CN115152527A - Method for cultivating high-quality cordyceps militaris sporocarp in short period - Google Patents
Method for cultivating high-quality cordyceps militaris sporocarp in short period Download PDFInfo
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- 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
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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
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Abstract
The invention discloses a method for cultivating high-quality cordyceps militaris sporocarp in a short period, which adopts crop wastes such as sweet potato vines and cordyceps militaris culture medium wastes as main raw materials of a fermentation culture medium, establishes a scientific and reasonable cultivation method, and further makes full use of nutrient substances such as starch, protein, saccharides and amino acids in the wastes and micromolecular substances generated by extracellular enzyme enzymolysis of cordyceps militaris. The method has simple process, rich and easily-obtained substituted raw material resources, can save 40-60% of grain for production, reduce the raw material cost by about 10%, realize the utilization of agricultural wastes, change waste into valuables, reduce environmental pollution, and accord with the concept of green, environment-friendly and sustainable development.
Description
Technical Field
The invention belongs to the field of agricultural production waste recycling and biotechnology, and particularly relates to a method for cultivating high-quality cordyceps militaris sporocarp in a short period, in particular to a method for preparing high-quality cordyceps militaris sporocarp with good biological form and functional indexes by utilizing crop waste sweet potato vine and cordyceps militaris culture medium waste in a shorter cultivation period.
Background
Cordyceps militaris (Cordyceps militaris) is also called Cordyceps militaris, and belongs to Cordyceps fungus of Clavicipitaceae of Hymenomycetes of Ascomycotina. As the traditional fungus used as both medicine and food, the fungus is popular with people due to the nutritional and economic values, the cordyceps militaris is discovered and separated in the eighties of the last century in China, the cultivation technology is rapidly developed, large-scale planting is preliminarily realized, and the fungus is not limited by seasons.
The cordyceps militaris cultivation raw materials are mainly rice and wheat grains, and the cost of the raw materials is far higher than that of other edible fungus varieties. However, the economic benefit of cordyceps militaris planting is higher than that of the traditional basic agriculture, the cordyceps militaris planting method becomes a part of the post industry for farmers to get rid of poverty and rich, along with improvement and maturity of the planting skills of farmers, the planting scale is continuously enlarged, a large amount of cordyceps militaris culture medium waste is generated, the quality is about 6-8 times of that of cordyceps militaris dry products, the waste of raw materials reaches 30%, great pressure and trouble are brought to the production environment, and the culture medium waste needs to be treated urgently. At present, a large amount of effective recycling ways of culture medium wastes are lacked, the natural ecological environment and the reproduction environment are polluted, and the sustainable development of the cordyceps industry is restricted.
The agricultural cultivated land area is continuously reduced due to the desertification of Chinese land, and the stress-resistant sweet potato can be used as staple foodThe planting of plants is continuously advancing. Sweet potatoes have been planted in more than 100 countries in the world due to the characteristics of drought resistance, barren resistance, strong environmental adaptability, high and stable yield and the like. The sweet potato is one of main cultivated crops in China, and the planting area is about 1100 ten thousand hm 2 The sweet potato cultivation area accounts for more than 65% of the sweet potato cultivation area in the world, and the cultivation area and the yield are in the first place in the world. The sweet potato vine produced in China every year is up to more than 150 hundred million tons. Most of the waste water is discarded beside the field or burned except for a small part of the waste water, so that the ecological environment is polluted, the production is inconvenient, and the resource waste is caused. In folk traditional Chinese medicine, sweet potato vines are often used as auxiliary therapeutic medicinal food materials for reducing blood fat and blood sugar, preventing and treating arteriosclerosis, cardiovascular and cerebrovascular diseases and the like, the effects are consistent with the research field effects of cordyceps militaris, and feasibility is provided for the sweet potato vines to participate in the culture of cordyceps militaris.
Currently, attempts have been made to reuse agricultural production by-products to increase economic value. For example, patent CN104969773A discloses a method for obtaining cordyceps militaris fermentation product or fruiting body by using sweet potato residue fermentation, the sweet potato residue used in the invention is mainly starch residue, which replaces grain for cordyceps militaris cultivation, the biological conversion rate is relatively low, which is not beneficial to industrial cultivation, the grain substitute material used as raw material is industrial waste, and the beneficiary is a production enterprise. The method utilizes the vines at the overground part of the sweet potatoes, has rich functional components, relatively high biological conversion rate of replacing the grains for cultivating the cordyceps sinensis, uses the vines as agricultural production waste, and is beneficial to agricultural production.
Disclosure of Invention
The method is simple in process, rich and easily available in culture substitute raw materials, low in cost, capable of saving production grains, avoiding waste of agricultural resources and reducing environmental pollution.
On one hand, the application provides a method for culturing high-quality cordyceps militaris sporocarp in a short period, which comprises the following steps:
the method comprises the following steps: inoculating the cordyceps militaris preservation strain into a primary culture medium for culture to obtain cordyceps militaris primary seeds;
step two: inoculating the first-stage cordyceps militaris seeds into a second-stage culture medium for culture to obtain second-stage cordyceps militaris seeds;
step three: inoculating the second-level cordyceps militaris seeds into a sporocarp culture medium, and culturing until cordyceps militaris sporocarps are obtained;
the first-stage culture medium, the second-stage culture medium and the fruiting body culture medium all contain cordyceps militaris culture medium waste and sweet potato vine, wherein the mass ratio of the cordyceps militaris culture medium waste to the sweet potato vine in the first-stage culture medium to the second-stage culture medium is (10-30): (4-12), wherein the mass ratio of cordyceps militaris culture medium waste to sweet potato vine in the sporocarp culture medium is (30-60): (20-10).
