CN106034738B - A kind of method with silkworm pupa as host artificial culture silkworm chrysalis cicada fungus - Google Patents

Abstract

The invention discloses a method for artificially cultivating cicadas with silkworm chrysalis as a host. The method comprises the following steps: separating and screening cicada strains from wild cicadas, inoculating them on culture medium, and cultivating them until bacteria grow out. Carry out alternating light and dark culture for the silk body, and after being covered with spores, wash the spores to make a spore suspension; use the live silkworm chrysalis as the host, and use the cicadae spore suspension to infuse the silkworm with the cicadae spore suspension by injection, soaking or body spraying. Live silkworm chrysalis infection; culture the infected silkworm chrysalis until the silkworm chrysalis hardens, and then carry out alternating light and dark culture. The present invention uses live silkworm chrysalis as a host to cultivate silkworm chrysalis cicadae, which has high nutritional content and is non-toxic, and can be developed into various food and health care products, which are safe and reliable to eat; the method for cultivating cicadae with silkworm silkworm chrysalis in the present invention has short cultivation period and technical process It is simple, suitable for large-scale production, and has broad application prospects.

Description

A method for artificially cultivating silkworm chrysalis cicadas with silkworm chrysalis as host

technical field

The invention belongs to a method for cultivating cicada flowers, in particular to a method for artificially cultivating silkworm chrysalis cicadas with silkworm chrysalis as a host.

Background technique

Cicadae (Cordyceps cicadae) is a composite of the subunit and nymph corpse of Cordyceps cicadae Shing parasitizing on the nymphs of Cicadae insects. It is a traditional precious Chinese medicinal material in my country. In China, cicadae has been used as a traditional Chinese medicine and food for more than 1500 years, long before Cordyceps sinensis. Modern medical research shows that cicadae contains a variety of active ingredients, which have the functions of treating convulsions in children, anti-tumor, analgesia and sedation, improving kidney function, anti-fatigue, immune regulation, etc. It is a precious medicinal material similar to Cordyceps sinensis, which can be used as replacement of.

The market demand for cicadae is increasing day by day, but the growth of cicadae requires a specific ecological environment and host insects, the natural yield is low, and people have been digging in large quantities for a long time, and the resources are decreasing day by day. In order to meet market and clinical needs, in recent years, researchers have carried out research on the artificial cultivation of cicadae, and have made some progress. At present, there are already some public reports about cicada flower artificial cultivation technology both at home and abroad. For example, "a method for cultivating cicadae (CN103392500A)", inoculate Paecilomyces cicadae on the culture medium after activation, cultivate in dark until bacteria buds grow, and ventilate and cultivate in the dark, and cultivate for 40 days Fruiting bodies can be harvested left and right. "Cicada flower fruiting body with cicada larvae as host and its cultivation method (CN102763560A)", the fruiting body of Cordyceps militaris is separated, purified and rejuvenated by spores on a solid medium to obtain the mycelium of Cordyceps militaris, which is cultured by shaking with a liquid medium, Make liquid strains, process the larvae of the cicada, prick a small hole on the body of the cicada larvae with a sterile needle, use a sterile syringe to inhale the liquid strain and inject it into the back of the body of the cicada larvae, place the infected cicada larvae on the In a sterile tissue culture bottle, inject 0.1ml of liquid bacteria into the outer body of cicada larvae with a syringe, cover the bottle cap, and cultivate until the fruiting body of cicada flower grows to 3cm long. "A bionic cultivation method of cicadae (CN103975765A)", based on the ecological environment and growth law of cicadae, adopts the methods of microbial engineering technology, ecology and cultivation to conduct systematic research on the bionic cultivation technology of cicadae, establish Cicada flower in the natural bamboo forest bionic cultivation technology operating procedures, the establishment of an imitation ecological system, so as to obtain high-quality cicada flower raw materials. "A production method of artificial cicadae (CN103598014A)", dehumidifying wild cicadae at low temperature, drying, collecting cicadae spore powder, mixing spore powder and culture medium in a weight ratio of 1:1000, mixing The latter culture medium was cultivated at 15-25° C. for 20-30 days to obtain cicadae. "A method for artificial cultivation of Paecilomyces cicadae and the application of its cultured products (CN102242070A)" Large-scale artificial cultivation of Paecilomyces cicadae through the steps of strain preparation, batching, boxing, sterilization, inoculation, solid fermentation, and harvesting , using corn flour, bran, wheat, barley, rice, millet, sorghum and other grains and bagasse, sucrose, shell powder, silkworm chrysalis powder, and potassium nitrate as the medium to cultivate Paecilomyces cicadae. "A method for artificial cultivation of cicada flowers (CN101225362A)", select Paecilomyces cicadae heterokaryon strain, culture Paecilomyces cicadae spores with 0.05% Tween 80 water after cultivating Paecilomyces cicadae for 1 week After washing and diluting into Paecilomyces cicadae spore liquid, the infected body (mature larvae of silkworm) is placed in the Paecilomyces cicadae spore liquid for infection, and cultivated at 20°C-28°C for 2-3 weeks to obtain cicada flowers.

