CN111758484A - Large-scale high-efficiency high-yield silkworm chrysalis cordyceps production and cultivation method - Google Patents
Large-scale high-efficiency high-yield silkworm chrysalis cordyceps production and cultivation method Download PDFInfo
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- CN111758484A CN111758484A CN202010668743.1A CN202010668743A CN111758484A CN 111758484 A CN111758484 A CN 111758484A CN 202010668743 A CN202010668743 A CN 202010668743A CN 111758484 A CN111758484 A CN 111758484A
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- 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
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- 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
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- 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
Abstract
The invention provides a large-scale high-efficiency high-yield silkworm chrysalis cordyceps production and cultivation method. According to the technical scheme, a silkworm pupa disinfection method is improved to ozone disinfection, and concentrated open type bacteria cultivation is adopted after inoculation. Specifically, the silkworm chrysalis is placed at the concentration of 30-40mg/m3Sterilizing for 10min in the ozone environment; after inoculation, a disc-shaped container is used for concentrated open type fungus cultivation, the environment temperature is 15-17 ℃, the relative air humidity is 50-60% RH, and light-shielding cultivation is carried out for 5 d. The invention adopts ozone for disinfection, has no disinfection dead angle, can treat a large amount of silkworm chrysalis at one time and has simple operation; and no reagent is required to be prepared, and the problems of chemical residue and environmental pollution are avoided. Meanwhile, the invention adopts centralized management open type fungus culture, the hypha can obtain sufficient oxygen in the growth process and dissipate the heat generated in respiration in time,can effectively shorten the rigidness time and improve the rigidness rate. Compared with the prior art, the invention can reduce the cost investment by 80 percent.
Description
Technical Field
The invention relates to the technical field of cordyceps militaris cultivation, in particular to a large-scale high-efficiency high-yield silkworm chrysalis cordyceps militaris production and cultivation method.
Background
Cordyceps militaris, also known as Cordyceps militaris, Cordyceps militaris. The school name Cordyceps mileris (L.) link. Belongs to the genus Cordyceps of Clavipitaceae of the order Sphaeriales of the subphylum Ascomycotina of the order Sclerotinia. Is a rare edible and medicinal fungus. Modern pharmaceutical research shows that the cordyceps militaris contains the same components as cordyceps sinensis, such as nucleosides, polysaccharides, cordycepin, sterols and the like. Has the treatment effect on diseases of immune system, nervous system, cardiovascular system and the like, has the anti-tumor and anti-cancer effects and has wide application prospect. At present, grains such as wheat, rice and the like are mostly adopted as culture mediums for cordyceps militaris. Although the production cost is low, the product function can not be compared with the cordyceps militaris cultivated by pupa bodies. The silkworm chrysalis cordyceps sinensis has a real medicinal value.
The silkworm industry is a special industry in southern areas of China, the silkworm pupa resources are very rich, and the development of the research on the efficient inoculation cultivation technology of the silkworm pupa cordyceps militaris living body has unique geographical conditions. At present, the conventional cultivation method has certain defects.
Firstly, the conventional method generally adopts chemical disinfectants for the disinfection of pupa bodies: for example, some researchers use 1% potassium permanganate solution to soak for 5min, and rinse the residual potassium permanganate solution with sterile water; or soaking in 75% ethanol for 5min, and washing with sterile water; or sterilizing with sodium dichloroisocyanurate smoke. The method has long disinfection time, and the cleaning of the disinfectant is labor-consuming and time-consuming and is easy to have chemical residues; moreover, such methods are relatively low in throughput and relatively cumbersome to operate. Other researchers have disinfected pupa bodies by high-temperature sterilization, however, high-temperature sterilization causes denaturation of proteins and saccharides, thereby destroying existing nutrients.
In addition, the conventional fungus culturing process is relatively complicated, more labor is used, the production efficiency is low, and large-area large-scale cultivation is not facilitated; meanwhile, the conventional fungus culturing process generally performs closed type fungus growing in a culture box, and the space is unreasonably utilized, so that the silkworm pupae is longer in rigidification time and lower in rigidification rate.
Disclosure of Invention
The invention aims to provide a large-scale high-efficiency high-yield silkworm chrysalis cordyceps production and cultivation method aiming at the technical defects of the prior art, and aims to solve the technical problems that a pupa disinfection method is low in efficiency, complicated to operate and easy to have chemical residues in the conventional cultivation process.
The invention also aims to solve the technical problem of improving the treatment efficiency, simplifying the operation steps and avoiding chemical residues on the premise of ensuring the disinfection effectiveness.