Further, the sweet potato vine is processed by adopting the following method: drying fresh sweet potato vine until the water content is 8-12%, and crushing to 4-40 meshes to obtain the sweet potato vine granules.
Preferably, the sweet potato vine treatment specifically comprises: selecting fresh, non-mouldy, non-rotten and non-lignified sweet potato vines after sweet potato picking in autumn, cleaning, draining water, putting into a pulverizer, pulverizing the sweet potato vines into 2-3cm small sections, then placing the pulverized sweet potato vines into a forced air drying oven, drying at 60-80 ℃ until the water content reaches 8-12%, and bagging for later use.
Wherein, the dried sweet potato vine is further crushed by a crusher to be sieved by a 4-8 mesh sieve, and coarse particles (namely the sweet potato vine with the particle size of 4-8 meshes) of the sweet potato vine are obtained; pulverizing the dried sweet potato vine by a pulverizer to pass through a 20-40 mesh sieve to obtain sweet potato vine powder particles (namely the sweet potato vine with the particle size of 20-40 meshes).
More preferably, the first-stage culture medium and the second-stage culture medium adopt the sweet potato vine powder with smaller particle size, so that active substances in the sweet potato vine can be fully extracted, and the application of the sweet potato vine powder in the culture media is more beneficial to domestication of cordyceps militaris strain culture; the sweet potato vine coarse particles with thicker particle sizes are adopted in the fruit body compost, the plant tissue structure of the sweet potato vine has good air permeability, the sweet potato vine coarse particles with thicker particle sizes are selected, the air permeability of the tissue structure of the sweet potato vine coarse particles can be kept, the sweet potato vine coarse particles with thicker particle sizes can also keep higher porosity after being mixed with cordyceps militaris culture medium waste, and simultaneously can show good water retention effect and slow release function.
Further, the cordyceps militaris culture medium waste is prepared by adopting the following method: the culture material of the collected cordyceps militaris sporocarp is crushed to the grain size of 0.4-0.6 cm and dried until the water content is 8-12% for later use.
The culture medium of fruiting bodies which are free from mildew, foreign fungus pollution, dirt and rich in cordyceps militaris hypha can be selected, fruiting body residual sections are selected and discarded, and the culture medium is subjected to extracellular enzymatic hydrolysis of fruiting bodies, so that the toughness is reduced, and the culture medium is easy to break.
In one embodiment, the cordyceps militaris fruiting body culture medium can be selected from cordyceps militaris fruiting body culture medium which is conventional in the field, and the cordyceps militaris fruiting body culture medium is used as the cordyceps militaris culture medium waste after the cordyceps militaris fruiting body is cultured and collected.
In a preferred embodiment, the culture medium of the cordyceps militaris, which only contains crop nutrients in the method, is used as a fresh culture medium, and the culture medium is used as the waste of the cordyceps militaris culture medium after the cordyceps militaris sporocarp is cultured and collected.
Preferably, the sweet potato vine powder and the cordyceps militaris culture medium waste with the relatively fine particle size (20-40 meshes) are applied to a primary culture medium and a secondary culture medium in the form of extracting solutions thereof, including but not limited to aqueous extracting solutions thereof. Therefore, active substances in the sweet potato vine and the cordyceps militaris culture medium waste can be fully extracted at the same time, and the domestication of cordyceps militaris strain culture is facilitated.
Preferably, the sweet potato vine coarse grains with the larger grain diameter (4-8 meshes) and the cordyceps militaris culture medium waste are applied to the sporocarp culture medium in the form of solid particles. At the moment, the air permeability of the sweet potato vine coarse grains can be utilized to improve the absorption of active substances in cordyceps militaris culture medium waste in the hypha growth process, and the quality of the final cordyceps militaris sporocarp is further improved.
Furthermore, the primary culture medium, the secondary culture medium and/or the fruiting body culture material also contain crop nutrients, wherein the crop nutrients are selected from one or more of potatoes, wheat grains, rice and corns.
Further, the primary culture medium, the secondary culture medium and/or the sporocarp culture medium also contain nutritional auxiliary materials, and the nutritional auxiliary materials are selected from glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate and vitamin B 1 Agar and glycine.
Further, the preparation method of the primary culture medium and the secondary culture medium comprises the following steps: mixing the crop nutrient, cordyceps militaris culture medium waste and sweet potato vine granules, adding water, boiling for 15-30 min, filtering to obtain a filtrate, and adding nutritional auxiliary materials into the filtrate to obtain the feed additive, wherein the addition amount of the crop nutrient is 30-200g/L; and/or the presence of a gas in the atmosphere,
the preparation method of the fruiting body culture material comprises the following steps: mixing the crop nutrient, the cordyceps militaris culture medium waste and the sweet potato vine granules to obtain a mixed material with the total mass of 100 parts, and adding 1.5-2.0 g/ml of nutritional auxiliary materials based on the mass of the mixed material to obtain the feed additive.