Bombyx mori silkworm chrysalis is very rich in nutrients, and its traditional utilization is mainly to develop into various food and health products. In recent years, the cultivation of Cordyceps militaris from silkworm chrysalis has gradually become a research hotspot in the field of multi-purpose utilization in sericulture. But, so far, also do not adopt the method for silkworm chrysalis to cultivate cicadae.

Contents of the invention

Purpose of the invention: Aiming at the problems existing in the prior art, the present invention provides a method for artificially cultivating silkworm chrysalis cicadas with the silkworm chrysalis as a host. The present invention adopts live silkworm chrysalis as a host to cultivate silkworm chrysalis cicadae, which has high nutritional content and is non-toxic, and can be developed into various food and health care products, and is safe and reliable to eat; the method for cultivating cicadae with silkworm chrysalis in the present invention has a short cultivation period and is technologically advanced. The process is simple and suitable for large-scale production.

Technical scheme: in order to achieve the above object, as described in the present invention, a method for artificially cultivating silkworm chrysalis cicadas with silkworm chrysalis as a host, comprises the following steps:

(1) Isolate and screen the cicadae strains from wild cicadae, cultivate the cicadae strains until mycelium grows, and then carry out alternating light and dark culture. After the spores are overgrown, wash the spores to make cicadae spore suspension liquid;

(2) disinfect the body surface of silkworm live silkworm chrysalis; infect silkworm silkworm chrysalis by cicada spore suspension;

(3) Cultivate the infected silkworm chrysalis until the silkworm chrysalis hardens, and white mycelium appears between the nodes;

(4) The hardened silkworm chrysalis is placed in a sterile environment, and light and dark are alternately cultivated.

Further, in the step (1), cultivating the cicadae strain until mycelium grows is to inoculate the cicadae strain on a PDA solid medium, and cultivate in dark at 20-22° C. for 8-10 days until the mycelium grows.

Further, washing down the spores in step (1) is washing down the spores with sterile water with 0.03-0.07% Tween 80.

Further, the specific steps of alternating light and dark cultivation in step (1) are alternating light and dark cultivation at 22-24°C, the cycle is 12-14h, the light intensity is 250-350Lx, the humidity is 85%-95%, and the cultivation time is 10-12d; step (4) The specific steps of the alternating light and dark culture are 20-24°C light and dark alternating culture, the cycle is 10-14h, the light intensity is 150-400Lx, the humidity is 85%-95%, and the culture time is 10-25d. The growth conditions simulate the natural growth conditions of wild cicadas, and the obtained cicadas are not only similar in appearance to wild cicadas, but also have comparable quality to wild cicadas.

Further, the concentration of the cicadae spore suspension in step (1) is 10 ⁴ -10 ⁷ spores/mL.

Further, infecting the silkworm chrysalis in the step (2) is to inject 5-10ul cicadae spore suspension into the subcutaneous tissue of the silkworm chrysalis with a syringe, and put it into a sterilized culture dish.

Further, the step (2) to infect the silkworm chrysalis is to put the silkworm chrysalis in the cicada spore suspension for 10-15 minutes and then take it out and put it into a sterilized petri dish.

Further, infecting the silkworm chrysalis in the step (2) is to spray the cicadae spore suspension on the body surface of the silkworm chrysalis by spraying, and put it into a sterilized petri dish.