The invention also aims to solve the technical problems that the conventional fungus culturing method is complicated to operate and low in efficiency, and silkworm pupas are long in rigidification time and low in rigidification rate.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a large-scale high-efficiency high-yield silkworm chrysalis cordyceps production and cultivation method comprises the following steps:
1) taking live silkworm pupae, and placing the live silkworm pupae at 30-40mg/m3Keeping for 10min in the ozone environment;
2) taking the silkworm chrysalis treated in the step 1), and injecting 0.2-0.3mL of inoculated liquid strain into each silkworm chrysalis under the aseptic condition;
3) placing the silkworm chrysalis inoculated in the step 2) into a disc-shaped container, and culturing for 5d in a dark place at the ambient temperature of 15-17 ℃ and the relative air humidity of 50-70% RH under the condition that the upper end of the container is not covered;
4) adjusting the culture temperature to 20-22 ℃ and the relative air humidity to be more than 80% RH, continuing culturing, ventilating every day, and illuminating for 10-12h every day;
5) harvesting when the length of the sporocarp reaches 5-7 cm;
6) drying with hot air at 55-60 ℃.
Preferably, the living pupae in the step 1) do not comprise dead pupae, diseased pupae, worm pupae, malformed pupae and soft pupae; the live silkworm chrysalis is peeled off when in use.
Preferably, the inoculation in step 2) is carried out using a continuous syringe.
Preferably, there are several disc-shaped containers in step 3), and several disc-shaped containers are longitudinally stacked, and the number of stacked layers is not more than 4.
Preferably, the liquid seed culture in step 2) is prepared by the following method: inoculating cordyceps militaris strains to a solid slant culture medium under an aseptic condition, and culturing for 7-10 days at 18-22 ℃ in a dark place; then, transferring the culture medium into a liquid culture medium, and performing shake culture at 18-20 ℃ in the dark for 5-7 days; the frequency of shaking culture is 130-150 r/min.
Preferably, the solid slant medium comprises the following components: 200g of peeled and boiled potato juice, 15-20g of glucose, 5-7g of peptone, 0.5-1g of monopotassium phosphate, 0.2-0.5g of magnesium sulfate, 18-20g of agar powder and 1000mL of water.
Preferably, the liquid medium comprises the following components: 200g of peeled and boiled potato juice, 15-20g of glucose, 5-7g of peptone, 0.5-1g of monopotassium phosphate, 0.2-0.5g of magnesium sulfate and 1000mL of water.
The invention provides a large-scale high-efficiency high-yield silkworm chrysalis cordyceps production and cultivation method. The technical scheme comprises the steps of silkworm chrysalis disinfection, inoculation, fungus cultivation, grass growing management, harvesting, drying and the like, wherein the silkworm chrysalis disinfection method is improved to ozone disinfection; after inoculation, concentrated open type bacteria cultivation is adopted. Specifically, the silkworm chrysalis is placed at the concentration of 30-40mg/m3Sterilizing for 10min in the ozone environment; after inoculation, a disc-shaped container is used for concentrated open type fungus cultivation, the environment temperature is 15-17 ℃, the relative air humidity is 50-60% RH, and light-shielding cultivation is carried out for 5 d.
The invention adopts ozone for disinfection, the ozone is gas, the fluidity is good, the wrapping property is strong, the ozone can be fully contacted with silkworm chrysalis, no disinfection dead angle exists, a large amount of silkworm chrysalis can be treated at one time, and the operation is simple. Compared with the traditional chemical disinfection method, the method has the advantages of strong operability, no need of reagent preparation, no chemical residue and environmental pollution, cleanness and low cost. Meanwhile, the invention adopts centralized management open type fungus culture, so that the hyphae can obtain sufficient oxygen in the growth process, and dissipate the heat generated during respiration in time, thereby avoiding 'fungus burning', effectively shortening the hardening time and improving the hardening rate. The space utilization rate is high when the centralized management is carried out, the operation is convenient, the production links are reduced, and the energy and labor cost consumption is effectively reduced. Compared with the prior art, the method can reduce 80% of cost investment, and has the specific and prominent technical advantages.
The invention can not only increase the added value and economic benefit of the silkworm chrysalis, but also improve the application level of the silkworm chrysalis, simultaneously solve the hidden trouble that the silkworm chrysalis hurts agriculture, and realize the fusion and promotion of economic value, social value and even cultural value.
Detailed Description
Hereinafter, specific embodiments of the present invention will be described in detail. Well-known structures or functions may not be described in detail in the following embodiments in order to avoid unnecessarily obscuring the details. Approximating language, as used herein in the following examples, may be applied to identify quantitative representations that could permissibly vary in number without resulting in a change in the basic function. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Example 1
A large-scale high-efficiency high-yield silkworm chrysalis cordyceps production and cultivation method comprises the following steps:
preparing strains:
preserving cordyceps militaris strains at low temperature: conventional Cordyceps militaris strains.
