CN105379562B - A method of the sterile silkworm grass of culture - Google Patents
A method of the sterile silkworm grass of culture Download PDFInfo
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- CN105379562B CN105379562B CN201510882691.7A CN201510882691A CN105379562B CN 105379562 B CN105379562 B CN 105379562B CN 201510882691 A CN201510882691 A CN 201510882691A CN 105379562 B CN105379562 B CN 105379562B
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- 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 present invention provides a kind of methods of the sterile silkworm grass of culture, including：By head pigmentation stage or the pervious silkworm seed sterilization of head pigmentation stage；The silkworm seed after the sterilization is placed in Sterilized isolator and is hatched；The sterile newly-hatched silkworm sterilizing man-made feeds hatched in the Sterilized isolator are fed；Produce Cordyceps militaris bacterium solution；Cordyceps militaris bacterium solution is injected to the abdominal cavity of the sterile silkworm in the 3rd~5 day 5 age；The dead stiff sterile silkworm for growing a small amount of mycelia is taken out from the feeding disk, it is transferred in 20 DEG C of 25 DEG C of illumination on daytime, night alternate gnotobasis of dark and cultivates 38-42 days, 95% or more the ambient humidity is kept around here, that is, obtains the sterile silkworm grass that whole body covers with the sporophore of mycelia and varying length.
Description
Technical field
The present invention relates to the culture of silkworm grass more particularly to sterile culture silkworm grass.
Background technology
Cordyceps sinensis also known as cordyceps sinensis are a kind of civil usual rare tonic herbs in China, and nutritional ingredient is higher than ginseng,
It can be used as medicine, also edible, be superior delicacies, there is very high nutritive value.Cordyceps sinensis due to cordycepin contained therein,
The immunity of body can be enhanced, nourishing lung and kidney has apparent inhibiting effect to lung cancer, liver cancer etc..It is clinically long to the deficiency syndrome of the lung
Cough is panted, hemoptysis of pulmonary tuberculosis, night sweat, kidney deficiency soreness of waist and knee joint, impotence and seminal emission, under the red blood cell after neurasthenia and chemotherapy, radiotherapy
Drop is all effective in cure.The important mark number of cordycepin content has become cordyceps sinensis quality good or not refers to.
However, cordyceps sinensis is due to its scarcity, price is very expensive, and it is rare to develop and use inexpensive high-quality silkworm grass replacement
Rare cordyceps sinensis has become the hot subject of silkworm grass research and development.Silkworm grass is also referred to as silkworm cordyceps sinensis or silkworm cordyceps sinensis, is to use pupa worm
Entomogenous fungi combination made of artificial culture on careless bacterial strain spore inoculating to silkworm.
The distribution of cordycepin is followed successively by Mycelium culture liquid, mycelium, stroma and gradually increases.The quality of cordyceps sinensis is improved,
Having only keeps mycelia growth vigorous, expands growth area；Promote stroma growth population and length.However, existing silkworm grass culture
In technology, obtained cordycepin content is relatively low, is unable to reach the mean level of cordyceps sinensis.Therefore, this field always exists
To the needs that silkworm grass cultural method is improved, to further increase the content of cordycepin in silkworm grass.
Invention content
The present invention provides a kind of methods of the sterile silkworm grass of culture, and sterile silkworm is raised by specific condition and feed
As the host of cordyceps growth, development and production third generation cordyceps sinensis, cordycepin content is the number of wild cordyceps sinensis
Ten times are apparently higher than cordyceps sinensis to hundreds of times, other active biomass generation.
