CN114073214B - 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 method for cultivating and cultivating 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 the parameters of the ultraviolet radiation mutagenesis are 40-70 seconds of ultraviolet irradiation, and the irradiation dose is 3-6 erg/second/mm 2 (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 the selenium absorption rate and the organic conversion rate of the selenium-rich oyster mushroom strains, and the organic selenium content of the selenium-rich oyster mushrooms is increased to 100-600mg/kg, and the organic selenium content of the selenium-rich oyster mushrooms obtained by the method accounts for more than 99 percent of the total selenium content.
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 strong selenium enrichment capability proved by the cultivation process. The cultivation of the functional selenium-rich oyster mushroom by 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 improve the economic value and the nutritional value of the oyster mushroom, thereby developing a new idea and developing a new economic growth point for the field of oyster mushroom cultivation.
The prior art adopts the traditional culture medium and cultivation material to cultivate the selenium-rich oyster mushroom, the utilization rate of selenium is relatively low, the oyster mushroom has a balance point for the absorption and conversion of selenium in the growth process, and the strain poisoning is caused by the overhigh inorganic selenium concentration in the cultivation material, 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 method for cultivating and cultivating selenium-rich oyster mushroom, which prepares and obtains a selenium-rich oyster mushroom cultivation strain through ultraviolet radiation mutagenesis and domestication screening, wherein the parameters of the ultraviolet radiation mutagenesis are 40-70 seconds of ultraviolet irradiation, and the irradiation dose is 3-6 erg/second/mm 2 (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, 5mm 2 The 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 radiation is carried out for 40 to 70 seconds, and the radiation dose is 3 to 6 ergs/second/mm 2 Placing the irradiated liquid culture medium I containing the spores in an incubator at 30 ℃ 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 potato, 2-50g 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, 1mg 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, 2-50g 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, 10mg of vitamin B, 20g of peptone, 1g of calcium carbonate, 2g of ferrous sulfate, 2g of zinc sulfate and 1000mL of distilled water, wherein the raw materials are mixed, heated and boiled for 5min, filtered to remove residues, and subpackaged by using 250mL triangular bottles, and the liquid filling amount of each bottle is 50mL, sterilized at 121 ℃ for 20min and cooled for standby.
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 neem leaf extract two 5-50g, neem seed extract 5-50g, sodium selenite 0.5-2g, agar 20g, glucose 18g, and distilled water 1L, the above raw materials are mixed, heated and boiled for 5min, filtered to remove residue, subpackaged into test tubes in a quantitative manner when the materials 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 20min; 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 preparation method of the second liquid culture medium comprises the following steps: dissolving 5-50g of second neem leaf extract, 5-50g of neem seed extract and 0.5-2g of sodium selenite in 1L of distilled water, heating, 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: the neem leaves are picked in 9-12 months, dried or dried in the sun, and crushed into 10-60 meshes according to the mass ratio of the neem leaves: water =1:10-20, adding water into neem leaf powder, heating to 50-80 ℃, extracting for 2-3h, filtering to obtain an extracting solution, using filter residues as raw materials of the solid cultivation material, concentrating filtrate to obtain a thick extract, baking the thick extract in an oven to make 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 neem leaves in 9-12 months, drying or sun-drying, crushing the neem leaves to 10-60 meshes, and mixing the neem leaves: 10-20 of water, adding water into the neem leaf powder, heating to 50-80 ℃, extracting for 2-3h, 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 3-5h, heating and boiling for 10min to inactivate enzyme after enzymolysis is completed, and obtaining 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 uniformly for 10-20 hours, leaching for 3-4 hours, filtering, wherein filter residues are neem seed cakes used as raw materials of solid cultivation materials in the following process, heating filtrate to 60-70 ℃, adding 1% -2% of papain or subtilisin into the filtrate, stirring and mixing uniformly, performing enzymolysis for 1 hour, boiling for 10min, and inactivating enzymes to obtain the 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 stems, 5-10% of neem leaf filter residues, 5-15% of neem seed cakes, 10-15% of corncobs, 0.5% of quicklime, 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 0.5-1cm, 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 of the invention adopts ultraviolet radiation to mutagenize oyster mushroom spores to produce mycelium with enhanced selenium enrichment capability, and strains with high organic selenium conversion rate are screened out under the condition of not introducing exogenous genes, thus having 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 the strains with strong selenium enrichment capacity and then expanding cultivation to obtain the selenium-enriched oyster mushroom, improves the selenium absorption rate and the organic conversion rate of the selenium-enriched oyster mushroom strains, and promotes the organic selenium content of the selenium-enriched oyster mushroom to 100-600mg/kg, and the organic selenium content of the selenium-enriched oyster mushroom 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 stopped growing on the 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 compost of example 2 at 26 days.