In a preferred embodiment, the above preparation method, step one, uses a primary culture medium, which has the following composition: 100g/L of crop nutrient, 10-30g/L of cordyceps militaris culture medium waste, 2-20g/L of sweet potato vine granules are boiled for 20-25 min, filter cloth is filtered to obtain filtrate, 10g/L of glucose, 2.5g/L of peptone, 1.5g/L of magnesium sulfate, 3g/L of monopotassium phosphate, 10mg/L of vitamin B1 and 10g/L of agar are added, and sterilization is carried out for 30min at 121 ℃;
the second-stage culture medium adopted in the second step comprises the following components: boiling 100g/L of crop nutrient, 10-30g/L of cordyceps militaris culture medium waste and 2-20g/L of sweet potato vine granules for 20-25 minFiltering with filter cloth to obtain filtrate, adding glucose 10g/L, peptone 2.5g/L, magnesium sulfate 1.5g/L, potassium dihydrogen phosphate 3g/L, and vitamin B10 mg/L 1 ；
The sporophore compost adopted in the third step comprises the following components: preparing a nutrient solution: magnesium sulfate 0.5g/L, potassium dihydrogen phosphate 2g/L, glycine 0.1g/L; 10-90 parts of crop nutrient, 8-80 parts of cordyceps militaris culture medium waste and 2-30 parts of sweet potato vine granules, wherein the total amount of the mixed materials is controlled to be 100 parts by mass, and the mixed materials and the nutrient solution are mixed according to the mixing ratio of 1: 1.5-2.0 g/ml.
Further, the culture method in the first step comprises: inoculating the cordyceps militaris preservation strain into the primary culture medium in an aseptic manner, and culturing at 18-22 ℃ in a dark place.
In one embodiment, step one is specifically prepared as follows: dissolving the first-stage culture medium in subpackaging test tubes, sterilizing at 121 deg.C for 30min, cooling to room temperature, cutting Cordyceps militaris preservation strain into 4 × 4mm hypha pieces under aseptic condition with inoculating hook, shoveling out the hypha pieces with inoculating shovel, placing in the middle lower part of the fresh first-stage seed slant, slightly pressing, culturing at 18-22 deg.C in dark place, stopping culturing after the tube is full, and storing at low temperature.
Further, the culture method in the second step comprises: inoculating the first-level cordyceps militaris seeds into the second-level culture medium in an aseptic manner, and culturing for 3-5 days at the temperature of 18-22 ℃ and the rotating speed of 120-150 rpm in a dark place.
In one embodiment, the specific preparation method of step two is as follows: subpackaging the secondary culture medium in triangular flasks, sterilizing at 121 ℃ for 30min, cooling to room temperature, dividing the primary cordyceps militaris seeds into 3-4 hypha pieces with the size of 2 x 2mm under the aseptic condition by using an inoculation hook, shoveling the hypha pieces by using an inoculation shovel, placing the hypha pieces in the secondary seed triangular flasks, culturing at 18-22 ℃ and 130-150rpm for 3-5 days, and stopping culturing when the ratio of the strong hypha to the rich volume of the hypha balls reaches 50-70%.
Preferably, the production of the third-level liquid strains in the fermentation tank can be synchronously realized according to production requirements so as to meet the requirement of large-scale cultivation. The culture material preparation method is the same as the culture material preparation method of the second-level liquid seed of cordyceps militaris, and the culture conditions are as follows: the charging volume accounts for 60 percent of the volume in the tank, the temperature is reduced to 22-24 ℃ after the material is consumed, cordyceps militaris secondary liquid seeds are inoculated according to the charging volume in the tank, the inoculation amount is 3-5 percent, the tank pressure is 0.02-0.06Mpa, the temperature is 20-22 ℃, the rotating speed is 130-150rpm, the fermentation time is 2-3 days, and the culture is stopped when the hyphae are strong and the ratio of the rich volume of the fungus balls reaches 50-70 percent.
Further, the culture method in the third step comprises: 6-8% of cordyceps militaris secondary seeds are aseptically inoculated into a fruiting body culture material, are cultured in a dark place at 18-22 ℃, enter a color conversion stage, the illumination intensity is controlled to be 200-400Lx, the air humidity is controlled to be 60-80%, when the color of hypha is converted to orange color, holes are punctured for ventilation to form cordyceps militaris fruiting body primordium, the air humidity is controlled to be 85-95%, and the cordyceps militaris fruiting body primordium is cultured until the cordyceps militaris fruiting body primordium can be harvested.
On the other hand, the application also provides a cordyceps militaris culture which comprises the cordyceps militaris sporocarp primordium prepared by the method.
On the other hand, the application also provides the cordyceps militaris sporocarp prepared by the cultivation method.
