 0.2 ~ 0.3 milliliter/bar, described aseptic silkworm after injection is still placed in described Sterilized isolator raise, described Sterilized isolator keeps feeding process to carry out in the dark state, described feeding process keeps the temperature of 27-29 DEG C, humidity is 70-80%, for whole described Sterilized isolator, transfer rate through the air of aseptic process is 250-350 liter/sky, until described aseptic silkworm is dead stiff and grow a small amount of mycelia;
(6) aseptic silkworm stiff for the death growing a small amount of mycelia obtained in step (5) is taken out from described raising dish, proceed to 25 DEG C of illumination on daytime, cultivate 38-42 days in gnotobasis that night, 20 DEG C of dark replaced, keep described ambient humidity more than 95% around here, namely obtain the aseptic silkworm Chinese caterpillar fungus that whole body covers with the sporophore of mycelia and varying length.
(7) water extraction method is adopted to extract cordycepin.
Comparative example 1
According to the step of (1) in embodiment 1-(3) obtain aseptic five the length of time silkworm;
(4) Cordyceps militaris is inoculated in solid culture medium, 20 DEG C of dark culturing 9 days; Then photosensitive cultivation 3 days, by mycelium inoculation in liquid spawn culture medium, under dark condition, every day shakes 3 hours with 200r/min on constant-temperature table, continues cultivation and obtains liquid spawn after 5 days, obtained inoculation bacterium liquid;
(5) aseptic injection mode is adopted the inoculation bacterium liquid that step (4) is obtained to be inoculated in the caudal horn of the obtained aseptic silkworm body of step (3), abdominal foot or internode, the inoculum concentration of the aseptic silkworm body of every bar is 0.1 milliliter, asepticly 70min is dried through 18 DEG C, normal sterile raises 2.5 days, after rigid silkworm body is removed sand, under dark condition, keep humidity 85%, temperature 20 DEG C, cultivate 7 days, then photosensitive go out grass cultivate 30 days, through the low temperature drying 2h of less than 100 DEG C, obtain aseptic silkworm Chinese caterpillar fungus.
(6) water extraction method identical with embodiment 1 (7) is adopted to extract cordycepin.
Comparative example 2
Select non-invention method raise obtain five the length of time silkworm; Adopt silkworm Chinese caterpillar fungus as obtained in step (4)-(6) identical in embodiment 1; And then adopt the water extraction method identical with embodiment 1 (7) to extract cordycepin.
Comparative example 3
According to the step of (1) in embodiment 1-(3) obtain aseptic five the length of time silkworm;
(4) by 19-3, MSl and 1-Y bacterial classification in mass ratio for 1:1:1 mixing after be inoculated on silkworm body (silkworm being supported to the silkworm body in the 2nd day 5 ages); The concentration of bacterial classification is 250mg/mL, and injection rate is 3mL; ; Then aseptic culture, goes out grass and namely obtains silkworm Chinese caterpillar fungus.
Inoculate as needle-penetration inoculation, aseptic culture temperature is 15 DEG C, and air humidity is 85%; Illumination requires: inoculate latter 20 days for cultivating early stage, to illumination no requirement (NR), within 20-60 days, is the middle and later periods, requires photosensitive 14 hours of every day after inoculation.
(5) water extraction method identical with embodiment 1 (7) is adopted to extract cordycepin.
Comparative example 4
According to the step of (1) in embodiment 1-(3) obtain aseptic five the length of time silkworm;
(4) preparation of Cordceps militaris spore suspension: make covering with mycelial Cordceps militaris bacterial classification sterile water wash-out the spore suspension that concentration is 300mg/mL, Cord blood is for subsequent use;
(5) hypothermic anesthesia before inoculation: live body silkworm puts into constant incubator, place 2 days for 4-6 DEG C, period is feeding not, and make silkworm enter semidormancy state because of low temperature and hunger, vital movement reduces;
(6) inoculate: use the syringe after sterilizing to extract spore suspension, carefully thrust by silkworm head, slowly inject about 3mL, and spray a small amount of spore suspension at silkworm body surface;
(7) silkworm still survived after process is put into incubator, 4-6 DEG C of cultivation, until silkworm death ossifys; The silkworm of death being ossify is taken out, and head upward, is embedded in not containing organic matter and in the sand of high temperature hot air sterilization, makes head expose 1mm slightly, airtight container, cultivating 1 week at 25 DEG C; Moved to by Chinese caterpillar fungus subsequently to cultivate in room and cultivate, every square metre keeps the illumination of a 20W electricity-saving lamp, and electricity-saving lamp hangs high 1.5 meters, and temperature 18 DEG C, being cultured to head under the condition of humidity 80% ~ 90% has obvious mycelial growth and have fruit body primordium to be differentiated to form; Keep 18 DEG C, humidity is down to 70% and is continued to be cultured to silkworm body head fruit body length more than 2cm, and cultivation completes, and takes out silkworm Chinese caterpillar fungus 45 DEG C of freeze-day with constant temperature.
(8) water extraction method identical with embodiment 1 (7) is adopted to extract cordycepin.
following table 1show the contrast of the cordycepin content of the embodiment of the present invention 1 and comparative example 1-4 gained silkworm Chinese caterpillar fungus:
table 1
As
upper table instituteshow, the silkworm Chinese caterpillar fungus obtained by the inventive method can significantly improve cordycepin content relative to prior art, the rate and go out carelessness and the silkworm keeping relatively high is ossify simultaneously.
