CN111713334A

CN111713334A - Ecological edible fungus cultivation method

Info

Publication number: CN111713334A
Application number: CN202010595668.0A
Authority: CN
Inventors: 吴小红
Original assignee: Lianyungang Yinfeng Edible Fungi Technology Co ltd
Current assignee: Lianyungang Yinfeng Edible Fungi Technology Co ltd
Priority date: 2020-06-28
Filing date: 2020-06-28
Publication date: 2020-09-29

Abstract

The invention relates to the technical field of edible fungus cultivation, in particular to a tissue separation method by a separation tissue separation method, which comprises the steps of cutting fresh and tender tissues of rhizomorph, sporocarp and sclerotium for separation to obtain pure hyphae, and the knots of the actual binuclear hyphae such as the sporocarp and the rhizomorph have strong regeneration capability. The mushroom residue obtained after fruiting is prepared into a fermented feed, is a new environment-friendly feed product which changes waste into valuable, is rich in mineral substances such as protein, amino acid, calcium, phosphorus and the like, can be used as a basic feed raw material, is added with proper auxiliary materials for nutrition balance, and is used for preparing functional feeds with different purposes.

Description

Ecological edible fungus cultivation method

Technical Field

The invention relates to the technical field of edible fungus culture, in particular to an ecological edible fungus culture method.

Background

The history of edible mushrooms cultivated in China is long, before 1949, two kinds of mushrooms, namely mushrooms and black fungi, are cultivated by the traditional cut-log cultivation method mainly, after the country is built, the cultivation amount of the agaricus bisporus is increased year by year, the main cultivation types in the 70 th century are increased to 10 (oyster mushrooms, straw mushrooms, pholiota nameko, flammulina velutipes, tremella fuciformis, auricularia polytricha and hericium erinaceus), the cultivation types in the 80 th century are increased to 14 (grifola frondosa, tremella aurantium, poria cocos and shoestring fungi are increased), the cultivation types in the 90 th century are increased to 44, the edible mushrooms cultivated in the current scale are agaricus bisporus, basi mushrooms, hypertrophic mushrooms, delicious mushrooms, pleurotus ostreatus, pleurotus cornucopiae, pleurotus pulmonarius, pleurotus eryngii, ferulae, pleurotus eryngii, pholiota, the agaricus bisporus, stropharia rugoso-annulata, tremella aurantialba, cordyceps militaris, ganoderma lucidum, ganoderma sinense, ganoderma capense, ganoderma tsugae, poria cocos, polyporus umbellatus, armillaria mellea (associated gastrodia elata), hypsizygus marmoreus, dictyophora indusiata, dictyophora phalloidea, tricholoma lobayense, boletus edulis and tricholoma fulvum, wherein 31 of the large-scale commercial cultivation are available.

From the point of view of the variety and variety of commercial cultivation, the variety of commercial cultivation in recent years is agaricus bisporus, basci mushroom, hypertrophic mushroom, delicacy mushroom, pleurotus ostreatus, fossa pleurotus ostreatus, lung pleurotus ostreatus, pleurotus cornucopiae, pleurotus citrinopileatus, pleurotus eryngii, pleurotus ferulae, black fungus, auricularia polytricha, flammulina velutipes, pholiota nameko, straw mushroom, hericium erinaceus, agrocybe aegerita, coprinus comatus, tremella, ganoderma lucidum, ganoderma sinensis, ganoderma capense, ganoderma lucidum, ganoderma tsumadai, poria cocos, gastrodia elata armillaria, hypsizygus marmoreus, dictyophora indusiata and the like, the variety and variety of cultivars ensure the yearly cultivation and continuous development of edible fungi in the agricultural production mode in China, and the traditional cultivation mode has the defects of low yield and poor quality in edible fungi cultivation, therefore, an ecological edible fungus culture method is provided.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides an ecological edible fungus culture method, which solves the problem of low yield of the existing edible fungus during planting.

