CN109644779B - Method for cultivating cordyceps militaris by inoculating 5-instar larvae of castor silkworms with cordyceps militaris - Google Patents

Method for cultivating cordyceps militaris by inoculating 5-instar larvae of castor silkworms with cordyceps militaris Download PDF

Info

Publication number
CN109644779B
CN109644779B CN201910098897.9A CN201910098897A CN109644779B CN 109644779 B CN109644779 B CN 109644779B CN 201910098897 A CN201910098897 A CN 201910098897A CN 109644779 B CN109644779 B CN 109644779B
Authority
CN
China
Prior art keywords
cordyceps militaris
silkworm
inoculating
castor
culturing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910098897.9A
Other languages
Chinese (zh)
Other versions
CN109644779A (en
Inventor
张俊
李一平
颜新培
廖模祥
钱荷英
张月华
李刚
张晓�
米雅兰
徐瑛
雷语
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hunan Institute Of Sericulture
Original Assignee
Hunan Institute Of Sericulture
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hunan Institute Of Sericulture filed Critical Hunan Institute Of Sericulture
Priority to CN201910098897.9A priority Critical patent/CN109644779B/en
Publication of CN109644779A publication Critical patent/CN109644779A/en
Application granted granted Critical
Publication of CN109644779B publication Critical patent/CN109644779B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • A01G18/50—Inoculation of spawn
    • 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
    • A01G18/20—Culture media, e.g. compost
    • 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
    • A01G18/30—Accessories for use before inoculation of spawn, e.g. sterilisers
    • 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
    • A01G18/60—Cultivation rooms; Equipment therefor
    • 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
    • A01G18/60—Cultivation rooms; Equipment therefor
    • A01G18/69—Arrangements for managing the environment, e.g. sprinklers

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Mycology (AREA)
  • Environmental Sciences (AREA)
  • Mushroom Cultivation (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

A method for cultivating cordyceps militaris by inoculating 5 th-instar larvae of castor silkworms with cordyceps militaris comprises the following steps: (1) selecting a cordyceps militaris host; (2) carrying out enlarged culture on a strain liquid preparation solution; (3) inoculating host ricinus communis silkworm for post-treatment; (4) culturing and managing the cordyceps militaris sporocarp: inoculating slant strain of Cordyceps militaris to solid plate culture medium, activating, inoculating to liquid culture medium, performing amplification culture, inoculating to healthy castor silkworm larva, and culturing and managing in sterile transparent plastic box. The invention fully utilizes the advantages of large individual, soft body, rich nutrition and the like of the castor silkworm larva to cultivate the fruiting body; the pollution rate of mixed bacteria is low, the infection and stiffness rate and the grass yield are high, the obtained castor silkworm cordyceps sinensis has good stability, high fruiting body yield and rich effective active ingredient content, is suitable for large-scale production and has wide development prospect.