The beneficial effects of the invention comprise at least one of the following:
1. according to the cultivation method, the method for producing the cordyceps militaris by utilizing the culture medium waste of the sweet potato vine and the cordyceps militaris enables nutrient substances such as starch, protein, saccharides and amino acids in the waste and micromolecule substances generated by enzymolysis of extracellular enzymes of the cordyceps militaris to be further fully utilized, the efficient ecological cyclic utilization of the current agricultural waste resources is promoted, the resource waste and the environmental pollution can be reduced, the grain for production raw materials can be saved, and the production cost is reduced; the sweet potato vine is light and rich in minerals, vitamins, flexible fibers and flavonoids, so that the quality of cordyceps militaris can be improved, the sweet potato vine is rich in resources, low in cost and high in nutrition, and has the unique advantage of sweet potato vine, the sweet potato vine is fully utilized, waste is turned into wealth, farmers can benefit from benefiting, the production enthusiasm of farmers is mobilized, and agricultural production is promoted; meanwhile, the cordyceps militaris culture medium waste is utilized, long-distance transportation is not needed, materials are taken in situ, the transportation cost is convenient to reduce, and the efficient sustainable development of the cordyceps militaris industrial chain is greatly promoted;
2. according to the method provided by the application, the sweet potato vine and cordyceps militaris culture medium waste is dried and dried at low temperature in a simple and easy way, so that the loss of nutrition is reduced, the waste can be treated quickly, the waste is easy to store, deterioration pollution and loss caused by long-term disposal are avoided, the sweet potato vine and cordyceps militaris culture medium waste is stored in a centralized collection way, the production and feeding time is adjusted, and multiple purposes can be met;
3. the method provided by the application reduces the grain consumption of the raw materials, reduces the waste pollution and improves the quality of the cordyceps militaris. The method mainly comprises the steps of optimizing culture conditions during the culture of first-level strains and second-level strains of cordyceps militaris, reducing conventional culture nutrient components such as potatoes and peptone, performing preliminary domestication by adding waste of a culture medium of the cordyceps militaris and the like, providing nutrition to strengthen the activity of the strains, and realizing large-scale third-level strain culture of a fermentation tank according to production needs; in the process of culturing sporocarp, proper culture conditions are adopted, the usage amount of raw material grain is reduced, and the production purpose is achieved by adding cordyceps militaris culture medium waste and sweet potato vine in different proportions and adopting a scientific culture method;
4. according to the method provided by the application, the sweet potato vine and the cordyceps militaris culture medium waste are used for replacing partial potatoes, glucose, peptone and the like in the strain culture process, so that the cost is reduced, strains are domesticated, and the activity is improved;
5. the method provided by the application can improve the quality and biological indexes of the cordyceps militaris, can improve the content of flavone and the like in the cordyceps militaris sporocarp and enhance the oxidation resistance;
6. the method provided by the application roughly performs benefit analysis, and the commercial cordyceps militaris fruiting body dry product is calculated by 70 yuan per kilogram, the wheat grain or rice is calculated by 2.2 yuan per kilogram, and the biological conversion rate of the cordyceps militaris fruiting body is 80%.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
FIG. 1 is a schematic view showing the growth of a first-stage Cordyceps militaris strain, wherein (a) corresponds to the first-stage Cordyceps militaris strain of comparative example 1, and (b) corresponds to the first-stage Cordyceps militaris strain of example 1;
FIG. 2 is a hypha micrograph of secondary liquid seeds of Cordyceps militaris, wherein (a) corresponds to comparative example 1, (b) corresponds to example 1, (c) corresponds to example 2, (d) corresponds to example 3, (e) corresponds to example 4, and (f) corresponds to example 5;
FIG. 3 is a schematic diagram showing germination of primordia of fruiting bodies of Cordyceps militaris, wherein (a) corresponds to comparative example 1, (b) corresponds to example 1, (c) corresponds to example 2, (d) corresponds to example 3, (e) corresponds to example 4, and (f) corresponds to example 5;
FIG. 4 is a schematic diagram showing the growth of fruiting bodies of Cordyceps militaris, wherein (a) corresponds to comparative example 1, (b) corresponds to example 1, (c) corresponds to example 2, (d) corresponds to example 3, (e) corresponds to example 4, and (f) corresponds to example 5;
FIG. 5 is a diagram illustrating various exemplary Cordyceps militaris fruiting body function indexes.
Detailed Description
The following detailed description is given by way of example in order to more clearly illustrate the general concept of the present application. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art, that the present invention may be practiced without one or more of these specific details. In other instances, well-known features have not been described in order to avoid obscuring the invention.
The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the starting materials, instruments and reagents mentioned in the following examples were all commercially available.
In the following examples, the waste culture medium of sweet potato vine and cordyceps militaris is prepared by the following method:
1. and (3) treating sweet potato vines:
selecting fresh, mildew-free, rot-free and non-lignified sweet potato vines, cleaning, draining, putting the sweet potato vines into a pulverizer, pulverizing the sweet potato vines into small segments of 2-3cm, putting the pulverized sweet potato vines into a blast drying oven, drying at 60-80 ℃ until the water content reaches 8-12%, and bagging for later use;
further pulverizing the dried sweet potato vine by a pulverizer to pass through a 4-8 mesh sieve to obtain coarse particles (i.e. sweet potato vine with particle size of 4-8 meshes) of the sweet potato vine; pulverizing the dried sweet potato vine by a pulverizer to pass through a 20-40 mesh sieve to obtain sweet potato vine powder particles (namely the sweet potato vine with the particle size of 20-40 meshes).
