CN115152527B - Method for cultivating high-quality cordyceps militaris fruiting bodies in short period - Google Patents
Method for cultivating high-quality cordyceps militaris fruiting bodies in short period Download PDFInfo
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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
- A01G18/40—Cultivation of spawn
Abstract
The invention discloses a method for cultivating high-quality cordyceps militaris fruiting bodies in a short period, which adopts crop waste sweet potato vine and cordyceps militaris culture medium waste as main raw materials of a fermentation culture medium, establishes a scientific and reasonable cultivation method, and further fully utilizes nutrient substances such as starch, protein, sugar, amino acid and the like in the waste and micromolecular substances generated by extracellular enzyme enzymolysis of cordyceps militaris. The invention has simple process, abundant and easily obtained substitute 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 environmental protection sustainable development.
Description
Technical Field
The invention belongs to the field of recycling and biotechnology of agricultural production waste, and particularly relates to a method for cultivating high-quality cordyceps militaris fruiting bodies in a short period, in particular to a method for preparing high-quality cordyceps militaris fruiting bodies with good biological morphology and function indexes in a shorter cultivation period by utilizing crop waste sweet potato vine and cordyceps militaris culture medium waste.
Background
Cordyceps militaris (Cordyceps militaris) is also called Cordyceps militaris, which belongs to Cordyceps genus of Clavipitaceae family of Ascomycotina order. As the traditional fungi for both medicine and food, the fungi are popular with people because of the nutrition and economic value, the cultivation technology is developed rapidly since the cordyceps militaris is discovered and separated in the eighties of the last century in China, and the large-scale cultivation is realized primarily without being limited by seasons.
The raw materials for cultivating the cordyceps militaris 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 cultivation method has become a prop industry for partial farmers to get rid of poverty and become rich, the cultivation scale is continuously enlarged along with the improvement and maturity of the planting skills of the farmers, a large amount of Cordyceps militaris culture medium waste is generated, the quality of the Cordyceps militaris culture medium waste is about 6-8 times of that of dried Cordyceps militaris products, the raw material waste reaches 30%, and huge pressure and trouble are brought to the production environment, so that the culture medium waste needs to be treated urgently. At present, a large number of effective recycling ways are lacking in the waste of the culture medium, so that the natural ecological environment and the reproduction environment are polluted, and the sustainable development of the cordyceps industry is restricted.
The desertification of the Chinese land causes the continuous reduction of agricultural cultivated land area, and the stress-resistant sweet potato crop which can be used as main food can be continuously planted. Sweet potato is planted in more than 100 countries in the world due to the characteristics of drought resistance, barren resistance, strong environmental adaptability, high yield, stable yield and the like. Sweet potato is one of the 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 world sweet potato cultivation area, and the cultivation area and the yield are the first place in the world. The sweet potato vine produced per year in China is up to more than 150 hundred million tons. Most of the waste water is discarded to the field or burnt out except that the little waste water is utilized, so that the ecological environment is polluted to cause production inconvenience and resource waste. The folk traditional Chinese medicine often uses sweet potato vine as an auxiliary therapeutic medicine and food material for reducing blood fat, reducing blood sugar, preventing and treating arteriosclerosis, cardiovascular and cerebrovascular diseases and the like, which is consistent with the efficacy of the research field of the cordyceps militaris, and provides feasibility for the sweet potato vine to participate in the cultivation of the cordyceps militaris.
At present, attempts have been made to reuse agricultural production byproducts to increase economic value. For example, patent CN104969773a discloses a method for obtaining cordyceps militaris fermentation product or fruiting body by fermenting sweet potato residues, the sweet potato residues used in the method are mainly starch residues, the grain for cordyceps militaris cultivation is replaced, the biological conversion rate is relatively low, the industrial cultivation is not facilitated, the grain substitute for the raw materials is industrial waste, and the profitable person is the production enterprise. The application utilizes the vines of the overground parts of the sweet potatoes, has rich functional components, relatively high biological conversion rate of grains for replacing cordyceps cultivation, and the vines are agricultural production waste, benefit to farmers and are beneficial to agricultural production.
Disclosure of Invention
The application aims at providing a method for preparing high-quality cordyceps militaris fruiting bodies rapidly and in high yield by utilizing a large amount of waste materials in the current agricultural planting production field and the cordyceps militaris culture field, namely sweet potato vine and cordyceps militaris culture medium waste as culture matrixes.
