CN106034738B - A kind of method with silkworm pupa as host artificial culture silkworm chrysalis cicada fungus - Google Patents
A kind of method with silkworm pupa as host artificial culture silkworm chrysalis cicada fungus Download PDFInfo
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Abstract
The invention discloses a kind of method with silkworm pupa as host artificial culture cicada fungus, methods described comprises the following steps：Cicada fungus bacterial strain is separated from wild cicada fungus, is inoculated on culture medium, culture is to growing mycelium, then carries out light and shade alternate culture, after spore is covered with, spore is washed down, is made spore suspension；With silkworm living silkworm chrysalises as host, the method sprayed using injection, immersion or body is infected silkworm living silkworm chrysalises using cicada fungus spore suspension；The silkworm pupa that will have been infected, culture is hardened to silkworm chrysalis, carries out light and shade alternate culture.The present invention uses living silkworm chrysalises to cultivate silkworm chrysalis cicada fungus for host, and nutritional ingredient is high, nontoxic, can develop into various food and health products, edible safety reliability；The method that the present invention cultivates cicada fungus with silkworm pupa, cultivation cycle is short, and technical matters is easy, it is adaptable to large-scale production, has broad application prospects.
Description
Technical field
Method the invention belongs to cultivate cicada fungus, and in particular to it is a kind of with silkworm pupa be host artificial culture silkworm chrysalis cicada fungus
Method.
Background technology
Cicada fungus (Cordyceps cicadae) is parasitic ergot University of Science and Technology cicada grass (Cordyceps cicadae Shing)
The complex of stroma and nymph corpse on the cicada nymph of cicada mountain, is a kind of traditional rare traditional Chinese medicine of China.In
State, cicada fungus is used as traditional Chinese medicine and food have been had more than 1500 years, much earlier than Cordyceps sinensis.Modern medicine study table
Bright cicada fungus composition containing various active, with treatment convulsion in childhood, antitumor, analgesia and it is calm, improve renal function, it is antifatigue, exempt from
The functions such as epidemic disease regulation, are a kind of valuable ingredients of traditional Chinese medicine close with Cordyceps sinensis, can be used as the substitute of Cordyceps sinensis.
Cicada fungus market demand increasingly increases, but the specific ecological environment of cicada fungus growth needs and host insect, it is natural
The rate of output is relatively low, and along with people largely excavate for a long time, resource is reduced increasingly.In order to meet the demand in market and clinic, in recent years
Come, scientific research personnel has carried out the artificial culture research of cicada fungus, and makes some progress.At present, some passes are had both at home and abroad
In the open report of the artificial culture technique of cicada fungus.For example, " a kind of cultural method of cicada fungus (CN103392500A) ", intends blue or green by cicada
It is inoculated on culture medium after mould (Paecilomyces cicadae) activation, to after growing button, lucifuge is aerated light culture
Culture, culture 40 days or so can harvest fructification." cicada fungus fructification and its breeding method with cicada larva as host
(CN102763560A) ", fruiting bodies of cordyceps militaris is purificated and rejuvenated on solid medium through spore separation, Cordyceps militaris mycelia is obtained
Body, through using fluid nutrient medium shaken cultivation, is made liquid spawn, and golden cicada larva is processed, and nothing is used on cicada larva body
Acicula pricks duck eye, and sucking liquid spawn with asepsis injector injects cicada larva body back, and the cicada larva of infectable infection is placed in
In aseptic tissue culture bottle, 0.1ml liquid spawns are injected into cicada larva ectosome with syringe, cover bottle cap, culture is real to cicada fungus
Body is long long to 3cm." the bionical cultural method of a kind of cicada fungus (CN103975765A) ", with the ecological environment and growth rhythm of cicada fungus
It is foundation, using microbial engineering, the method for ecological and cultivation, carries out the bionical culture cultivation technique system of cicada fungus and grind
