CN106034738A - Method for manually cultivating silkworm chrysalis cordyceps cicadae by using silkworm chrysalis of domestic silkworms as hosts - Google Patents
Method for manually cultivating silkworm chrysalis cordyceps cicadae by using silkworm chrysalis of domestic silkworms as hosts Download PDFInfo
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Abstract
The invention discloses a method for manually cultivating silkworm chrysalis cordyceps cicadae by using silkworm chrysalis of domestic silkworms as hosts. The method comprises the following steps of separating and screening out cordyceps cicadae bacterial strains from wild cordyceps cicadae, inoculating the screened cordyceps cicadae bacterial strains to culture mediums, performing culturing until mycelium grows out, then performing light-shade alternate culturing, after spores overgrow, picking the spores in a washing manner, and making spore suspension; using in-vivo silkworm chrysalis of domestic silkworms as hosts, and enabling the in-vivo silkworm chrysalis of the domestic silkworms to be infected through cordyceps cicadae spore suspension by a method of injection, soaking or body spraying; and culturing the infected silkworm chrysalis of the domestic silkworms until the silkworm chrysalis is hardened, and then performing light-shade alternate culturing. According to the method disclosed by the invention, the in-vivo silkworm chrysalis is used as the hosts for cultivating the silkworm chrysalis cordyceps cicadae which is high in content of nutrient components and free from toxins, and the silkworm chrysalis cordyceps cicadae can be developed into various foods and health-care products, which are safe and reliable to eat. According to the method disclosed by the invention, the silkworm chrysalis of the domestic silkworms is used for cultivating the cordyceps cicadae, the culturing cycle is short, the technical technology is simple and convenient, and the method is suitable for large-scale production and has wide application prospects.
Description
Technical field
The method that the invention belongs to cultivate Periostracum cicadae, is specifically related to a kind of with silkworm pupa for host artificial culture Pupa bombycis Periostracum cicadae
Method.
Background technology
Periostracum cicadae (Cordyceps cicadae) is that Clavicipitaceae University of Science and Technology Cicadae grass (Cordyceps cicadae Shing) is parasitic
Stroma on the Cicadae nymph of cicada mountain and the complex of nymph corpse, be the traditional a kind of rare Chinese medicine of China.In
State, Periostracum cicadae is used as traditional Chinese crude drug and food has had more than 1500 year, far away early than Cordyceps.Modern medicine study table
Bright Periostracum cicadae composition Han various active, has treatment convulsion in childhood, antitumor, analgesia and calmness, improves renal function, resisting fatigue, exempts from
The functions such as epidemic disease regulation, are a kind of valuable ingredients of Chinese medicine close with Cordyceps, can be as the succedaneum of Cordyceps.
Periostracum cicadae market demand increases day by day, but the specific ecological environment of Periostracum cicadae growth needs and host insect, natural
The rate of output is relatively low, adds people and excavates the most in a large number, and resource reduces increasingly.In order to meet market and clinical demand, in recent years
Coming, scientific research personnel has carried out artificial culture's research of Periostracum cicadae, and makes some progress.At present, have some both at home and abroad to close
Open report in Periostracum cicadae artificial culture technology.Such as, " cultural method (CN103392500A) of a kind of Periostracum cicadae ", Cicadae is intended green grass or young crops
Being inoculated in culture medium after mould (Paecilomyces cicadae) activation, light culture is to after growing button, and lucifuge is aerated
Cultivate, cultivate and can gather in the crops sporophore in about 40 days." the Periostracum cicadae sporophore with Cicadae larva as host and breeding method thereof
(CN102763560A) ", Cordyceps militaris (L.) Link.sporophore is purificated and rejuvenated through spore separation on solid medium, obtains Cordyceps militaris (L.) Link. mycelia
Body, through using fluid medium shaken cultivation, makes liquid spawn, processes golden cicada larva, uses nothing on Cicadae larva health
Acicula pricks duck eye, sucks liquid spawn with asepsis injector and injects Cicadae larva body back, is placed in by the Cicadae larva of infectable infection
In aseptic tissue culture bottle, with syringe, 0.1ml liquid spawn is injected Cicadae larva ectosome, cover bottle cap, cultivate to Periostracum cicadae real
Body length is to 3cm length." cultural method (CN103975765A) that a kind of Periostracum cicadae is bionical ", with ecological environment and the growth rhythm of Periostracum cicadae
For foundation, use microbial engineering, ecology and the method for cultivation, carry out Periostracum cicadae bionical cultivation cultivation technique system and grind