(II) technical scheme

In order to achieve the purpose, the invention provides the following technical scheme: the method for cultivating the ecological edible fungus strain is characterized by comprising the following analysis steps

Step one separation

Tissue separation method the tissue separation method is to cut fresh tender tissue of the strongylocentrotus, the sporocarp and the sclerotium for separation to obtain pure hyphae, the actually binuclear hypha knots such as the sporocarp and the strongylocentrotus, and the method can be divided into the strongylocentrotus tissue separation method, the sporocarp tissue separation method and the sclerotium tissue separation method because the selected materials are different during separation.

Spore separation method is to germinate mature ascospore or sexual spore in ascocarp or sporocarp into pure hypha, and further culture into bacteria.

Screening in step two

The method for screening antibiotic producing bacteria includes separating pure strain from soil, culturing on plate containing agar culture medium, transferring the strain block to agar culture medium containing test bacteria by means of hole puncher, culturing at proper temperature for a certain time and taking out, if the periphery of the strain block has transparent bacteriostatic ring, it shows that said strain possesses the capability of producing antibiotic substance for inhibiting growth of test bacteria, and the obtained strain is undergone the process of shake flask liquid fermentation, and the content of antibiotic in the fermentation liquor or mycelium can be measured, so that the strain with high production capacity can be selected.

On the other hand, the extracted antibiotics are subjected to structural analysis, pharmacological test, clinical test and the like, and the antibiotics can be used as production strains, and the lipase production strains can be screened by coating the separated mould on an agar culture medium containing tallow, culturing for a certain time at constant temperature, and if transparent circles appear around bacterial colonies, the bacteria have the capability of decomposing fat, further performing shake flask fermentation to determine the enzyme activity, and selecting the strains with high yield as starting strains of the production strains.

Improvement of step three

The genetic breeding method is used for mutating and recombining genetic factor DNA of wild strains (original strains separated and screened from the nature) so as to select excellent strains which have high yield, good quality of finished products or new culture characteristics such as product inhibition resistance, low-cost raw materials and the capability of producing new varieties.

Subsequently, a mutant strain is selected, and a high-producing strain is selected therefrom. In recent years, along with the development of fermentation metabolism control research, yield or other characteristics can be improved by changing reaction points in biosynthetic pathways according to the reaction points, such as breeding of mutant strains resistant to product feedback inhibition, mutant strains capable of increasing cell permeability, mutant strains with auxotrophy and the like, and the rational screening method is widely applied to breeding of amino acid producing bacteria.

Step four preparation

The method is characterized in that a strain is subjected to amplification culture under a certain condition to obtain a preparation process of pure production strains with a certain quantity and quality, the preparation process is used for further amplifying the strain quantity and synthesizing products in a fermentation tank, seed preparation comprises spore preparation and mycelium preparation, the strain is stored in a production strain in a sandy soil pipe or a freezing pipe, a small amount of strain is picked in an aseptic operation, the strain is inoculated on an agar slant culture medium and cultured for 5-7 days (or a longer time) at 25 ℃ (or a higher temperature), the obtained spores are further cultured for more spores by using a solid culture medium with a larger surface area, for preparation of mould spores, most of natural culture media such as rice and millet are adopted, the mature slant spores are prepared into a suspension, the suspension is inoculated on a flat bottle solid culture medium and cultured for 14 days at 25-28 ℃, the mature flat bottle spores are dried in vacuum, and the water content is reduced to be below 10%, putting the mixture into a refrigerator at 4 ℃ for standby, wherein a spore bottle prepared at one time can be used for about half a year in production, most of culture mediums of actinomycetes spores are prepared by bran or pea leachate, beef extract, inorganic salt and agar, slant spores are inoculated into an expanded flat bottle spore culture medium and are cultured for 5-14 days at 28-37 ℃, a large number of obtained spores can be directly used as seeds of a seed tank, if some production strains do not produce spores, such as gibberellin-producing bacteria or few spore-producing bacteria, a shake flask liquid culture can be adopted to prepare mycelia serving as seeds of the seed tank, the seed tank aims to ensure that the inoculated limited spores or mycelia rapidly germinate, grow and reproduce into a large number of thalli, and the components of the culture mediums are carbon sources (such as glucose), nitrogen sources (such as corn steep liquor and the like) and inorganic salt (such as phosphate) which are easy to be utilized by the thalli, the seeds used as the fermentation tank (7) are vigorous in vitality, deep in dyeing, thick and strong in hypha, free of mixed bacteria and abnormal bacteria, and the inoculation amount is generally 10-20%.