2. Preparing cordyceps militaris culture medium waste:
preparing a nutrient solution: magnesium sulfate 0.5g/L, potassium dihydrogen phosphate 2g/L, glycine 0.1g/L;
preparing a solid material: putting 100 parts of wheat grains into a 500ml culture bottle;
adding the solid material into the mixture according to the proportion of 1: adding nutrient solution in a proportion of 1.6-1.8 g/ml, and mixing, wherein the mass of each bottle is 30g; covering a polyethylene film on the bottle mouth, fastening with a rubber band, placing the culture bottle into a steam sterilization pot, sterilizing at 121 ℃ for 60-70 min, and cooling to room temperature to obtain a fresh culture material;
inoculating cordyceps militaris seeds (cordyceps militaris preservation strains, which can also be conventional culture mediums in other embodiments, such as the first-level culture medium or the second-level culture medium without sweet potato vine and waste in the application, to a fresh culture material in a sterile environment, wherein the inoculation volume of the strains is 6-8% of the volume of the culture medium, and the strains are cultured in a dark place at the temperature of 18-22 ℃; after the white hypha grows over the surface of the culture material in the covering bottle, the culture is carried out in a color conversion stage, the illumination intensity of the culture is 200-400Lx, and the air humidity is 65-75%; when the color of hyphae turns to orange, uniformly pricking holes on a culture bottle mouth by using a sterile needle for ventilation, and allowing primordium to grow out, culturing in an environment with air humidity of 85-95%, and harvesting when the top end of the fruiting body of cordyceps militaris slightly expands to the bottle mouth;
the culture materials left after the cordyceps militaris sporocarp is collected are called cordyceps militaris culture medium waste, the cordyceps militaris culture medium waste which is free from mildew, foreign bacteria pollution, dirt and rich in cordyceps militaris hypha is selected, sporocarp residual sections are selected and discarded (the culture materials are reduced in toughness and easy to break through exoenzyme enzymolysis of sporocarp), the materials are rubbed and dispersed by a rubbing machine, the particle size is 0.4-0.6 cm, the materials are placed in a blast drying box at the temperature of 60-80 ℃ to be dried until the water content is 8-12%, and the materials are bagged for later use.
Example 1
In the embodiment, the method for culturing cordyceps militaris by using the sweet potato vine and cordyceps militaris culture medium waste as main raw materials comprises the following steps:
step one, preparation of first-grade cordyceps militaris seeds
Mixing and boiling 100g/L of potatoes, 10g/L of cordyceps militaris culture medium waste and 5g/L of sweet potato vine powder particles (the particle size is 20-40 meshes) for 20-25 min, filtering by using filter cloth to obtain filtrate, and adding 10g/L of glucose, 2.5g/L of peptone, 1.5g/L of magnesium sulfate, 3g/L of monopotassium phosphate and 10mg/L of vitamin B into the filtrate 1 And 10g/L agar, uniformly mixing and then placing the mixture into a test tube, then placing the test tube into a steam sterilization pot for sterilization at 121 ℃ for 30min, cooling to room temperature, using an inoculation hook to cut the cordyceps militaris preservation strain into 4 multiplied by 4mm hypha pieces away from the original inoculation point under the aseptic condition, using an inoculation shovel to scoop out the hypha pieces, placing the hypha pieces on the middle lower part of the inclined plane of the fresh first-class seed, slightly pressing, culturing in a dark place at 18-22 ℃, stopping culturing after the tube is filled, and storing at low temperature to obtain the cordyceps militaris first-class seed.
Step two, preparation of cordyceps militaris secondary liquid seeds
Mixing 100g/L of potatoes, 10g/L of cordyceps militaris culture medium waste and 4g/L of sweet potato vine powder particles (the particle size is 20-40 meshes) and boiling for 20-25 min, filtering by using filter cloth to obtain filtrate, and adding 10g/L of glucose, 2.5g/L of peptone, 1.5g/L of magnesium sulfate, 3g/L of monopotassium phosphate and 10mg/L of vitamin B into the filtrate 1 Mixing, placing into a triangular flask, inoculating the first-stage Cordyceps militaris seeds cultured in the first step in sterile environment, placing in a constant temperature shaking table at 18-22 deg.C and 140 rpm, culturing in dark for 3.5-4.5 days until the mycelia are strong and the ratio of fungus balls is 50-70%, and stopping culturing to obtain Cordyceps militarisSecondary liquid seeds.
In this example, the second-level liquid seed of Cordyceps militaris was used for growing grass.
Optionally, according to the requirement, the production of the cordyceps militaris three-level liquid strain in the fermentation tank can be synchronously realized, so as to meet the requirement of large-scale cultivation. The culture material preparation method is the same as the culture material preparation method of the second-level liquid seed of cordyceps militaris, and the culture conditions are as follows: the charging volume accounts for 60 percent of the inner volume of the tank, the Cordyceps militaris secondary liquid seeds are inoculated according to the charging volume in the tank after the Cordyceps militaris secondary liquid seeds are cooled to 22-24 ℃ after the Cordyceps militaris secondary liquid seeds are actually consumed, the inoculation amount is 3-5 percent, the tank pressure is 0.02-0.06Mpa, the rotating speed is 130-150rpm at 20-22 ℃, the fermentation time is 2-3 days, and the culture is stopped when the ratio of the strong hyphae and the rich fungus balls reaches 50-70 percent. The growth of the third-order liquid strain is shown in Table 2.
Step three, culturing cordyceps militaris sporocarp
Preparing a sporophore culture material:
preparing a nutrient solution: 0.5g/L magnesium sulfate, 2g/L monopotassium phosphate and 0.1g/L glycine;
preparing a solid material: putting 50 parts of wheat grains, 40 parts of cordyceps militaris culture medium waste and 10 parts of sweet potato vine coarse grains (the grain size is 4-8 meshes) into a 500ml culture bottle;
adding the solid material into the mixture according to the proportion of 1: adding the nutrient solution in a proportion of 1.6-1.8 g/ml, and mixing, wherein the mass of each bottle is 30g; covering a polyethylene film on the bottle mouth, fastening the bottle mouth by using a rubber band, placing the culture bottle into a steam sterilization pot, sterilizing the culture bottle for 60 to 70min at the temperature of 121 ℃, and cooling the culture bottle to room temperature to obtain a sporophore culture material;
inoculating the cordyceps militaris secondary liquid seed obtained in the step two into a sporocarp culture material in an aseptic environment, wherein the inoculation amount is 6-8%, and culturing in a dark place at the temperature of 18-22 ℃; after the white hypha grows over the surface of the culture material in the covering bottle, the culture is carried out in a color conversion stage, the illumination intensity of the culture is 200 to 400Lx, and the air humidity is 65 to 75 percent; when the color of hyphae turns to orange, uniformly pricking holes on a culture bottle mouth by using a sterile needle for ventilation, and allowing primordium to grow out, culturing in an environment with air humidity of 85-95%, and harvesting when the top end of the fruiting body of Cordyceps militaris slightly expands to the bottle mouth.
In one embodiment, in the cordyceps militaris sporocarp culture process in the third step, when the protoplasm protrusion is just formed after the color of the hypha is changed, the culture can be stopped according to the production application, and the cordyceps militaris sporocarp primordium is formed, and at the moment, the culture material is rich in the hypha, rich in nutritional ingredients such as protein, sugar and amino acid, and can be used for developing downstream products such as health care products.
Example 2
The preparation method of the embodiment is the same as the embodiment 1, and the difference is that in the preparation of the second-level cordyceps militaris liquid seed in the step two, 15g/L of cordyceps militaris culture medium waste and 6g/L of sweet potato vine powder particles are obtained; the solid materials in the fruiting body culture medium in the third step adopt 40 parts of wheat grains, 45 parts of cordyceps militaris culture medium waste and 15 parts of sweet potato vine coarse grains.
Example 3
The preparation method of the embodiment is the same as the embodiment 1, and the difference is that in the preparation of the second-level cordyceps militaris liquid seed in the step two, 20g/L of cordyceps militaris culture medium waste and 8g/L of sweet potato vine powder particles are obtained; and solid materials in the fruiting body culture medium in the third step are 30 parts of wheat grains, 50 parts of cordyceps militaris culture medium waste and 20 parts of sweet potato vine coarse grains.
Example 4
The preparation method of the embodiment is the same as the embodiment 1, except that 25g/L of cordyceps militaris culture medium waste and 10g/L of sweet potato vine granules are prepared in the second step of preparing the cordyceps militaris secondary liquid seeds; in the third step, the solid materials in the sporocarp culture medium are 10 parts of wheat grains, 70 parts of cordyceps militaris culture medium waste and 20 parts of sweet potato vine coarse grains.
Example 5
The preparation method of the embodiment is the same as the embodiment 1, and the difference is that in the preparation of the second-level cordyceps militaris liquid seed in the step two, 30g/L of cordyceps militaris culture medium waste and 12g/L of sweet potato vine powder particles are obtained; and solid materials in the sporocarp culture material in the third step are 10 parts of wheat grains, 80 parts of cordyceps militaris culture medium waste and 10 parts of sweet potato vine coarse grains.
Comparative example 1
This comparative example is prepared in substantially the same manner as example 1, except that:
set of primary mediaThe compositions are as follows: boiling potato at a concentration of 200g/L for 20-25 min, filtering with filter cloth to obtain filtrate, adding glucose at a concentration of 20g/L, peptone at a concentration of 5g/L, magnesium sulfate at a concentration of 1.5g/L, potassium dihydrogen phosphate at a concentration of 3g/L, and vitamin B 1 10mg/L, agar 10g/L.
The composition of the secondary medium was as follows: adding water into potato at a ratio of 200g/L, boiling for 20-25 min, filtering with filter cloth to obtain filtrate, respectively adding glucose at a ratio of 20g/L, peptone at a ratio of 5.0g/L, magnesium sulfate at a ratio of 1.5g/L, potassium dihydrogen phosphate at a ratio of 3g/L, and vitamin B 1 10 mg/L。
In the culture material of the sporocarp, 100 parts of wheat grains are adopted as solid materials, and the parameters of the other steps are the same.
And (3) comparing culture results:
the main nutrient components of the cordyceps militaris culture medium waste in each example are shown in table 1; the growth vigor and culture condition of the obtained first-stage Cordyceps militaris seed are shown in figure 1; the culture conditions of the second-level liquid seeds of Cordyceps militaris are shown in Table 2 and figure 2; the culture conditions of Cordyceps militaris fruiting body are shown in Table 3-Table 5 and figure 3-5. Wherein, 30 bottles of cordyceps militaris sporocarp are cultivated simultaneously in each example, and the data in the following table, such as the growth period in table 2 and each index in tables 3-5, are the average value of the data of the 30 bottles of cordyceps militaris sporocarp test.
TABLE 1
| Crude protein | Coarse fiber | Starch | Calcium (ll) containing calcium (II) | Total phosphorus | Amino acids | |
| Content (mg/g) | 184 | 49 | 430 | 0.9 | 4.6 | 81.7 |
As can be seen from FIG. 1, the first-grade cordyceps militaris seeds prepared in comparative example 1 and examples 1-5 have strong, compact and neat mycelia and good quality.
The growth conditions of the second-level liquid seeds of cordyceps militaris in the above examples are shown in table 2:
TABLE 2
| The diameter/cm of the fungus ball | Uniformity of fungus ball | Suspension property | Growth cycle/d | |
| Examples 1 to 5 | 0.18～0.22 | Uniformity | **** | 4.5 |
| Three-stage liquid strain | 0.20～0.22 | Uniformity | **** | 2.5 |
| Comparative example 1 | 0.25 | Is relatively uniform | *** | 5.0 |
Table 2 shows that the second-stage liquid seeds and the third-stage liquid strains in the fermentation tank in examples 1 to 5 have slightly smaller pellets than that in comparative example 1, better pellet uniformity and better suspension property (more suspension property is better), which indicates that the strains have good growth state and are easier to disperse during use, so that the fruiting bodies are easier to uniformly distribute, and meanwhile, the strain growth cycles of the third-stage liquid strains in examples 1 to 5 and the fermentation tank are shorter than that in comparative example 1, which indicates that the waste of the culture medium of sweet potato vine and cordyceps militaris can promote the growth of the strains to a certain extent, so that the quality of the strains is better.
As can be seen from FIG. 2, in both examples 1-5 and comparative example 1, the mycelia of Cordyceps militaris are thicker, have enlarged mycelia, have branches and separate mycelia, indicating strong reproductive capacity and good activity, and the images show that the mycelia in examples 1-5 are richer and have better strain quality than comparative example 1.
The fruiting situation of cordyceps militaris in the above example is shown in table 3:
TABLE 3
| Hypha germination/h | Time of full charge level/h | Color conversion/d | Percentage of grass output% | Formation of primordia/d | |
| Example 1 | 24 | 132 | 4 | 100 | 13 |
| Example 2 | 24 | 120 | 4 | 100 | 12 |
| Example 3 | 24 | 108 | 3.5 | 100 | 12.5 |
| Example 4 | 24 | 132 | 3.5 | 100 | 13 |
| Example 5 | 24 | 132 | 4 | 100 | 14 |
| Comparative example 1 | 28 | 144 | 4.5 | 100 | 13 |
From table 3 and fig. 3, it can be seen that in examples 1-5, the mycelia of the fruiting body begin to germinate faster than the mycelia of comparative example 1, which is 24h and 28h respectively, and the time for covering the material surface is shorter than that of comparative example 1, which indicates that the growth speed of the cordyceps militaris mycelia on the substituted material of the culture medium waste of potato vine and cordyceps militaris is faster; the hypha of the comparative example 1 has long color conversion time under visible light, and the formation time of the orange primordium of the examples 2 and 3 is earlier than that of the comparative example 1, which shows that the cordyceps militaris hypha grows well on the substitute materials with different proportions, and meanwhile, the growth cycle is shortened, so that the manpower and material resources can be reduced to a certain extent, and the energy is saved.
The biological indicators of cordyceps militaris in the above example are shown in table 4:
TABLE 4
It can be seen from table 4 and fig. 4 that the diameter of the fruiting bodies in examples 1-5 is slightly smaller than that in comparative example 1, but the number of the fruiting bodies is higher than that in comparative example 1, and the biological efficiency in examples 1-3 is slightly higher than that in comparative example 1, which indicates that the culture medium waste of sweet potato vine and cordyceps militaris has a certain influence on the yield of the fruiting bodies, the yield is slightly increased, the distortion rate is low, the fruiting bodies in the examples are good, the commodity value is high, and the growth cycle of examples 1-3 is shorter than that in comparative example 1.
The functional indexes of the cordyceps militaris fruiting body in the example are shown in table 5:
TABLE 5
The index detection methods in table 5 and fig. 5 are respectively as follows: the cordycepin adopts high performance liquid chromatography, and the test method comprises the following steps: accurately weighing 1.0g of cordyceps militaris sporocarp powder, placing the cordyceps militaris sporocarp powder into a round-bottom flask respectively, adding 20mL of 90% methanol solution, heating and refluxing for 45min, cooling, fixing the volume to 50mL, shaking up, filtering, discarding primary filtrate, taking subsequent filtrate, filtering by using a microporous membrane to obtain a test solution, wherein a chromatographic column is C18, and a mobile phase is methanol: water =15: 85, the detection wavelength is 258nm, the sample injection amount is 5 mu L, and the flow rate is 1mL/min. The flavone is obtained by sodium nitrite-aluminum nitrate method, total oxidation resistance is accurately measured by ABTS method and using a kit with a product number of G0142F.