Study carefully, establish Periostracum cicadae bionic cultivation regulations for technical operation under natural bamboo forest, set up Ecology system, thus it is former to obtain high-quality Periostracum cicadae
Material." production method (CN103598014A) of a kind of artificial isaria cicadae miq ", by wild Cordyceps cicadae Shing low temperature dehumidification, is dried, and receives
Collection Periostracum cicadae spore powder, mixes with weight ratio for 1:1000 ratio with culture medium according to spore powder, by mixed culture medium at 15-
Cultivate at 25 DEG C 20-30 days and obtain Periostracum cicadae." a kind of method of Paecilomyces cicadidae(Miquel)Samson artificial culture and the application of cultured products thereof
(CN102242070A) " prepare through strain, dispensing mounted box, sterilizing, inoculation, solid fermentation, the step such as gather the most artificial
Cultivate Paecilomyces cicadidae(Miquel)Samson, use the corn such as Semen Maydis powder, wheat bran, Semen Tritici aestivi, Fructus Hordei Vulgaris, rice, foxtail millet, Sorghum vulgare Pers. and bagasse, sucrose, shell
Powder, dried silkworm chrysalis meal, potassium nitrate are culture medium culturing Paecilomyces cicadidae(Miquel)Samson." artificial culture method (CN101225362A) of a kind of Periostracum cicadae ",
Select Paecilomyces cicadidae(Miquel)Samson heterokaryotic isolations, through with after culture medium culturing Paecilomyces cicadidae(Miquel)Samson 1 week, with 0.05% Tween 80 water by Paecilomyces cicadidae(Miquel)Samson
Spore is washed down and is diluted to paecilomyces cicada spore liquid, and infectosome (silkworm mature larva) is placed in paecilomyces cicada spore liquid infection
After, 20 DEG C-28 DEG C cultivations obtain Periostracum cicadae in 2-3 week.
Silkworm pupa nutritional labeling is the abundantest, and tradition utilizes and mainly develops into various food and health product.In recent years,
Pupa bombycis cultivation Cordyceps militaris (L.) Link. is increasingly becoming silkworm industry multipurpose and utilizes the study hotspot in field, and " silkworm chrysalis Cordyceps sinensis wine " is exactly with silkworm chrysalis Cordyceps sinensis
Form for major ingredient.But, up to now, the most do not use the method that Pupa bombycis cultivates Periostracum cicadae.
Summary of the invention
Goal of the invention: the problem existed for prior art, the invention provides a kind of artificial with silkworm pupa for host
The method cultivating Pupa bombycis Periostracum cicadae.The present invention uses silkworm living silkworm chrysalises to be that host cultivates Pupa bombycis Periostracum cicadae, and nutritional labeling is high, nontoxic,
Can develop into various food and health product, edible safety is reliable；The method that the present invention cultivates Periostracum cicadae with silkworm pupa, cultivates week
Phase is short, and technical matters is easy, it is adaptable to large-scale production.
Technical scheme: to achieve these goals, a kind of with silkworm pupa for host artificial culture silkworm as described herein
The method of pupa Periostracum cicadae, comprises the steps:
(1) from wild Periostracum cicadae, separate Periostracum cicadae bacterial strain, by Periostracum cicadae strain culturing to growing mycelium, then carry out bright
Dark alternate culture, after covering with spore, washes down spore, makes Periostracum cicadae spore suspension；
(2) silkworm living silkworm chrysalises body surface is carried out disinfection；By Periostracum cicadae spore suspension, silkworm pupa is infected；
(3) silkworm pupa that will have infected, cultivates hardening to Pupa bombycis, and white hypha body occurs in internode；
(4) by hardening Pupa bombycis, it is placed in gnotobasis, carries out light and shade alternate culture.
Further, step (1) described by Periostracum cicadae strain culturing to growing mycelium for Periostracum cicadae inoculation is solid in PDA
In body culture medium, 20-22 DEG C of light culture 8-10 days is to growing mycelium.
Further, step (1) is described is washed down spore as to be washed by spore by the sterilized water of 0.03-0.07% Tween 80
Under.
Further, step (1) described light and shade alternate culture concretely comprises the following steps 22-24 DEG C of light and shade alternate culture, and the cycle is
12-14h, intensity of illumination 250-350Lx, humidity 85%-95%, incubation time is 10-12d；The alternately training of step (4) described light and shade
Supporting and concretely comprise the following steps 20-24 DEG C of light and shade alternate culture, the cycle is 10-14h, intensity of illumination 150-400Lx, humidity 85%-95%,
Incubation time is 10-25d.This growth conditions simulates wild Periostracum cicadae natural growthing condition, and the Periostracum cicadae obtained not only profile is with wild
Periostracum cicadae is similar, and quality is suitable with wild Periostracum cicadae.