Further, in step (3), culturing until the silkworm chrysalis hardens is cultured in the dark at 20-24° C. until the silkworm chrysalis hardens.

Further, the sterile environment in step (4) is sand or a sterile plastic box that is sterilized by boiling for 0.5-1.5 hours.

Beneficial effects: Compared with the prior art, the method for artificially cultivating silkworm chrysalis cicadas with silkworm chrysalis as a host has the following advantages: the live silkworm chrysalis is used as a host to cultivate silkworm chrysalis cicadas, which has high nutritional components and is non-toxic, and can be developed into various Food and health products, safe and reliable to eat; the cultivation method of the present invention has short cultivation period, simple and convenient technical process, and is suitable for large-scale production; on the one hand, the present invention provides a new way for the development and utilization of silkworm chrysalis resources with high added value. It is conducive to improving the economic benefits of sericulture; on the other hand, due to the sharp increase in demand for Cordyceps in recent years, the price of Cordyceps has risen sharply, which in turn has promoted the demand for cicadae. Facing the problem of depletion, the present invention has potential social benefits for protecting the wild resources of cicadae and maintaining ecological balance.

Description of drawings

Fig. 1 is the cicadae sample cultivated under the conditions of embodiment 1;

Fig. 2 is the cicada flower sample cultivated under the conditions of embodiment 2;

Fig. 3 is the cicada flower sample cultivated under the conditions of embodiment 3;

Fig. 4 is the cicada flower sample cultivated under the conditions of embodiment 4.

detailed description

The present invention will be further described below in conjunction with drawings and embodiments.

Example 1

1. Isolate from the wild cicadae collected in the bamboo forest of Anji, Zhejiang, and obtain the cicadae strain capable of infecting silkworm chrysalis after multiple generations of screening, inoculate it on PDA solid medium, and culture it in dark at 20°C for 10 days until the mycelium grows Afterwards, alternate light and dark cultivation was carried out at 24° C., the cycle was 14 hours, the light intensity was 350 Lx, the humidity was 95%, and the cultivation time was 10 days. After being covered with spores, wash the spores with 0.03% Tween 80 sterile water to prepare a cicadae spore suspension and dilute to a concentration of 10 ⁴ /mL.

2. Disinfect the silkworm chrysalis body surface with 75% alcohol by spraying, inject 5ul of cicadae spore suspension into the subcutaneous tissue of silkworm chrysalis with a micro-syringe, put it into a sterilized petri dish quickly after inoculation, and infect .

3. Cultivate the infected silkworm chrysalis in the dark in an incubator at 20°C until the chrysalis hardens and white mycelium appears between the nodes, generally about 8 days.

4. Bury the hardened silkworm chrysalis in the sand sterilized by boiling for 0.5 hours, transfer it to a biochemical incubator, and carry out alternating light and dark cultivation at 24°C. The cycle is 14 hours, the light intensity is 400Lx, and the humidity is 95%. After 10 days of cultivation, Figure 1 is obtained. A sample of the cicada flower shown.

Example 2

1. Isolate from the wild cicadae collected in the bamboo forest of Anji, Zhejiang, and obtain the cicadae strain capable of infecting silkworm chrysalis through multi-generation screening, inoculate it on PDA solid medium, and cultivate it in dark at 21°C for 9 days until the mycelium grows After that, alternate light and dark cultivation was carried out at 23° C., the cycle was 12 hours, the light intensity was 300 Lx, the humidity was 90%, and the cultivation time was 11 days. After being covered with spores, wash the spores with 0.05% Tween 80 sterile water to prepare a cicadae spore suspension and dilute to a concentration of 10 ⁶ /mL.

2. Disinfect the body surface of the silkworm chrysalis with 75% alcohol by spraying, put the silkworm chrysalis in the cicada spore suspension and soak for 10 minutes, take it out, put it into a sterilized petri dish, and infect it.

3. Cultivate the infected silkworm chrysalis in the dark in an incubator at 22°C until the chrysalis hardens and white mycelium appears between the nodes, generally about 7 days.

4. Put the hardened silkworm chrysalis into a sterile plastic box, cover it with a layer of plastic wrap, transfer it to a biochemical incubator, and carry out alternating light and dark cultivation at 22°C. The cycle is 12h, the light intensity is 300Lx, and the humidity is 90%. 22d obtains the cicadae flower sample shown in FIG. 2 .