The method described in the present invention includes：
(1) head pigmentation stage or 75% ethyl alcohol of the pervious silkworm seed of head pigmentation stage and 4% formalin soaking disinfection are sterilized；
(2) silkworm seed after the sterilization is placed in Sterilized isolator and is hatched, the Sterilized isolator
Including several horizontal positioned feeding disks, the Sterilized isolator keeps hatching process to carry out in the dark state, described to incubate
Change process keeps 25 DEG C of steady temperature, humidity 70-80%, for the entire Sterilized isolator, through aseptic process
The transmission rate of air be 250-350 liters/day, until obtaining sterile newly-hatched silkworm；
(3) the sterile newly-hatched silkworm sterilizing man-made feeds hatched in the Sterilized isolator are fed, it is described to go out
Bacterium man-made feeds are man-made feeds through high pressure steam sterilization, then supply because the vitamin of sterilizing loss obtains, go out wherein described
Bacterium man-made feeds include：Based on the total weight of the sterilizing man-made feeds, dried mulberry leaf powder 36.5wt%, defatted soy flour
35.4wt%, Sang Lvzhi powder 5wt%, cornstarch 11.0wt%, sucrose 4.5%, vitamin B1 .5wt%, vitamin
C1.5wt%, peaceful lemon acid 2.0wt%, crotonic acid 0.4wt%, choline chloride 0.2wt%, the total weight of the sterilizing man-made feeds
For 100wt%, the Sterilized isolator keeps feeding process to carry out in the dark state, and the feeding process is kept for 27-29 DEG C
Temperature, humidity 70-80%, for the entire Sterilized isolator, the transmission rate of the air through aseptic process is
250-350 liters/day, until obtaining the sterile silkworm in the 3rd~5 day 5 age；
(4) Cordyceps militaris bacterium solution is produced, including by Natural C.militaris strain transferred species potato fluid nutrient medium, and will be described
Potato fluid nutrient medium, which is placed in fungal culture case, is protected from light shaking culture 5 to 7 days at 20 DEG C；
(5) by the abdominal cavity of the sterile silkworm in the 3rd~5 day 5 age obtained in the Cordyceps militaris bacterium solution injection step (3),
Injection volume is 0.2~0.3 milliliter/item, and the sterile silkworm after injection is still placed in the Sterilized isolator and is raised, described
Sterilized isolator keeps feeding process to carry out in the dark state, the temperature of 27-29 DEG C of the feeding process holding, and humidity is
70-80%, for the entire Sterilized isolator, the transmission rate of the air through aseptic process is 250-350 liters/day,
Until the sterile silkworm is dead stiff and grows a small amount of mycelia；
(6) the stiff sterile silkworm of the death for growing a small amount of mycelia obtained in step (5) is taken out from the feeding disk,
It is transferred in 20 DEG C of 25 DEG C of illumination on daytime, night alternate gnotobasis of dark and cultivates 38-42 days, keep the ambient humidity around here
95% or more, that is, obtain the sterile silkworm grass that whole body covers with the sporophore of mycelia and varying length.
In an embodiment of the invention, further include inorganic salts in the sterilizing man-made feeds, the inorganic salts choosing
From the arbitrary combination of following substance：Calcium carbonate, sodium chloride, potassium sulfate, manganese sulfate, calcium phosphate, magnesium sulfate, hydrated copper sulfate, water
Close ferric sulfate and sodium fluoride.
In an embodiment of the invention, the method for the culture silkworm grass, wherein the feeding disk is drawer
Formula raises disk.
The cultural method of silkworm grass based on the present invention, the cordycepin extracted in the silkworm grass of obtained specific yield
Amount greatly improve, advantageously provide the cheap substitute products of cordyceps sinensis.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Embodiment 1
(1) head pigmentation stage or 75% ethyl alcohol of the pervious silkworm seed of head pigmentation stage and 4% formalin soaking disinfection are sterilized；
(2) silkworm seed after the sterilization is placed in Sterilized isolator and is hatched, the Sterilized isolator
Including several horizontal positioned feeding disks, the Sterilized isolator keeps hatching process to carry out in the dark state, described to incubate
Change process keeps 25 DEG C of steady temperature, humidity 70-80%, for the entire Sterilized isolator, through aseptic process
The transmission rate of air be 250-350 liters/day, until obtaining sterile newly-hatched silkworm；
(3) the sterile newly-hatched silkworm sterilizing man-made feeds hatched in the Sterilized isolator are fed, it is described to go out
Bacterium man-made feeds are man-made feeds through high pressure steam sterilization, then supply because the vitamin of sterilizing loss obtains, go out wherein described
Bacterium man-made feeds include：Based on the total weight of the sterilizing man-made feeds, dried mulberry leaf powder 36.5wt%, defatted soy flour
35.4wt%, Sang Lvzhi powder 5wt%, cornstarch 11.0wt%, sucrose 4.5%, vitamin B1 .5wt%, vitamin
C1.5wt%, peaceful lemon acid 2.0wt%, crotonic acid 0.4wt%, choline chloride 0.2wt%, the total weight of the sterilizing man-made feeds
For 100wt%, the Sterilized isolator keeps feeding process to carry out in the dark state, and the feeding process is kept for 27-29 DEG C
Temperature, humidity 70-80%, for the entire Sterilized isolator, the transmission rate of the air through aseptic process is
250-350 liters/day, until obtaining the sterile silkworm in the 3rd~5 day 5 age；
(4) Cordyceps militaris bacterium solution is produced, including by Natural C.militaris strain transferred species potato fluid nutrient medium, and will be described
Potato fluid nutrient medium, which is placed in fungal culture case, is protected from light shaking culture 5 to 7 days at 20 DEG C；
(5) by the abdominal cavity of the sterile silkworm in the 3rd~5 day 5 age obtained in the Cordyceps militaris bacterium solution injection step (3),
Injection volume is 0.2~0.3 milliliter/item, and the sterile silkworm after injection is still placed in the Sterilized isolator and is raised, described
Sterilized isolator keeps feeding process to carry out in the dark state, the temperature of 27-29 DEG C of the feeding process holding, and humidity is
70-80%, for the entire Sterilized isolator, the transmission rate of the air through aseptic process is 250-350 liters/day,
Until the sterile silkworm is dead stiff and grows a small amount of mycelia；
(6) the stiff sterile silkworm of the death for growing a small amount of mycelia obtained in step (5) is taken out from the feeding disk,
It is transferred in 20 DEG C of 25 DEG C of illumination on daytime, night alternate gnotobasis of dark and cultivates 38-42 days, keep the ambient humidity around here
95% or more, that is, obtain the sterile silkworm grass that whole body covers with the sporophore of mycelia and varying length.