Further, step (1) described Periostracum cicadae spore suspension concentration is 104-107Individual/mL.
Further, described carry out silkworm pupa of step (2) infects for injecting 5-with syringe at Pupa bombycis subcutaneous tissue
10ul Periostracum cicadae spore suspension, puts in sterilized culture dish.
Further, described carry out silkworm pupa of step (2) infects for Pupa bombycis is placed in Periostracum cicadae spore suspension immersion
Take out after 10-15min, put in sterilized culture dish.
Further, described carry out silkworm pupa of step (2) is infected for being sprayed at Pupa bombycis body surface by the method for spraying
Periostracum cicadae spore suspension, puts in sterilized culture dish.
Further, step (3) described cultivation is hardening hardening to Pupa bombycis for 20-24 DEG C of light culture to Pupa bombycis.
Further, step (4) described gnotobasis is to boil sand or the aseptic plastic box of 0.5-1.5h sterilizing.
Beneficial effect: compared with prior art, the method that the present invention is host artificial culture Pupa bombycis Periostracum cicadae with silkworm pupa
Having the advantage that employing silkworm living silkworm chrysalises is that host cultivates Pupa bombycis Periostracum cicadae, nutritional labeling is high, nontoxic, can develop into each
Planting food and health product, edible safety is reliable；The breeding method of the present invention, cultivation cycle is short, and technical matters is easy, it is adaptable to big
Large-scale production；One aspect of the present invention is that the exploitation of Pupa bombycis resource high added value provide new way, is conducive to improving and supports
The economic benefit of silkworm industry；On the other hand, increase due to Cordyceps demand in recent years, cause Cordyceps price to ramp, enter
And promoted the demand to Periostracum cicadae, simultaneously along with Periostracum cicadae environmental destruction, Periostracum cicadae resource will face the problem of exhaustion, because of
This, the present invention has potential social benefit to protection Periostracum cicadae wild resource, maintenance ecological balance.
Accompanying drawing explanation
Fig. 1 is the Periostracum cicadae sample cultivated under the conditions of embodiment 1；
Fig. 2 is the Periostracum cicadae sample cultivated under the conditions of embodiment 2；
Fig. 3 is the Periostracum cicadae sample cultivated under the conditions of embodiment 3；
Fig. 4 is the Periostracum cicadae sample cultivated under the conditions of embodiment 4.
Detailed description of the invention
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment 1
1, separate from the wild Periostracum cicadae being collected in Anji County, Zhejiang Province bamboo grove, obtain the Cicadae that can infect Pupa bombycis through many generation screenings
Flower bacterial strain, is inoculated on PDA solid medium, and 20 DEG C of light culture 10 days are to after growing mycelium, then carry out 24 DEG C of light and shades
Alternate culture, the cycle is 14h, intensity of illumination 350Lx, humidity 95%, and incubation time is 10d.After covering with spore, with 0.03%
Spore is washed down by the sterilized water of Tween 80, makes Periostracum cicadae spore suspension, and being diluted to concentration is 104Individual/mL.
2, with 75% ethanol, silkworm pupa body surface is carried out disinfection, with microsyringe silkworm silkworm by the method for spraying
The Periostracum cicadae spore suspension of pupa subcutaneous tissue injection 5ul, puts into rapidly in sterilized culture dish after inoculation, infects.
3, the silkworm pupa that will have infected, in 20 DEG C of incubators, light culture is hardening to Pupa bombycis, and white hypha occurs in internode
Body, typically about 8d.
4, by hardening silkworm pupa, embedment is boiled in the sand of 0.5h sterilizing, proceeds to biochemical cultivation case, carry out 24 DEG C bright
Dark alternate culture, the cycle is 14h, intensity of illumination 400Lx, humidity 95%, cultivates 10d and obtains the Periostracum cicadae sample shown in Fig. 1.
Embodiment 2
1, separate from the wild Periostracum cicadae being collected in Anji County, Zhejiang Province bamboo grove, obtain the Cicadae that can infect Pupa bombycis through many generation screenings
Flower bacterial strain, is inoculated on PDA solid medium, and 21 DEG C of light culture 9 days are to after growing mycelium, then carry out 23 DEG C of light and shades and hand over
For cultivating, the cycle is 12h, intensity of illumination 300Lx, humidity 90%, and incubation time is 11d.After covering with spore, tell with 0.05%
Spore is washed down by the sterilized water of temperature 80, makes Periostracum cicadae spore suspension, and being diluted to concentration is 106Individual/mL.