Study carefully, establish cicada fungus bionic cultivation regulations for technical operation under natural bamboo forest, set up Ecology system, so that it is former to obtain high-quality cicada fungus
Material." a kind of production method of artificial isaria cicadae miq (CN103598014A) ", low temperature dehumidification is spent by wild golden cicada, is dried, and is received
Collection cicada fungus conidia powder, according to conidia powder and culture medium with weight ratio be 1:1000 ratios mix, by mixed culture medium in 15-
Cultivated at 25 DEG C 20-30 days and obtain cicada fungus.A kind of " the application of method and its cultured products that Paecilomyces cicadae is manually cultivated
(CN102242070A) it is " artificial on a large scale by steps such as strain preparation, dispensing mounted box, sterilizing, inoculation, solid fermentation, harvestings
Culture Paecilomyces cicadae, using the cereal such as corn flour, wheat bran, wheat, barley, rice, grain, sorghum and bagasse, sucrose, shell
Powder, dried silkworm chrysalis meal, potassium nitrate are medium culture Paecilomyces cicadae." a kind of artificial culture method of cicada fungus (CN101225362A) ",
Selection Paecilomyces cicadae heterokaryotic isolationses, after with medium culture Paecilomyces cicadae 1 week, with 0.05% Tween 80 water by Paecilomyces cicadae
Spore is washed down and is diluted to Paecilomyces cicadae spore liquid, infectosome (silkworm mature larva) is placed in Paecilomyces cicadae spore liquid and is infected
Afterwards, 20 DEG C of -28 DEG C of cultures obtain cicada fungus in 2-3 weeks.
Silkworm pupa nutritional ingredient very abundant, tradition is utilized mainly develops into various food and health products.In recent years,
Silkworm chrysalis culture Cordyceps militaris is increasingly becoming study hotspot of the silkworm industry multipurpose using field, and " silkworm chrysalis Cordyceps sinensis wine " is exactly with silkworm chrysalis Cordyceps sinensis
For major ingredient is formed.But, so far, the method for not cultivating cicada fungus using silkworm chrysalis also.
The content of the invention
Goal of the invention：It is artificial by host of silkworm pupa the invention provides one kind for the problem that prior art is present
The method for cultivating silkworm chrysalis cicada fungus.The present invention uses silkworm living silkworm chrysalises to cultivate silkworm chrysalis cicada fungus for host, and nutritional ingredient is high, nontoxic,
Various food and health products, edible safety reliability can be developed into；The method that the present invention cultivates cicada fungus with silkworm pupa, culture week
Phase is short, and technical matters is easy, it is adaptable to large-scale production.
Technical scheme：To achieve these goals, as described herein it is a kind of with silkworm pupa be host artificial culture silkworm
The method of pupa cicada fungus, comprises the following steps：
(1) cicada fungus bacterial strain is separated from wild cicada fungus, by cicada fungus strain culturing to growing mycelium, then is carried out bright
Dark alternate culture, after spore is covered with, spore is washed down, is made cicada fungus spore suspension；
(2) silkworm living silkworm chrysalises body surface is carried out disinfection；Silkworm pupa is infected by cicada fungus spore suspension；
(3) silkworm pupa that will have been infected, culture to silkworm chrysalis is hardened, and white hypha body occurs in internode；
(4) by the silkworm chrysalis being hardened, it is placed in gnotobasis, carries out light and shade alternate culture.
Further, it is to consolidate cicada fungus inoculation in PDA to mycelium is grown by cicada fungus strain culturing that step (1) is described
On body culture medium, 20-22 DEG C of light culture 8-10 days is to growing mycelium.
Further, described wash down spore of step (1) is to be washed spore by the sterilized water of 0.03-0.07% Tween 80s
Under.
Further, step (1) the 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) light and shade is alternately trained
Support 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.The growth conditions simulates wild cicada fungus natural growthing condition, the cicada fungus for obtaining not only profile with it is wild
Cicada fungus is similar, and quality is suitable with wild cicada fungus.
Further, step (1) the cicada fungus spore suspension concentration is 104-107Individual/mL.
Further, step (2) it is described silkworm pupa is carried out infection be with syringe silkworm chrysalis hypodermis inject 5-
10ul cicada fungus spore suspensions, are put into sterilized culture dish.