Cordyceps militaris liquid strain: the culture steps are as follows:
(1) test tube slant strain activation
Preparing a culture medium: 200g of potato (peeled and boiled juice), 20g of glucose, 5g of peptone, 1g of monopotassium phosphate, 0.5g of magnesium sulfate, 20g of agar powder and 1000ml of water. Subpackaging in test tubes, sterilizing at 121 deg.C for 20min, and placing the slant. Then inoculating the preserved Cordyceps militaris strain into a test tube slant under aseptic condition, and culturing at 18-20 deg.C in dark for 7-10 days.
(2) Culturing triangle liquid seeds:
preparing a culture medium: 200g of potato (peeled and boiled juice), 20g of glucose, 5g of peptone, 1g of monopotassium phosphate and 0.5g of magnesium sulfate, and 1000ml of water. Subpackaging the prepared culture medium into triangular flasks, and sealing with cotton plugs or double-layer polypropylene films. Placing into autoclave, and sterilizing at 121 deg.C for 20 min. After the preparation of the culture medium is finished, inoculating the slant test tube strains under the aseptic condition, culturing at the temperature of 18-20 ℃, and carrying out vibration culture in a dark place for 5-7 days, wherein the vibration frequency is 130-.
(II) silkworm pupa disinfection:
selecting fresh silkworm pupa, ozone concentration is 30-40mg/m3, and treatment time is 10 min.
(III) inoculation:
under aseptic conditions, each silkworm pupa is injected with 0.2-0.3ml of inoculated liquid strain by using a continuous injector.
(IV) cultivating bacteria:
the inoculated silkworm chrysalis is placed in a bacteria culture tray, and the single stack does not exceed four layers. The bacteria culture plate is not required to be covered and is directly placed in a bacteria culture room for culture. The environmental temperature is 15-17 ℃, the relative air humidity is 50% -70% RH, the culture time is 5 days in a dark place.
(V) grass emergence management:
the culture temperature is 20-22 ℃, the relative air humidity is above 80% RH, the air is ventilated every day, the air is kept fresh, and the illumination time is 10-12 h.
(VI) harvesting:
harvesting when the sporophytes grow to 5-7 cm.
(VII) drying:
drying with hot air at 55-60 deg.C.
Example 2
A large-scale high-efficiency high-yield silkworm chrysalis cordyceps production and cultivation method comprises the following steps:
1. silkworm chrysalis disinfection: the silkworm pupae are peeled off at present according to the inoculation amount so as to ensure the quality of the silkworm pupae, and the silkworm pupae are moderate in size and fresh and alive. The dead pupae, the diseased pupae, the worm pupae, the malformed pupae and the soft pupae can not be used after being uniformly removed. The pupa Bombycis disinfection is carried out with ozone concentration of 30-40mg/m3 and treatment time of 10 min.
2. Inoculation: under aseptic conditions, each silkworm pupa is injected with 0.2-0.3ml of inoculated liquid strain by using a continuous injector.
3. And (3) cultivating bacteria: the inoculated silkworm chrysalis is placed in a bacteria cultivation plate, the bacteria cultivation plate can be stacked, and the number of single stacked plates is not more than four. The bacteria culture plate is not required to be covered and is directly placed in a bacteria culture room for culture. The environmental temperature is 15-17 ℃, the relative air humidity is 50% -70% RH, the culture time is 5 days in a dark place.
4. Grass growing management: the culture temperature is 20-22 ℃, the relative air humidity is above 80% RH, the air is ventilated every day, the air is kept fresh, and the illumination time is 10-12 h.
5. Harvesting: harvesting when the sporophytes grow to 5-7 cm.
6. Drying: drying with hot air at 55-60 deg.C.
The cordyceps militaris strain inoculated in the step 2 is a cordyceps militaris liquid strain, and the strain is prepared by test tube slant strain culture and triangular flask liquid strain culture, and the specific process steps are as follows:
and (3) culturing strains on test tube inclined planes:
preparing a culture medium: the proportion of each component is as follows: 200g of potato (peeled and boiled juice), 15-20g of glucose, 5-7g of peptone, 0.5-1g of monopotassium phosphate, 0.2-0.5g of magnesium sulfate, 18-20g of agar powder and 1000ml of water. After the slant culture medium is prepared, Cordyceps militaris strain is inoculated under aseptic condition, and is cultured at 18-22 deg.C in dark for 7-10 days.