2, with 75% ethanol, silkworm pupa body surface is carried out disinfection by the method for spraying, Pupa bombycis is placed on Periostracum cicadae spore and hangs
Take out after liquid soaks 10min, put in sterilized culture dish, infect.
3, the silkworm pupa that will have infected, in 22 DEG C of incubators, light culture is hardening to Pupa bombycis, and white hypha occurs in internode
Body, typically about 7d.
4, by hardening silkworm pupa, put in aseptic plastic box, and cover one layer of preservative film, proceed to biochemical cultivation case,
Carrying out 22 DEG C of light and shade alternate cultures, the cycle is 12h, intensity of illumination 300Lx, humidity 90%, cultivates 22d and obtains Periostracum cicadae shown in Fig. 2
Sample.
Embodiment 3
1, separate from the wild Periostracum cicadae being collected in Anji County, Zhejiang Province bamboo grove, obtain the Cicadae that can infect Pupa bombycis through many generation screenings
Flower bacterial strain, is inoculated on PDA solid medium, and 22 DEG C of light culture 8 days are to after growing mycelium, then carry out 22 DEG C of light and shades and hand over
For cultivating, the cycle is 12h, intensity of illumination 250Lx, humidity 85%, and incubation time is 12d.After covering with spore, tell with 0.07%
Spore is washed down by the sterilized water of temperature 80, makes Periostracum cicadae spore suspension, and being diluted to concentration is 107Individual/mL.
2, with 75% ethanol, silkworm pupa body surface is carried out disinfection, with microsyringe silkworm silkworm by the method for spraying
The Periostracum cicadae spore suspension of pupa subcutaneous tissue injection 10ul, puts into rapidly in sterilized culture dish after inoculation, infects.
3, the silkworm pupa that will have infected, in 24 DEG C of incubators, light culture is hardening to Pupa bombycis, and white hypha occurs in internode
Body, typically about 6d.
4, by hardening silkworm pupa, embedment is boiled in the sand of 1.5h sterilizing, proceeds to biochemical cultivation case, carry out 20 DEG C bright
Dark alternate culture, the cycle is 10h, intensity of illumination 150Lx, humidity 85%, cultivates 25d and obtains the Periostracum cicadae sample shown in Fig. 3.
Embodiment 4
1, separate from the wild Periostracum cicadae being collected in Anji County, Zhejiang Province bamboo grove, obtain the Cicadae that can infect Pupa bombycis through many generation screenings
Flower bacterial strain, is inoculated on PDA solid medium, and 22 DEG C of light culture 9 days are to after growing mycelium, then carry out 22 DEG C of light and shades and hand over
For cultivating, the cycle is 12h, intensity of illumination 300Lx, humidity 90%, and incubation time is 10d.After covering with spore, tell with 0.05%
Spore is washed down by the sterilized water of temperature 80, makes Periostracum cicadae spore suspension, and being diluted to concentration is 105Individual/mL.
2, with 75% ethanol, silkworm pupa body surface is carried out disinfection by the method for spraying, Pupa bombycis is placed on Periostracum cicadae spore and hangs
Take out after liquid soaks 15min, put in sterilized culture dish, infect.
3, the silkworm pupa that will have infected, in 22 DEG C of incubators, light culture is hardening to Pupa bombycis, and white hypha occurs in internode
Body, typically about 7d.
4, by hardening silkworm pupa, embedment is boiled in the sand of 1h sterilizing, proceeds to biochemical cultivation case, carries out 22 DEG C of light and shades
Alternate culture, the cycle is 12h, intensity of illumination 200Lx, humidity 90%, cultivates 20d and obtains the Periostracum cicadae sample shown in Fig. 4.
Embodiment 5
1, separate from the wild Periostracum cicadae being collected in Anji County, Zhejiang Province bamboo grove, obtain the Cicadae that can infect Pupa bombycis through many generation screenings
Flower bacterial strain, is inoculated on PDA solid medium, and 21 DEG C of light culture 10 days are to after growing mycelium, then carry out 23 DEG C of light and shades
Alternate culture, the cycle is 13h, intensity of illumination 300Lx, humidity 90%, and incubation time is 12d.After covering with spore, with 0.05%
Spore is washed down by the sterilized water of Tween 80, makes Periostracum cicadae spore suspension, and being diluted to concentration is 105Individual/mL.
2, silkworm pupa body surface is carried out disinfection with 75% ethanol by the method for spraying, in the method by spraying silkworm
Pupal cell table injection Periostracum cicadae spore suspension, puts in sterilized culture dish, infects.