Further, described infection is carried out to silkworm pupa is that silkworm chrysalis is placed in cicada fungus spore suspension to soak to step (2)
Taken out after 10-15min, be put into sterilized culture dish.
Further, step (2) it is described silkworm pupa infect be by spray method silkworm chrysalis body surface spray
Cicada fungus spore suspension, is put into sterilized culture dish.
Further, step (3) culture to silkworm chrysalis is hardened as 20-24 DEG C of light culture to silkworm chrysalis is hardened.
Further, step (4) described gnotobasis is to boil the sand or aseptic plastic box of 0.5-1.5h sterilizings.
Beneficial effect：Compared with prior art, the present invention is the method for host artificial culture silkworm chrysalis cicada fungus with silkworm pupa
Have the following advantages that：Silkworm living silkworm chrysalises are used to cultivate silkworm chrysalis cicada fungus for host, nutritional ingredient is high, nontoxic, can develop into each
Plant food and health products, edible safety reliability；Breeding method of the invention, cultivation cycle is short, and technical matters is easy, it is adaptable to big
Large-scale production；One aspect of the present invention provides new way for the exploitation of silkworm chrysalis resource high added value, is conducive to improving and supports
The economic benefit of silkworm industry；On the other hand, in recent years because Chinese caterpillar fungus demand increases, cause Cordyceps sinensis price to ramp, enter
And the demand to cicada fungus has been promoted, while with cicada fungus environmental destruction, cicada fungus resource will face the problem of exhaustion, because
This, the present invention has potential social benefit to protection cicada fungus wild resource, the maintenance ecological balance.
Brief description of the drawings
Fig. 1 is the cicada fungus sample of cultivation under the conditions of embodiment 1；
Fig. 2 is the cicada fungus sample of cultivation under the conditions of embodiment 2；
Fig. 3 is the cicada fungus sample of cultivation under the conditions of embodiment 3；
Fig. 4 is the cicada fungus sample of cultivation under the conditions of embodiment 4.
Specific embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment 1
1st, separated from the wild cicada fungus for being collected in Anji County, Zhejiang Province bamboo grove, the cicada that can infect silkworm chrysalis is obtained through many generation screenings
Flower bacterial strain, is inoculated on PDA solid mediums, and 20 DEG C of light cultures 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, and humidity 95%, incubation time is 10d.After spore is covered with, with 0.03%
The sterilized water of Tween 80 washes down spore, is made cicada fungus spore suspension, and it is 10 to be diluted to concentration4Individual/mL.
2nd, the method by spraying is carried out disinfection with 75% alcohol to silkworm pupa body surface, with micro syringe in silkworm silkworm
Pupa hypodermis injects the cicada fungus spore suspension of 5ul, is put into rapidly in sterilized culture dish after inoculation, is infected.
3rd, the silkworm pupa that will have been infected, light culture to silkworm chrysalis is hardened in 20 DEG C of incubators, and white hypha occurs in internode
Body, typically about 8d.
4th, by the silkworm pupa being hardened, embedment boil 0.5h sterilizing sand in, be transferred to biochemical cultivation case, carry out 24 DEG C it is bright
Dark alternate culture, the cycle is 14h, and intensity of illumination 400Lx, humidity 95%, culture 10d obtains the cicada fungus sample shown in Fig. 1.
Embodiment 2
1st, separated from the wild cicada fungus for being collected in Anji County, Zhejiang Province bamboo grove, the cicada that can infect silkworm chrysalis is obtained through many generation screenings
Flower bacterial strain, is inoculated on PDA solid mediums, and 21 DEG C of light cultures 9 days are to after growing mycelium, then carry out 23 DEG C of light and shades and hand over
For culture, the cycle is 12h, intensity of illumination 300Lx, and humidity 90%, incubation time is 11d.After spore is covered with, told with 0.05%
The sterilized water of temperature 80 washes down spore, is made cicada fungus spore suspension, and it is 10 to be diluted to concentration6Individual/mL.