Step five storage

The method comprises the following steps of putting an agar slant spore culture, a hypha suspension and a spore culture prepared from cereal raw materials such as bran, rice, millet and the like into a refrigerator for storage, wherein the storage time is not more than 1-2 months, and if a spore bottle prepared from the cereal raw materials is vacuumized and wax-dipped on a cotton plug, the external air and water vapor are isolated, and the storage time can reach 3-4 months;

secondly, a liquid nitrogen ultra-low temperature preservation method, which is to suspend the cells in the growth stabilization period in 10 percent of glycerin or other low freezing point liquid, seal the cells in an ampoule tube, control the cooling speed to ensure that the temperature of the ampoule tube is gradually reduced to-35 ℃, and then the ampoule tube can be placed in a liquid nitrogen tank at the temperature of-150 to-196 ℃ for preservation, and most microorganisms such as viruses, phages, various bacteria, actinomycetes, yeasts and protozoa, especially some microorganisms which are difficult to freeze drying can be preserved for a long time by the method.

A sand-soil preservation method for culturing the spore-forming bacteria, actinomycetes and mould includes such steps as proportionally mixing sand with soil (3: 2), diluted acid treating, washing, sieving, loading in small test tube (1 cm in height), sterilizing for 2-3 times, baking to obtain spore suspension, adding 2-2.5 ml of sterile distilled water, sucking a little sand-soil tube, vacuum pumping to obtain loose appearance, and preserving in refrigerator at 4 deg.C for 5-7 years.

Preferably, the strain in the fourth step is cultured and grown to 13-15 cm.

Preferably, the illumination culture in the fourth step is performed by using a fluorescent lamp.

Preferably, the five-point refrigerator temperature of the step is 4 ℃.

Preferably, the fungus bag is cultured in a greenhouse with the temperature of 15-17 ℃ and the humidity of 65-75%, the temperature in the greenhouse is adjusted to be 19-28 ℃ and the humidity of 80-85% after fruiting, and meanwhile, the greenhouse is ventilated for 2-3 hours every day.

Preferably, the temperature of the culture medium in the fourth step is reduced to be below 20 ℃ for inoculation of the strain.

(III) advantageous effects

The invention provides an ecological edible fungus cultivation method. The method has the following beneficial effects:

the method is characterized in that the original greenhouse is checked and repaired for standby in advance, the greenhouse is built before fruiting when the greenhouse is not built, the site is selected when the greenhouse is built, the greenhouse is generally selected to be capable of draining when soil is loose, semi-sunny and waterlogging, and watering when drought occurs, the water resource is rich, the road traffic is convenient, the greenhouse is built at a place where various production data and products are convenient to transport, forests with early germination and late leaf fall are selected as cultivation places under the forest, and the conditions of roads, water wells, electric power facilities and the like in the forest are preferably considered under the forest. The edible fungi cultivated by the nutrient medium for cultivating the edible fungi have good flavor and rich nutrition; the mushroom residue obtained after fruiting is prepared into a fermented feed, is a new environment-friendly feed product which changes waste into valuable, is rich in mineral substances such as protein, amino acid, calcium, phosphorus and the like, can be used as a basic feed raw material, is added with proper auxiliary materials for nutrition balance, and is used for preparing functional feeds with different purposes. Can also replace part of concentrated feed such as fish meal and bean cake, etc., to reduce cost.

Detailed Description

The invention provides a technical scheme that: the method for cultivating the ecological edible fungus strain is characterized by comprising the following analysis steps

Step one separation

Tissue isolation method the tissue isolation method is to cut fresh tender tissue of the strongylocentrotus, the sporocarp and the sclerotium for isolation to obtain pure hyphae, the actually binuclear hypha knots such as the sporocarp and the strongylocentrotus, and the method can be divided into the strongylocentrotus tissue isolation method, the sporocarp tissue isolation method and the sclerotium tissue isolation method due to different materials selected during isolation, and in practice, the sporocarp tissue isolation method is the most effective and commonly used method. However, for the colloid fungi, because the amount of hypha contained in the fruit body is low, the difficulty of tissue isolation and culture is high for the black fungus, the white fungus and the like. Tissue separation belongs to the category of asexual propagation, can maintain the characteristics of original strains, has small genetic stability variation, is not only suitable for edible fungi with difficult collection or germination of spores, but also suitable for stabilizing the genetics of excellent varieties.

The spore separating method is to germinate mature ascospore or sexual spore in ascocarp or sporophore into pure hypha and further culture into hypha with strong activity and short fungus age. Only the individual difference and the common natural differentiation phenomenon exist among spores, the variation is large, and the spores can be used for producing medium-sized balls after fruiting tests. For spore-forming fungi, spore isolation is generally used.

Screening in step two

On the other hand, the extracted antibiotics are subjected to structural analysis, pharmacological tests, clinical tests and the like, and are determined to be effective, and can be used as production strains. And if the fungus strain obtained by the separation is coated on an agar culture medium containing tallow, the fungus strain is cultured for a certain time at constant temperature, and if transparent circles appear around the colony, the fungus strain has the capability of decomposing fat, and is further subjected to shake flask fermentation to determine the enzyme activity, and the fungus strain with high yield is selected as the starting strain of the production strain.

Improvement of step three

Step four preparation

It is a preparation process of pure production strains with certain quantity and quality through the amplification culture of strains under certain conditions. For inoculating into a fermentation tank to further expand the amount of bacteria and synthesize the product. The seed preparation includes spore preparation and mycelium preparation. The strain is preserved in a production strain in a sandy soil tube or a freezing tube, a little strain is picked in an aseptic operation, is inoculated into an agar slant culture medium, and is cultured for 5-7 days (or a longer time) at 25 ℃ (or a higher temperature). The obtained spores need to be further cultured in a solid medium with a larger surface area to obtain more spores, and for the preparation of mold spores, natural culture mediums such as rice and millet are mostly adopted. And preparing the mature bevel spores into a suspension, inoculating the suspension onto a flat bottle solid culture medium, and culturing for 14 days at 25-28 ℃. The mature oblate spores are dried in vacuum to reduce the water content to below 10 percent and put into a refrigerator at 4 ℃ for standby. The spore bottle prepared at one time can be used for about half a year in production. The culture medium of actinomycetes spore is prepared with bran or pea extract, beef extract, inorganic salt and agar. Inoculating the slant spores into an expanded flat flask spore culture medium, and culturing for 5-14 days at 28-37 ℃. The obtained large amount of spores can be directly used as seeds of a seeding tank, if some production strains do not produce spores, such as gibberellin-producing bacteria or few spore-producing bacteria, mycelium can be prepared by adopting liquid culture in a shaking flask and used as seeds of the seeding tank, the seeding tank aims to ensure that the inoculated limited spores or mycelium rapidly germinates, grows and breeds into a large amount of thalli, wherein the components of a culture medium comprise a carbon source (such as glucose) and a nitrogen source (such as corn steep liquor) which are easily utilized by the thalli, inorganic salt (such as phosphate) and the like, the seeds used as a fermentation tank need to be vigorous in vitality, deep in dyeing, thick in hypha, free of infectious microbes and abnormal thalli, and the inoculation amount is generally 10-20%.

Step five storage

Preferably, the strain in the fourth step is cultured and grown to 13-15 cm.

Preferably, the five-point refrigerator temperature of the step is 4 ℃.

Example 1

Culturing at 20 deg.C for 5-7 days (or longer). The obtained spores need to be further cultured in a solid medium with a larger surface area to obtain more spores, and for the preparation of mold spores, natural culture mediums such as rice and millet are mostly adopted. And preparing the mature bevel spores into a suspension, inoculating the suspension onto a flat bottle solid culture medium, and culturing for 14 days at the temperature of 20-25 ℃. The mature oblate spores are dried in vacuum to reduce the water content to below 10 percent and put into a refrigerator at 4 ℃ for standby. The spore bottle prepared at one time can be used for about half a year in production.

Example 2

Culturing at 25 deg.C (or higher temperature) for 5-7 days (or longer time). The obtained spores need to be further cultured in a solid medium with a larger surface area to obtain more spores, and for the preparation of mold spores, natural culture mediums such as rice and millet are mostly adopted. And preparing the mature bevel spores into a suspension, inoculating the suspension onto a flat bottle solid culture medium, and culturing for 14 days at the temperature of 20-25 ℃. The mature oblate spores are dried in vacuum to reduce the water content to below 10 percent and put into a refrigerator at 4 ℃ for standby. The spore bottle prepared at one time can be used for about half a year in production.

Example 3

Culturing at 30 deg.C (or higher temperature) for 5-7 days (or longer time). The obtained spores need to be further cultured in a solid medium with a larger surface area to obtain more spores, and for the preparation of mold spores, natural culture mediums such as rice and millet are mostly adopted. And preparing the mature bevel spores into a suspension, inoculating the suspension onto a flat bottle solid culture medium, and culturing for 14 days at 25-30 ℃. The mature oblate spores are dried in vacuum to reduce the water content to below 10 percent and put into a refrigerator at 4 ℃ for standby. The spore bottle prepared at one time can be used for about half a year in production.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Claims

1. The method for cultivating the ecological edible fungus strain is characterized by comprising the following analysis steps

Step one separation

The tissue separation method is to cut fresh tender tissues of the strongylocentrotus, the sporocarp and the sclerotium for separation so as to obtain pure hyphae, wherein the sporocarp, the strongylocentrotus and the like are actually knots of binuclear hyphae and have strong regeneration capacity;

spore separation method is to germinate mature ascospore or sexual spore in ascocarp or sporocarp into pure hypha, and further culture into bacteria;

screening in step two

The method for screening antibiotic producing bacteria includes separating pure strain from soil, culturing on plate with agar culture medium, transferring the strain block to agar culture medium with test bacteria with hole puncher, culturing at proper temperature for certain time and taking out, and if there is transparent bacteriostasis ring around the strain block, the strain is proved to have the capacity of producing antibiotic matter for inhibiting the growth of the test bacteria;

on the other hand, the extracted antibiotics are subjected to structural analysis, pharmacological test, clinical test and the like, and the antibiotics are effective and can be used as production strains, and for example, the method for screening lipase production strains is to coat the separated mould on an agar culture medium containing tallow, and culture the mould at constant temperature for a certain time, if transparent circles appear around bacterial colonies, the mould has the capability of decomposing fat, further performing shake flask fermentation to determine the enzyme activity, and selecting the strains with high yield as the starting strains of the production strains;

improvement of step three

The genetic breeding method is adopted to mutate and recombine the genetic factor DNA of a wild strain (an original strain separated and screened from the nature) so as to select a good strain which has high yield, good quality of finished products or new culture characteristics such as product inhibition resistance, low-price raw materials and the capability of producing new varieties, and the method comprises the steps of mutation breeding, crossbreeding, cell fusion technology and recombinant DNA technology, wherein the mutation breeding is to treat a monospore suspension of a production strain by using physical or chemical mutagens such as ultraviolet rays, cobalt-60, ethylene imine and the like so as to obtain an induced mutant strain;