Description

Method for cultivating cordyceps militaris by inoculating 5-instar larvae of castor silkworms with cordyceps militaris
Technical Field
The invention relates to a method for cultivating silkworm cordyceps by inoculating 5-instar larvae of castor silkworms with cordyceps militaris, belonging to the technical field of cordyceps cultivation.
Background
Cordyceps sinensis is a kind of homologous fungi with medicine and food with obvious nourishing efficacy and has the reputation of 'live gold'. Ancient cloud: "Ningyichongcao one bundle, don't require jinyuman car". Cordyceps sinensis, cordyceps militaris and cordyceps cicadae are superior to Chinese medicinal cordyceps sinensis. Cordyceps militaris (Cordyceps militaris), also called as Cordyceps militaris, is a model species of Cordyceps in Clavipitaceae, which is an insect-bacterium complex formed by infecting insect larvae in different ways by taking lepidoptera as hosts and breaking through the body surface of the insects through continuous growth and development, wherein fungi and insect shells coexist. The medicinal value of the cordyceps militaris is recorded in medical books, the medicinal effect is mild, the cordyceps militaris has wide medicinal and edible properties, and no side effect, and the cordyceps militaris is a high-grade tonic used as both medicine and food in China. The research proves that the cordyceps militaris contains active ingredients such as cordycepin, pentostatin, cordyceps polysaccharide and the like, has the effects of resisting fatigue and aging, improving the immunity of the organism, inhibiting tumors and the like, can be comparable with ginseng and pilose antler, and is a rare edible fungus and a precious anti-cancer traditional Chinese medicinal material.
In 1950, scientists found and identified adenosine cordycepin in Cordyceps militaris for the first time, and discovered its biological activities such as antibacterial, anti-insect and anticancer in the subsequent period. The cordycepin content is the most important index for measuring the quality of the cordyceps sinensis. In 2011, the Shanghai plant physiological and ecological research institute of Chinese academy of sciences has successively completed the genome research of 18 cordyceps sinensis, cordyceps militaris, cordyceps sobolifera and the like. On the basis, the molecular mechanism of cordycepin biosynthesis is completely analyzed. And the cordyceps militaris can synthesize the anticancer drug pentostatin, molecular evidence is provided for the anticancer activity of the cordyceps militaris, and a new molecular index is provided for the quality standard of the cordyceps militaris.
Because the cordyceps militaris has incomparable advantages of other cordyceps militaris, the cordyceps militaris has good development and utilization prospects in the aspects of functional foods, medicines and the like, and is approved by the national ministry of science and technology to be a substitute and a new resource food of the cordyceps militaris at present. In recent years, the technology for artificially cultivating the cordyceps militaris by taking lepidoptera economic insect silkworm larvae as hosts is mature, the cultivated cordyceps militaris has the form similar to that of natural cordyceps militaris, and the content of active ingredients is higher than that of the cordyceps militaris cultivated by other hosts. The ricinus is a large lepidoptera silk secreting insect, similar to silkworm, the silkworm contains rich crude fat, protein, amino acid, vitamin and mineral elements, the fatty acid composition is reasonable, the content of unsaturated fatty acid is high, and the ricinus also contains rich linolenic acid, and is a potential source of low-cholesterol food. In the middle and later period of the 20 th century, Guangxi Ricinus communis silkworm raisers try to cook silkworm chrysalis as a dish due to the lack of food and meat, and gradually form a distinctive diet culture in the local place over time, and the original edible chrysalis is developed into edible mature silkworms and preputial. The stress resistance and disease resistance of the castor silkworm are stronger than those of the silkworm, the castor silkworm is large in size, the silkworm body is soft, and the nutrition is rich. The castor silkworm is taken as a host, so that large-scale production inoculation is facilitated, the yield of the silkworm cordyceps sinensis is increased, and the active ingredient accumulation is promoted, so that the additional value of the health food is increased, and the large-scale popularization is facilitated.
The castor silkworm cordyceps sinensis is one of cordyceps militaris, and at present, few reports of successful artificial cultivation cordyceps militaris related to castor silkworm are reported. The Chinese patent application 201910113947.2 discloses a cassava silkworm pupa cordyceps sinensis cultured by taking cassava silkworm pupas as hosts and a culturing method thereof: the invention relates to a method for culturing cordyceps militaris strains into cordyceps militaris liquid strains, injecting the strains into cassava silkworm pupa bodies by using a sterile injector, and culturing the cordyceps militaris under the environmental conditions of temperature, humidity, light and the like. The cassava silkworm chrysalis cordyceps product has various active ingredients and nutrient components, has similar efficacy to cordyceps sinensis, can be used as a medicine, a health-care product and a food, has simple cultivation method, short period and low cost, and is a practical method for developing novel cordyceps militaris. The Chinese patent application 201811136359.6 discloses an artificial cultivation method of castor-oil plant, silkworm and cordyceps sinensis, which comprises the following steps: comprises the breeding of castor silkworms, and is characterized by also comprising the steps of host silkworm breeding and selection, cordyceps mother strain selection, host silkworm inoculation, spawn running and the like. The method combines the silkworm larva of Ricinus communis as host with Cordyceps militaris by liquid strain, and the cultured Cordyceps militaris has high content of effective components, especially SOD content higher than that of Cordyceps militaris. Liyajie et al (2011) inoculate silkworm pupa with Cordyceps militaris strain to culture Cordyceps militaris, and has cordycepin content higher than that of wild Cordyceps militaris by 2 times and adenosine content equivalent to that of wild Cordyceps militaris.