2nd, the method by spraying is carried out disinfection with 75% alcohol to silkworm pupa body surface, and silkworm chrysalis is placed on into cicada fungus spore hangs
Taken out after 10min is soaked in liquid, be put into sterilized culture dish, infected.
3rd, the silkworm pupa that will have been infected, light culture to silkworm chrysalis is hardened in 22 DEG C of incubators, and white hypha occurs in internode
Body, typically about 7d.
4th, by the silkworm pupa being hardened, it is put into aseptic plastic box, and covers one layer of preservative film, is transferred to biochemical cultivation case,
22 DEG C of light and shade alternate cultures are carried out, the cycle is 12h, and intensity of illumination 300Lx, humidity 90%, culture 22d obtains cicada fungus shown in Fig. 2
Sample.
Embodiment 3
1st, separated from the wild cicada fungus for being collected in Anji County, Zhejiang Province bamboo grove, the cicada that can infect silkworm chrysalis is obtained through many generation screenings
Flower bacterial strain, is inoculated on PDA solid mediums, and 22 DEG C of light cultures 8 days are to after growing mycelium, then carry out 22 DEG C of light and shades and hand over
For culture, the cycle is 12h, intensity of illumination 250Lx, and humidity 85%, incubation time is 12d.After spore is covered with, told with 0.07%
The sterilized water of temperature 80 washes down spore, is made cicada fungus spore suspension, and it is 10 to be diluted to concentration7Individual/mL.
2nd, the method by spraying is carried out disinfection with 75% alcohol to silkworm pupa body surface, with micro syringe in silkworm silkworm
Pupa hypodermis injects the cicada fungus spore suspension of 10ul, is put into rapidly in sterilized culture dish after inoculation, is infected.
3rd, the silkworm pupa that will have been infected, light culture to silkworm chrysalis is hardened in 24 DEG C of incubators, and white hypha occurs in internode
Body, typically about 6d.
4th, by the silkworm pupa being hardened, embedment boil 1.5h sterilizing sand in, be transferred to biochemical cultivation case, carry out 20 DEG C it is bright
Dark alternate culture, the cycle is 10h, and intensity of illumination 150Lx, humidity 85%, culture 25d obtains the cicada fungus sample shown in Fig. 3.
Embodiment 4
1st, separated from the wild cicada fungus for being collected in Anji County, Zhejiang Province bamboo grove, the cicada that can infect silkworm chrysalis is obtained through many generation screenings
Flower bacterial strain, is inoculated on PDA solid mediums, and 22 DEG C of light cultures 9 days are to after growing mycelium, then carry out 22 DEG C of light and shades and hand over
For culture, the cycle is 12h, intensity of illumination 300Lx, and humidity 90%, incubation time is 10d.After spore is covered with, told with 0.05%
The sterilized water of temperature 80 washes down spore, is made cicada fungus spore suspension, and it is 10 to be diluted to concentration5Individual/mL.
2nd, the method by spraying is carried out disinfection with 75% alcohol to silkworm pupa body surface, and silkworm chrysalis is placed on into cicada fungus spore hangs
Taken out after 15min is soaked in liquid, be put into sterilized culture dish, infected.
3rd, the silkworm pupa that will have been infected, light culture to silkworm chrysalis is hardened in 22 DEG C of incubators, and white hypha occurs in internode
Body, typically about 7d.
4th, by the silkworm pupa being hardened, embedment is boiled in the sand of 1h sterilizings, is transferred to biochemical cultivation case, carries out 22 DEG C of light and shades
Alternate culture, the cycle is 12h, and intensity of illumination 200Lx, humidity 90%, culture 20d obtains the cicada fungus sample shown in Fig. 4.
Embodiment 5
1st, separated from the wild cicada fungus for being collected in Anji County, Zhejiang Province bamboo grove, the cicada that can infect silkworm chrysalis is obtained through many generation screenings
Flower bacterial strain, is inoculated on PDA solid mediums, and 21 DEG C of light cultures 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, and humidity 90%, incubation time is 12d.After spore is covered with, with 0.05%
The sterilized water of Tween 80 washes down spore, is made cicada fungus spore suspension, and it is 10 to be diluted to concentration5Individual/mL.