in recent years, along with the development of fermentation metabolism control research, yield or other characteristics can be improved by changing reaction points in a biosynthesis pathway according to the reaction points and through the change of the reaction points, such as breeding of mutant strains resistant to product feedback inhibition, mutant strains increasing cell permeability, auxotrophic mutant strains and the like, and the rational screening method is widely applied to breeding of amino acid producing bacteria;

step four preparation

The method is characterized in that a strain is subjected to amplification culture under a certain condition to obtain a preparation process of pure production strains with a certain quantity and quality, the preparation process is used for further amplifying the strain quantity and synthesizing products in a fermentation tank, seed preparation comprises spore preparation and mycelium preparation, the strain is stored in a production strain in a sandy soil pipe or a freezing pipe, a small amount of strain is picked in an aseptic operation, the strain is inoculated on an agar slant culture medium and cultured for 5-7 days (or a longer time) at 25 ℃ (or a higher temperature), the obtained spores are further cultured for more spores by using a solid culture medium with a larger surface area, for preparation of mould spores, most of natural culture media such as rice and millet are adopted, the mature slant spores are prepared into a suspension, the suspension is inoculated on a flat bottle solid culture medium and cultured for 14 days at 25-28 ℃, the mature flat bottle spores are dried in vacuum, and the water content is reduced to be below 10%, putting the mixture into a refrigerator at 4 ℃ for standby, wherein a spore bottle prepared at one time can be used for about half a year in production, most of culture mediums of actinomycetes spores are prepared by bran or pea leachate, beef extract, inorganic salt and agar, slant spores are inoculated into an expanded flat bottle spore culture medium and are cultured for 5-14 days at 28-37 ℃, a large number of obtained spores can be directly used as seeds of a seed tank, if some production strains do not produce spores, such as gibberellin-producing bacteria or few spore-producing bacteria, a shake flask liquid culture can be adopted to prepare mycelia serving as seeds of the seed tank, the seed tank aims to ensure that the inoculated limited spores or mycelia rapidly germinate, grow and reproduce into a large number of thalli, and the components of the culture mediums are carbon sources (such as glucose), nitrogen sources (such as corn steep liquor and the like) and inorganic salt (such as phosphate) which are easy to be utilized by the thalli, the seeds used as the fermentation tank have vigorous vitality, deep dyeing, thick and strong hyphae, no mixed bacteria and abnormal thalli, and the inoculation amount is generally 10 to 20 percent;

step five storage

secondly, a liquid nitrogen ultralow temperature preservation method is adopted, cells in a growth stabilization period are suspended in 10% glycerol or other low freezing point liquid and sealed in an ampoule tube, then the cooling speed is controlled, the temperature of the ampoule tube is gradually reduced to-35 ℃, and the ampoule tube can be placed in a liquid nitrogen tank at the temperature of-150 to-196 ℃ for preservation, and most microorganisms such as viruses, phages, various bacteria, actinomycetes, yeasts and protozoa, particularly some microorganisms which are difficult to freeze drying can be preserved for a long time by the method;

2. The method for cultivating ecological edible fungus strains according to claim 1, wherein the method comprises the following steps: and culturing and growing the strains to 13-15 cm.

3. The method for cultivating ecological edible fungus strains according to claim 1, wherein the method comprises the following steps: and in the fourth step, fluorescent lamp illumination culture is selected during illumination culture.

4. The method for cultivating ecological edible fungus strains according to claim 1, wherein the method comprises the following steps: the temperature of the five-point refrigerator in the step is 4 ℃.

5. The method for cultivating ecological edible fungus strains according to claim 1, wherein the method comprises the following steps: and step four, culturing the fungus bags in a greenhouse with the temperature of 15-17 ℃ and the humidity of 65-75%, adjusting the temperature in the greenhouse to be 19-28 ℃ and the humidity to be 80-85% after fruiting, and ventilating the greenhouse for 2-3 hours every day.

6. The method for cultivating ecological edible fungus strains according to claim 1, wherein the method comprises the following steps: and in the fourth step, the temperature of the culture medium is reduced to be below 20 ℃ for inoculation of strains.