The castor silkworm is a wild silkworm, the stress resistance and the disease resistance of the castor silkworm are much stronger than those of the silkworm, the key problem to be solved in the artificial cultivation of the castor silkworm is how to improve the infection capacity of the cordyceps militaris on the castor silkworm and how to prevent the silkworm bodies from being easily infected by other germs and becoming black and softened with rotten odor during the infection and the rigidification of the cordyceps militaris.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide the method for artificially culturing the cordyceps militaris by inoculating the 5 th larva of the castor silkworm with the cordyceps militaris, wherein the method has low infectious microbe pollution rate, high infection rigidness rate and high grass yield, and is stable.
The technical scheme adopted for solving the technical problems is that the method for cultivating the cordyceps militaris by inoculating the cordyceps militaris to the 5 th-instar larvae of the castor silkworms comprises the following steps:
(1) selecting a cordyceps militaris host: selecting healthy silkworms of 3-5 d castor silkworms with five instars, feeding castor leaves continuously after eating at the age of 5, under the environment of the temperature of 25-30 ℃ and the relative humidity of 60-70%, ensuring ventilation, removing sand in time, and selecting robust silkworms of castor silkworms for later use;
(2) and (3) expanding culture of a strain liquid preparation solution: selecting cordyceps militaris slant mother strains, inoculating the cordyceps militaris slant mother strains on a prepared solid plate culture medium added with 5-8 wt% of silkworm chrysalis powder, and activating for 7-10 days; under aseptic operation, uniformly cutting strains into soybean sizes, inoculating 7-9 soybean-sized strains into 1L of liquid culture medium added with 6-10 wt% of silkworm chrysalis powder in time, standing and culturing for 1 day under dark conditions at the temperature of 20-22 ℃, starting oscillation culture on day 2, oscillating for 3-5 hours and 130-140 r/min every day, culturing for 1 day under the illumination intensity of 100-150 lx on day 5, and filtering with 3-4 layers of sterile gauze to remove mycelia to obtain a strain liquid preparation solution;
the slant mother strain of Cordyceps militaris is preferably a first-generation hybrid silkworm larva strain with strong disease resistance, strong infectivity, stable grass generation, and high yield of fruiting body.
Further, in the step (2), the slant mother strain of the cordyceps militaris is cordyceps militaris, and after activation, the first-generation hybrid variety silkworm 5-instar larva with strong disease resistance is subjected to living rejuvenation and is preferably separated, purified and stored for later use.
The in-situ prepared solid plate culture medium added with 5-8 wt% of silkworm chrysalis powder and the liquid culture medium added with 6-10 wt% of silkworm chrysalis powder are common solid plate culture medium and liquid culture medium respectively added with silkworm chrysalis powder.
The weight ratio of the raw materials of the in-situ prepared solid plate culture medium added with 5-8 wt% of silkworm chrysalis powder is as follows: 25% of potato, 2% of glucose, 0.2% of peptone, 0.1% of yeast extract, 5-8 wt% of silkworm chrysalis powder, 1.5% of agar powder, KH2PO40.15%, MgSO40.05%, 1000 mL of water and pH 7.0; the liquid culture medium added with 6-10 wt% of silkworm chrysalis powder comprises the following raw materials in parts by weight: 25% of potato, 2% of glucose, 0.25% of peptone, 0.2% of yeast extract, 6-10 wt% of silkworm chrysalis powder, KH2PO40.2%, MgSO40.1%, VB 10.01%, 1000 mL of water and pH 7.5.
(3) Host castor silkworm inoculation post-treatment: taking the robust ricinus silkworm larvae obtained in the step (1), injecting 0.45-0.60 ml of strain liquid preparation liquid obtained in the step (2) into each head, immediately placing silkworm bodies on plant ash to freely crawl after injection (the plant ash is treated by high-temperature sterilization at the temperature of more than 100 ℃ for 8-10 hours), culturing for 3 days under ventilation conditions at the temperature of 21-24 ℃ and the relative humidity of 60-70%, placing the silkworm bodies into a gauze wood frame after the inoculated wounds completely heal, covering the silkworm bodies with gauze, culturing for 7-9 days under ventilation conditions at the temperature of 21-24 ℃ and the relative humidity of 60-70%, collecting and arranging silkworm bodies which are infected and stiffened but not inoculated with bacteria after more than 95% of silkworm bodies are infected and stiffened, and removing silkworm bodies which are black and softened or not infected and stiffened due to infectious microbes;
the plant ash is preferably black ash obtained by burning rice chaff;
(4) culturing and managing the cordyceps militaris sporocarp: placing the silkworm bodies which are subjected to infection and hardening and do not grow fungi in the step (3) in a transparent plastic box with a cover (the transparent plastic box with the cover is subjected to ultraviolet lamp sterile treatment for more than 3 hours), and culturing for 7-10 days in a dark environment with the temperature of 18-22 ℃ and the relative humidity of 60-70%; then culturing for 4-6 days under the conditions that the light intensity is 100-260 lx, the temperature is 20-22 ℃ in the daytime, the temperature is 16-18 ℃ at night and the relative humidity is 70-75%; after the transparent plastic box cover with the cover is punched for ventilation, the box is cultured for 40 to 50 days under the conditions that the light intensity is 150 to 300lx, the temperature is 18 to 23 ℃ and the relative humidity is 70 to 80 percent, and then the mature cordyceps militaris of the castor silkworm is obtained.
Further, in the step (2), the preparation method of the silkworm chrysalis powder comprises the following steps: drying and crushing the silkworm pupae which are yellow-brown, hard and not eclosion before the silkworm pupae are at 60-70 ℃, and sieving the crushed silkworm pupae with a 80-mesh sieve to obtain the silkworm pupae.
Further, in the step (3), the inoculation is slowly and stably carried out, so that the mechanical injury to the silkworm body is reduced as much as possible, and the premature death of the silkworm body without infection or rigor is avoided.
After the injection is finished, the silkworm body is immediately placed on the plant ash to freely crawl, and the plant ash is beneficial to the dryness and the wound healing of the surface of the castor silkworm and the absorption of water and urine discharged after the castor silkworm is inoculated, so that the mixed bacteria pollution can be effectively prevented.