2nd, the method by spraying is carried out disinfection with 75% alcohol to silkworm pupa body surface, in the method by spraying in silkworm
Pupal cell table sprays cicada fungus spore suspension, is put into sterilized culture dish, is infected.
3rd, the silkworm pupa that will have been infected, light culture to silkworm chrysalis is hardened in 22 DEG C of incubators, and white hypha occurs in internode
Body, typically about 7d.
4th, by the silkworm pupa being hardened, it is put into aseptic plastic box, and covers one layer of preservative film, is transferred to biochemical cultivation case,
22 DEG C of light and shade alternate cultures are carried out, the cycle is 12h, intensity of illumination 250Lx, and humidity 90%, incubation time is 18d.
Claims (9)
1. a kind of method with silkworm pupa as host artificial culture silkworm chrysalis cicada fungus, it is characterised in that comprise the following steps：
(1) cicada fungus bacterial strain is separated from wild cicada fungus, by cicada fungus inoculation on PDA solid mediums, 20-22 DEG C
Light culture 8-10 days is to growing mycelium, then carries out light and shade alternate culture, after spore is covered with, spore is washed down, is made cicada fungus
Spore suspension；
(2) silkworm living silkworm chrysalises body surface is carried out disinfection；Silkworm pupa is infected by cicada fungus spore suspension；
(3) silkworm pupa that will have been infected, culture to silkworm chrysalis is hardened, and white hypha body occurs in internode；
(4) by the silkworm chrysalis being hardened, it is placed in gnotobasis, carries out light and shade alternate culture.
2. with silkworm pupa it is according to claim 1 the method for host artificial culture silkworm chrysalis cicada fungus, it is characterised in that step
(1) described wash down spore is for the sterilized water of 0.03-0.07% Tween 80s washes down spore by mass fraction.
3. with silkworm pupa it is according to claim 1 the method for host artificial culture silkworm chrysalis cicada fungus, it is characterised in that step
(1) the 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) the 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, and humidity 85%-95%, incubation time is 10-25d.
4. with silkworm pupa it is according to claim 1 the method for host artificial culture silkworm chrysalis cicada fungus, it is characterised in that step
(1) the cicada fungus spore suspension concentration is 104-107Individual/mL.
5. with silkworm pupa it is according to claim 1 the method for host artificial culture silkworm chrysalis cicada fungus, it is characterised in that step
(2) it is described silkworm pupa is carried out infection be with syringe silkworm chrysalis hypodermis inject 5-10ul cicada fungus spore suspensions, be put into
In sterilized culture dish.
6. with silkworm pupa it is according to claim 1 the method for host artificial culture silkworm chrysalis cicada fungus, it is characterised in that step
(2) it is described silkworm pupa is carried out infection be silkworm chrysalis is placed in cicada fungus spore suspension soak 10-15min after take out, be put into
In the culture dish of sterilizing.
7. with silkworm pupa it is according to claim 1 the method for host artificial culture silkworm chrysalis cicada fungus, it is characterised in that step
(2) it is described silkworm pupa infect be by spray method silkworm chrysalis body surface spray cicada fungus spore suspension, be put into and gone out
In the culture dish of bacterium.
8. with silkworm pupa it is according to claim 1 the method for host artificial culture silkworm chrysalis cicada fungus, it is characterised in that step
(3) culture to silkworm chrysalis is hardened as 20-24 DEG C of light culture to silkworm chrysalis is hardened.
9. with silkworm pupa it is according to claim 1 the method for host artificial culture silkworm chrysalis cicada fungus, it is characterised in that step
(4) gnotobasis is to boil the sand or aseptic plastic box of 0.5-1.5h sterilizings.
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| CN108901587B (en) * | 2017-04-18 | 2021-02-09 | 浙江泛亚生物医药股份有限公司 | Solid culture method of cordyceps sobolifera |
| 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 |
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