Example 3

1. Isolate from the wild cicadae collected in the bamboo forest of Anji, Zhejiang, and obtain the cicadae strain capable of infecting silkworm chrysalis through multi-generation screening, inoculate it on PDA solid medium, and cultivate it in dark at 22°C for 8 days until the mycelium grows Afterwards, alternate light and dark cultivation was carried out at 22° C., with a period of 12 hours, a light intensity of 250 Lx, a humidity of 85%, and a cultivation time of 12 days. After being covered with spores, wash the spores with 0.07% Tween 80 sterile water to prepare a cicadae spore suspension and dilute to a concentration of 10 ⁷ /mL.

2. Disinfect the silkworm chrysalis body surface with 75% alcohol by spraying, inject 10ul of cicada spore suspension into the subcutaneous tissue of the silkworm chrysalis with a micro-injector, put it into a sterilized petri dish quickly after inoculation, and infect .

3. Cultivate the infected silkworm chrysalis in the dark in a 24°C incubator until the chrysalis hardens and white mycelium appears between the nodes, generally about 6 days.

4. Bury the hardened silkworm chrysalis in the sterilized sand boiled for 1.5 hours, transfer it to a biochemical incubator, and carry out alternating light and dark cultivation at 20°C. The cycle is 10 hours, the light intensity is 150Lx, and the humidity is 85%. After 25 days of cultivation, Figure 3 is obtained. A sample of the cicada flower shown.

Example 4

1. Isolate from the wild cicadae collected in the bamboo forest of Anji, Zhejiang, and obtain the cicadae strain capable of infecting silkworm chrysalis after multiple generations of screening, inoculate it on PDA solid medium, and cultivate it in dark at 22°C for 9 days until the mycelium grows Afterwards, alternate light and dark cultivation was carried out at 22° C., with a period of 12 hours, a light intensity of 300 Lx, a humidity of 90%, and a cultivation time of 10 days. After the spores are overgrown, the spores are washed with 0.05% Tween 80 sterile water to prepare a cicadae spore suspension, which is diluted to a concentration of 10 ⁵ /mL.

2. Disinfect the body surface of silkworm silkworm chrysalis with 75% alcohol by spraying, put the silkworm chrysalis in the cicada spore suspension and soak it for 15 minutes, take it out, put it into a sterilized petri dish, and infect it.

3. Cultivate the infected silkworm chrysalis in the dark in an incubator at 22°C until the chrysalis hardens and white mycelium appears between the nodes, generally about 7 days.

4. Bury the hardened silkworm chrysalis in the sand sterilized by boiling for 1 hour, transfer it to a biochemical incubator, and carry out alternating light and dark cultivation at 22°C. The cycle is 12 hours, the light intensity is 200Lx, and the humidity is 90%. After culturing for 20 days, the results shown in Figure 4 are obtained. The cicada flower sample shown.

Example 5

1. Isolate from the wild cicadae collected in the bamboo forest of Anji, Zhejiang, and obtain the cicadae strain capable of infecting silkworm chrysalis through multi-generation screening, inoculate it on PDA solid medium, culture in dark at 21°C for 10 days until the mycelium grows After that, alternate light and dark cultivation was carried out at 23° C., with a period of 13 hours, a light intensity of 300 Lx, a humidity of 90%, and a cultivation time of 12 days. After the spores are overgrown, the spores are washed with 0.05% Tween 80 sterile water to prepare a cicadae spore suspension, which is diluted to a concentration of 10 ⁵ /mL.

2. Disinfect the body surface of silkworm silkworm chrysalis with 75% alcohol by spraying, spray cicada spore suspension on the surface of silkworm chrysalis by spraying, put it into a sterilized petri dish, and infect.

3. Cultivate the infected silkworm chrysalis in the dark in an incubator at 22°C until the chrysalis hardens and white mycelium appears between the nodes, generally about 7 days.

4. Put the hardened silkworm chrysalis into a sterile plastic box, cover it with a layer of plastic wrap, transfer it to a biochemical incubator, and carry out alternating light and dark cultivation at 22°C. The cycle is 12h, the light intensity is 250Lx, and the humidity is 90%. The 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.