In the step (4), different light intensities and temperature differences are adopted for treatment, because the color change of surface hyphae needs illumination induction after the castor silkworm body grows, and meanwhile, certain temperature difference stimulation is beneficial to the formation of fruiting body primordium.
The invention has the advantages that:
1. the invention primarily screens and improves the infection and rigor ability of the cordyceps militaris by rejuvenating cordyceps militaris strains of first-generation hybrid varieties silkworm 5-instar larvae with strong disease resistance, and effectively prevents strain degeneration caused by long-term low-temperature storage and multiple passages.
2. The silkworm chrysalis powder with the weight of 5-10 wt% is added into the solid culture medium and the liquid culture medium, so that the activity and the infection capacity of cordyceps militaris strains can be further maintained and improved, the rigidification time is shortened, the loss of nutrient components of silkworm bodies during infection rigidification is reduced, and the grass growing stability and yield are improved.
3. The castor silkworm is a comparatively large-sized omnivorous wild silkworm, the silkworm body contains rich nutrient substances and the disease resistance of the silkworm larva is strong, after the 5-instar castor silkworm larva is inoculated with cordyceps militaris, the infection and the rigidification of the castor silkworm body are stable, the grass yield is high, the worm part of the cordyceps militaris of the castor silkworm is neat and full, the length of a fruit body is more than 3cm, and the content of active ingredients such as cordycepin, pentostatin and the like is rich.
4. After a large amount of cordyceps militaris liquid strain is inoculated to the silkworm body of the castor bean, the silkworm body is placed on plant ash which is subjected to high-temperature sterilization for 8-10 hours at 100 ℃ and cultured for 3 days under the ventilation condition of the temperature of 21-24 ℃ and the relative humidity of 60-70%, and the silkworm body is placed into a gauze wood frame for continuous culture after the inoculated wound is completely healed, so that the probability of septicemia of the silkworm body due to infectious microbe infection is greatly reduced.
The invention fully utilizes the advantages of large individual, soft body, rich nutrition and the like of the castor silkworm larva to cultivate the fruiting body; the method has the advantages of simple operation, easy popularization, low infectious microbe pollution rate, high infection and rigidness rate and grass yield, good stability of the obtained cordyceps militaris, high fruiting body yield, rich effective active ingredients, low cost, plump appearance of the cordyceps militaris, and suitability for large-scale production. The method disclosed by the invention is beneficial to the diversified development of the castor-silkworm industry, improves the comprehensive utilization additional value, improves the quality and grade of related products of the silkworm cordyceps sinensis and has a wide development prospect.
Drawings
FIG. 1 is a graph showing the infection and stiffness of Cordyceps militaris cultivated in example 2 of the present invention;
FIG. 2 is a fungus growth map of Cordyceps militaris cultured in example 2 of the present invention;
FIG. 3 is a diagram of management of silkworm cordyceps sinensis growth in example 2 of the present invention.
Detailed Description
The present invention will be further described with reference to the following examples.
The first embodiment is as follows:
the embodiment comprises the following steps:
(1) selecting a cordyceps militaris host: selecting healthy silkworms of 3d castor silkworms of five ages, feeding castor leaves continuously in the environment of 25 ℃ and 60% relative humidity after eating in the area of 5 ages, ensuring ventilation, removing sand in time, and selecting robust silkworm larvae of the castor silkworms for later use;
(2) and (3) expanding culture of a strain liquid preparation solution: selecting a cordyceps militaris slant mother strain which has strong infectivity on first-generation hybrid varieties of silkworm larvae with strong disease resistance, stable grass production and high-quality and high-yield fruiting bodies, inoculating 1 soybean-sized cordyceps militaris slant mother strain on 1L of a prepared solid plate culture medium added with 5wt% of silkworm chrysalis powder, activating for 10 days, uniformly dividing the strain into soybean sizes under aseptic operation, timely inoculating 9 soybean-sized strain in 1L of a liquid culture medium added with 6wt% of silkworm chrysalis powder, standing and culturing for 1d under dark conditions and at the temperature of 22 ℃, starting to perform shaking culture on day 2, shaking for 3h and 130r/min every day, culturing for 1d under the illumination intensity of 100lx on day 5, and filtering out hyphae by using 3 layers of aseptic gauze to obtain a strain liquid preparation solution;
the slant mother strain of Cordyceps militaris is Cordyceps militaris, and is activated, and separated, purified and stored for later use after rejuvenation and optimization of the first-generation hybrid variety silkworm 5 th larva living body with strong disease resistance.
The weight ratio of the raw materials of the prepared solid plate culture medium added with 5wt% of silkworm chrysalis powder is as follows: 25% of potato, 2% of glucose, 0.2% of peptone, 0.1% of yeast extract, 5wt% of silkworm chrysalis powder, 1.5% of agar powder, KH2PO40.15%, MgSO40.05%, 1000 mL of water and pH 7.0;
the liquid culture medium added with 6wt% of silkworm chrysalis powder comprises the following raw materials in parts by weight: 25% of potato, 2% of glucose, 0.25% of peptone, 0.2% of yeast extract, 6wt% of silkworm chrysalis meal, KH2PO40.2%, MgSO40.1%, VB 10.01%, 1000 mL of water and pH 7.5.
In the step (2), the preparation method of the silkworm chrysalis powder comprises the following steps: drying pupa Bombycis before hardening and feathering, pulverizing at 70 deg.C, and sieving with 80 mesh sieve.