Detailed Description
The present invention is further illustrated by the following examples.
Example 1: comparative 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 A 0 (ii) a Candidate strain A 0 Generating 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, 10mg of vitamin B, 20g of peptone, 1g of calcium carbonate, 2g of ferrous sulfate, 2g of zinc sulfate and 1000mL of distilled water. Heating above materials to boil for 5min, filtering to remove residue, sterilizing at 121 deg.C for 20min. 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 neem leaves in 9-12 months, drying or sun-drying, crushing the neem leaves to 10-60 meshes, and mixing the neem leaves: 10-20 parts of water, namely adding water into neem leaf powder, heating to 50-80 ℃, extracting for 2-3 hours, filtering to obtain an extracting solution, using filter residues as raw materials of a solid cultivation material, concentrating 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 A 0 Inoculating on the plate, culturing in an incubator at 30 deg.C for 20-30 days to produce fruiting body, and forming spore on the cover of the culture dish;
(3) Preparing a first liquid culture medium, wherein the preparation method comprises the following steps: 200g of peeled potatoes, 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, 10mg 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 250mL triangular bottles, filling the liquid volume of each bottle of 50mL,121 ℃, sterilizing 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 5mm 2 The spores of (1) are treated by ultrasonic for 3min in the ultrasonic wave in the triangular flask with diluted oyster mushroom spores, so that the spores are well dispersed to form monospores, the ultrasonic 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/mm 2 And 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 spore at 30 deg.c for 5-10 days to obtain mutagenic strain.
TABLE 1 ultraviolet radiation orthogonal experimental design for selenium-enriched Pleurotus ostreatus mutagenic strains
The treatment was carried out 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 B 6 、B 8 、B 9 Poor growth and marked atrophy observed starting on day 3, B 1 、B 2 、B 3 、B 4 、B 5 Normal spore development, B 7 The 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 by 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 20min; 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 neem leaves in 9-12 months, drying or sun-drying, crushing the neem leaves to 10-60 meshes, and mixing the neem leaves: 10-20 of water, adding water into the neem leaf powder, heating to 50-80 ℃, extracting for 2-3h, 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 3-5h, heating and boiling for 10min to inactivate enzyme after enzymolysis is completed, and obtaining a neem leaf extract II.
The preparation method of the neem seed extract comprises the following steps: collecting mature neem fruits, removing pericarp, drying seeds in the sun, shelling, removing oil from kernels by a squeezing method, crushing kernel cakes without neem oil, screening by a standard sieve of 20 meshes, adding water into the kernel cakes, wherein the mass ratio of the water addition is as follows: neem cake powder: water =1, uniformly stirring and mixing, leaching for 3-4 hours, 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, uniformly stirring and mixing, performing enzymolysis for 1 hour, boiling for 10min, and inactivating enzyme to obtain a neem seed extract.
Culturing the mutagenized strain with selenium-rich plate, self-inoculating, and observing 1 、B 2 、B 4 、B 5 The hyphae grow well and white and healthy single colonies are obtained. B is 3 、B 7 The growth was slow and the hyphae were weak.B 6 、B 8 、B 9 No mycelium was obtained. The results show that the high radiation dose and the long radiation time cause irreversible damage to some strains, and reduce the activity of the strains, so that the strains cannot adapt to high-concentration inorganic selenium.
(6) Starting from this experimental procedure, candidate species A 0 And mutagenized species B were treated in the same manner. Picking B obtained on the flat plate in the step (5) 1 、B 2 、B 4 、B 5 、B 3 、B 7 Respectively inoculating microcolonies to the selenium-rich slant surfaces prepared in the step (5), and simultaneously inoculating the candidate strain A 0 Respectively inoculating to selenium-rich slant, culturing at 30 deg.C for 5-20 days to obtain first-stage selenium-rich strain, observing during culture period A 0 、B 1 、B 2 、B 4 、B 5 Good growth, full of tubes on day 8 after inoculation, B 3 、B 7 The growth was relatively slow, the hyphae were fine, and the tubes were full on day 17 after inoculation. Respectively selecting the first-stage selenium-rich mycelia which grow vigorously to inoculate on a selenium-rich slant, placing the first-stage selenium-rich mycelia in an incubator to be cultured for 5-20 days at 30 ℃ to obtain second-stage selenium-rich strains, then inoculating and rejuvenating the well-grown selenium-rich strains on the slant once every 3 months, and after inoculation, keeping the mycelia in a clean place at room temperature after the mycelia grow over a test tube. Multiple acclimatization to obtain selenium-rich Pleurotus Ostreatus test tube acclimatized seed (shown in figure 1), wherein A 0 Is a candidate strain, A is A 0 Domesticated strain without ultraviolet mutagenesis, B is A 0 Domesticating the strain of selenium-rich oyster mushroom through ultraviolet mutagenesis.