Claims (3)
1. cultivate a method for aseptic silkworm Chinese caterpillar fungus, described method comprises:
(1) by silkworm seed 75% ethanol and the 4% formalin soaking disinfection sterilizing before head pigmentation stage or head pigmentation stage;
(2) the described silkworm seed after described sterilization is placed in Sterilized isolator to hatch, what described Sterilized isolator comprised several horizontal positioned raises dish, described Sterilized isolator keeps hatching process to carry out in the dark state, described hatching process keeps the steady temperature of 25 DEG C, humidity is 70-80%, for whole described Sterilized isolator, the transfer rate through the air of aseptic process is 250-350 liter/sky, until obtain aseptic newly-hatched silkworm;
(3) the described aseptic newly-hatched silkworm sterilizing artificial feed hatched in described Sterilized isolator are fed, described sterilizing artificial feed are that artificial feed are through high pressure steam sterilization, the vitamin then supplied because of sterilizing loss obtains, wherein said sterilizing artificial feed comprise: based on the gross weight of described sterilizing artificial feed, dried mulberry leaf powder 36.5wt%, defatted soy flour 35.4wt%, mulberry green branch powder 5wt%, corn starch 11.0wt%, sucrose 4.5%, vitamin B1 .5wt%, vitamin C 1.5wt%, peaceful lemon acid 2.0wt%, crotonic acid 0.4wt%, Choline Chloride 0.2wt%, the gross weight of described sterilizing artificial feed is 100wt%, described Sterilized isolator keeps feeding process to carry out in the dark state, described feeding process keeps the temperature of 27-29 DEG C, humidity is 70-80%, for whole described Sterilized isolator, transfer rate through the air of aseptic process is 250-350 liter/sky, until obtain the aseptic silkworm in 3rd ~ 5 days 5 ages,
(4) produce cordyceps liquid, comprising by Natural C.militaris bacterial classification transferred species potato liquid nutrient medium, and described potato liquid nutrient medium is placed in the inherent 20 DEG C of lucifuge joltings of fungal culture case and cultivates 5 to 7 days;
(5) by the abdominal cavity of the aseptic silkworm in described 3rd ~ 5 days 5 ages of acquisition in described cordyceps liquid implantation step (3), injection volume is 0.2 ~ 0.3 milliliter/bar, described aseptic silkworm after injection is still placed in described Sterilized isolator raise, described Sterilized isolator keeps feeding process to carry out in the dark state, described feeding process keeps the temperature of 27-29 DEG C, humidity is 70-80%, for whole described Sterilized isolator, transfer rate through the air of aseptic process is 250-350 liter/sky, until described aseptic silkworm is dead stiff and grow a small amount of mycelia;
(6) aseptic silkworm stiff for the death growing a small amount of mycelia obtained in step (5) is taken out from described raising dish, proceed to 25 DEG C of illumination on daytime, cultivate 38-42 days in gnotobasis that night, 20 DEG C of dark replaced, keep described ambient humidity more than 95% around here, namely obtain the aseptic silkworm Chinese caterpillar fungus that whole body covers with the sporophore of mycelia and varying length.
2. the method for the aseptic silkworm Chinese caterpillar fungus of cultivation according to claim 1, also comprise mineral salt in wherein said sterilizing artificial feed, described mineral salt are selected from any combination of following material: calcium carbonate, sodium chloride, potassium sulfate, manganese sulphate, calcium phosphate, magnesium sulfate, hydrated copper sulfate, ferric sulfate hydrate and sodium fluoride.
3. the method for cultivation silkworm Chinese caterpillar fungus according to claim 1 and 2, wherein said raising is coiled as drawer type raises dish.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105940956A (en) * | 2016-06-06 | 2016-09-21 | 江苏科技大学 | Artificial silkworm chrysalis cordyceps sobolifera cultivation method achieved by taking tussah silkworm chrysalises as hosts |
| CN106034738A (en) * | 2016-06-06 | 2016-10-26 | 江苏科技大学 | Method for manually cultivating silkworm chrysalis cordyceps cicadae by using silkworm chrysalis of domestic silkworms as hosts |
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| CN1167825A (en) * | 1996-05-27 | 1997-12-17 | 日原町 | Method for artificial culture of cordyceps, e.g. Cordyceps mititaris |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105940956A (en) * | 2016-06-06 | 2016-09-21 | 江苏科技大学 | Artificial silkworm chrysalis cordyceps sobolifera cultivation method achieved by taking tussah silkworm chrysalises as hosts |
| CN106034738A (en) * | 2016-06-06 | 2016-10-26 | 江苏科技大学 | Method for manually cultivating silkworm chrysalis cordyceps cicadae by using silkworm chrysalis of domestic silkworms as hosts |
| CN106034738B (en) * | 2016-06-06 | 2017-06-16 | 江苏科技大学 | A kind of method with silkworm pupa as host artificial culture silkworm chrysalis cicada fungus |
| CN105940956B (en) * | 2016-06-06 | 2017-06-20 | 江苏科技大学 | A kind of method with tussah silkworm chrysalis as host artificial culture silkworm chrysalis cicada fungus |
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