3, the silkworm pupa that will have infected, in 22 DEG C of incubators, light culture is hardening to Pupa bombycis, and white hypha occurs in internode
Body, typically about 7d.
4, by hardening silkworm pupa, put in aseptic plastic box, and cover one layer of preservative film, proceed to biochemical cultivation case,
Carrying out 22 DEG C of light and shade alternate cultures, the cycle is 12h, intensity of illumination 250Lx, humidity 90%, and incubation time is 18d.
Claims (10)
1. the method that a kind is host artificial culture Pupa bombycis Periostracum cicadae with silkworm pupa, it is characterised in that comprise the steps:
(1) from wild Periostracum cicadae, separate Periostracum cicadae bacterial strain, by Periostracum cicadae strain culturing to growing mycelium, then carry out light and shade friendship
For cultivating, after covering with spore, spore is washed down, make Periostracum cicadae spore suspension；
(2) silkworm living silkworm chrysalises body surface is carried out disinfection；By Periostracum cicadae spore suspension, silkworm pupa is infected；
(3) silkworm pupa that will have infected, cultivates hardening to Pupa bombycis, and white hypha body occurs in internode；
(4) by hardening Pupa bombycis, it is placed in gnotobasis, carries out light and shade alternate culture.
The method being host artificial culture Pupa bombycis Periostracum cicadae with silkworm pupa the most according to claim 1, it is characterised in that step
(1) described by Periostracum cicadae strain culturing to grow mycelium for by Periostracum cicadae inoculation on PDA solid medium, 20-22 DEG C is dark
Cultivate 8-10 days to growing mycelium.
The method being host artificial culture Pupa bombycis Periostracum cicadae with silkworm pupa the most according to claim 1, it is characterised in that step
(1) described spore is washed down as to be washed down by spore by the sterilized water that mass fraction is 0.03-0.07% Tween 80.
The method being host artificial culture Pupa bombycis Periostracum cicadae with silkworm pupa the most according to claim 1, it is characterised in that step
(1) described light and shade alternate culture concretely comprises the following steps 22-24 DEG C of light and shade alternate culture, and the cycle is 12-14h, intensity of illumination 250-
350Lx, humidity 85%-95%, incubation time is 10-12d；Step (4) described light and shade alternate culture concretely comprises the following steps 20-24 DEG C
Light and shade alternate culture, the cycle is 10-14h, intensity of illumination 150-400Lx, humidity 85%-95%, and incubation time is 10-25d.
The method being host artificial culture Pupa bombycis Periostracum cicadae with silkworm pupa the most according to claim 1, it is characterised in that step
(1) described Periostracum cicadae spore suspension concentration is 104-107Individual/mL.
The method being host artificial culture Pupa bombycis Periostracum cicadae with silkworm pupa the most according to claim 1, it is characterised in that step
(2) described carry out silkworm pupa infects for injecting 5-10ul Periostracum cicadae spore suspension with syringe at Pupa bombycis subcutaneous tissue, puts into
In sterilized culture dish.
The method being host artificial culture Pupa bombycis Periostracum cicadae with silkworm pupa the most according to claim 1, it is characterised in that step
(2) described carry out silkworm pupa infects for taking out after Pupa bombycis is placed in Periostracum cicadae spore suspension immersion 10-15min, puts into
In the culture dish of sterilizing.
The method being host artificial culture Pupa bombycis Periostracum cicadae with silkworm pupa the most according to claim 1, it is characterised in that step
(2) described carry out silkworm pupa infects for spraying Periostracum cicadae spore suspension by the method for spraying at Pupa bombycis body surface, puts into and goes out
In the culture dish of bacterium.
The method being host artificial culture Pupa bombycis Periostracum cicadae with silkworm pupa the most according to claim 1, it is characterised in that step
(3) described cultivation is hardening hardening to Pupa bombycis for 20-24 DEG C of light culture to Pupa bombycis.
The method being host artificial culture Pupa bombycis Periostracum cicadae with silkworm pupa the most according to claim 1, it is characterised in that step
Suddenly (4) described gnotobasis is to boil sand or the aseptic plastic box of 0.5-1.5h sterilizing.
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| CN107637390A (en) * | 2017-11-03 | 2018-01-30 | 江苏农林职业技术学院 | A kind of live body silkworm and the method for the wild cicada fungus of silkworm chrysalis culture |
| CN108770595A (en) * | 2018-06-22 | 2018-11-09 | 贵州大学 | A kind of Ecology soil covering culture method of cicada fungus |
| CN108901587A (en) * | 2017-04-18 | 2018-11-30 | 浙江泛亚生物医药股份有限公司 | A kind of solid culture method of cicada fungus |
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