(3) Host castor silkworm inoculation post-treatment: taking the robust ricinus silkworm larvae obtained in the step (1), injecting 0.45ml of strain liquid preparation liquid obtained in the step (2) into each head, immediately putting the silkworm bodies on plant ash subjected to high-temperature sterilization at 100 ℃ for 8 hours to freely crawl after injection, culturing for 3d under ventilation conditions at 21 ℃ and 60% relative humidity, putting the silkworm bodies into a gauze wood frame after an inoculated wound is completely healed, covering the silkworm bodies with a gauze, culturing for 9d under ventilation conditions at 21 ℃ and 70% relative humidity, collecting and sorting silkworm bodies which are infected and stiffened but not infected and stiffened after more than 95% of silkworm bodies are infected and stiffened, and removing silkworm bodies which are infected by infectious microbes, blackened and softened or not infected and stiffened;
in the step (3), the silkworm bodies are slowly and stably propelled during inoculation, so that mechanical wounds to the silkworm bodies are reduced as much as possible, and premature death of the silkworm bodies without infection or stiffness is avoided.
(4) Culturing and managing the cordyceps militaris sporocarp; placing the silkworm bodies which are subjected to infection and hardening and do not grow bacteria in the step (3) in a transparent plastic box with a cover and subjected to ultraviolet lamp sterile treatment for 3 hours, and culturing for 10 days in a dark environment with the temperature of 18 ℃ and the relative humidity of 60%; culturing for 6 days under the conditions of light intensity of 100lx, temperature of 22 ℃ in the daytime, 16 ℃ at night and relative humidity of 70%; after the transparent plastic box cover with the cover is punched for ventilation, the box is cultured for 50 days under the conditions that the light intensity is 150lx, the temperature is 18 ℃ and the relative humidity is 70 percent, and then the mature cordyceps militaris, namely the castor silkworm chrysalis, is obtained.
The breeding index statistics in this embodiment: the infection rate of the castor silkworm body is 92%, the non-infection rate is 8%, the total loss of the miscellaneous bacteria pollution, the mechanical death and the like is 2%, the grass yield is 93%, the length of the fruiting body is 2-5 cm, and through determination, the cordycepin content in the obtained castor silkworm cordyceps is 8.42mg/g, the pentostatin content is 8.16mg/g, and the adenosine content is 5.07 mg/g.
Example two:
the embodiment comprises the following steps:
(1) selecting a cordyceps militaris host: selecting healthy castor silkworm bodies of five-instar 4d, feeding castor leaves continuously in an environment with the temperature of 28 ℃ and the relative humidity of 65% after eating in the area of 5 instar, ensuring ventilation, removing sand in time, and selecting robust castor silkworm larvae for later use;
(2) and (3) expanding culture of a strain liquid preparation solution: selecting a cordyceps militaris slant mother strain which has strong infectivity on first-generation hybrid varieties, strong disease resistance, stable grass production and high-quality and high-yield fruiting bodies, inoculating 1 soybean-sized cordyceps militaris slant mother strain on 1L of a prepared solid plate culture medium added with 7wt% of silkworm chrysalis powder, activating for 9 days, uniformly dividing the strain into soybean sizes under aseptic operation, timely inoculating 8 soybean-sized strain in 1L of a liquid culture medium added with 8wt% of silkworm chrysalis powder, standing and culturing for 1d under dark conditions and at the temperature of 20 ℃, starting to perform shaking culture on day 2, performing shaking for 4h and 135r/min every day, performing culturing for 1d under the light intensity of 130lx on day 5, and filtering out hyphae by using 4 layers of sterile gauze to obtain a strain liquid preparation solution;
the slant mother strain of Cordyceps militaris is Cordyceps militaris, and is activated, and separated, purified and stored for later use after rejuvenation and optimization of the first-generation hybrid variety silkworm 5 th larva living body with strong disease resistance.
The weight ratio of the raw materials of the prepared solid plate culture medium added with 7wt% of silkworm chrysalis powder is as follows: 25% of potato, 2% of glucose, 0.2% of peptone, 0.1% of yeast extract, 7wt% of silkworm chrysalis powder, 1.5% of agar powder, KH2PO40.15%, MgSO40.05%, 1000 mL of water and pH 7.0;
the liquid culture medium added with 8wt% of silkworm chrysalis powder comprises the following raw materials in parts by weight: 25% of potato, 2% of glucose, 0.25% of peptone, 0.2% of yeast extract, 8wt% of silkworm chrysalis powder, KH2PO40.2%, MgSO40.1%, VB 10.01%, 1000 mL of water and pH 7.5.
In the step (2), the preparation method of the silkworm chrysalis powder comprises the following steps: drying pupa Bombycis before hardening and feathering, pulverizing at 70 deg.C, and sieving with 80 mesh sieve.
(3) Inoculating host ricinus communis silkworm for post-treatment; selecting the strong ricinus silkworm larvae obtained in the step (1), injecting 0.50ml of strain liquid preparation liquid obtained in the step (2) into each head, immediately placing the silkworm bodies on plant ash subjected to high-temperature sterilization at 100 ℃ for 9 hours to freely crawl after injection, culturing for 3d under the ventilation condition with the temperature of 23 ℃ and the relative humidity of 70%, placing the silkworm bodies into a gauze wood frame after the inoculated wounds are completely healed, covering the silkworm bodies with a gauze, continuously culturing for 8d under the ventilation condition with the temperature of 24 ℃ and the relative humidity of 70%, collecting and sorting silkworm bodies which are infected and stiffened but not fermented after more than 95% of silkworm bodies are infected and stiffened, and removing the silkworm bodies which are infected by infectious microbes, blackened and softened or not infected and stiffened;
in the step (3), the silkworm bodies are slowly and stably propelled during inoculation, so that mechanical wounds to the silkworm bodies are reduced as much as possible, and premature death of the silkworm bodies without infection or stiffness is avoided.
(4) Culturing and managing the cordyceps militaris sporocarp; placing the silkworm bodies which are subjected to infection and hardening and do not grow bacteria in the step (3) in a transparent plastic box with a cover and subjected to ultraviolet lamp sterile treatment for 3 hours, and culturing for 8 days in a dark environment with the temperature of 20 ℃ and the relative humidity of 70%; culturing for 5 days under the conditions of light intensity of 200lx, temperature of 22 ℃ in the day, 16 ℃ at night and relative humidity of 75%; after the transparent plastic box cover with the cover is punched for ventilation, the box is cultured for 45 days under the conditions that the light intensity is 250lx, the temperature is 20 ℃ and the relative humidity is 75 percent, and then the mature cordyceps militaris, namely the castor silkworm and the silkworm are obtained.