In one aspect, the present application provides a method for cultivating high-quality cordyceps militaris fruiting bodies in a short period, comprising the following steps:
step one: inoculating the cordyceps militaris preserved 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 seeds of the cordyceps militaris into a fruiting body culture material, and culturing until obtaining cordyceps militaris fruiting bodies;
the primary culture medium, the secondary 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 primary culture medium to the secondary culture medium is (10-30): (4-12), wherein the mass ratio of the cordyceps militaris culture medium waste to the sweet potato vine in the fruiting body culture medium is (30-60): (20-10).
Further, the sweet potato vine adopts sweet potato vine particles treated by the following method: and (3) drying fresh sweet potato vine until the water content is 8-12%, and crushing the fresh sweet potato vine to 4-40 meshes to obtain the sweet potato vine particles.
Preferably, the processing of the sweet potato vine specifically comprises: selecting fresh, mildew-free, rot-free and non-lignified sweet potato vine after harvesting sweet potato in autumn, cleaning, draining water, putting into a pulverizer, pulverizing the sweet potato vine into small sections of 2-3cm, putting the pulverized sweet potato vine 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 until the sweet potato vine can pass through a 4-8 mesh sieve, thus obtaining coarse grains of the sweet potato vine (namely the sweet potato vine with the grain size of 4-8 meshes); further pulverizing the dried sweet potato vine with pulverizer to obtain sweet potato vine powder (i.e. sweet potato vine with particle size of 20-40 meshes) which can pass through 20-40 meshes.
More preferably, sweet potato vine particles with smaller particle sizes are adopted in the primary culture medium and the secondary culture medium, so that active substances in the sweet potato vine can be fully extracted, and the sweet potato vine particles are applied to the culture medium to be more beneficial to domestication of cordyceps militaris strain culture; the coarse grain of the sweet potato vine is adopted in the fruiting body culture material, and has good air permeability due to the plant tissue structure of the sweet potato vine, the coarse grain of the sweet potato vine can keep the air permeability of the tissue structure of the sweet potato vine, and also can keep high porosity after being mixed with the cordyceps militaris culture medium waste due to the coarse grain of the sweet potato vine, and meanwhile, the fruiting body culture material can also show good water retention effect and sustained release function, so that on one hand, the ventilation effect of hypha growth is greatly improved when the cordyceps militaris fruiting body is cultivated, particularly after fruiting body primordium grows out, the growth speed of the cordyceps militaris is improved, the cultivation period is shortened, and on the other hand, the absorption of nutrient substances in the sweet potato vine in the growth process is facilitated, and the quality of biological indexes and functional indexes of the final cordyceps militaris fruiting body is improved.
Further, the cordyceps militaris culture medium waste is prepared and obtained by the following method: crushing the culture material of the collected cordyceps militaris fruiting bodies to the particle size of 0.4-0.6 cm, and drying until the water content is 8-12% for standby.
Wherein, can choose the culture material of the fruiting body that does not have mildewing, no mixed bacterial pollution, no filth, cordyceps militaris mycelium are abundant, and choose the fruiting body and discard the incomplete section, this culture material has reduced the toughness through the extracellular enzymatic hydrolysis of fruiting body, easy to break.
In one embodiment, the cordyceps militaris fruiting body culturing material which is conventional in the art can be selected and used as the cordyceps militaris culture medium waste after the cordyceps militaris fruiting body is cultured and collected.
In a preferred embodiment, the fruiting body culture medium containing only crop nutrients in the method can be used as fresh culture medium, and after the fruiting body of Cordyceps militaris is cultured and collected, the fruiting body culture medium can be used as Cordyceps militaris culture medium waste.
Preferably, the sweet potato vine particles with a smaller particle size (20-40 meshes) and the cordyceps militaris culture medium waste are applied to the primary culture medium and the secondary culture medium in the form of extracting solutions, wherein the extracting solutions comprise, but are not limited to, aqueous extracting solutions. So that 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 the cordyceps militaris strain culture is facilitated.
Preferably, coarse sweet potato vine particles with a larger particle size (4-8 meshes) and cordyceps militaris culture medium waste are applied to the fruiting body culture medium in the form of solid particles. At the moment, the air permeability of the coarse particles of the sweet potato vine can be utilized to improve the absorption of active substances in the cordyceps militaris culture medium waste in the mycelium growth process, and the quality of the final cordyceps militaris fruiting body is further improved.
Further, the primary culture medium, the secondary culture medium and/or the fruiting body culture medium 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 fruiting body culture medium also contain nutritional auxiliary materials selected from glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate and vitamin B 1 One or more of agar, glycineA kind of module is assembled in the module and the module is assembled in the module.
Further, the preparation method of the primary culture medium and the secondary culture medium comprises the following steps: mixing the crop nutrients, the cordyceps militaris culture medium waste and the sweet potato vine particles, adding water, boiling for 15-30 min, filtering to obtain filtrate, and adding nutrition auxiliary materials into the filtrate to obtain the crop nutrient, wherein the adding amount of the crop nutrients is 30-200g/L; and/or the number of the groups of groups,
the preparation method of the fruiting body culture material comprises the following steps: mixing the crop nutrients, the cordyceps militaris culture medium waste and the sweet potato vine particles to obtain a mixed material with the total mass of 100 parts, and adding nutritional auxiliary materials with the mass of 1:1.5-2.0 g/ml based on the mixed material.
In a preferred embodiment, the preparation method, the first stage medium used in the first step, comprises the following components: boiling 100g/L of crop nutrients, 10-30g/L of cordyceps militaris culture medium waste and 2-20g/L of sweet potato vine particles for 20-25 min, filtering with a filter cloth to obtain filtrate, adding 10g/L of glucose, 2.5g/L of peptone, 1.5g/L of magnesium sulfate, 3g/L of potassium dihydrogen phosphate, 10mg/L of vitamin B1 and 10g/L of agar, and sterilizing at 121 ℃ for 30min;
the second-stage culture medium adopted in the second step comprises the following components: 100g/L of crop nutrient, 10-30g/L of cordyceps militaris culture medium waste and 2-20g/L of sweet potato vine particles are boiled together for 20-25 min, filtered by a filter cloth to obtain filtrate, and 10g/L of glucose, 2.5g/L of peptone, 1.5g/L of magnesium sulfate, 3g/L of potassium dihydrogen phosphate and 10mg/L of vitamin B are respectively added 1 ；
The fruiting body culture material adopted in the step three comprises the following components: preparing a nutrient solution: 0.5g/L of magnesium sulfate, 2g/L of monopotassium phosphate and 0.1g/L of glycine; 10-90 parts of crop nutrients, 8-80 parts of cordyceps militaris culture medium waste and 2-30 parts of sweet potato vine particles are mixed, the total amount of the mixed materials is controlled to be 100 parts by mass, and the mixing ratio of the mixed materials and nutrient solution is 1: 1.5-2.0 g/ml.
Further, the culturing method in the first step includes: aseptically inoculating Cordyceps militaris preservation strain into the primary culture medium, and culturing at 18-22 ℃ in dark.
In one embodiment, step one specific method of preparation is as follows: dissolving the first-stage culture medium fully in a split charging test tube, sterilizing at 121deg.C for 30min, cooling to room temperature, inoculating Cordyceps militaris preserved strain under aseptic condition, cutting into 4×4mm mycelium slices away from the original inoculating point, shoveling out the mycelium slices with an inoculating shovel, placing the mycelium slices in the middle lower part of fresh first-stage seed inclined plane, slightly pressing, culturing at 18-22deg.C in dark place, stopping culturing after full tube, and preserving at low temperature.
Further, the culturing method in the second step comprises the following steps: aseptically inoculating the first-stage seeds of Cordyceps militaris into the second-stage culture medium, and culturing for 3-5 days at 18-22 ℃ and rotating speed of 120-150 rpm in dark.
In one embodiment, the specific preparation method of step two is as follows: sub-packaging the secondary culture medium in a triangular flask, sterilizing at 121 ℃ for 30min, cooling to room temperature, dividing the first-class seeds of Cordyceps militaris into 3-4 mycelium slices of 2X 2mm away from the original inoculation point by using an inoculation hook under aseptic condition, shoveling out the mycelium slices by using an inoculation shovel, placing the mycelium slices into the secondary seed triangular flask, culturing for 3-5 days at 18-22 ℃ and 130-150rpm, and stopping culturing until the mycelium is robust and the enriched volume ratio of the mycelium balls reaches 50-70%.
Preferably, three-level liquid strains can be synchronously produced in the fermentation tank according to production requirements, so that the requirement of large-scale cultivation is met. The preparation method of the culture material is the same as that of the second-stage liquid seeds of the cordyceps militaris, and the culture conditions are as follows: the charging volume is 60% of the internal volume of the tank, the temperature is reduced to 22-24 ℃ after actual elimination, the second-level liquid seeds of Cordyceps militaris are inoculated according to the charging volume of the tank, the inoculation amount is 3-5%, 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 cultivation is stopped when the hypha is strong and the enriched volume ratio of the fungus balls reaches 50-70%.
Further, the culturing method in the third step comprises the following steps: aseptically inoculating 6-8% of cordyceps militaris secondary seeds into the fruiting body culture material, culturing at 18-22 ℃ in the dark, then entering a color conversion stage, controlling the illumination intensity to be 200-400Lx, controlling the air humidity to be 60-80%, and culturing until mycelia change to orange color, forming cordyceps militaris fruiting body primordium, controlling the air humidity to be 85-95%, and harvesting.
On the other hand, the application also provides a cordyceps militaris culture, which comprises cordyceps militaris fruiting body primordium prepared by the method.
On the other hand, the application also provides the cordyceps militaris fruiting body prepared and obtained by the cultivation method.
The beneficial effects of the invention include at least one of the following:
1. according to the cultivation method provided by the application, the sweet potato vine and cordyceps militaris culture medium waste is utilized to produce cordyceps militaris, so that nutrient substances such as starch, protein, sugar and amino acid in the waste and micromolecular substances generated by extracellular enzyme enzymolysis of cordyceps militaris are further fully utilized, the efficient ecological recycling 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, can improve the quality of cordyceps militaris, is rich in resources, low in cost and high in nutrition, is an unattainable advantage of the sweet potato vine, is fully utilized, changes waste into valuable, can be beneficial to farmers, mobilizes the production enthusiasm of the farmers, and promotes agricultural production; meanwhile, the cordyceps militaris culture medium waste is utilized, long-distance transportation is not needed, and the materials are taken in situ, so that the transportation cost is convenient, the transportation cost is reduced, and the efficient and sustainable development of the cordyceps militaris industrial chain is greatly promoted;
2. according to the method, the sweet potato vine and cordyceps militaris culture medium waste is subjected to simple and feasible airing and low-temperature drying, so that nutrition loss is reduced, the quick treatment can be realized, the sweet potato vine and cordyceps militaris culture medium 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 reserved in a centralized collection mode, the production feeding time is regulated, and the method can meet multiple purposes;
3. the method reduces grain consumption of raw materials, reduces waste pollution and improves quality of cordyceps militaris. The method adopts the measures that the culture conditions are optimized when the first-stage strain and the second-stage strain of the cordyceps militaris are cultured, the conventional culture nutrient components such as potatoes, peptone and the like are reduced, the primary domestication is carried out by adding the waste of the culture medium of the cordyceps militaris of the sweet potato vine and the like, the vigor of the strain which is strong in nutrition is provided, and the large-scale three-stage strain culture of the fermentation tank can be realized according to the production requirement; in the fruiting body culture process, proper culture conditions are adopted, the use amount of raw materials and grains is reduced, and the production purpose is achieved by adding different proportions of cordyceps militaris culture medium waste and sweet potato vine and a scientific culture method;
4. according to the method provided by the application, in the strain culture process, the sweet potato vine and cordyceps militaris culture medium waste is used for replacing part of potatoes, glucose, peptone and the like, so that the cost is reduced, the strain is domesticated, and the activity is improved;
5. the method provided by the application can improve the quality and biological index of the cordyceps militaris, can increase the content of flavone and the like in the fruiting body of the cordyceps militaris, and can enhance the antioxidation capability;
6. according to the method provided by the application, benefit analysis is roughly performed, and the biological conversion rate of the cordyceps militaris fruiting body is 80% based on 70 yuan per kilogram of a commercial cordyceps militaris fruiting body dry product and 2.2 yuan per kilogram of wheat grain or rice.
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 embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:
FIG. 1 is a schematic diagram showing the growth of the 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 a Cordyceps militaris secondary liquid seed, 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 of the primordial germination 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 schematic diagram of functional index of fruiting bodies of Cordyceps militaris in various examples.
Detailed Description
In order to more clearly illustrate the general concepts of the present application, the following detailed description is given by way of example. 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 invention may be practiced without one or more of these details. In other instances, well-known features have not been described in detail in order to avoid obscuring the invention.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise indicated, all materials, instruments and reagents used in the examples below were obtained commercially.
In the following examples, sweet potato vine and cordyceps militaris medium waste are prepared by the following methods:
1. treatment of sweet potato vine:
selecting fresh, mildew-free, rot-free and non-lignified sweet potato vine, cleaning, draining water, putting the sweet potato vine into a pulverizer, pulverizing the sweet potato vine into small sections of 2-3cm, putting the pulverized sweet potato vine 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 with a pulverizer until the sweet potato vine can pass through a 4-8 mesh sieve to obtain coarse grains (i.e. sweet potato vine with a particle size of 4-8 meshes); further pulverizing the dried sweet potato vine with pulverizer to obtain sweet potato vine powder (i.e. sweet potato vine with particle size of 20-40 meshes) which can pass through 20-40 meshes.
2. Preparing cordyceps militaris culture medium waste:
preparing a nutrient solution: 0.5g/L of magnesium sulfate, 2g/L of monopotassium phosphate and 0.1g/L of glycine;
preparing solid materials: 100 parts of wheat grains are put into a 500ml capacity culture flask;
the weight ratio of the raw materials to the solid materials is 1: adding the nutrient solution into the mixture according to the 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 by using rubber bands, putting the culture bottle into a steam sterilization pot for sterilization at 121 ℃ for 60-70 min, and cooling to room temperature to obtain fresh culture materials;
inoculating Cordyceps militaris seeds (Cordyceps militaris preserved strains, or conventional culture mediums in other embodiments, such as primary culture medium or secondary culture medium without sweet potato vine and waste in the application) to the fresh culture medium under aseptic environment, wherein the inoculation volume of the strains is 6-8% of the volume of the culture medium, and culturing in dark at 18-22deg.C; after the white hypha grows fully to cover the surface of the culture material in the 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 the mycelium is changed into orange, uniformly puncturing holes and ventilating the mycelium at a culture bottle opening by using a sterile needle, growing out primordium protrusions, culturing under the environment with the air humidity of 85-95%, and harvesting when the top end of the cordyceps militaris fruiting body slightly expands and is up to the bottle opening;
the culture material left after harvesting the cordyceps militaris fruiting body is called cordyceps militaris culture medium waste, the cordyceps militaris culture medium waste which is free of mildew, pollution and dirt and rich in cordyceps militaris mycelia is selected, fruiting body residues are selected and discarded (the culture material is subjected to extracellular enzymatic hydrolysis of the fruiting body to reduce toughness and is easy to break), the fruiting body residues are crushed and dispersed by a rubbing machine, the particle size is 0.4-0.6 cm, and the fruiting body residues are placed in a blast drying oven with the temperature of 60-80 ℃ to be dried until the water content is 8-12%, and are packaged for standby.
Example 1
The method for culturing cordyceps militaris by using sweet potato vine and cordyceps militaris culture medium waste as main raw materials in the embodiment comprises the following steps of:
step one, preparation of first-class seeds of cordyceps militaris
Mixing 100g/L potato, 10g/L Cordyceps militaris culture medium waste, 5g/L sweet potato vine powder particles (particle size 20-40 mesh), boiling for 20-25 min, and filteringFiltering with cloth to obtain filtrate, adding 10g/L glucose, 2.5g/L peptone, 1.5g/L magnesium sulfate, 3g/L potassium dihydrogen phosphate, 10mg/L vitamin B 1 Mixing 10g/L agar, loading into test tube, sterilizing the test tube in steam sterilizing pot at 121deg.C for 30min, cooling to room temperature, inoculating Cordyceps militaris preservation strain under aseptic condition, cutting into 4×4mm mycelium slices with inoculating hook at a position far away from the original inoculating point, shoveling out the mycelium slices with inoculating shovel, placing into the middle lower part of fresh first-stage seed inclined plane, slightly pressing, culturing at 18-22deg.C in dark place, stopping culturing after full tube, and preserving at low temperature to obtain Cordyceps militaris first-stage seed.
Step two, preparation of cordyceps militaris secondary liquid seeds
Mixing 100g/L potato, 10g/L Cordyceps militaris culture medium waste, 4g/L sweet potato vine particles (particle size 20-40 mesh) and boiling for 20-25 min, filtering with filter cloth to obtain filtrate, adding 10g/L glucose, 2.5g/L peptone, 1.5g/L magnesium sulfate, 3g/L potassium dihydrogen phosphate, 10mg/L vitamin B 1 Mixing uniformly, filling into a triangular flask, inoculating the first-stage cordyceps militaris seeds cultured in the first step under aseptic environment, placing into a constant-temperature shaking table with the temperature of 18-22 ℃ and the rotating speed of 140 rpm, and culturing for 3.5-4.5 days in a dark place until mycelia are robust and the enriched ratio of fungus balls reaches 50-70%, and stopping culturing to obtain the second-stage cordyceps militaris liquid seeds.
In this example, the second liquid seeds of Cordyceps militaris were used for grass-growing cultivation.
Optionally, the three-level liquid strain of the cordyceps militaris can be synchronously produced in the fermentation tank according to the requirement, so that the requirement of large-scale cultivation is met. The preparation method of the culture material is the same as that of the second-stage liquid seeds of the cordyceps militaris, and the culture conditions are as follows: the filling volume is 60% of the internal volume of the tank, the temperature is reduced to 22-24 ℃ after actual elimination, the second-level liquid seeds of Cordyceps militaris are inoculated according to the filling volume of the tank, the inoculation amount is 3-5%, the tank pressure is 0.02-0.06mpa, the rotating speed is 130-150rpm, the fermentation time is 2-3 days, and the cultivation is stopped when the hypha is strong and the enriched volume of the fungus balls reaches 50-70%. The growth of the three-stage liquid strain is shown in Table 2.
Step three, culturing cordyceps militaris fruiting bodies
Preparing a fruiting body culture material:
preparing a nutrient solution: 0.5g/L of magnesium sulfate, 2g/L of monopotassium phosphate and 0.1g/L of glycine;
preparing solid materials: 50 parts of wheat grains, 40 parts of cordyceps militaris culture medium waste and 10 parts of sweet potato vine coarse particles (with the particle size of 4-8 meshes) are placed into a 500ml capacity culture bottle;
the weight ratio of the raw materials to the solid materials is 1: adding the nutrient solution into the mixture according to the 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 rubber band, sterilizing the culture bottle in a steam sterilizing pot at 121deg.C for 60-70 min, and cooling to room temperature to obtain fruiting body culture material;
inoculating the cordyceps militaris secondary liquid seed prepared in the second step into a fruiting body culture material under a sterile environment, and culturing in a dark place at the temperature of 18-22 ℃ with the inoculum size of 6-8%; after the white hypha grows fully to cover the surface of the culture material in the 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 the mycelium is changed into orange, uniformly puncturing holes and ventilating the mycelium at a culture bottle opening by using a sterile needle, growing out primordium protrusions, culturing under the environment with the air humidity of 85-95%, and harvesting when the top end of the cordyceps militaris fruiting body slightly expands and is up to the bottle opening.
In an embodiment, in the culturing process of the cordyceps militaris fruiting body in the third step, when the primordial protuberance is just formed after the color of the mycelium is changed, the culturing can be stopped according to the production purpose, namely, the cordyceps militaris fruiting body primordium is formed, and the mycelium in the culturing material is rich at the moment and is rich in nutritional ingredients such as protein, sugar and amino acid, and the cordyceps militaris fruiting body culturing method can be used for developing downstream products such as health-care products.
Example 2
The preparation method of the embodiment is approximately the same as that of the embodiment 1, except that 15g/L of cordyceps militaris medium waste and 6g/L of sweet potato vine powder particles are used for preparing the second-stage liquid seeds of cordyceps militaris; the solid materials in the culture material of the fruiting body in the step three are 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 approximately the same as that of the embodiment 1, except that in the preparation of the second-stage liquid seed of the cordyceps militaris, 20g/L of waste of a cordyceps militaris culture medium and 8g/L of sweet potato vine particles are adopted; the solid materials in the culture material of the fruiting body in the step three 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 approximately the same as that of the embodiment 1, except that 25g/L of cordyceps militaris medium waste and 10g/L of sweet potato vine particles are used for preparing the second-stage liquid seeds of cordyceps militaris; the solid materials in the culture material of the fruiting body in the step three 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 approximately the same as that of the embodiment 1, except that 30g/L of cordyceps militaris medium waste and 12g/L of sweet potato vine powder particles are used for preparing the second-stage liquid seeds of cordyceps militaris; the solid materials in the culture material of the fruiting body in the step three 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 substantially identical to the preparation method of example 1, except that:
the composition of the primary medium was as follows: boiling potato according to 200g/L for 20-25 min, filtering with filter cloth to obtain filtrate, adding glucose 20g/L, peptone 5g/L, magnesium sulfate 1.5g/L, potassium dihydrogen phosphate 3g/L, and vitamin B respectively 1 10mg/L, agar 10g/L.
The composition of the secondary medium was as follows: adding 200g/L potato into water, boiling for 20-25 min, filtering with filter cloth to obtain filtrate, adding 20g/L glucose, 5.0g/L peptone, 1.5g/L magnesium sulfate, 3g/L potassium dihydrogen phosphate and vitamin B respectively 1 10 mg/L。
In the sporophore culture material, 100 parts of wheat grains are used as solid materials, and the parameters of the rest steps are the same.
Comparison of culture results:
the main nutritional ingredients of the cordyceps militaris culture medium waste in each example are shown in table 1; the growth vigor and the culture condition of the obtained first-stage cordyceps militaris seeds are shown in the figure 1; the culture conditions of the cordyceps militaris secondary liquid seeds are shown in table 2 and figure 2; the culture conditions of Cordyceps militaris fruiting body are shown in tables 3-5 and figures 3-5. Wherein, each example simultaneously cultivates 30 bottles of cordyceps militaris fruiting bodies, and the data in the following table, such as the growth period in table 2 and various indexes in tables 3-5, are all average values of 30 bottles of cordyceps militaris fruiting body test data.
TABLE 1
| Crude protein | Coarse fibers | Starch | Calcium | 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 seeds of Cordyceps militaris prepared in comparative example 1 and examples 1-5 are thick, dense, neat and good in quality.
The growth conditions of the cordyceps militaris secondary liquid seeds in the examples are shown in table 2:
TABLE 2
| The size diameter/cm of the fungus ball | Uniformity of fungus ball | Suspension property | Growth cycle/d | |
| Examples 1 to 5 | 0.18～0.22 | Uniformity of | **** | 4.5 |
| Three-stage liquid strain | 0.20～0.22 | Uniformity of | **** | 2.5 |
| Comparative example 1 | 0.25 | More uniform | *** | 5.0 |
From table 2, it can be seen that the secondary liquid seeds of examples 1 to 5 and the third liquid strain of the fermenter, the fungus balls are slightly smaller than those of comparative example 1, the uniformity of the fungus balls is better, the suspension property is better (the more suspension property is better), which means that the strain growth state is better, the strain is easier to disperse during use, the fruiting body is easier to uniformly distribute, and the strain growth period of examples 1 to 5 and the third liquid strain of the fermenter is shorter than that of comparative example 1, which means that the strain growth can be promoted to a certain extent in the waste of sweet potato vine and Cordyceps militaris medium, and the strain quality is better.
As can be seen from FIG. 2, in both examples 1 to 5 and comparative example 1, the mycelia of Cordyceps militaris are relatively strong, the mycelia are enlarged, the mycelia are branched, the mycelia are separated, the reproduction capability is strong, the vitality is good, and the mycelia in examples 1 to 5 are richer than those in comparative example 1 and the quality of the strain is better.
The fruiting body of Cordyceps militaris in the above example shows the grass conditions in Table 3:
TABLE 3 Table 3
| Hypha germination/h | Full charge level time/h | Color conversion/d | Yield of grass percent | Primordium formation/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 the fruiting body mycelia in examples 1-5 start to germinate faster than the mycelia in comparative example 1, respectively for 24h and 28h, and the time of covering the material surface is shorter than that in comparative example 1, which indicates that the growth speed of Cordyceps militaris mycelia on the waste substitute material of potato vine and Cordyceps militaris culture medium is faster; the color conversion time of the mycelium of the comparative example 1 is long, and the formation time of the orange color primordium of the examples 2 and 3 is earlier than that of the comparative example 1, which shows that the cordyceps militaris mycelium grows well on substitutes with different proportions, and meanwhile, the growth period is shortened, so that the manpower and material resources can be reduced to a certain extent, and the energy is saved.
The biological index of Cordyceps militaris in the above example is shown in table 4:
TABLE 4 Table 4
As can be seen from Table 4 and FIG. 4, the fruit body diameters of examples 1 to 5 are slightly lower than that of comparative example 1, but the fruit body numbers are higher than that of comparative example 1, and the biological efficiency of examples 1 to 3 is slightly higher than that of comparative example 1, which shows that the waste of the culture medium of sweet potato vine and Cordyceps militaris has a certain influence on the fruit body yield, the yield is slightly increased, the deformity rate is low, the fruit bodies in the examples are good in quality and high in commodity value, and the growth period of examples 1 to 3 is shorter than that of comparative example 1.
The functional indexes of the cordyceps militaris fruiting bodies in the above examples are shown in table 5:
TABLE 5
The index detection methods in table 5 and fig. 5 are respectively: the cordycepin adopts a high performance liquid chromatography method, and the testing method comprises the following steps: accurately weighing 1.0g of cordyceps militaris fruiting body powder respectively, placing the cordyceps militaris fruiting body powder into round-bottom flasks, adding 20mL of 90% methanol solution, heating and reflux-extracting for 45min, cooling, fixing the volume to 50mL, shaking uniformly, filtering, discarding the primary filtrate, taking the subsequent filtrate, filtering with a microporous filter membrane, taking the filtrate as 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 sodium nitrite-aluminum nitrate method, the total antioxidant capacity is measured accurately by ABTS method and product number G0142F kit.
From the above graphs, the cordycepin content of the cordyceps militaris fruiting bodies in 5 examples is similar to that of the comparative examples, and is slightly improved; the flavonoid content is obviously higher than that of the comparative example, the total antioxidant capacity is obviously higher than that of the comparative example, and the functional index of the cordyceps militaris fruiting body cultivated in the examples 1-5 is higher than that of the comparative example in the comprehensive view, which shows that the quality of the cordyceps militaris fruiting body is improved by taking agricultural waste sweet potato vine and cordyceps militaris culture medium waste as substitutes. However, comparing with Table 4 and FIG. 4, it is found that examples 1 to 3 are preferable in terms of growth cycle, biological efficiency and functional index, namely, 10g/L of cordyceps militaris medium waste and 5g/L of sweet potato vine powder particles are added to the first seed, 10 to 30g/L of cordyceps militaris medium waste and 4 to 12g/L of sweet potato vine powder particles are added to the second seed, 30 to 60 parts of cordyceps militaris medium waste and 10 to 20 parts of sweet potato vine coarse particles are contained in cordyceps militaris fruiting body cultivation material, and the cultivation effect is good through a scientific cultivation method. The cultivation effect of examples 4 to 5 is not ideal and needs to be improved. In the comprehensive view, the invention successfully uses the cordyceps militaris culture medium waste and the sweet potato vine to culture cordyceps militaris for the first time, replaces the grain for culture materials, has certain economic benefit and has more obvious ecological benefit and social benefit.
The foregoing is merely exemplary of the present invention and is not intended to limit the present invention. Various modifications and variations of the present invention will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are to be included in the scope of the claims of the present invention.
Claims (9)
1. The method for cultivating high-quality cordyceps militaris fruiting bodies in a short period is characterized by comprising the following steps of:
step one: inoculating the cordyceps militaris preserved 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 seeds of the cordyceps militaris into a fruiting body culture material, and culturing until obtaining cordyceps militaris fruiting bodies;
the primary culture medium, the secondary 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 primary culture medium and the secondary culture medium is (10-30): (4-12), wherein the mass ratio of the cordyceps militaris culture medium waste to the sweet potato vine in the fruiting body culture medium is (30-60): (20-10);
the sweet potato vine adopts sweet potato vine particles treated by the following method: drying fresh sweet potato vine until the water content is 8% -12%, and crushing the fresh sweet potato vine to 4-40 meshes to obtain sweet potato vine particles; and/or the number of the groups of groups,
the cordyceps militaris culture medium waste is prepared by the following method: crushing the culture material of which the fruiting bodies of the cordyceps militaris are collected to have the particle size of 0.4-0.6 cm, and drying the culture material until the water content is 8-12% for later use;
the primary medium comprises: 100g/L crop nutrition, 10-30g/L Cordyceps militaris culture medium waste, 2-20g/L sweet potato vine particles are boiled for 20-25 min, filtered by filter cloth to obtain filtrate, 10g/L glucose, 2.5g/L peptone, 1.5g/L magnesium sulfate, 3g/L potassium dihydrogen phosphate, 10mg/L vitamin B are respectively added 1 10g/L agar;
the secondary medium comprises: 100 Boiling together with crop nutrition, cordyceps militaris culture medium waste 10-30g/L and sweet potato vine particles 2-20g/L for 20-25 min, filtering with filter cloth to obtain filtrate, and adding glucose 10g/L, peptone 2.5g/L, magnesium sulfate 1.5g/L, potassium dihydrogen phosphate 3g/L and vitamin B10 mg/L respectively 1 ；
The fruiting body culture material comprises: nutritional adjuvants, crop nutrients, cordyceps militaris culture medium waste, and sweet potato vine granule.
2. The method according to claim 1, wherein the crop nutrition in the primary medium, the secondary medium and/or the fruit body culture is selected from any one or more of potato, wheat, rice, corn.
3. The method according to claim 2, characterized in that the nutritional adjuvants in the fruiting body cultivation material are magnesium sulfate, potassium dihydrogen phosphate and glycine.
4. A method according to claim 3, characterized in that the method for preparing the fruiting body culture material comprises: mixing the crop nutrients, the cordyceps militaris culture medium waste and the sweet potato vine particles to obtain a mixed material with the total mass of 100 parts, and adding nutritional auxiliary materials with the mass of 1:1.5-2.0 g/ml based on the mixed material.
5. The method according to claim 1, wherein the culturing method in the first step comprises: aseptically inoculating cordyceps militaris preservation strains into the primary culture medium, and culturing at 18-22 ℃ in a dark place.
6. The method according to claim 1, wherein the culturing method in the second step comprises: and aseptically inoculating the first-stage cordyceps militaris seeds into the second-stage culture medium, and culturing for 3-5 days at the temperature of 18-22 ℃ and the rotating speed of 130-150rpm in a dark place.
7. The method according to claim 1, wherein the culturing method in the third step comprises: aseptically inoculating 6% -8% of cordyceps militaris secondary seeds into the fruiting body culture material, culturing at 18-22 ℃ in the dark, entering a color conversion stage, controlling the illumination intensity to be 200-400lx, controlling the air humidity to be 60% -80%, and culturing until mycelia are converted into orange colors, forming cordyceps militaris fruiting body primordia, controlling the air humidity to be 85% -95%, and harvesting.
8. A cordyceps militaris culture, which comprises cordyceps militaris fruiting body primordia prepared by the method of claim 7.
9. A cordyceps militaris fruit body prepared by the method of any one of claims 1-7.
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