According to the chart, the cordycepin content in the cordyceps militaris sporocarp in 5 embodiments is similar to that in the comparative example and is slightly improved; the contents of flavonoids are obviously higher than the comparative example, the total antioxidant capacity is obviously higher than the comparative example, and the function indexes of the cordyceps militaris sporocarp cultured in the examples 1-5 are higher than the comparative example comprehensively, which shows that the quality of the cordyceps militaris sporocarp is improved by taking agricultural wastes, namely sweet potato vine and cordyceps militaris culture medium wastes as substitute materials. However, as compared with Table 4 and FIG. 4, it is found that the growth cycle, biological efficiency and functional indexes show that the embodiment 1-3 is better, namely, 10g/L of cordyceps militaris culture medium waste and 5g/L of sweet potato vine powder are added into the first-level seeds, 10-30g/L of cordyceps militaris culture medium waste and 4-12 g/L of sweet potato vine powder are added into the second-level seeds, 30-60 parts of cordyceps militaris culture medium waste and 10-20 parts of sweet potato vine coarse particles are added into the cordyceps militaris sporocarp culture medium, and the culture effect is good through a scientific culture method. Examples 4 to 5 were unsatisfactory in cultivation and were in need of improvement. In conclusion, the invention successfully uses the cordyceps militaris culture medium waste and the sweet potato vine to culture the cordyceps militaris for the first time, replaces the culture material for grain, and has certain economic benefit and more obvious ecological benefit and social benefit.
The above description is only an example of the present invention, and is not intended to limit the present invention. Various modifications and alterations to this invention will become apparent to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Claims (10)
1. A method for culturing high-quality cordyceps militaris sporocarp in a short period is characterized by comprising the following steps:
the method comprises the following steps: inoculating the cordyceps militaris preservation strain into a primary culture medium for culturing to obtain cordyceps militaris primary seeds;
step two: inoculating the first-stage cordyceps militaris seeds into a second-stage culture medium for culture to obtain second-stage cordyceps militaris seeds;
step three: inoculating the second-level cordyceps militaris seeds into a sporocarp culture medium, and culturing until cordyceps militaris sporocarps are obtained;
the first-stage culture medium, the second-stage culture medium and the sporocarp culture medium all contain cordyceps militaris culture medium waste and sweet potato vine, wherein the mass ratio of the cordyceps militaris culture medium waste to the sweet potato vine in the first-stage culture medium and the second-stage culture medium is (10-30): (4-12), wherein the mass ratio of cordyceps militaris culture medium waste to sweet potato vine in the sporocarp culture medium is (30-60): (20-10).
2. The method according to claim 1, characterized in that the sweet potato vine is a sweet potato vine granulate treated with: drying fresh sweet potato vine until the water content is 8-12%, and crushing to 4-40 meshes to obtain the sweet potato vine granules; and/or the presence of a gas in the gas,
the cordyceps militaris culture medium waste is prepared by adopting the following method: the culture material of the collected cordyceps militaris sporocarp is crushed to the grain size of 0.4-0.6 cm and dried until the water content is 8-12% for later use.
3. The method of claim 1, wherein the primary medium, secondary medium and/or fruiting body culture medium further comprises a crop nutrient selected from one or more of potato, wheat, rice and corn.
4. The method according to claim 3, wherein the primary culture medium, the secondary culture medium and/or the culture medium for fruit body further comprises a nutritional supplement selected from glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate, vitamin B 1 Agar and glycine.
5. The method of claim 4, wherein the primary and secondary culture media are prepared by a method comprising: mixing the crop nutrient, cordyceps militaris culture medium waste and sweet potato vine particles, adding water, boiling for 15-30 min, filtering to obtain a filtrate, and adding nutritional auxiliary materials into the filtrate to obtain the compound fertilizer, wherein the addition amount of the crop nutrient is 30-200g/L; and/or the presence of a gas in the atmosphere,
the preparation method of the fruiting body culture material comprises the following steps: mixing the crop nutrient, the cordyceps militaris culture medium waste and the sweet potato vine granules to obtain a mixed material with the total mass of 100 parts, and adding 1.5-2.0 g/ml of nutritional auxiliary materials based on the mass of the mixed material to obtain the feed additive.
6. The method according to claim 1, wherein the culturing method in the first step comprises: inoculating Cordyceps militaris preservation strain into the primary culture medium under aseptic condition, and culturing at 18-22 deg.C in dark.
7. The method according to claim 1, wherein the culturing method in the second step comprises: inoculating the first-level seed of Cordyceps militaris into the second-level culture medium under sterile conditions, and culturing at 18-22 deg.C and 130-150rpm in dark condition for 3-5 days.
8. The method according to claim 1, wherein the culturing in step three comprises: 6-8% of cordyceps militaris secondary seeds are aseptically inoculated into a fruiting body culture material, are cultured in a dark place at 18-22 ℃, enter a color conversion stage, the illumination intensity is controlled to be 200-400Lx, the air humidity is controlled to be 60-80%, when the color of hypha is converted to orange color, holes are punctured for ventilation to form cordyceps militaris fruiting body primordium, the air humidity is controlled to be 85-95%, and the cordyceps militaris fruiting body primordium is cultured until the cordyceps militaris fruiting body primordium can be harvested.
9. A Cordyceps militaris culture, comprising the cordyceps militaris fruiting body primordium prepared by the method of claim 8.
10. A fruiting body of Cordyceps militaris obtained by the method of any one of claims 1 to 8.
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