The breeding index statistics in this embodiment: the infection rate of the castor silkworm body is 94 percent, the non-infection rate is 3 percent, the total loss of the miscellaneous bacteria pollution, the mechanical death and the like is 4 percent, the grass yield is 92 percent, the length of the fruiting body is 3-6 cm, and the cordycepin content, the pentostatin content and the adenosine content in the obtained castor silkworm cordyceps are respectively 8.52mg/g, 8.46mg/g and 5.27 mg/g through determination.
EXAMPLE III
The embodiment comprises the following steps:
(1) selecting a cordyceps militaris host; selecting healthy silkworm bodies of 5d castor silkworms, feeding castor leaves continuously after eating at the age of 5d in an environment with the temperature of 30 ℃ and the relative humidity of 70%, ensuring ventilation and timely removing sand, and selecting robust silkworm larvae of the castor silkworms for later use;
(2) performing enlarged culture on a strain liquid preparation solution, selecting a cordyceps militaris slant mother strain which has strong infectivity on a first-generation hybrid variety silkworm larva, is stable in grass generation and has high-quality and high-yield fruiting bodies, inoculating 1 soybean-sized cordyceps militaris slant mother strain on 1L of a prepared solid plate culture medium added with 8wt% of silkworm chrysalis powder, after activating for 7 days, uniformly dividing the strain into soybean sizes under aseptic operation, timely inoculating 7 soybean-sized strain in 1L of a liquid culture medium added with 10wt% of silkworm chrysalis powder, performing static culture for 1d under dark conditions and at the temperature of 21 ℃, starting oscillation culture on the 2 nd day, oscillating for 4h and 140r/min every day, culturing for 1d under the illumination intensity of 150lx on the 5 th day, and removing hypha by using 4 layers of sterile filtration gauze to obtain a strain liquid preparation solution;
the slant mother strain of Cordyceps militaris is Cordyceps militaris, and is activated, and separated, purified and stored for later use after rejuvenation and optimization of the first-generation hybrid variety silkworm 5 th larva living body with strong disease resistance.
The weight ratio of the raw materials of the in-situ prepared solid plate culture medium added with 8wt% of silkworm chrysalis powder is as follows: 25% of potato, 2% of glucose, 0.2% of peptone, 0.1% of yeast extract, 8wt% of silkworm chrysalis powder, 1.5% of agar powder, KH2PO40.15%, MgSO40.05%, 1000 mL of water and pH 7.0;
the liquid culture medium added with 10wt% of silkworm chrysalis powder comprises the following raw materials in parts by weight: 25% of potato, 2% of glucose, 0.25% of peptone, 0.2% of yeast extract, 10wt% of silkworm chrysalis meal, KH2PO40.2%, MgSO40.1%, VB 10.01%, 1000 mL of water and pH 7.5.
In the step (2), the preparation method of the silkworm chrysalis powder comprises the following steps: drying pupa Bombycis before hardening and feathering, pulverizing at 70 deg.C, and sieving with 80 mesh sieve. A
(3) Inoculating host ricinus communis silkworm for post-treatment; selecting healthy and clean castor silkworm larvae obtained in the step (1), injecting 0.60ml of strain liquid preparation liquid obtained in the step (2) into each head, immediately putting silkworm bodies on plant ash subjected to high-temperature sterilization at 100 ℃ for 10 hours after injection, freely crawling, culturing for 3d under ventilation conditions at 24 ℃ and 65% relative humidity, putting the silkworm bodies into a gauze frame after an inoculated wound is completely healed, covering the silkworm bodies with a gauze, culturing for 7d under ventilation conditions at 23 ℃ and 65% relative humidity, collecting and sorting silkworm bodies which are infected and stiffened but not fermented after more than 95% of silkworm bodies are infected and stiffened, and removing the silkworm bodies infected, blackened and softened or not infected and stiffened due to infectious microbes;
in the step (3), the silkworm bodies are slowly and stably propelled during inoculation, so that mechanical wounds to the silkworm bodies are reduced as much as possible, and premature death of the silkworm bodies without infection or stiffness is avoided.
(4) Culturing and managing the cordyceps militaris sporocarp; placing the silkworm bodies which are subjected to infection and hardening and do not grow bacteria in the step (3) in a transparent plastic box with a cover and subjected to ultraviolet lamp sterile treatment for 3 hours, and culturing for 7 days in a dark environment at the temperature of 23 ℃ and the relative humidity of 65%; culturing for 4 days under the conditions that the light intensity is 260lx, the temperature is 21 ℃ in the day, the temperature is 17 ℃ at night and the relative humidity is 73%; after the transparent plastic box cover with the cover is punched for ventilation, the light intensity is 300lx, the temperature is 23 ℃, and the relative humidity is 80 percent, and the mature cordyceps militaris, namely the castor silkworm, silkworm and the castor silkworm are cultured for 40 days.
The breeding index statistics in this embodiment: the infection rate of the castor silkworm body is 91 percent, the non-infection rate is 5 percent, the total loss of the miscellaneous bacteria pollution, the mechanical death and the like is 4 percent, the grass yield is 95 percent, the length of sporocarp is 3-6 cm, and the cordycepin content, the pentostatin content and the adenosine content in the obtained silkworm cordyceps are respectively 8.19mg/g, 8.32mg/g and 5.13 mg/g through determination.
Comparative experiment
The prior art is adopted: the Chinese patent application 201811136359.6 discloses an artificial cultivation method of castor silkworm cordyceps (comparative example 1) and Liyajie (2011) for cultivating castor silkworm cordyceps and silkworm chrysalis by inoculating cordyceps militaris strains to castor silkworm chrysalis and cultivating the castor silkworm cordyceps (comparative example 2), wherein the infection rate, the grass yield and the active ingredients are shown in the following table 1: it can be seen that the infection rate of the invention is close to that of the prior art, but the weed yield is higher than that of the prior art, and the main active ingredients are far higher than that of the prior art.
Figure DEST_PATH_IMAGE002