Culturing liquid strains in a triangular flask: preparing a culture medium: the proportion of each component is as follows: 200g of potato (peeled and boiled juice), 15-20g of glucose, 5-7g of peptone, 0.5-1g of monopotassium phosphate, 0.2-0.5g of magnesium sulfate and 1000ml of water. Subpackaging the prepared culture medium into triangular flasks, and sealing with cotton plugs or double-layer polypropylene films. Placing into autoclave, and sterilizing at 121 deg.C for 20 min. After the preparation of the culture medium is finished, inoculating the slant test tube strains under the aseptic condition, culturing at the temperature of 18-20 ℃, and carrying out vibration culture in a dark place for 5-7 days, wherein the vibration frequency is 130-.
Example 3
A large-scale high-efficiency high-yield silkworm chrysalis cordyceps production and cultivation method comprises the following steps:
1) taking live silkworm pupae, and placing the live silkworm pupae at 30-40mg/m3Keeping for 10min in the ozone environment;
2) taking the silkworm chrysalis treated in the step 1), and injecting 0.2-0.3mL of inoculated liquid strain into each silkworm chrysalis under the aseptic condition;
3) placing the silkworm chrysalis inoculated in the step 2) into a disc-shaped container, and culturing for 5d in a dark place at the ambient temperature of 15-17 ℃ and the relative air humidity of 50-70% RH under the condition that the upper end of the container is not covered;
4) adjusting the culture temperature to 20-22 ℃ and the relative air humidity to be more than 80% RH, continuing culturing, ventilating every day, and illuminating for 10-12h every day;
5) harvesting when the length of the sporocarp reaches 5-7 cm;
6) drying with hot air at 55-60 ℃.
The embodiments of the present invention have been described in detail, but the description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention. Any modification, equivalent replacement, and improvement made within the scope of the application of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A large-scale high-efficiency high-yield silkworm chrysalis cordyceps production and cultivation method is characterized by comprising the following steps:
1) taking live silkworm pupae, and placing the live silkworm pupae at 30-40mg/m3Keeping for 10min in the ozone environment;
2) taking the silkworm chrysalis treated in the step 1), and injecting 0.2-0.3mL of inoculated liquid strain into each silkworm chrysalis under the aseptic condition;
3) placing the silkworm chrysalis inoculated in the step 2) into a disc-shaped container, and culturing for 5d in a dark place at the ambient temperature of 15-17 ℃ and the relative air humidity of 50-70% RH under the condition that the upper end of the container is not covered;
4) adjusting the culture temperature to 20-22 ℃ and the relative air humidity to be more than 80% RH, continuing culturing, ventilating every day, and illuminating for 10-12h every day;
5) harvesting when the length of the sporocarp reaches 5-7 cm;
6) drying with hot air at 55-60 ℃.
2. The method for producing and cultivating the large-scale high-efficiency high-yield silkworm chrysalis cordyceps sinensis according to claim 1, wherein the live silkworm chrysalis in the step 1) does not include dead chrysalis, diseased chrysalis, worm chrysalis, malformed chrysalis and soft chrysalis; the live silkworm chrysalis is peeled off when in use.
3. The method for producing and cultivating silkworm chrysalis cordyceps militaris on a large scale with high efficiency and high yield according to claim 1, wherein the inoculation in the step 2) is performed by using a continuous injector.
4. The method for producing and cultivating the silkworm chrysalis cordyceps militaris on a large scale with high efficiency and high yield according to claim 1, wherein a plurality of disc-shaped containers are arranged in the step 3), the disc-shaped containers are longitudinally stacked, and the number of stacked layers is not more than 4.
5. The method for producing and cultivating silkworm chrysalis cordyceps militaris on a large scale with high efficiency and high yield according to claim 1, wherein the liquid spawn in the step 2) is prepared by the following method: inoculating cordyceps militaris strains to a solid slant culture medium under an aseptic condition, and culturing for 7-10 days at 18-22 ℃ in a dark place; then, transferring the culture medium into a liquid culture medium, and performing shake culture at 18-20 ℃ in the dark for 5-7 days; the frequency of shaking culture is 130-150 r/min.
6. The method for producing and cultivating silkworm chrysalis cordyceps militaris on a large scale with high efficiency and high yield according to claim 5, wherein the solid slant culture medium comprises the following components: 200g of peeled and boiled potato juice, 15-20g of glucose, 5-7g of peptone, 0.5-1g of monopotassium phosphate, 0.2-0.5g of magnesium sulfate, 18-20g of agar powder and 1000mL of water.
7. The method for producing and cultivating silkworm chrysalis cordyceps militaris on a large scale with high efficiency and high yield according to claim 5, wherein the liquid culture medium comprises the following components: 200g of peeled and boiled potato juice, 15-20g of glucose, 5-7g of peptone, 0.5-1g of monopotassium phosphate, 0.2-0.5g of magnesium sulfate and 1000mL of water.
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