(7) Preparing a second liquid culture medium for cultivating strains according to the following method, wherein the formula of the second liquid culture medium is as follows: 10g of two neem leaf extracts, 10g of neem seed extracts, 0.5g of sodium selenite and 1L of distilled water. Boiling the above materials, filtering to remove residue, subpackaging with 250mL triangular flask, liquid loading amount of 80mL, plugging with cotton plug, sterilizing at 121 deg.C for 20min, and cooling for use. Picking untreated candidate species A 0 Step (6) obtaining A 0 Test tube domesticated strains A and A without ultraviolet mutagenesis 0 The selenium-rich Pleurotus Ostreatus domesticated strains B subjected to ultraviolet mutagenesis are each about 5mm 2 Respectively inoculated in triangular flasksAnd (3) performing shake culture at 30 ℃ and 120r/min for 10 days in the liquid culture medium II to obtain the selenium-rich oyster mushroom liquid culture strain (shown in figure 3).
(8) Preparing the 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 quick lime, 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 0.5-1cm 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 (17X 33mm), packaging with 1kg of cultivation material per bag, tying the bag opening with newspaper and cotton thread, and sterilizing at 121 deg.C for 20min.
(9) A obtained in the step (7) 0 、A、B 1 、B 2 、B 4 、B 5 、B 3 、B 7 Respectively inoculating selenium-rich oyster mushroom liquid culture strains into a selenium-containing solid culture material of neem, controlling the inoculation amount of each bag to be about 10mL, respectively inoculating 100 fungus bags for each strain, keeping the inoculated fungus bags at 20-30 ℃ and the humidity of 80% to enable the fungus bags to spawn, observing and recording the pollution number of the fungus bags, the health condition of hyphae during spawn running, recording spawn running time after the fungus bags are full of hyphae, observing that all the fungus bags are pollution-free, and A and B 1 、B 2 、B 4 、B 5 The 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 is 3 、B 7 The growth is slow, the hyphae are white and sparse, and the fungus bags are full of hyphae 41 days after inoculation; a. The 0 When the fungus grows abnormally, the hyphae stop growing after growing inwards to 2-4 cm from the bag opening, the color of the hyphae is gradually changed from white to light yellow, the fungus bag cannot be filled, and finally the mushroom does not grow. Opening the bag mouth of the full fungus bag, keeping the temperature at 15-30 ℃, keeping the humidity at 80%, and illuminating properly,Ventilating to allow fruiting body to grow out, and obtaining selenium-rich Pleurotus Ostreatus. 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 result shows 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 detoxification capability is higher than that of the strain without 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/mm 2 It is more suitable. In particular B5, at 6 ergs/sec/mm 2 When the radiation dose of the selenium-enriched oyster mushroom is irradiated for 70 seconds for mutagenesis, the selenium-enriched oyster mushroom mycelia are fast in development speed, the mycelia are white and strong, 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 (819 g) which are not subjected to ultraviolet mutagenesis; the selenium content in the dry product is 283.1mg/kg, which is higher than that of the strain without ultraviolet mutagenesis (89 mg/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-rich oyster mushroom liquid culture strain B5 obtained by domestication 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 corncobs, 8% of corn flour, 5% of cottonseed hulls, 11% of wood chips, 0.5% of gypsum, 0.5% of quick lime, 0.02% of sodium selenite and 59.98% of water. Preparing raw materials according to the content of the components, fully and uniformly mixing other raw materials except sodium selenite and 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 ℃,20 min, 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 the 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 bag to grow bacteria, and observing and recording the pollution number of the bag and the health condition of hyphae during the bacteria growing period. In the case of the fungus sack of the selenium-containing solid neem compost at 26 days, as shown in fig. 4, and in the case of the fungus sack of the conventional selenium-containing solid compost, as shown in fig. 5, it was seen that the mycelia of the selenium-containing solid neem compost grew faster. And recording the spawn running time when hypha grows over the fungus bags, opening the bag openings, keeping the temperature at 15-30 ℃, keeping the humidity at 80%, and properly illuminating and ventilating to enable fruiting bodies to grow out to obtain the selenium-enriched oyster mushrooms. And collecting mature sporocarps, weighing the mature sporocarps respectively, recording the fresh weight, and calculating the 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 acclimation-screening media on mutagenized species
(1) The same procedure as in example 1 was repeated to prepare selenium-rich plates and selenium-rich slant cultures.