Claims (7)

1. A method for cultivating cordyceps militaris by inoculating 5 th-instar larvae of castor silkworms with cordyceps militaris is characterized by comprising the following steps of:
(1) selecting a cordyceps militaris host: selecting healthy silkworms of 3-5 d castor silkworms with five instars, feeding castor leaves continuously after eating at the age of 5, under the environment of the temperature of 25-30 ℃ and the relative humidity of 60-70%, ensuring ventilation, removing sand in time, and selecting robust silkworms of castor silkworms for later use;
(2) and (3) expanding culture of a strain liquid preparation solution: selecting cordyceps militaris slant mother strains, inoculating the cordyceps militaris slant mother strains on a prepared solid plate culture medium added with 5-8 wt% of silkworm chrysalis powder, and activating for 7-10 days; under aseptic operation, uniformly cutting strains into soybean sizes, inoculating 7-9 soybean-sized strains into 1L of liquid culture medium added with 6-10 wt% of silkworm chrysalis powder in time, standing and culturing for 1 day under dark conditions at the temperature of 20-22 ℃, starting oscillation culture on day 2, oscillating for 3-5 hours and 130-140 r/min every day, culturing for 1 day under the illumination intensity of 100-150 lx on day 5, and filtering with 3-4 layers of sterile gauze to remove mycelia to obtain a strain liquid preparation solution;
(3) host castor silkworm inoculation post-treatment: taking the robust ricinus silkworm larvae obtained in the step (1), injecting 0.45-0.60 ml of strain liquid preparation liquid obtained in the step (2) into each head, immediately placing the silkworms on plant ash to freely crawl after injection, culturing for 3 days under ventilation conditions at the temperature of 21-24 ℃ and the relative humidity of 60-70%, placing the silkworms into a gauze wood frame after the inoculated wound is completely healed, covering the silkworms with a gauze, culturing for 7-9 days under ventilation conditions at the temperature of 21-24 ℃ and the relative humidity of 60-70%, collecting and sorting silkworm bodies which are infected and rigidified but not inoculated after more than 95% of the silkworms are infected and rigidified, and removing the silkworm bodies which are infected by infectious microbes, blackened and softened or not infected and rigidified;
(4) culturing and managing the cordyceps militaris sporocarp: placing the silkworm bodies which are subjected to infection and hardening and do not grow bacteria in the step (3) in a transparent plastic box with a cover in order, and culturing for 7-10 days in a dark environment with the temperature of 18-22 ℃ and the relative humidity of 60-70%; then culturing for 4-6 days under the conditions that the light intensity is 100-260 lx, the temperature is 20-22 ℃ in the daytime, the temperature is 16-18 ℃ at night and the relative humidity is 70-75%; after punching a hole on a transparent plastic box cover with a cover for ventilation, culturing for 40-50 days under the conditions that the light intensity is 150-300 lx, the temperature is 18-23 ℃ and the relative humidity is 70-80% to obtain mature cordyceps bombycis mori;
the cordyceps militaris slant mother strain in the step (2) is cordyceps militaris, is a mother strain which has strong infectivity, stable grass production and high yield of fruiting bodies for the first generation hybrid variety silkworm larva with strong disease resistance, and is separated, purified and stored for later use after being activated and optimized for rejuvenation of the first generation hybrid variety silkworm larva of 5 th age with strong disease resistance.
2. The method for cultivating cordyceps militaris by inoculating 5 th-instar larvae of ricinus communis with cordyceps militaris, as claimed in claim 1, wherein in the step (2), the weight ratio of the raw materials of the prepared solid plate culture medium added with 5-8 wt% of silkworm chrysalis meal is as follows: 25% of potato, 2% of glucose, 0.2% of peptone, 0.1% of yeast extract, 5-8 wt% of silkworm chrysalis powder, 1.5% of agar powder and KH2PO4 0.15%、MgSO40.05%, 1000 mL of water, pH 7.0; the liquid culture medium added with 6-10 wt% of silkworm chrysalis powder comprises the following raw materials in parts by weight: 25% of potato, 2% of glucose, 0.25% of peptone, 0.2% of yeast extract, 6-10 wt% of silkworm chrysalis powder and KH2PO4 0.2%、MgSO40.1%、VB10.01%, 1000 mL of water, pH 7.5.
3. The method for cultivating cordyceps militaris by inoculating 5 th-instar larvae of ricinus communis with cordyceps militaris as claimed in claim 1, wherein in the step (2), the preparation method of silkworm chrysalis powder comprises the following steps: drying and crushing the silkworm pupae which are yellow-brown, hard and not eclosion before the silkworm pupae are at 60-70 ℃, and sieving the crushed silkworm pupae with a 80-mesh sieve to obtain the silkworm pupae.
4. The method for cultivating cordyceps militaris by inoculating 5 th-instar larvae of ricinus communis with cordyceps militaris as claimed in claim 1, wherein in the step (3), the inoculation is slowly and smoothly carried out.
5. The method for cultivating cordyceps militaris by inoculating 5 th-instar larvae of ricinus communis with cordyceps militaris as claimed in claim 1, wherein in the step (3), the plant ash is black ash obtained by burning rice chaff.
6. The method for cultivating cordyceps militaris by inoculating 5 th-instar larvae of ricinus communis with cordyceps militaris as claimed in claim 1, wherein in the step (3), the plant ash is treated by high-temperature sterilization at a temperature of more than 100 ℃ for 8-10 hours.
7. The method for cultivating cordyceps militaris by inoculating 5 th-instar larvae of ricinus communis with cordyceps militaris as claimed in claim 1, wherein in the step (4), the transparent plastic box with the cover is subjected to aseptic treatment for more than 3 hours by an ultraviolet lamp.
CN201910098897.9A 2019-01-31 2019-01-31 Method for cultivating cordyceps militaris by inoculating 5-instar larvae of castor silkworms with cordyceps militaris Active CN109644779B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910098897.9A CN109644779B (en) 2019-01-31 2019-01-31 Method for cultivating cordyceps militaris by inoculating 5-instar larvae of castor silkworms with cordyceps militaris