(7) water extraction method is used to extract cordycepin.
Comparative example 1
According to sterile five ages silkworm is obtained in embodiment 1 the step of (1)-(3)；
(4) Cordyceps militaris is inoculated in solid medium, 20 DEG C of dark culturings 9 days；Then photosensitive culture 3 days, by mycelia
It is inoculated in liquid spawn culture medium, under dark condition, is shaken 3 hours with 200r/min on constant-temperature table daily, continue culture 5
Liquid spawn is obtained after it, and inoculation bacterium solution is made；
(5) use aseptic injection mode that step (4) inoculation bacterium solution obtained is inoculated in sterile silkworm body made from step (3)
Caudal horn, abdominal foot or internode, the inoculum concentration of every sterile silkworm body is 0.1 milliliter, sterile dries 70min, normal sterile through 18 DEG C
Raising 2.5 days, after rigid silkworm body is removed sand, under dark condition, holding humidity 85%, 20 DEG C of temperature, culture 7 days, then
It is photosensitive go out grass culture 30 days, through 100 DEG C of low temperature drying 2h below, sterile silkworm grass is made.
(6) cordycepin is extracted using the identical water extraction method with embodiment 1 (7).
Comparative example 2
Select the five age silkworms that non-present invention method is raised；Using step (4)-(6) system identical in such as embodiment 1
Obtain silkworm grass；And then cordycepin is extracted using the identical water extraction method with embodiment 1 (7).
Comparative example 3
According to sterile five ages silkworm is obtained in embodiment 1 the step of (1)-(3)；
(4) it is 1 in mass ratio by 19-3, MSl and 1-Y strain:1:Silkworm body is inoculated into after 1 mixing (to support silkworm to 5 ages the 2nd
It silkworm body) on；A concentration of 250mg/mL of strain, injection rate 3mL；；Then sterile culture goes out grass up to silkworm grass.
Inoculation is that needle-penetration is inoculated with, and sterile culture temperature is 15 DEG C, air humidity 85%；Illumination requirement：20 after inoculation
It is cultivates early period, and to illumination no requirement (NR), the 20-60 days after inoculation are the middle and later periods, it is desirable that photosensitive 14 hours daily.
(5) cordycepin is extracted using the identical water extraction method with embodiment 1 (7).
Comparative example 4
According to sterile five ages silkworm is obtained in embodiment 1 the step of (1)-(3)；
(4) preparation of Cordceps militaris spore suspension：To cover with mycelial Cordceps militaris strain be made of sterile water elution it is dense
Degree is the spore suspension of 300mg/mL, and Cord blood is spare；
(5) hypothermic anesthesia before being inoculated with：Live body silkworm is put into constant incubator, and 4-6 DEG C is placed 2 days, and during which not feeding, makes
Because low temperature and starvation enter semidormancy state, vital movement reduces silkworm；
(6) it is inoculated with：Spore suspension is extracted using the syringe after sterilizing, is carefully pierced into, is slowly injected by silkworm head
3mL or so, and spray a small amount of spore suspension in silkworm body surface；
(7) silkworm still to survive after processing is put into incubator, 4-6 DEG C of culture, until silkworm death ossifys；It will be dead
Rigid silkworm is taken out, and head upward, is embedded in the sand without organic matter and through high temperature hot air sterilization, head is made to reveal slightly
Go out 1mm, sealing container is cultivated 1 week at 25 DEG C；Then cordyceps sinensis is moved in culture room and is cultivated, every square metre keeps a 20W section
Can light shine, energy-saving lamp hang it is 1.5 meters high, under conditions of 18 DEG C of temperature, humidity 80%~90% culture have apparent bacterium to head
Silk grows and has fruit body primordium to be differentiated to form；Keep 18 DEG C, humidity be down to 70% continue culture it is long to silkworm body head fructification
Degree is completed more than 2cm, culture, takes out the careless 45 DEG C of freeze-day with constant temperature of silkworm.
(8) cordycepin is extracted using the identical water extraction method with embodiment 1 (7).
The following table 1 shows the comparison of the cordycepin content of silkworm grass obtained by the embodiment of the present invention 1 and comparative example 1-4：
Table 1
As shown above, silkworm grass obtained can significantly improve cordycepin compared with the existing technology by the method for the invention
Content, and keep relatively high silkworm to ossify simultaneously and rate and go out carelessness.
Claims (1)
1. a kind of method of the sterile silkworm grass of culture, the method includes：
(1) head pigmentation stage or 75% ethyl alcohol of the pervious silkworm seed of head pigmentation stage and 4% formalin soaking disinfection are sterilized；
(2) silkworm seed after the sterilization is placed in Sterilized isolator and is hatched, the Sterilized isolator includes
Several horizontal positioned feeding disks, the Sterilized isolator keeps hatching process to carry out in the dark state, described to hatch
The steady temperature of 25 DEG C of journey holding, humidity 70-80%, for the entire Sterilized isolator, the sky through aseptic process
The transmission rate of gas is 250-350 liters/day, until obtaining sterile newly-hatched silkworm；
(3) the sterile newly-hatched silkworm sterilizing man-made feeds hatched in the Sterilized isolator are fed, the sterilizing people
Work feed be man-made feeds through high pressure steam sterilization, then supply because sterilizing loss vitamin obtain, wherein the sterilizing people
Work feed includes：Based on it is described sterilizing man-made feeds total weight, dried mulberry leaf powder 36.5wt%, defatted soy flour 35.4wt%,
Sang Lvzhi powder 5wt%, cornstarch 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 total weight of the sterilizing man-made feeds is 100wt%, described
Sterilized isolator keeps feeding process to carry out in the dark state, the temperature of 27-29 DEG C of the feeding process holding, and humidity is
70-80%, for the entire Sterilized isolator, the transmission rate of the air through aseptic process is 250-350 liters/day,
Until obtaining the sterile silkworm in the 3rd~5 day 5 age；
(4) Cordyceps militaris bacterium solution is produced, including by Natural C.militaris strain transferred species potato fluid nutrient medium, and by the potato
Fluid nutrient medium, which is placed in fungal culture case, is protected from light shaking culture 5 to 7 days at 20 DEG C；
(5) by the abdominal cavity of the sterile silkworm in the 3rd~5 day 5 age obtained in the Cordyceps militaris bacterium solution injection step (3), injection
Amount is 0.2~0.3 milliliter/item, and the sterile silkworm after injection is still placed in the Sterilized isolator and is raised, described sterile
Isolator keeps feeding process to carry out in the dark state, and the feeding process keeps 27-29 DEG C of temperature, humidity 70-
80%, for the entire Sterilized isolator, the transmission rate of the air through aseptic process is 250-350 liters/day, until
The sterile silkworm is dead stiff and grows a small amount of mycelia；
(6) the stiff sterile silkworm of the death for growing a small amount of mycelia obtained in step (5) is taken out from the feeding disk, is transferred to
It is cultivated 38-42 days in 20 DEG C of 25 DEG C of illumination on daytime, night alternate gnotobasis of dark, keeps the ambient humidity 95% around here
More than, that is, obtain the sterile silkworm grass that whole body covers with the sporophore of mycelia and varying length；
Further include inorganic salts in the wherein described sterilizing man-made feeds, the inorganic salts are selected from the arbitrary combination of following substance：Carbonic acid
Calcium, sodium chloride, potassium sulfate, manganese sulfate, calcium phosphate, magnesium sulfate, hydrated copper sulfate, ferric sulfate hydrate and sodium fluoride；
The wherein described feeding disk is that drawer type raises disk.
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| 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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| JP3593429B2 (en) * | 1996-05-27 | 2004-11-24 | 日原町 | Artificial culture method of Cordyceps |
| CN102919588A (en) * | 2012-11-02 | 2013-02-13 | 韩益飞 | Flacherie-preventing feed |
| CN103798058B (en) * | 2014-02-26 | 2016-06-29 | 山东省蚕业研究所 | A kind of breeding method of aseptic silkworm grass |
| CN104285820B (en) * | 2014-10-13 | 2016-09-21 | 海南森瑞谱生命科学药业股份有限公司 | One strain selenium-enriched cordceps militaris strain and application thereof |
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