(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 20min; 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 mutagenic 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) C1, preparation of culture strains: inoculating C1 into liquid culture medium II to obtain C1 culture strain, and culturing with the same method as in example 1
(5) C2, preparation of culture strains: inoculating C2 into a PD liquid culture medium to obtain a C2 cultivated strain, wherein the preparation method of the PD liquid culture medium comprises the following steps: cleaning and cutting 200g of potatoes, cooking the potatoes thoroughly, filtering the potatoes by using 4 layers of gauze, adding 18g of glucose and 1g of sodium selenite (the mass fraction is 0.1 percent), complementing 1L of distilled water, heating and boiling the potatoes, subpackaging the potatoes in a 250mL triangular flask, filling 80mL of solution in each flask, plugging the bottles by using a cotton plug, sterilizing the potatoes at the temperature of 121 ℃, carrying out 20min, and cooling the potatoes for later use. Selecting the domesticated strain C2 obtained in the step (3) to be about 5mm 2 Inoculating into PD liquid culture medium in a triangular flask, and shake culturing at 30 deg.C and 120r/min for 7-15 days to obtain 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 stems, 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 0.5-1cm, spraying water on the neem leaves and neem stems, wherein the water spraying amount is 10% of the dry neem material, 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 C1 cultivation strains into a neem selenium-containing solid cultivation material, inoculating C2 cultivation strains into the conventional selenium-containing solid cultivation material in the same embodiment 2, controlling the inoculation amount of each bag to be about 10mL, keeping the temperature to be 20-30 ℃, keeping the humidity to be 80%, enabling the bags to grow fungi, observing and recording the pollution number of the bags, the health condition of hyphae during the fungus growing period, recording the fungus growing time when the hyphae fully grow in the bags, dismantling the bag openings, keeping the temperature to be 15-30 ℃, keeping the humidity to be 80%, and enabling the fruiting bodies to grow, thereby obtaining the selenium-rich oyster mushrooms. And collecting mature sporocarps, weighing the mature sporocarps respectively, recording the fresh weight, and calculating the 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 (7)
1. A method for cultivating selenium-rich Pleurotus Ostreatus comprises ultraviolet radiation mutagenesis and domestication screening to obtain selenium-rich Pleurotus Ostreatus culture strain, wherein the ultraviolet radiation mutagenesis parameters are ultraviolet irradiation for 40-70 s, and irradiation dose is 3-6 erg/s/mm 2 (ii) a Then inoculating the selenium-rich oyster mushroom cultivation strain into the selenium-containing solid neem cultivation material, opening the bag opening after hypha grows over the fungus bag, and growing out sporocarp to obtain the selenium-rich oyster mushroom;
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 obtained Pleurotus Ostreatus spore with liquid culture medium one, 5mm 2 The spores of (2) are diluted in 50mL of the first liquid medium, the first liquid medium containing the spores is placed in a dark box, and ultraviolet irradiation is carried out for 40-70 seconds at a dose of 3-6 erg/sec/mm 2 Placing the irradiated liquid culture medium I containing spores in an incubator for culturing for 5-10 days at 30 ℃ to obtain a mutagenic strain; the preparation method of the flat plate comprises the following steps: the culture medium is 200g of peeled potato, 2-50g 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, 1mg 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 first liquid culture medium comprises the following steps: to200g of skin potatoes, 2-50g 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, 10mg 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 residues, subpackaging by using 250mL triangular bottles, bottling each liquid amount, sterilizing at 50mL and 121 ℃ for 20min, and cooling for later use;
step two, domestication and screening: preparing a selenium-rich plate and a selenium-rich slant, inoculating the mutagenic strain obtained in the first step to the selenium-rich plate, placing the plate in an incubator for 5-10 days at 30 ℃, then selecting microcolonies with good growth and white and healthy hyphae on the plate, inoculating the microcolonies to the selenium-rich slant, 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, inoculating the mycelia to a new selenium-rich slant, culturing the mycelia at 30 ℃ for 5-20 days to obtain a second-stage selenium-rich strain, screening the selenium-rich strain with good growth every 3 months, inoculating the mycelia to the new selenium-rich slant for rejuvenation once, and obtaining a domesticated selenium-rich oyster mushroom strain 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 composed of two 5-50g of neem leaf extract, 5-50g of neem seed extract, 0.5-2g of sodium selenite, 20g of agar, 18g of glucose and 1L of distilled water, the above raw materials are mixed, heated and boiled for 5min, filtered to remove residues, subpackaged into test tubes in a quantitative manner when the materials are hot, and the mouth of each test tube is plugged by a cotton plug; sterilizing the rest culture medium and test tube at 121 deg.C for 20min; 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: and (3) selecting the domesticated selenium-rich oyster mushroom strains obtained in the second step, inoculating the domesticated selenium-rich oyster mushroom strains into a second liquid culture medium, and performing shake culture at 30 ℃ and 120r/min for 7-15 days to obtain selenium-rich oyster mushroom strains, wherein the second liquid culture medium is prepared by the following steps: dissolving second 5-50g of neem leaf extract, 5-50g of neem seed extract and 0.5-2g of sodium selenite in 1L of distilled water, heating and boiling, filtering to remove residues, subpackaging with triangular flask, plugging with cotton plug, sterilizing at 121 deg.C for 20min, and cooling;
the preparation method of the first neem leaf extract comprises the following steps: the neem leaves are picked in 9-12 months, dried or dried in the sun, and crushed into 10-60 meshes according to the mass ratio of the neem leaves: water =1:10-20, adding water into neem leaf powder, heating to 50-80 ℃, extracting for 2-3h, filtering to obtain an extracting solution, using filter residues as a raw material of a solid cultivation material, concentrating filtrate to obtain a thick extract, baking the thick extract in an oven to make fragrance, taking out, cooling, and crushing to obtain a neem leaf extract I;
the preparation method of the second neem leaf extract comprises the following steps: picking neem leaves in 9-12 months, drying or sun-drying, crushing the neem leaves into 10-60 meshes, and mixing the neem leaves: 10-20 of water =1, adding water into neem leaf powder, heating to 50-80 ℃, extracting for 2-3h, filtering to obtain an extracting solution, using filter residue as a raw material of a solid cultivation material, heating filtrate to 40-50 ℃, adding 1% of compound protease into the extracting solution, stirring at a constant speed of 80r/min, keeping for 3-5h, heating and boiling for 10min to inactivate enzyme after enzymolysis is completed, and obtaining a neem leaf extract II.
2. The method for cultivating and cultivating selenium-rich Pleurotus ostreatus as claimed in claim 1, 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 frequency is 53KHz and the temperature is 25 ℃.
3. The cultivation method of selenium-enriched Pleurotus Ostreatus according to claim 1, 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: and (2) stirring and mixing uniformly 10-20% with water =1, leaching for 3-4h, filtering, wherein filter residues are neem seed cakes used as raw materials of solid cultivation materials in the following process, the filtrate is heated to 60-70 ℃, papain or subtilisin is added into the filtrate in an amount of 1% -2%, stirring and mixing uniformly, performing enzymolysis for 1h, boiling for 10min, and inactivating enzymes to obtain the neem seed extract.
4. The cultivation method of selenium-enriched oyster mushroom according to claim 3, wherein the preparation method of the Neem selenium-containing solid cultivation material comprises the following steps: the feed is prepared from the following raw materials in percentage by mass: 10-20% of neem leaves and neem stems, 5-10% of neem leaf filter residues, 5-15% of neem seed cakes, 10-15% of corncobs, 0.5% of quicklime, 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 (17X 33mm), tying with cotton thread, sterilizing at 121 deg.C for 20min, and cooling.
5. A cultivation method of selenium-enriched Pleurotus Ostreatus according to claim 4, wherein the leaves and stems of Azadirachta indica are harvested in 9-12 months, sun-dried, and pulverized to 0.5-1 cm.
6. The method for cultivating and cultivating selenium-enriched Pleurotus ostreatus according to claim 4, wherein the neem leaves and neem stems are picked in 9-12 months, dried in the sun, crushed to 0.5-1cm, sprayed with water in an amount of 10% of the dry materials, piled up for 3-4 days, fermented, and spread out and dried in the sun to obtain the selenium-enriched Pleurotus ostreatus.
7. Use of the cultivation and culture method of selenium-enriched Pleurotus ostreatus as claimed in any of claims 1-6 in the production of selenium-enriched Pleurotus ostreatus.
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