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910098897.9A CN109644779B (en) 2019-01-31 2019-01-31 Method for cultivating cordyceps militaris by inoculating 5-instar larvae of castor silkworms with cordyceps militaris

Publications (2)

Publication Number Publication Date
CN109644779A CN109644779A (en) 2019-04-19
CN109644779B true CN109644779B (en) 2021-07-13

Family

ID=66122010

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910098897.9A Active CN109644779B (en) 2019-01-31 2019-01-31 Method for cultivating cordyceps militaris by inoculating 5-instar larvae of castor silkworms with cordyceps militaris

Country Status (1)

Country Link
CN (1) CN109644779B (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021025103A1 (en) * 2019-08-07 2021-02-11 株式会社 沖縄Ｕｋａｍｉ養蚕 Composition for inhibiting reduction of testosterone and/or dihydrotestosterone
CN110616154A (en) * 2019-09-27 2019-12-27 江西省农业科学院农产品质量安全与标准研究所 Cordyceps militaris cultured by taking hermetia illucens pupae as host and culturing method thereof
CN113455278B (en) * 2021-06-29 2023-03-07 江苏省农业科学院 Identification device and identification method for anti-Tyrophagus putrescentiae of oyster mushroom
CN114457172A (en) * 2022-03-21 2022-05-10 江苏科技大学 Castor silkworm EST-SSR molecular marker and application thereof

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102612985A (en) * 2011-08-29 2012-08-01 何寒 Production technology for cordyceps militaris mycelium
CN102440149B (en) * 2011-10-10 2013-01-23 江苏科技大学 Method for breeding cordyceps militaris by utilizing silkworm larvae
CN103215250A (en) * 2013-03-30 2013-07-24 徐州鸿宇农业科技有限公司 Protoplast mutation breeding method for improving cordycepin content of cordyceps militaris
CN105359825A (en) * 2013-05-15 2016-03-02 刘东波 Silkworm insect grass cultivating method of infecting bombyxmori larva by cordyceps fungus strain
CN103704022A (en) * 2013-12-20 2014-04-09 凌中鑫 Method for cultivating artificial cordyceps sinensis with five-instar silkworms as carrier
CN108112423A (en) * 2017-12-27 2018-06-05 湖南省蚕桑科学研究所 A kind of method that emulated ecological environment earthing cultivates silkworm grass
CN109042067B (en) * 2018-09-28 2021-02-12 广西罗塞塔生物工程有限公司 Artificial cultivation method of castor-oil plant, silkworm and cordyceps

Also Published As

Publication number Publication date
CN109644779A (en) 2019-04-19

Similar Documents

Publication Publication Date Title
CN109644779B (en) Method for cultivating cordyceps militaris by inoculating 5-instar larvae of castor silkworms with cordyceps militaris
CN1029382C (en) Method for artificially cultivating north Chinese caterpiller fungus daughter vaccine
CN101215527A (en) Method for cultivating silkworm chrysalis Cordyceps sinensis
CN103270887B (en) Silkworm chrysalis northern Chinese caterpillar Fungus industrial cultivation technique
CN104303820A (en) Large-scale production method of living silkworm cordyceps militaris
CN104350942A (en) Method for preparing cordyceps mycelia through mulberry twig powder and silkworm chrysalis powder
CN104641943A (en) Method for cultivating pleurotu comucopiae on mulberry twigs
CN104969911A (en) Silkworm breeding technology of green silk
CN108112423A (en) A kind of method that emulated ecological environment earthing cultivates silkworm grass
CN107950288B (en) Straw mushroom cultivation process
KR100276842B1 (en) Culture medium composition for snow Cordyceps sinensis and its culture method
CN1100754A (en) Culture method of Cordyceps sinensis and its product
CN107853081B (en) Inoculation method of mushroom culture medium
KR20200071427A (en) Feed composition for the larva of Protaetia brevitarsis comprising barley sprout or breeding method of the larva using the same
CN109042067B (en) Artificial cultivation method of castor-oil plant, silkworm and cordyceps
KR100925705B1 (en) Cultivation Method of Vegetable Worm Enzyme Using White Grub
CN110616154A (en) Cordyceps militaris cultured by taking hermetia illucens pupae as host and culturing method thereof
CN103704022A (en) Method for cultivating artificial cordyceps sinensis with five-instar silkworms as carrier
KR100723068B1 (en) Method for culturing flammulina velutipes including ginseng saponin
JP2003116522A (en) Medium for culturing cordyceps sinensis sacc. and method for culturing cordyceps sinensis sacc.
CN108094047B (en) Strain suitable for infecting parent strain with hepialus armoricanus larvae and cordyceps militaris cultivation method
CN108432544A (en) A kind of cultural method of ramulus mori cloud ear
KR100644243B1 (en) Composition for cultivating cordyceps and cultivating process using thereof
KR20120077691A (en) A cordycepin producing method using unhatched egg
KR100509686B1 (en) Method for producing cordyceps sinensis using larvaes of fly

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant