CN114073214A - Cultivation and cultivation method of selenium-rich oyster mushrooms - Google Patents
Cultivation and cultivation method of selenium-rich oyster mushrooms Download PDFInfo
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Abstract
The invention relates to a cultivation and cultivation method of selenium-rich oyster mushroom, which is characterized in that a selenium-rich oyster mushroom cultivation strain is prepared and obtained after ultraviolet radiation mutagenesis and domestication screening, wherein parameters of the ultraviolet radiation mutagenesis are 40-70 seconds of ultraviolet irradiation, and the irradiation dose is 3-6 erg/second/mm2(ii) a And then inoculating the selenium-rich oyster mushroom cultivation strain into the neem selenium-containing solid cultivation material, and taking apart the bag opening after hypha grows over the fungus bag, so as to grow out the fruiting body, thereby obtaining the selenium-rich oyster mushroom. According to the method, ultraviolet radiation is adopted to mutagenize oyster mushroom spores to generate mycelia with enhanced selenium enrichment capacity, and neem is added in the processes of domestication and cultivation of selenium-enriched oyster mushroom strains, so that the whole production process of the selenium-enriched oyster mushrooms is organized, and the safety is guaranteed; the invention improves selenium-rich oyster mushroomThe selenium absorption rate and the organic conversion rate of the strain increase the content of the selenium-rich oyster mushroom organic selenium to 600mg/kg of 100-.
Description
Technical Field
The invention relates to the technical field of modern agricultural cultivation, in particular to a cultivation and cultivation method of selenium-rich oyster mushrooms.
Technical Field
Selenium is a trace element which is required to be obtained from the outside by a human body and cannot be synthesized by the human body. Selenium has effects in resisting oxidation, resisting tumor, prolonging life, preventing cardiovascular and cerebrovascular diseases, enhancing immunity, antagonizing heavy metals, and preventing and treating diabetes. Selenium deficiency in human body can cause muscular dystrophy, myocardial degeneration, hepatic necrosis, immunologic function reduction, keshan disease, Kaschin-Beck disease, etc. Selenium exists in the earth crust but is relatively rare, and selenium resources are not uniformly distributed, so that most of the countries and most of the areas in China are still in a state of selenium deficiency. Selenium exists in nature in the form of inorganic selenium and organic selenium, but the toxicity of inorganic selenium is high, and the bioavailability is low. Inorganic selenium can be converted into organic selenium by organism, and can be combined with macromolecular substances in organism to form organic selenium polysaccharide, organic selenium protein, organic selenium amino acid, organic selenium nucleic acid, etc. Compared with inorganic selenium, the biological source organic selenium has the advantages of high absorption and utilization rate, safety, no toxicity and the like.
The oyster mushroom has the advantages of multiple varieties, fast growth, large biomass, convenience for large-scale cultivation, no influence from seasons, rich nutrient components such as protein, amino acid, polysaccharide, vitamin, mineral elements and the like, delicious taste and very strong selenium enrichment capability proved by the cultivation process. The cultivation of the functional selenium-rich oyster mushroom by using the selenium-rich culture medium is beneficial to improving the current situation of selenium deficiency in 70% of areas in China, is beneficial to improving the dietary structure of residents in China, and can also improve the economic value and the nutritional value of the oyster mushroom, thereby developing a new thought and developing a new economic growth point for the field of oyster mushroom cultivation.
The method for cultivating selenium-rich oyster mushrooms by adopting the traditional culture medium and cultivation materials in the prior art has the advantages that the utilization rate of selenium is relatively low, the oyster mushrooms have a balance point on the absorption and conversion of selenium in the growth process, and the too high inorganic selenium concentration in the cultivation materials can cause strain poisoning, so that the low yield and the low organic selenium content in the product are caused. Experiments show that in the domestication process of strains, the concentration of inorganic selenium in a culture medium of oyster mushroom hypha bred by ultraviolet mutation reaches 0.25%, and the growth and development of the hypha are inhibited. When the concentration of inorganic selenium in the culture medium of the ordinary non-mutagenized oyster mushroom hypha is 0.06%, the growth and development of the hypha are inhibited. According to the invention, through ultraviolet radiation mutation breeding and the combination of applying a selenium-containing culture medium in the process of subculturing oyster mushroom mycelia, high-yield and stable-yield selenium-rich oyster mushroom strains are domesticated, and the method of adding the neem into the solid culture material can greatly improve the contents of organic selenium and total selenium in the oyster mushrooms and improve the disease and insect resistance of the selenium-rich oyster mushrooms.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a novel method for cultivating and cultivating selenium-rich oyster mushrooms, which can greatly improve the total selenium content and the organic selenium content in the oyster mushrooms, improve the selenium absorption rate and the conversion rate of the oyster mushrooms and improve the disease and insect resistance of the oyster mushrooms.
The invention provides a cultivation and cultivation method of selenium-rich oyster mushrooms, which is characterized in that selenium-rich oyster mushroom cultivation strains are prepared and obtained after ultraviolet radiation mutagenesis and domestication screening, wherein parameters of the ultraviolet radiation mutagenesis are 40-70 seconds of ultraviolet irradiation, and the irradiation dose is 3-6 erg/second/mm2(ii) a And then inoculating the selenium-rich oyster mushroom cultivation strain into the neem selenium-containing solid cultivation material, and taking apart the bag opening after hypha grows over the fungus bag, so as to grow out the fruiting body, thereby obtaining the selenium-rich oyster mushroom.
Preferably, the cultivation method of the selenium-rich oyster mushroom cultivation strain comprises the following steps:
step one, ultraviolet radiation mutagenesis: inoculating candidate Pleurotus Ostreatus mycelium onto flat plate, culturing at 30 deg.C for 20-30 days to obtain large amount of Pleurotus Ostreatus spore, diluting with liquid culture medium, and culturing to 5mm2The spores of (1) are diluted in 50mL of the first liquid medium, the first liquid medium containing the spores is placed in a dark box, and ultraviolet radiation is 40 ∞70 seconds, and the irradiation dose is 3-6 erg/s/mm2Placing the irradiated liquid culture medium I containing the spores in an incubator at 30 ℃ for culturing for 5-10 days to obtain a mutagenic strain; the preparation method of the flat plate comprises the following steps: the culture medium is 200g of peeled potatoes, 2-50 g of neem leaf extract, 20g of agar, 18g of glucose, 1.0g of monopotassium phosphate, 1.0g of dipotassium phosphate, 2g of magnesium sulfate, 110 mg of vitamin B, 20g of peptone, 1g of calcium carbonate, 2g of ferrous sulfate, 2g of zinc sulfate and 1000mL of distilled water, the raw materials are mixed, heated and boiled for 5min, filtered to remove residues, sterilized at 121 ℃ for 20min, poured on a sterile culture dish in an ultraclean workbench while hot, sealed, stood and cooled to obtain a flat plate; the preparation method of the liquid medium I comprises the following steps: 200g of peeled potato, 2-50 g of first neem leaf extract, 0.1g of sodium selenite, 18g of glucose, 1.0g of monopotassium phosphate, 1.0g of dipotassium phosphate, 2g of magnesium sulfate, 110 mg of vitamin B, 20g of peptone, 1g of calcium carbonate, 2g of ferrous sulfate, 2g of zinc sulfate and 1000mL of distilled water, mixing the raw materials, heating and boiling for 5min, filtering and removing slag, subpackaging by using 250mL triangular bottles, filling 50mL of liquid in each bottle, sterilizing at 121 ℃ for 20min, and cooling for later use.
Step two, domestication and screening: preparing a selenium-rich flat plate and a selenium-rich inclined surface, inoculating mutagenic strains obtained in the first step to the selenium-rich flat plate, placing the plate in an incubator for 5-10 days at 30 ℃, then selecting microcolonies with good growth and white and healthy hypha on the plate to be inoculated to the selenium-rich inclined surface, placing the plate in the incubator for 5-20 days at 30 ℃ to obtain a first-stage selenium-rich strain, screening mycelia of the first-stage selenium-rich strain with vigorous growth to be inoculated to a new selenium-rich inclined surface, culturing the plate at 30 ℃ for 5-20 days to obtain a second-stage selenium-rich strain, then screening the selenium-rich strain with good growth to be inoculated to the new selenium-rich inclined surface for rejuvenation once every 3 months, and obtaining domesticated strains of selenium-rich oyster mushrooms after multiple domestication and screening; the preparation method of the selenium-rich flat plate and the selenium-rich inclined plane comprises the following steps: the culture medium is two 5-50 g of neem leaf extract, 5-50 g of neem seed extract, 0.5-2 g of sodium selenite, 20g of agar, 18g of glucose and 1L of distilled water, the raw materials are mixed, heated and boiled for 5min, filtered to remove residues, quantitatively packaged into test tubes when the test tubes are hot, and the test tube openings are plugged by cotton plugs; sterilizing the rest culture medium and test tube at 121 deg.C for 20 min; taking out the test tube, swinging the test tube into an inclined plane, and cooling to obtain a selenium-rich inclined plane; pouring the rest culture medium into a sterile culture dish in an ultra-clean workbench while the culture medium is hot, and sealing and cooling to obtain a selenium-rich flat plate;
step three, preparing a culture strain: selecting the domesticated strain of the selenium-enriched oyster mushrooms obtained in the second step, inoculating the domesticated strain of the selenium-enriched oyster mushrooms into a second liquid culture medium, and performing shake cultivation at 30 ℃ and 120r/min for 7-15 days to obtain a cultivated strain of the selenium-enriched oyster mushrooms, wherein the second liquid culture medium is prepared by the following steps: dissolving 5-50 g of second neem leaf extract, 5-50 g of neem seed extract and 0.5-2 g of sodium selenite in 1L of distilled water, heating and boiling, filtering to remove residues, subpackaging with a triangular flask, plugging with a cotton plug, sterilizing at 121 ℃ for 20min, and cooling for later use.
Preferably, the liquid culture medium containing spores in the step one is subjected to ultrasonic treatment in ultrasonic waves for 3min before ultraviolet irradiation, so that the liquid culture medium is well dispersed to form monospores, and the ultrasonic power is 53KHz and the temperature is 25 ℃.
Preferably, the preparation method of the first neem leaf extract comprises the following steps: picking the neem leaves in 9-12 months, drying or sun-drying the neem leaves, crushing the neem leaves to 10-60 meshes, and mixing the neem leaves in mass ratio: adding water into the neem leaf powder at a ratio of 1: 10-20, heating to 50-80 ℃, extracting for 2-3 h, filtering to obtain an extracting solution, using filter residues as raw materials of the solid cultivation material, concentrating the filtrate to obtain a thick extract, baking the thick extract in an oven to obtain fragrance, taking out, cooling, and crushing to obtain the neem leaf extract.
Preferably, the preparation method of the second neem leaf extract comprises the following steps: picking the neem leaves in 9-12 months, drying or sun-drying the neem leaves, crushing the neem leaves to 10-60 meshes, and mixing the neem leaves in mass ratio: adding water into the neem leaf powder at a ratio of 1: 10-20, heating to 50-80 ℃, extracting for 2-3 h, filtering to obtain an extracting solution, using filter residues as raw materials of a solid cultivation material, heating the filtrate to 40-50 ℃, adding 1% of compound protease into the filtrate, stirring at a constant speed of 80r/min, keeping for 3-5 h, heating to boil for 10min after enzymolysis is completed, and inactivating enzymes to obtain the neem leaf extract.
Preferably, the preparation method of the neem seed extract comprises the following steps: collecting mature neem fruits, removing peel, drying seeds in the sun and shelling, removing oil from kernels by a squeezing method, crushing the kernel cakes without neem oil, screening by a standard sieve of 20 meshes, adding water into the kernel cakes, wherein the water addition amount is as follows by mass: neem cake powder: stirring and mixing water 1: 10-20 uniformly, leaching for 3-4 h, filtering, using filter residue, namely neem seed cake as a raw material of a solid cultivation material, heating filtrate to 60-70 ℃, adding 1% -2% of papain or subtilisin into the filtrate, stirring and mixing uniformly, performing enzymolysis for 1h, boiling for 10min, and inactivating enzyme to obtain a neem seed extract.
Preferably, the preparation method of the neem selenium-containing solid cultivation material comprises the following steps: the raw materials are prepared according to the following mass ratio: 10-20% of neem leaves and neem rods, 5-10% of neem leaf filter residues, 5-15% of neem seed cakes, 10-15% of corncobs, 0.5% of quick lime, 0.5% of gypsum, 0.005-0.2% of sodium selenite and 57-69% of water; fully mixing other raw materials except sodium selenite and water uniformly, dissolving sodium selenite with proper amount of water, adding into the mixed raw materials, adding water, fully stirring and mixing uniformly, subpackaging with polypropylene fungus bags with specification of 17 x 33mm, fastening bag openings with cotton threads, sterilizing at 121 deg.C for 20min, and cooling for later use;
preferably, the neem leaves and the neem stems are picked in 9-12 months, dried in the sun and crushed to 0.5-1 cm.
Preferably, the neem leaves and the neem stems are picked in 9-12 months, dried in the sun, crushed to 0.5-1 cm, sprayed with water with the amount of 10% of the dry materials, piled, fermented for 3-4 days, and spread out and dried in the sun to obtain the neem leaf and neem stems.
The cultivation and cultivation method of the selenium-rich oyster mushroom provided by the invention is applied to the production of the selenium-rich oyster mushroom.
The invention has the following beneficial effects:
(1) the method provided by the invention adopts ultraviolet radiation to mutagenize oyster mushroom spores to produce mycelia with enhanced selenium enrichment capability, and strains with high organic selenium conversion rate are screened under the condition that no exogenous gene is introduced, so that the method has the characteristics of safety and high efficiency. The inorganic selenium tolerance of the conventional strain is 0.06%, the inorganic selenium tolerance of the mutagenized oyster mushroom strain can reach 0.25%, the organic selenium conversion rate of the conventional oyster mushroom strain in oyster mushroom sporocarp is 81.2%, and the organic selenium conversion rate of the mutagenized oyster mushroom strain is 99.7%;
(2) according to the method, the neem is added in the process of domesticating and cultivating the selenium-rich oyster mushroom strains, and the anti-pollution and anti-pest capacities of the culture medium containing neem components in the process of domesticating the strains and cultivating the selenium-rich oyster mushroom are enhanced, so that the whole production process of the selenium-rich oyster mushroom is organized, and the safety is ensured;
(3) the invention adopts a cultivation method of domesticating and screening strains with strong selenium enrichment capacity and then obtaining the selenium-enriched oyster mushrooms through enlarged cultivation, improves the selenium absorption rate and the organic conversion rate of the selenium-enriched oyster mushroom strains, increases the organic selenium content of the selenium-enriched oyster mushrooms to 600mg/kg and ensures that the organic selenium content of the selenium-enriched oyster mushrooms obtained by the method accounts for more than 99 percent of the total selenium content.
Drawings
FIG. 1 shows healthy mutagenized Pleurotus ostreatus strains grown vigorously on a selenium-rich slant in example 1;
FIG. 2 is the mutagenized Pleurotus ostreatus strain of example 1 stopping its growth on a selenium rich slant;
FIG. 3 shows the liquid culture of selenium-enriched Pleurotus ostreatus obtained in example 1;
FIG. 4 shows the fungus sack of neem selenium-containing solid cultivation material in example 2 for 26 days;
FIG. 5 shows the fungus sack of the conventional selenium-containing solid cultivation material in example 2 at 26 days.
Detailed Description
The present invention is further illustrated by the following examples.
Example 1: contrast test for mutagenizing selenium-rich oyster mushroom strains under different ultraviolet radiation conditions
(1) Selecting oyster mushroom strain with strong stress resistance, fast growth and high yield as candidate strain A0(ii) a Candidate Strain A0Generating mutagenic bacteria B1-B6 by the following steps (2) - (5);
(2) preparing a plate culture medium, wherein the formula is as follows: 200g of peeled potato, 10g of first neem leaf extract, 20g of agar, 18g of glucose, 1.0g of monopotassium phosphate, 1.0g of dipotassium phosphate, 2g of magnesium sulfate, 110 mg of vitamin B, 20g of peptone, 1g of calcium carbonate, 2g of ferrous sulfate, 2g of zinc sulfate and 1000mL of distilled water. Heating the above materials to boil for 5min, filtering to remove residue, sterilizing at 121 deg.C for 20 min. Pouring the hot liquid into a sterile culture dish in an ultra-clean workbench, standing and cooling for later use; the preparation method of the first neem leaf extract comprises the following steps: picking the neem leaves in 9-12 months, drying or sun-drying the neem leaves, crushing the neem leaves to 10-60 meshes, and mixing the neem leaves in mass ratio: adding water into neem leaf powder at a ratio of 1: 10-20, heating to 50-80 ℃, extracting for 2-3 h, filtering to obtain an extracting solution, using filter residues as raw materials of a solid cultivation material, concentrating the filtrate to obtain a thick extract, baking the thick extract in an oven to obtain fragrance, taking out, cooling, and crushing to obtain a neem leaf extract I.
Mixing Pleurotus Ostreatus strain A0Inoculating the seeds on the flat plate, culturing the seeds in an incubator at 30 ℃ for 20-30 days to generate sporophores, and forming spores on a cover of the culture dish;
(3) preparing a first liquid culture medium, wherein the preparation method comprises the following steps: 200g of peeled potato, 10g of first neem leaf extract, 0.1g of sodium selenite, 18g of glucose, 1.0g of monopotassium phosphate, 1.0g of dipotassium phosphate, 2g of magnesium sulfate, 110 mg of vitamin B, 20g of peptone, 1g of calcium carbonate, 2g of ferrous sulfate, 2g of zinc sulfate and 1000mL of distilled water, heating and boiling the raw materials for 5min, filtering and removing residues, subpackaging by using a 250mL triangular flask, filling 50mL of liquid in each flask, sterilizing at 121 ℃, standing for 20min, and cooling for later use.
(4) Selecting spores formed in the step (2) and diluting the spores in the triangular flasks prepared in the step (3) in the liquid culture medium, wherein each triangular flask is diluted by about 5mm2The spores of the oyster mushroom are well dispersed by placing the triangular flask diluted with the spores of the oyster mushroom in ultrasonic waves for ultrasonic treatment for 3min to form monospores, wherein the ultrasonic wave power is 53KHz, and the temperature is 25 ℃. Placing the ultrasonic triangular flask in a dark box, performing ultraviolet radiation mutagenesis, selecting irradiation time of 3 levels of 40 seconds, 70 seconds and 100 seconds, and selecting irradiation dose of 3, 6 and 9 ergs/second/mm2And 3 levels, designing a two-factor three-level orthogonal test, and totally 9 tests, wherein the experimental design is shown in table 1. Culturing the irradiated spores at 30 ℃ for 5-10 days to obtain a mutagenic strain.
TABLE 1 orthogonal experimental design for ultraviolet radiation of selenium-enriched Pleurotus ostreatus mutagenic strains
The treatment was performed according to the experimental design of Table 1, and the mutagenized spores were incubated in an incubator at 30 ℃ for 5-10 days, during which time they were observed continuously, where B6、B8、B9Poor growth and marked atrophy observed starting on day 3, B1、B2、B3、B4、B5Normal spore development, B7The growth was slightly worse.
(5) And (3) preparing a selenium-rich flat plate and a selenium-rich slant culture medium. Respectively inoculating the mutagenized oyster mushroom strains obtained in the step (4) to a selenium-rich flat plate, and placing the flat plate in an incubator at 30 ℃ for 5-10 days to obtain single colonies with good hypha growth, white color and health, namely the domesticated first-level strains B.
The selenium-rich flat plate and the selenium-rich inclined plane are manufactured according to the following method: the formula of the culture medium is as follows: the traditional Chinese medicine composition comprises 10g of two neem leaf extracts, 10g of neem seed extracts, 0.5g of sodium selenite, 20g of agar, 18g of glucose and 1L of distilled water. Weighing the above raw materials, boiling for 5min, filtering to remove residue, quantitatively packaging into test tubes, and plugging the test tube with cotton plug; sterilizing the rest culture medium and test tube at 121 deg.C for 20 min; taking out the test tube, placing into an inclined plane to obtain a selenium-rich inclined plane, pouring the rest culture medium into a sterile culture dish in a super clean bench while the culture medium is hot, sealing, and cooling to obtain a selenium-rich flat plate.
The preparation method of the second neem leaf extract comprises the following steps: picking the neem leaves in 9-12 months, drying or sun-drying the neem leaves, crushing the neem leaves to 10-60 meshes, and mixing the neem leaves in mass ratio: adding water into the neem leaf powder at a ratio of 1: 10-20, heating to 50-80 ℃, extracting for 2-3 h, filtering to obtain an extracting solution, using filter residues as raw materials of the solid cultivation material, heating the filtrate to 40-50 ℃, adding 1% of compound protease into the filtrate, stirring at a constant speed of 80r/min, keeping for 3-5 h, heating to boil for 10min after enzymolysis is completed, and inactivating enzymes to obtain a neem leaf extract II.
The preparation method of the neem seed extract comprises the following steps: collecting mature neem fruits, removing peel, drying seeds in the sun and shelling, removing oil from kernels by a squeezing method, crushing the kernel cakes without neem oil, screening by a standard sieve of 20 meshes, adding water into the kernel cakes, wherein the water addition amount is as follows by mass: neem cake powder: stirring and mixing water 1: 10-20 uniformly, leaching for 3-4 h, filtering, using filter residue, namely neem seed cake as a raw material of a solid cultivation material, heating filtrate to 60-70 ℃, adding 1% -2% of papain or subtilisin into the filtrate, stirring and mixing uniformly, performing enzymolysis for 1h, boiling for 10min, and inactivating enzyme to obtain a neem seed extract.
Culturing mutagenic strain with selenium-rich plate, self-inoculating, and observing1、B2、B4、B5The hyphae grow well and white and healthy single colonies are obtained. B is3、B7The growth was slow and the hyphae were weak. B is6、B8、B9No mycelium was obtained. The results show that the high radiation dose and the long radiation time cause irreversible damage to some strains, and the activity of the strains is reduced, so that the strains cannot adapt to the high-concentration inorganic selenium.
(6) Starting from this experimental procedure, candidate species A0And mutagenized species B were treated in the same manner. Picking B obtained on the flat plate in the step (5)1、B2、B4、B5、B3、B7Respectively inoculating the microcolonies to the selenium-rich slant prepared in the step (5), and simultaneously inoculating the candidate strain A0Respectively inoculating to selenium-rich slant, culturing at 30 deg.C for 5-20 days in incubator to obtain first-stage selenium-rich strain, observing during culture period A0、B1、B2、B4、B5Good growth, full of tubes on day 8 after inoculation, B3、B7The growth was relatively slow, the hyphae were fine, and the tubes grew full on day 17 after inoculation. Respectively selecting first-stage selenium-rich mycelia with vigorous growth, inoculating to a selenium-rich slant, and culturing in an incubator at 30 deg.C for 5-20 days to obtainAnd (3) second-stage selenium-rich strains, then grafting and rejuvenating the well-grown selenium-rich strains on the inclined plane once every 3 months, and preserving the strains in a clean place at room temperature after the mycelia grow over the test tube. Multiple acclimatization to obtain selenium-rich Pleurotus Ostreatus test tube acclimatized seed (shown in figure 1), wherein A0Is a candidate strain, A is A0Domesticated strain without ultraviolet mutagenesis, B is A0Domesticating the strain of selenium-rich oyster mushroom through ultraviolet mutagenesis.
(7) Preparing a second liquid culture medium for culturing the strains according to the following method: 10g of two neem leaf extracts, 10g of neem seed extracts, 0.5g of sodium selenite and 1L of distilled water. Heating the above materials to boil, filtering to remove residue, subpackaging with 250mL triangular flask, liquid loading amount of 80mL per bottle, plugging with cotton plug, sterilizing at 121 deg.C for 20min, and cooling for use. Picking untreated candidate species A0Step (6) obtaining A0Test tube domesticated strains A and A without ultraviolet mutagenesis0The domesticated strain B of selenium-rich Pleurotus Ostreatus subjected to ultraviolet mutagenesis is about 5mm each2Respectively inoculating the culture materials into a second liquid culture medium in a triangular flask, and performing shake culture at 30 ℃ and 120r/min for 10 days to obtain selenium-enriched oyster mushroom liquid culture strains (shown in figure 3).
(8) Preparing a neem selenium-containing solid cultivation material: preparing a solid cultivation material according to the following raw materials by mass: 10% of neem leaves and neem stems, 5% of neem leaf filter residues, 5% of neem seed cakes, 10% of corncobs, 0.5% of quicklime, 0.5% of gypsum, 0.01% of sodium selenite and 68.99% of water. The preparation method of the neem leaves and the neem stems comprises the following steps: picking neem leaves and neem stems in 9-12 months, drying in the sun, and crushing to 0.5-1 cm to obtain the neem leaf and neem stems; the neem leaf filter residue is the filter residue generated when the first neem leaf extract and the second neem leaf extract are prepared by the method; the neem seed cake is the filter residue generated when the neem seed extract is prepared by the method. Mixing the above materials except sodium selenite and water, dissolving sodium selenite in appropriate amount of water, adding into the mixture, adding water, stirring, mixing, packaging with polypropylene bags (17 × 33 mm), packaging with 1kg of cultivation material per bag, tying the bag opening with newspaper and cotton thread, and sterilizing at 121 deg.C for 20 min.
(9) A obtained in the step (7)0、A、B1、B2、B4、B5、B3、B7Respectively inoculating selenium-rich liquid culture strains of oyster mushroom into a selenium-containing solid culture medium of neem, controlling the inoculation amount of each bag to be about 10mL, respectively inoculating 100 fungus bags to each strain, keeping the temperature of 20-30 ℃ and the humidity of 80% after inoculation, allowing the fungus bags to grow fungi, observing and recording the pollution number of the fungus bags, the health condition of hyphae during the fungus growing period, recording the fungus growing time after the hyphae grow to full of the fungus bags, observing that all the fungus bags are pollution-free, A, B1、B2、B4、B5The growth is good, the spawn running is fast, the hypha is white and strong, and the fungus bags are full of hypha after inoculation for 26 days; b is3、B7The growth is slow, the hyphae are white and sparse, and the fungus bags are full of hyphae 41 days after inoculation; a. the0And (3) abnormal spawn running, hypha stops growing after growing inwards to 2-4 cm from the bag opening, the color of the hypha is gradually changed from white to light yellow, the fungus bag cannot be full of the hypha, and no mushroom is produced finally. And (3) opening the bag mouth of the full fungus bag, keeping the temperature of 15-30 ℃ and the humidity of 80%, and properly illuminating and ventilating to allow fruiting bodies to grow out to obtain the selenium-rich oyster mushrooms. Collecting mature sporocarp, weighing respectively, recording fresh weight, and calculating average yield of each fungus bag. And detecting the total selenium content and the organic selenium content in the selenium-rich oyster mushrooms after drying. The results are shown in Table 2:
TABLE 2 Experimental results of selenium-enriched Pleurotus ostreatus produced by different strains
Through orthogonal tests of different ultraviolet radiation time and radiation dose, the results show that the selenium-enriched oyster mushroom strain B1/B2/B4/B5 subjected to ultraviolet mutagenesis domestication has the advantages that hyphae grow well in a high-concentration inorganic selenium cultivation material, the detoxifying capacity is higher than that of a strain not subjected to ultraviolet mutagenesis, the selenium absorption rate and the organic conversion rate are remarkably superior, the ultraviolet radiation time is selected to be 50-70 seconds, and the ultraviolet radiation dose is about 6 ergs/second/mm2It is more suitable. In particular B5, at 6 ergs/sec/mm2When the radiation dose of the selenium-enriched oyster mushroom is irradiated for 70 seconds for mutagenesis, the selenium-enriched oyster mushroom hyphae have high development speed and hyphaeThe body is white and healthy, the spawn running time is short, and the average spawn running time is 26 days; the yield of the selenium-rich oyster mushroom products is relatively high, and the average fresh weight per bag is 831g, which is higher than that of strains (819g) without ultraviolet mutagenesis; the selenium content in the dry product is 283.1mg/kg, which is higher than that of the strain without ultraviolet mutagenesis (89mg/kg), the organic conversion rate is 99.7 percent, and is higher than that of the strain without ultraviolet mutagenesis (81.2 percent).
EXAMPLE 2 comparative testing of different selenium-containing solid cultures
(1) The selenium-enriched oyster mushroom liquid culture strain B5 obtained by domestication and preparation by the method is used as a culture strain used in the following steps of the comparative experiment.
(2) The formula and the preparation method of the conventional selenium-containing solid cultivation material are as follows: 15% of corncob, 8% of corn meal, 5% of cottonseed hull, 11% of wood chip, 0.5% of gypsum, 0.5% of quicklime, 0.02% of sodium selenite and 59.98% of water. Preparing raw materials according to the content of the components, fully and uniformly mixing the other raw materials except the sodium selenite and the water, dissolving the sodium selenite with a proper amount of water, adding the dissolved sodium selenite into the mixed raw materials, adding the prepared water, fully and uniformly mixing, subpackaging by using polypropylene fungus bags with the specification of 17 x 33mm, 1kg of cultivation material in each bag, totally packaging 50 bags, tightly binding the bag openings by newspaper and cotton threads, sterilizing at 121 ℃ for 20min, and cooling for later use.
(3) The neem selenium-containing solid cultivation material of the invention is the same as the neem selenium-containing solid cultivation material in the embodiment 1.
(4) Inoculating B5 liquid culture strain into the selenium-containing solid culture material obtained in the steps (2) and (3), controlling the inoculation amount of each bag to be about 10mL, keeping the temperature to be 20-30 ℃ and the humidity to be 80%, enabling the bags to grow bacteria, and observing and recording the pollution number of the bags and the health condition of hyphae during the bacteria growing period. In 26 days, the fungus sack of the solid neem selenium-containing cultivation material is shown in fig. 4, and the fungus sack of the conventional solid neem selenium-containing cultivation material is shown in fig. 5, so that the mycelium of the solid neem selenium-containing cultivation material grows faster. And (3) recording the spawn running time when hyphae grow to full of the fungus bags, opening the bag openings, keeping the temperature of 15-30 ℃, keeping the humidity of 80%, and properly illuminating and ventilating to allow fruiting bodies to grow out, thereby obtaining the selenium-enriched oyster mushrooms. Collecting mature sporocarp, weighing respectively, recording fresh weight, and calculating average yield of each fungus bag. And detecting the total selenium content and the organic selenium content in the selenium-rich oyster mushrooms after drying. The results are shown in Table 3:
TABLE 3 Experimental results of selenium-enriched Pleurotus ostreatus produced with different selenium-containing solid cultivation materials
The solid cultivation material containing the neem has the advantages that the fungus bags are free of pollution in fungus growing and fruiting processes, the average yield is high, and due to the fact that the neem is used as a main component of the cultivation material, hypha can grow well in the high-concentration inorganic selenium cultivation material, the detoxification capability is strong, the total selenium content and the organic selenium content in the fruiting bodies are high, and the solid cultivation material has remarkable advantages in selenium absorption rate and organic conversion rate.
Example 3 comparative experiment of the Effect of different acclimatization-screening media on mutagenized species
(1) The same procedure as in example 1 was repeated to prepare selenium-rich plates and selenium-rich slant culture media.
(2) And preparing PD plates and slant culture media. The formula of the culture medium is as follows: cleaning and cutting 200g of potatoes, boiling, filtering with 4 layers of gauze, adding 20g of agar, 18g of glucose and 1g of sodium selenite (the mass fraction is 0.1%), adding 1000mL of distilled water, heating and boiling for 5min, quantitatively subpackaging test tubes when the test tubes are hot, and plugging the test tube openings with tampons; sterilizing the rest culture medium and test tube at 121 deg.C for 20 min; taking out the test tube, placing the test tube into an inclined plane to obtain a PD selenium-rich inclined plane culture medium, pouring the rest culture medium into a sterile culture dish in an ultra-clean workbench while the rest culture medium is hot, sealing, and cooling to obtain the PD selenium-rich flat culture medium.
(3) The mutagenized strain B5 obtained in the step 4 in the example 1 is respectively inoculated to the culture medium in the steps (1) and (2) in the example for domestication, the domestication process is the same as the example 1, the domesticated strain C1 is obtained after multiple domestication of a selenium-rich plate and a selenium-rich slant, and the domesticated strain C2 is obtained after multiple domestication of a PD plate and the slant.
(4) Preparation of C1 cultivated strain: inoculating C1 into liquid culture medium II to obtain C1 cultivated strain, and culturing the cultured strain in the same manner as in example 1
(5) Preparation of C2 cultivated strain: inoculating C2 into PD liquid culture medium to obtain C2 cultivated strain, wherein the PD liquid culture medium is prepared by the following steps: cleaning and cutting 200g of potatoes, boiling, filtering with 4 layers of gauze, adding 18g of glucose and 1g of sodium selenite (the mass fraction is 0.1%), supplementing 1L of distilled water, heating and boiling, subpackaging with a 250mL triangular flask, wherein the liquid loading amount of each flask is 80mL, plugging with a cotton plug, sterilizing at 121 ℃ for 20min, and cooling for later use. Selecting the domesticated strain C2 of about 5mm obtained in the step (3)2Inoculating the strain into a PD liquid culture medium in a triangular flask, and performing shake culture at 30 ℃ and 120r/min for 7-15 days to obtain the C2 cultivated strain.
(6) Preparing a neem selenium-containing solid cultivation material: preparing a solid cultivation material according to the following raw materials by mass: 15% of neem leaves and neem rods, 5% of neem leaf filter residues, 5% of neem seed cakes, 10% of corncobs, 0.5% of quick lime, 0.5% of gypsum, 0.03% of sodium selenite and 63.97% of water. The preparation method of the neem leaves and the neem stems comprises the following steps: picking neem leaves and neem stems in 9-12 months, drying in the sun, crushing to 0.5-1 cm, spraying water on the neem leaves and neem stems, wherein the water spraying amount is 10% of the dry neem materials, stacking, fermenting for 3-4 days, spreading and drying in the sun to obtain the neem tea. The rest is the same as example 1.
(8) Inoculation: inoculating a C1 culture strain into a neem selenium-containing solid culture material, inoculating a C2 culture strain into the conventional selenium-containing solid culture material in the same embodiment 2, controlling the inoculation amount of each bag to be about 10mL, keeping the temperature at 20-30 ℃ and the humidity at 80%, allowing the culture strain to grow, observing and recording the pollution number of the culture bag, the health condition of hypha during the fungus growing period, recording the fungus growing time after the hypha grows to fill the culture bag, detaching the bag opening, keeping the temperature at 15-30 ℃ and the humidity at 80%, and allowing fruiting bodies to grow under proper illumination and ventilation, thereby obtaining the selenium-rich oyster mushrooms. Collecting mature sporocarp, weighing respectively, recording fresh weight, and calculating average yield of each fungus bag. And detecting the total selenium content and the organic selenium content in the selenium-rich oyster mushrooms after drying.
The results are shown in Table 4:
TABLE 4 influence of Neem medium and PD medium on the experimental results for cultivation of selenium-enriched Pleurotus ostreatus
The method uses the neem as an important raw material in the whole processes of strain domestication, cultivar preparation and solid cultivation in the production of the selenium-rich oyster mushroom, and due to the use of the neem, the selenium-rich oyster mushroom is pollution-free, the hypha grows quickly, the spawn running time is short, the selenium absorption rate and the organic conversion rate are high, and the total amount of the obtained organic selenium has remarkable advantages compared with other methods.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. A cultivation and cultivation method of selenium-rich oyster mushrooms is characterized in that selenium-rich oyster mushroom cultivation strains are obtained through ultraviolet radiation mutagenesis and domestication screening, wherein parameters of the ultraviolet radiation mutagenesis are that ultraviolet irradiation is 40-70 seconds, and irradiation dose is 3-6 erg/second/mm2(ii) a And then inoculating the selenium-rich oyster mushroom cultivation strain into the neem selenium-containing solid cultivation material, and taking apart the bag opening after hypha grows over the fungus bag, so as to grow out the fruiting body, thereby obtaining the selenium-rich oyster mushroom.
2. The cultivation method of selenium-enriched oyster mushroom according to claim 1, wherein the cultivation method of the selenium-enriched oyster mushroom cultivation spawn comprises the following steps:
step one, ultraviolet radiation mutagenesis: inoculating candidate Pleurotus Ostreatus mycelium onto flat plate, culturing at 30 deg.C for 20-30 days to obtain large amount of Pleurotus Ostreatus spore, diluting with liquid culture medium, and culturing to 5mm2The spores are diluted in 50mL of the first liquid culture medium, the first liquid culture medium containing the spores is placed in a dark box, ultraviolet irradiation is carried out for 40-70 seconds, and the irradiation dose is 3-6 erg/s/mm2Placing the irradiated liquid culture medium I containing the spores in an incubator at 30 ℃ for culturing for 5-10 days to obtain a mutagenic strain; the preparation method of the flat plate comprises the following steps: the culture medium is 200g of peeled potatoes, 2-50 g of neem leaf extract, 20g of agar, 18g of glucose, 1.0g of monopotassium phosphate, 1.0g of dipotassium phosphate, 2g of magnesium sulfate, 110 mg of vitamin B, 20g of peptone, 1g of calcium carbonate, 2g of ferrous sulfate, 2g of zinc sulfate and 1000mL of distilled water, the raw materials are mixed, heated and boiled for 5min, filtered to remove residues, sterilized at 121 ℃ for 20min, poured on a sterile culture dish in an ultraclean workbench while hot, sealed, stood and cooled to obtain a flat plate; the preparation method of the liquid medium I comprises the following steps: 200g of peeled potato, 2-50 g of first neem leaf extract, 0.1g of sodium selenite, 18g of glucose, 1.0g of monopotassium phosphate, 1.0g of dipotassium phosphate, 2g of magnesium sulfate, 110 mg of vitamin B, 20g of peptone, 1g of calcium carbonate, 2g of ferrous sulfate, 2g of zinc sulfate and 1000mL of distilled water, mixing the raw materials, heating and boiling for 5min, filtering and removing slag, subpackaging by using 250mL triangular bottles, filling 50mL of liquid in each bottle, sterilizing at 121 ℃ for 20min, and cooling for later use.
Step two, domestication and screening: preparing a selenium-rich flat plate and a selenium-rich inclined surface, inoculating mutagenic strains obtained in the first step to the selenium-rich flat plate, placing the plate in an incubator for 5-10 days at 30 ℃, then selecting microcolonies with good growth and white and healthy hypha on the plate to be inoculated to the selenium-rich inclined surface, placing the plate in the incubator for 5-20 days at 30 ℃ to obtain a first-stage selenium-rich strain, screening mycelia of the first-stage selenium-rich strain with vigorous growth to be inoculated to a new selenium-rich inclined surface, culturing the plate at 30 ℃ for 5-20 days to obtain a second-stage selenium-rich strain, then screening the selenium-rich strain with good growth to be inoculated to the new selenium-rich inclined surface for rejuvenation once every 3 months, and obtaining domesticated strains of selenium-rich oyster mushrooms after multiple domestication and screening; the preparation method of the selenium-rich flat plate and the selenium-rich inclined plane comprises the following steps: the culture medium is two 5-50 g of neem leaf extract, 5-50 g of neem seed extract, 0.5-2 g of sodium selenite, 20g of agar, 18g of glucose and 1L of distilled water, the raw materials are mixed, heated and boiled for 5min, filtered to remove residues, quantitatively packaged into test tubes when the test tubes are hot, and the test tube openings are plugged by cotton plugs; sterilizing the rest culture medium and test tube at 121 deg.C for 20 min; taking out the test tube, swinging the test tube into an inclined plane, and cooling to obtain a selenium-rich inclined plane; pouring the rest culture medium into a sterile culture dish in an ultra-clean workbench while the culture medium is hot, and sealing and cooling to obtain a selenium-rich flat plate;
step three, preparing a culture strain: selecting the domesticated strain of the selenium-enriched oyster mushrooms obtained in the second step, inoculating the domesticated strain of the selenium-enriched oyster mushrooms into a second liquid culture medium, and performing shake cultivation at 30 ℃ and 120r/min for 7-15 days to obtain a cultivated strain of the selenium-enriched oyster mushrooms, wherein the second liquid culture medium is prepared by the following steps: dissolving 5-50 g of second neem leaf extract, 5-50 g of neem seed extract and 0.5-2 g of sodium selenite in 1L of distilled water, heating and boiling, filtering to remove residues, subpackaging with a triangular flask, plugging with a cotton plug, sterilizing at 121 ℃ for 20min, and cooling for later use.
3. The method for cultivating and cultivating selenium-rich Pleurotus ostreatus as claimed in claim 2, wherein the first liquid culture medium containing spores in the first step is subjected to ultrasonic treatment in ultrasonic wave for 3min before ultraviolet irradiation to disperse well and form monospores, and the ultrasonic power is 53KHz and the temperature is 25 ℃.
4. The cultivation method of selenium-enriched oyster mushroom according to claim 2, wherein the first neem leaf extract is prepared by the following steps: picking the neem leaves in 9-12 months, drying or sun-drying the neem leaves, crushing the neem leaves to 10-60 meshes, and mixing the neem leaves in mass ratio: adding water into neem leaf powder at a ratio of 1: 10-20, heating to 50-80 ℃, extracting for 2-3 h, filtering to obtain an extracting solution, using filter residues as raw materials of a solid cultivation material, concentrating the filtrate to obtain a thick extract, baking the thick extract in an oven to obtain fragrance, taking out, cooling, and crushing to obtain a neem leaf extract I.
5. The cultivation method of selenium-enriched oyster mushroom according to claim 4, wherein the second neem leaf extract is prepared by the following steps: picking the neem leaves in 9-12 months, drying or sun-drying the neem leaves, crushing the neem leaves to 10-60 meshes, and mixing the neem leaves in mass ratio: adding water into the neem leaf powder at a ratio of 1: 10-20, heating to 50-80 ℃, extracting for 2-3 h, filtering to obtain an extracting solution, using filter residues as raw materials of the solid cultivation material, heating the filtrate to 40-50 ℃, adding 1% of compound protease into the filtrate, stirring at a constant speed of 80r/min, keeping for 3-5 h, heating to boil for 10min after enzymolysis is completed, and inactivating enzymes to obtain a neem leaf extract II.
6. The cultivation method of selenium-enriched Pleurotus Ostreatus according to claim 5, wherein the neem seed extract is prepared by: collecting mature neem fruits, removing peel, drying seeds in the sun and shelling, removing oil from kernels by a squeezing method, crushing the kernel cakes without neem oil, screening by a standard sieve of 20 meshes, adding water into the kernel cakes, wherein the water addition amount is as follows by mass: neem cake powder: stirring and mixing water 1: 10-20 uniformly, leaching for 3-4 h, filtering, using filter residue, namely neem seed cake as a raw material of a solid cultivation material, heating filtrate to 60-70 ℃, adding 1% -2% of papain or subtilisin into the filtrate, stirring and mixing uniformly, performing enzymolysis for 1h, boiling for 10min, and inactivating enzyme to obtain a neem seed extract.
7. The cultivation method of selenium-enriched oyster mushroom according to claim 6, wherein the preparation method of the Neem selenium-containing solid cultivation material comprises the following steps: the raw materials are prepared according to the following mass ratio: 10-20% of neem leaves and neem rods, 5-10% of neem leaf filter residues, 5-15% of neem seed cakes, 10-15% of corncobs, 0.5% of quick lime, 0.5% of gypsum, 0.005-0.04% of sodium selenite and 57-69% of water; mixing the raw materials except sodium selenite and water, dissolving sodium selenite in appropriate amount of water, adding into the mixed raw materials, adding water, stirring, packaging with polypropylene bags (17 × 33 mm), tying with cotton thread, sterilizing at 121 deg.C for 20min, and cooling.
8. A cultivation method of selenium-enriched Pleurotus ostreatus as claimed in claim 7, wherein the leaves and stems of Azadirachta indica are picked in 9-12 months, dried in the sun and ground to 0.5-1 cm.
9. A cultivation method of selenium-enriched Pleurotus ostreatus as claimed in claim 7, wherein the leaves and stems of Azadirachta indica are picked in 9-12 months, dried in the sun, pulverized to 0.5-1 cm, sprayed with water at a spray rate of 10% of the dry material, stacked and fermented for 3-4 days, and spread out and dried in the sun to obtain the selenium-enriched Pleurotus ostreatus.
10. Use of the method for cultivating and cultivating selenium-enriched Pleurotus ostreatus as claimed in any one of claims 1-9 in the production of selenium-enriched Pleurotus ostreatus.
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