A method for preparing a cultivation substrate for stropharia rugosoannulata by using highland barley straw

Abstract

The present application relates to the field of agricultural cultivation technology, and specifically relates to a kind of preparation of big ball cover mushroom cultivation substrate using highland barley straw and preparation method, including highland barley straw, modified bentonite, modified beta-cyclodextrin, wheat bran, dry cow dung, superphosphate, potassium dihydrogen phosphate and gypsum.In the present application, modified bentonite adsorbs phenolic substances, the borate groups on the surface of modified beta-cyclodextrin form covalent bonds with ortho-diphenol hydroxyl groups, double-locking them, and modified beta-cyclodextrin cavities are wrapped, preventing phenolic substances from decomposing at high temperatures.Potassium dihydrogen phosphate forms a phosphate buffer system at the initial stage of mycelium outgrowth, resisting the acidification of the initial stage of mycelial metabolism and inhibiting the hydrolysis of borate ester bonds.As mycelial metabolism produces acid and consumes phosphate, the buffering capacity of potassium dihydrogen phosphate is lost, and at the late stage of mycelium outgrowth to the primordium stage, the pH naturally decreases, the borate ester bond hydrolyzes, the hydrophobicity of cetyltrimethylammonium bromide weakens, and both of them release phenolic substances.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural cultivation, and in particular to a method for preparing a Stropharia ocellaris cultivation substrate by utilizing highland barley straw. Background Art

[0002] Stropharia capillaris fruiting bodies have a rich texture, a crisp and smooth taste, and are rich in nutrients. They are a typical high-protein, low-fat, high-quality edible fungus. They also contain bioactive ingredients such as polysaccharides, sterols, flavonoids, phenolic substances, and lectins, which have pharmacological effects such as antioxidant, antibacterial, tumor suppression, and hypoglycemic effects. Stropharia capillaris can be cultivated using agricultural and forestry waste resources such as corn stalks, corn cobs, and highland barley straw, offering significant economic, ecological, and social benefits. Furthermore, the used cultivation substrate can be converted into high-quality fertilizer through composting, achieving resource recycling. However, conventional straw cultivation substrates are prone to the growth of bacteria such as Trichoderma and Aspergillus, which not only hinder the subsequent recycling of the cultivation substrate but also inhibit the growth of Stropharia capillaris.

[0003] Highland barley straw contains specific phenolic compounds, such as caffeic acid and ferulic acid. Ferulic acid is abundant in high concentrations, about 30% higher than wheat. These phenolic compounds exhibit antibacterial activity, achieving an inhibition rate of up to 80% against fungi such as Trichoderma and Aspergillus. This reduces the risk of contamination in the cultivation medium while creating a cleaner growth environment for Stropharia. However, phenolic compounds like caffeic acid and ferulic acid have a dual effect on the growth of Stropharia. During the early stages of silk emergence, young mycelium, either newly germinated or undergoing rapid elongation, has thin cell walls, an underdeveloped membrane system, and incomplete defense mechanisms. These compounds are highly susceptible to phenolic toxicity. Furthermore, high concentrations of phenolic compounds bind to the active centers of lignin-degrading enzymes, hindering the mycelium's ability to decompose and utilize nutrients such as cellulose. However, during the late stages of silk emergence or during primordium formation, when mycelium is fully encapsulated, the phenolic tolerance threshold increases. At this stage, phenolic compounds not only inhibit contamination but also scavenge free radicals, reducing oxidative damage to the mycelium, thereby enhancing mycelial viability and promoting growth. Summary of the Invention

[0004] (1) Technical problems to be solved

[0005] The present invention aims to provide a method for preparing a Stropharia capillaris cultivation substrate using highland barley straw, so as to solve the problem of bacterial contamination in existing Stropharia capillaris cultivation substrates, selectively retain phenolic substances, make them become natural antibacterial components in the substrate, reduce later pollution, and promote the growth of Stropharia capillaris.

[0006] (2) Technical solution

[0007] To achieve the above object, in one aspect, the application provides a kind of with highland barley straw preparation Kuehnera grandis cultivation substrate, by mass fraction includes raw material: highland barley straw 60~100 parts, modified bentonite 8~12 parts, modified β-cyclodextrin 5~8 parts, wheat bran 15~20 parts, dry cow dung 10~15 parts, calcium superphosphate 1~2 parts;

[0008] The cultivation substrate further includes: potassium dihydrogen phosphate and gypsum;

[0009] The mass of the potassium dihydrogen phosphate is 0.3%~0.5% of the dry weight of the cultivation substrate, and the mass of the gypsum is 0.1% of the dry weight of the cultivation substrate.

[0010] Further, the preparation method of the modified bentonite includes the following steps:

[0011] S11. Take bentonite through a 200 mesh sieve, and place it in a 105°C oven for drying to remove adsorbed water;

[0012] S12. Dissolve cetyltrimethylammonium bromide in deionized water, heat in a water bath, add bentonite, and react in a 60°C constant-temperature oscillator for 2 hours. Centrifuge the reaction solution at 4000 rmp for 10 minutes, collect the precipitate, and wash the precipitate with 60°C deionized water until there is no foam to remove free cetyltrimethylammonium bromide, to obtain a first compound;

[0013] S13. Dissolve citric acid in deionized water, add the first compound, and react under ultrasonic for 30 minutes to obtain a reaction solution. Cool the reaction solution to below 40°C in an ice bath, add NaOH to adjust the pH to neutral, centrifuge to collect the precipitate, and place it in a 60°C oven for drying to constant weight to obtain a second compound;

[0014] S14. Ultrasonically disperse ZnO nano powder in anhydrous ethanol, add the second compound, and stir at 45°C. Collect the solid by suction filtration, wash with ethanol, and dry in a 60°C vacuum drying oven. Crush and pass through a 200 mesh sieve to obtain modified bentonite.

[0015] Further, the mass of the cetyltrimethylammonium bromide, citric acid, and ZnO nano powder is 15%, 8%, and 3% of the mass of the bentonite, respectively.

[0016] Further, the preparation method of the modified β-cyclodextrin includes the following steps:

[0017] S21. Dissolve β-cyclodextrin in a NaOH solution, magnetically stir until completely transparent, add sodium chloroacetate solution dropwise, and stir at a 60°C constant-temperature oil bath for 6 hours. Adjust the pH to neutral with glacial acetic acid, purify by dialysis, and freeze-dry to obtain a third compound;

[0018] S22. A third compound is added to a 2-morpholinoethanesulfonic acid buffer solution at pH = 6, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide are added, and stirring is activated for 30 minutes to obtain a fourth compound;

[0019] S23. 3-aminobenzene boronic acid is dissolved in N,N-dimethylformamide and added to the fourth compound, and reacted at 45 DEG C under nitrogen protection in the dark, and then purified by dialysis with deionized water and HCl in sequence, and freeze-dried to obtain the modified beta-cyclodextrin.

[0020] Further, the mass ratio of the beta-cyclodextrin to sodium chloroacetate is 5:3, and the mass ratio of the fourth compound to 3-aminobenzene boronic acid is 4:1.

[0021] On the other hand, based on the same inventive concept, the application also provides a preparation method for preparing a large ball-cap mushroom cultivation substrate from highland barley straw, which is applied to the large ball-cap mushroom cultivation substrate prepared from highland barley straw and comprises the following steps:

[0022] S1. The highland barley straw is crushed to 3-5 cm, sieved and dusted, soaked in lime water for 24 hours, washed with clean water until neutral, steam exploded at 1.8 MPa and 185 DEG C, the silicon wax layer of the crushed highland barley straw is washed with oxalic acid solution, and then washed with running water until neutral, filtered by a centrifuge to obtain a first mixture;

[0023] S2. The wheat bran, dry cow dung and superphosphate are weighed according to the proportion, added to the first mixture, uniformly mixed, layered and laid, sprayed and moisturized, and then fermented at 60-65 DEG C for 2 days;

[0024] S3. The modified bentonite and modified beta-cyclodextrin are ultrasonically mixed in water according to the proportion, sodium alginate is added, stirred to form a gel, CaCl2 solution is added for solidification, the product is dried in an oven, and then crushed to obtain a second mixture;

[0025] S4. The first turning is carried out on the 3rd day of fermentation, and the second mixture is uniformly layered and scattered, sprayed and moisturized, and then continuously fermented at 60-65 DEG C for 2 days;

[0026] S5. The second turning is carried out on the 5th day of fermentation, and the potassium dihydrogen phosphate and gypsum are uniformly layered and scattered, and then continuously fermented at 50-55 DEG C for 2 days;

[0027] S6. After the 7th day, the maturation is stable, the pile temperature is reduced to below 45 DEG C, the pile is scattered and aired, and the moisture content is adjusted to 60-62%.

[0028] In the application, the highland barley straw, wheat bran, dry cow dung and superphosphate are the basic raw materials of the large ball-cap mushroom cultivation substrate, and on this basis, the modified bentonite, modified beta-cyclodextrin, potassium dihydrogen phosphate and gypsum are added.

[0029] Bentonite is a layered silicate with natural cation exchange capacity, but conventional bentonite has poor adsorption capacity for phenolic compounds. Therefore, it is modified with cetyltrimethylammonium bromide. Its long-chain alkyl groups form hydrophobic microdomains that capture phenolic molecules, increasing the bentonite's adsorption capacity. Citric acid increases the bentonite's specific surface area and porosity. Nano-zinc oxide provides a pH-responsive site. During the early stages of fermentation, the pH is neutral, and the modified bentonite firmly locks in phenols due to hydrophobic interactions. As fermentation progresses, microbial metabolism produces acid, and the quaternary ammonium groups of cetyltrimethylammonium bromide become protonated, shrinking the interlayer spacing and squeezing out the phenols.

[0030] After the modified bentonite adsorbs phenolic compounds on its surface, as the fermentation temperature rises, the phenolic compounds will decompose and oxidize. The present invention adds modified β-cyclodextrin. The hydrophobic cavity of β-cyclodextrin encapsulates phenols. The carboxymethylated β-cyclodextrin improves thermal stability and further prevents the decomposition and oxidation of phenolic compounds at high temperatures. In addition, boric acid groups are introduced into β-cyclodextrin. When the concentration of phenols is too high, the hydrophobic microdomains of cetyltrimethylammonium bromide preferentially adsorb a large amount of phenols. The boric acid groups form covalent bonds with the ortho-diphenolic hydroxyl groups of phenolic substances such as caffeic acid, performing a double lock. As the mycelium grows and metabolizes acid, it triggers the cleavage of the borate ester bond. At this time, the hydrophobicity of cetyltrimethylammonium bromide weakens, jointly promoting the release of phenolic substances. As natural antibacterial ingredients, phenolic substances not only reduce contamination of the cultivation medium and provide a clean environment for the growth of Stropharia rugosa, but also can scavenge free radicals and promote mycelial growth.

[0031] At the initial stage of substrate composting fermentation or shortly after inoculation, the microbial activity is vigorous, and macromolecular substances such as lignocellulose are largely decomposed, which often releases high-concentration free phenolic substances. The modified bentonite and modified β-cyclodextrin can adsorb free phenolic substances, but due to the metabolic acid production of mycelium during the out-silk process, the pH of the cultivation substrate environment decreases rapidly from neutral to below 5.0. The hydrophobicity of cetyltrimethylammonium bromide is weakened, and the borate ester bond is hydrolyzed in an acidic environment. Phenolic compounds are released too early. At this time, the young mycelium that has just germinated or is in the rapid extension stage has a thin cell wall, an imperfect membrane system and an incomplete defense mechanism, and is very susceptible to phenolic substances, which can inhibit mycelial growth at the initial stage of inoculation, and the late phenolic concentration is insufficient, and the pollution rate of Trichoderma, Aspergillus and the like increases linearly. By adding potassium dihydrogen phosphate and gypsum, at the initial stage of out-silk, the gypsum enhances the structural stability of the modified bentonite, preventing its premature disintegration at the initial stage, and the potassium dihydrogen phosphate forms a phosphate buffer system to resist the acidification at the initial stage of mycelial metabolism and inhibit the hydrolysis of the borate ester bond. As the mycelial metabolic acid consumption consumes phosphate, the potassium dihydrogen phosphate buffer capacity is disabled, and the pH naturally decreases at the late out-silk stage to the primordium stage, the borate ester bond is hydrolyzed, and the hydrophobicity of cetyltrimethylammonium bromide is weakened, both of which release phenolic substances. And the phosphate is the necessary nutrition for the mycelium, which is actively absorbed and utilized by the mycelium in the later stage, and will not cause residual effects.

[0032] (3) Beneficial effects

[0033] Compared with the prior art, the beneficial effects of the present application are:

[0034] 1. By the synergistic effect of modified bentonite and modified β-cyclodextrin, when the concentration of phenolic compounds is high, cetyltrimethylammonium bromide adsorbs a large amount of phenols, and the boronic acid group forms a covalent bond with the ortho-diphenolic hydroxyl group of phenolic substances such as caffeic acid, double-locking phenolic compounds. As the mycelium grows and consumes phenolic compounds, the metabolic acid production of the mycelium causes the borate ester bond to break, and the hydrophobicity of cetyltrimethylammonium bromide is weakened, which together promotes the release of phenolic substances. Thus, the free phenolic compounds are intelligently controlled, the growth of Trichoderma, Aspergillus and the like is inhibited, and the growth of Pleurotus eryngii mycelium is promoted.

[0035] 2. After the modified bentonite adsorbs phenols, as the fermentation temperature rises, phenols are easily decomposed and oxidized, while the hydrophobic cavity of β-cyclodextrin can wrap phenols. After carboxymethylation modification, the thermal stability of β-cyclodextrin is improved, further preventing the decomposition and oxidation of phenolic compounds at high temperatures.

[0036] 3. The mycelium metabolic acid production process can cause the pH of the substrate to drop sharply, leading to premature hydrolysis of the boronic acid group, releasing phenolic compounds, and inhibiting early mycelial growth. Subsequent phenolic supply is not available, and the addition of potassium dihydrogen phosphate forms a phosphate buffer system at the early stage of mycelium growth, resisting the early mycelial metabolic acidification and inhibiting the hydrolysis of boronic acid ester bonds. With the consumption of phosphate by mycelial metabolic acid production, the pH naturally decreases from the late mycelial growth to the primordium stage, and the boronic acid ester bond is hydrolyzed to release phenolic substances. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 SEM image of modified bentonite of example 1 of the present application;

[0038] Figure 2 SEM image of modified β-cyclodextrin of example 1 of the present application. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0040] Example 1: The present embodiment discloses a kind of preparation of big ball cover mushroom cultivation substrate using highland barley straw, including raw materials: highland barley straw 60 parts, modified bentonite 8 parts, modified β-cyclodextrin 5 parts, wheat bran 15 parts, dry cow dung 10 parts, superphosphate 1 part by mass fraction;

[0041] The cultivation substrate further comprises: potassium dihydrogen phosphate and gypsum;

[0042] The mass fraction of the potassium dihydrogen phosphate is 0.3% of the dry weight of the cultivation substrate, and the mass fraction of the gypsum is 0.1% of the dry weight of the cultivation substrate.

[0043] In the big ball cover mushroom cultivation substrate, modified bentonite and modified β-cyclodextrin work together, as shown in the scanning electron micrograph of bentonite after modification, the specific surface area is increased, and the phenolic substances can be more effectively adsorbed, Figure 1 Figure 2 ​The scanning electron micrograph of modified β-cyclodextrin clearly shows geometric crystals of varying sizes, with relatively large particles and good thermal stability. The cavities encapsulate phenolic compounds, protecting them from decomposition and oxidation at high temperatures. Boric acid groups are grafted onto the surface, forming borate ester bonds with the o-diphenolic hydroxyl groups, doubly locking the phenolic compounds in place. This prevents the toxic effects of phenolic compounds during the early stages of silk emergence, when the mycelium is young, with thin cell walls, an underdeveloped membrane system, and incomplete defense mechanisms. These compounds are highly susceptible to phenolic toxicity, which inhibits mycelial growth. However, as mycelial metabolism produces acid in the early stages of silk emergence, the pH drops sharply to below 5, leading to premature hydrolysis of the borate ester bonds and the rapid release of phenolic compounds. This results in a lack of antibacterial efficacy during the late stages of silk emergence and the primordium stage. Therefore, potassium dihydrogen phosphate is added to form a phosphate buffer system during the early stages of silk emergence, counteracting the acidification during early mycelial metabolism and inhibiting borate ester hydrolysis. As the mycelium metabolizes to produce acid and consumes phosphate, the pH naturally decreases from the late silk emergence stage to the primordium stage, the borate bond is hydrolyzed, and the hydrophobicity of hexadecyltrimethylammonium bromide is weakened, which together promotes the release of phenolic substances.

[0044] The preparation method of the modified bentonite comprises the following steps:

[0045] S11. Pass bentonite through a 200-mesh sieve and dry in an oven at 105°C to remove adsorbed water.

[0046] S12. 15 g of hexadecyltrimethylammonium bromide was dissolved in 500 ml of deionized water, heated in a water bath, and bentonite was added. The mixture was reacted in a constant temperature shaker at 60 ° C for 2 hours. The reaction solution was centrifuged at 4000 rpm for 10 minutes, and the precipitate was collected and washed with deionized water at 60 ° C until there was no foam to remove free hexadecyltrimethylammonium bromide to obtain the first compound;

[0047] S13. Dissolve citric acid in 200 ml of deionized water, add the first compound, and react under ultrasound for 30 minutes to obtain a reaction solution. Cool the reaction solution in an ice bath to below 40°C, add NaOH to adjust the pH to neutral, collect the precipitate by centrifugation, and dry it in an oven at 60°C to constant weight to obtain a second compound.

[0048] S14. Ultrasonic dispersion of ZnO nanopowder in 100 ml of anhydrous ethanol, addition of the second compound, constant temperature stirring at 45°C, collection of solid by filtration, washing with ethanol, drying in a vacuum drying oven at 60°C, and crushing through a 200-mesh sieve to obtain modified bentonite.

[0049] The masses of the hexadecyltrimethylammonium bromide, citric acid and ZnO nanopowder are 15%, 8% and 3% of the mass of the bentonite respectively.

[0050] The preparation method of the modified β-cyclodextrin comprises the following steps:

[0051] S21. 50g of β-cyclodextrin is dissolved in 200ml of NaOH solution, magnetically stirred until completely transparent, and sodium chloroacetate solution is added dropwise, 60℃ constant temperature oil bath, stirring reaction for 6 hours, adjust pH to neutral with glacial acetic acid, dialysis purification, freeze-drying to obtain the third compound;

[0052] S22. In 200ml of 2-morpholine ethanesulfonic acid buffer solution with pH=6, add 20g of the third compound, add 1.2g of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and 0.7g of N-hydroxysuccinimide, stir for 30 minutes to obtain the fourth compound;

[0053] S23. 3-Aminobenzene boronic acid is dissolved in N,N-dimethylformamide and added to the fourth compound, and reacted at 45℃ under nitrogen protection for 12 hours in the dark. Purification is performed by dialysis with deionized water and HCl in sequence, and freeze-drying to obtain the modified β-cyclodextrin.

[0054] The mass ratio of the β-cyclodextrin to sodium chloroacetate is 5:3, and the mass ratio of the fourth compound to 3-aminobenzene boronic acid is 4:1.

[0055] The preparation method of the cultivation substrate for the large milk-cap mushroom prepared from highland barley straw, comprises the following steps:

[0056] S1. Highland barley straw is crushed to 3cm, sieved and dusted, soaked in lime water for 24 hours, washed with clean water until neutral, steam exploded at 1.8MPa and 185℃, the silicon wax layer of the crushed highland barley straw is washed with oxalic acid solution, and then washed with running water until neutral, and filtered by a centrifugal machine to obtain a first mixture;

[0057] S2. Wheat bran, dry cow dung and superphosphate are weighed according to the proportion, added to the first mixture, uniformly mixed, layered and laid, and then sprayed with water to keep moist, and fermented at 60℃ for 2 days;

[0058] S3. Modified bentonite and modified β-cyclodextrin are ultrasonically mixed in water according to the proportion, 2% of sodium alginate based on the total mass of the modified bentonite and the modified β-cyclodextrin is added, stirred to form a gel, 2% of CaCl2 solution based on the total mass of the modified bentonite and the modified β-cyclodextrin is added, and the product is solidified for 30 minutes, then dried in an oven, and crushed to obtain a second mixture;

[0059] S4. The first turning is performed on the third day of fermentation, and the second mixture is uniformly layered and scattered, and then sprayed with water to keep moist, and the fermentation is continued at 60℃ for 2 days;

[0060] S5. The second turning is performed on the fifth day of fermentation, and potassium dihydrogen phosphate and gypsum are uniformly layered and scattered, and the fermentation is continued at 50℃ for 2 days;

[0061] S6. After 7 days, the mature stability is stable, the pile temperature is reduced to below 45℃, and the pile is spread and aired, and the moisture content is adjusted to 60%.

[0062] In the embodiment, the modified bentonite and the modified β-cyclodextrin are prepared by the method of the embodiment 1.

[0063] The cultivation substrate further comprises potassium dihydrogen phosphate and gypsum.

[0064] The mass of the potassium dihydrogen phosphate is 0.4% of the dry weight of the cultivation substrate, and the mass of the gypsum is 0.1% of the dry weight of the cultivation substrate.

[0065] In the embodiment, the modified bentonite and the modified β-cyclodextrin are prepared by the method of the embodiment 1.

[0066] In the embodiment, the modified bentonite and the modified β-cyclodextrin are prepared by the method of the embodiment 1.

[0067] The cultivation substrate further comprises potassium dihydrogen phosphate and gypsum.

[0068] The mass of the potassium dihydrogen phosphate is 0.5% of the dry weight of the cultivation substrate, and the mass of the gypsum is 0.1% of the dry weight of the cultivation substrate.

[0069] In the embodiment, the modified bentonite and the modified β-cyclodextrin are prepared by the method of the embodiment 1.

[0070] In the embodiment, the modified bentonite and the modified β-cyclodextrin are prepared by the method of the embodiment 1.

[0071] The cultivation substrate further comprises potassium dihydrogen phosphate and gypsum.

[0072] The mass of the potassium dihydrogen phosphate is 0.3% of the dry weight of the cultivation substrate, and the mass of the gypsum is 0.1% of the dry weight of the cultivation substrate.

[0073] The preparation method of the cultivation substrate for the cultivation of the large ball head mushroom by using the highland barley straw comprises the following steps.

[0074] S1. Take the highland barley straw, crush to 5 cm, sieve and remove dust, soak in lime water for 24 hours, rinse with clean water until neutral, steam explosion at 1.8 MPa and 185℃, wash the silicon wax layer of the crushed highland barley straw with oxalic acid solution, rinse with running water until neutral, filter with a centrifuge, and obtain a first mixture;

[0075] S2. Take the wheat bran, dry cow dung and superphosphate according to the proportion, add to the first mixture, mix uniformly, layer the materials, spray water to keep moist, and pile up to ferment at 65℃ for 2 days;

[0076] S3. Mix the modified bentonite and modified β-cyclodextrin in water by ultrasonic, add 2% of sodium alginate based on the total mass of the modified bentonite and modified β-cyclodextrin, stir to form a gel, add 2% of CaCl2 solution based on the total mass of the modified bentonite and modified β-cyclodextrin, solidify for 30 min, dry the product in an oven, and crush to obtain a second mixture;

[0077] S4. Carry out the first turning on the third day of fermentation, uniformly layer the second mixture, spray water to keep moist, and continue to ferment at 65℃ for 2 days;

[0078] S5. Carry out the second turning on the fifth day of fermentation, uniformly layer the potassium dihydrogen phosphate and gypsum, and continue to ferment at 55℃ for 2 days;

[0079] S6. After the seventh day of maturation and stabilization, the pile temperature is reduced to below 45℃, the pile is spread out to air, and the moisture content is adjusted to 62%.

[0080] The preparation method of the modified bentonite and modified β-cyclodextrin of the embodiment is consistent with that of example 1.

[0081] Example 5: The difference between this example and example 1 is that no cetyltrimethylammonium bromide is added in the preparation of the modified bentonite.

[0082] The example discloses a kind of highland barley straw for preparing large ball cap mushroom cultivation substrate, by mass fraction including raw material: highland barley straw 60 parts, modified bentonite 8 parts, modified β-cyclodextrin 5 parts, wheat bran 15 parts, dry cow dung 10 parts, superphosphate 1 part;

[0083] The cultivation substrate further includes: potassium dihydrogen phosphate and gypsum;

[0084] The mass of the potassium dihydrogen phosphate is 0.3% of the dry weight of the cultivation substrate, and the mass of the gypsum is 0.1% of the dry weight of the cultivation substrate.

[0085] The preparation method of the modified bentonite includes the following steps:

[0086] S11. Take bentonite through a 200 mesh sieve, dry in a 105℃ oven, and remove adsorbed water;

[0087] S12. Dissolve citric acid in 200ml deionized water, add bentonite, and react for 30 minutes under ultrasonic to obtain a reaction solution. The reaction solution is cooled to below 40℃ in an ice bath, NaOH is added to adjust the pH to neutral, and the precipitate is collected by centrifugation and dried to constant weight in a 60℃ oven to obtain the fifth compound;

[0088] S13. Ultrasonically disperse ZnO nano powder in 100ml anhydrous ethanol, add the fifth compound, and stir at 45℃. Collect the solid by suction filtration, wash with ethanol, dry in a 60℃ vacuum drying oven, crush through a 200 mesh sieve, and obtain the modified bentonite.

[0089] The mass of the citric acid and ZnO nano powder is 8% and 3% of the mass of the bentonite, respectively.

[0090] The preparation method of the modified β-cyclodextrin of the present embodiment is consistent with that of Example 1. The present embodiment is one kind of preparation method for preparing a large Stropharia rugoso-annulata cultivation substrate using highland barley straw.

[0091] Example 6: The difference between the present embodiment and Example 1 is that no ZnO nano powder is added in the preparation of the modified bentonite.

[0092] The present embodiment discloses a large Stropharia rugoso-annulata cultivation substrate prepared from highland barley straw, which comprises, by mass fraction, raw materials: highland barley straw 60 parts, modified bentonite 8 parts, modified β-cyclodextrin 5 parts, wheat bran 15 parts, dry cow manure 10 parts, and superphosphate 1 part.

[0093] The cultivation substrate further comprises potassium dihydrogen phosphate and gypsum.

[0094] The mass of the potassium dihydrogen phosphate is 0.3% of the dry weight of the cultivation substrate, and the mass of the gypsum is 0.1% of the dry weight of the cultivation substrate.

[0095] The preparation method of the modified bentonite comprises the following steps:

[0096] S11. Take bentonite through a 200 mesh sieve and dry in a 105℃ oven to remove adsorbed water;

[0097] S12. Dissolve 15g cetyltrimethylammonium bromide in 500ml deionized water, heat in a water bath, add bentonite, and react in a 60℃ constant temperature oscillator for 2 hours. Centrifuge the reaction solution at 4000rmp for 10 minutes, wash the precipitate with 60℃ deionized water until no foam is generated, remove the free cetyltrimethylammonium bromide, and obtain the first compound;

[0098] S13. Dissolve citric acid in 200ml deionized water, add the first compound, and react for 30 minutes under ultrasonic to obtain a reaction solution. Cool the reaction solution to below 40℃ in an ice bath, add NaOH to adjust the pH to neutral, centrifuge to collect the precipitate, and dry in a 60℃ vacuum drying oven. Crush the precipitate through a 200 mesh sieve to obtain the modified bentonite.

[0099] The mass of the cetyltrimethylammonium bromide and the citric acid is 15% and 8% of the mass of the bentonite, respectively.

[0100] The preparation method of the modified beta-cyclodextrin of the present embodiment is consistent with that of Embodiment 1. The present embodiment is one kind of preparation method for preparing a large ball-cap mushroom cultivation substrate using highland barley straw.

[0101] Embodiment 7: The difference between the present embodiment and Embodiment 1 is that the modified beta-cyclodextrin is not grafted with carboxymethyl.

[0102] The present embodiment discloses a large ball-cap mushroom cultivation substrate prepared from highland barley straw, which comprises, by mass fraction, raw materials: highland barley straw 60 parts, modified bentonite 8 parts, modified beta-cyclodextrin 5 parts, wheat bran 15 parts, dry cow manure 10 parts, and superphosphate 1 part.

[0103] The cultivation substrate further comprises potassium dihydrogen phosphate and gypsum.

[0104] The mass of the potassium dihydrogen phosphate is 0.3% of the dry weight of the cultivation substrate, and the mass of the gypsum is 0.1% of the dry weight of the cultivation substrate.

[0105] The preparation method of the modified beta-cyclodextrin comprises the following steps:

[0106] S21. Add 20g beta-cyclodextrin, 1.2g 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide, and 0.7g N-hydroxysuccinimide into 200ml pH=6 2-morpholinoethanesulfonic acid buffer, and stir for 30 minutes to activate to obtain a sixth compound.

[0107] S22. Dissolve 3-aminobenzene boronic acid in N,N-dimethylformamide and add it to the sixth compound. React at 45℃ under nitrogen protection for 12 hours in the dark, sequentially purify by dialysis with deionized water and HCl, and freeze-dry to obtain the modified beta-cyclodextrin.

[0108] The preparation method of the modified bentonite of the present embodiment is consistent with that of Embodiment 1. The present embodiment is one kind of preparation method for preparing a large ball-cap mushroom cultivation substrate using highland barley straw.

[0109] Embodiment 8: The difference between the present embodiment and Embodiment 1 is that the modified beta-cyclodextrin is not grafted with boronic acid groups.

[0110] The embodiment discloses a cultivation substrate for Stropharia rugosoannulata prepared from highland barley straw, which comprises the following raw materials in parts by mass: highland barley straw 60 parts, modified bentonite 8 parts, modified beta-cyclodextrin 5 parts, wheat bran 15 parts, dry cow manure 10 parts and superphosphate 1 part.

[0111] The cultivation substrate further comprises potassium dihydrogen phosphate and gypsum.

[0112] The mass of the potassium dihydrogen phosphate is 0.3% of the dry weight of the cultivation substrate, and the mass of the gypsum is 0.1% of the dry weight of the cultivation substrate.

[0113] The preparation method of the modified beta-cyclodextrin comprises the following steps.

[0114] S21. 50g of beta-cyclodextrin is dissolved in 200ml of NaOH solution, magnetically stirred until completely transparent, and sodium chloroacetate solution is added dropwise, 60℃ constant temperature oil bath, stirring reaction for 6 hours, adjusting pH to neutral with glacial acetic acid, dialysis purification, freeze-drying to obtain modified beta-cyclodextrin.

[0115] The preparation method of the modified bentonite of the embodiment is consistent with that of embodiment 1. The preparation method of the cultivation substrate for Stropharia rugosoannulata prepared from highland barley straw of the embodiment is consistent with that of embodiment 1.

[0116] Control group 1: the difference between the embodiment and embodiment 1 is that the modified bentonite is not contained.

[0117] The embodiment discloses a cultivation substrate for Stropharia rugosoannulata prepared from highland barley straw, which comprises the following raw materials in parts by mass: highland barley straw 60 parts, modified beta-cyclodextrin 5 parts, wheat bran 15 parts, dry cow manure 10 parts and superphosphate 1 part.

[0118] The cultivation substrate further comprises potassium dihydrogen phosphate and gypsum.

[0119] The mass of the potassium dihydrogen phosphate is 0.3% of the dry weight of the cultivation substrate, and the mass of the gypsum is 0.1% of the dry weight of the cultivation substrate.

[0120] The preparation method of the modified beta-cyclodextrin of the embodiment is consistent with that of embodiment 1. The preparation method of the cultivation substrate for Stropharia rugosoannulata prepared from highland barley straw of the embodiment is consistent with that of embodiment 1.

[0121] Control group 2: the difference between the embodiment and embodiment 1 is that the modified beta-cyclodextrin is not contained.

[0122] The embodiment discloses a cultivation substrate for Stropharia rugosoannulata prepared from highland barley straw, which comprises the following raw materials in parts by mass: highland barley straw 60 parts, modified bentonite 8 parts, wheat bran 15 parts, dry cow manure 10 parts and superphosphate 1 part.

[0123] The cultivation substrate further comprises potassium dihydrogen phosphate and gypsum.

[0124] The mass of the potassium dihydrogen phosphate is 0.3% of the dry weight of the cultivation substrate, and the mass of the gypsum is 0.1% of the dry weight of the cultivation substrate.

[0125] The preparation method of the modified bentonite of the embodiment is consistent with that of Embodiment 1. The preparation method of the cultivation substrate for the Stropharia rugosoannulata prepared by using the highland barley straw of the embodiment is consistent with that of Embodiment 1.

[0126] The control group 3 is different from Embodiment 1 in that the control group 3 does not contain potassium dihydrogen phosphate and gypsum.

[0127] The cultivation substrate for the Stropharia rugosoannulata prepared by using the highland barley straw disclosed in the embodiment comprises, by mass fraction, raw materials: 60 parts of highland barley straw, 8 parts of modified bentonite, 5 parts of modified β-cyclodextrin, 15 parts of wheat bran, 10 parts of dry cow manure, and 1 part of superphosphoric acid calcium.

[0128] The preparation method of the modified bentonite and the modified β-cyclodextrin of the embodiment is consistent with that of Embodiment 1. The preparation method of the cultivation substrate for the Stropharia rugosoannulata prepared by using the highland barley straw of the embodiment is consistent with that of Embodiment 1.

[0129] The control group 4 is a blank control group.

[0130] The cultivation substrate for the Stropharia rugosoannulata prepared by using the highland barley straw disclosed in the embodiment comprises, by mass fraction, raw materials: 60 parts of highland barley straw, 15 parts of wheat bran, 10 parts of dry cow manure, and 1 part of superphosphoric acid calcium.

[0131] The preparation method of the cultivation substrate for the Stropharia rugosoannulata prepared by using the highland barley straw comprises the following steps:

[0132] S1. The highland barley straw is crushed to 3 cm, screened and dusted, soaked in lime water for 24 hours, washed with clean water until neutral, steam exploded at 1.8 MPa and 185°C, the silicon wax layer of the crushed highland barley straw is washed with an oxalic acid solution, washed with running water until neutral, filtered by a centrifuge, and a first mixture is obtained;

[0133] S2. The wheat bran, dry cow manure, and superphosphoric acid calcium are weighed according to the proportion, added to the first mixture, uniformly mixed, layered and laid, sprayed with water to keep moist, and stacked and fermented at 60°C for 2 days;

[0134] S3. The first turning is performed on the third day of fermentation, and the fermentation is continued at 60°C for 2 days;

[0135] S5. The second turning is performed on the fifth day of fermentation, sprayed with water to keep moist, and the fermentation is continued at 50°C for 2 days;

[0136] S6. After 7 days, the mature stability is stable, the pile temperature is reduced to below 45℃, the pile is spread and aired, and the moisture content is adjusted to 60%.

[0137] Test verification:

[0138] 1. Bacteriostatic rate and pH value determination: Take the cultivation substrate at the early and late stages of filamentation, prepare the extract, test the inhibition rate of Trichoderma and Aspergillus spore germination, measure the pH value with a pH meter, and record the test results as shown in Table 1.

[0139]

[0140] Table 1 is a bacteriostatic rate and pH value statistical table of the cultivation substrate of each test group of Stropharia rugosoannulata at the early and late stages of filamentation. Compared with control groups 1, 2, 3, and 4, the effect of Example 1 is better, indicating that the addition of modified bentonite, modified β-cyclodextrin, potassium dihydrogen phosphate, and gypsum enhances the bacteriostatic effect of the cultivation substrate at the late stage of filamentation. The bacteriostatic rate of Example 5, Example 8, and control groups 1, 2, 3, and 4 at the early stage of filamentation is significantly higher than that at the late stage of filamentation, indicating that there are more free phenolic substances in the cultivation substrate at the early stage of filamentation, so that the phenolic substances decrease at the late stage, failing to achieve the bacteriostatic effect. This indicates that the synergistic effect of modified bentonite, modified β-cyclodextrin, potassium dihydrogen phosphate, and gypsum, at the early stage of filamentation, the cetyltrimethylammonium bromide on the surface of modified bentonite adsorbs a large amount of phenolic substances, the borate groups on the surface of modified β-cyclodextrin form borate ester bonds with ortho-diphenol hydroxyl groups, double-locking phenolic substances, gypsum enhances the structural stability of modified bentonite, avoiding its premature disintegration at the early stage, potassium dihydrogen phosphate forms a phosphate buffer system to resist the acidification at the early stage of mycelial metabolism, inhibiting the hydrolysis of borate ester bonds, so that phenolic substances are not released when mycelium is relatively fragile at the early stage of filamentation, avoiding the toxic effect on mycelium and inhibiting its growth. With the consumption of phosphate by mycelial metabolic acid production, the buffering capacity of potassium dihydrogen phosphate is lost, and the pH naturally decreases at the late stage of filamentation to the primordium stage, causing the hydrolysis of borate ester bonds and the weakening of the hydrophobicity of cetyltrimethylammonium bromide, both of which release phenolic substances. Compared with control group 3, the pH of Example 1 at the early stage of filamentation is slightly acidic, while control group 3 without the addition of potassium dihydrogen phosphate and gypsum is more acidic, which can weaken the hydrophobicity of cetyltrimethylammonium bromide and hydrolyze borate ester bonds in an acidic environment, causing the premature release of phenolic compounds and leading to a decrease in the bacteriostatic rate at the late stage of filamentation.

[0141] 2. The fruiting time, yield, height, and diameter of the test group of Stropharia rugosoannulata are compared, and the test results are recorded as shown in Table 2.

[0142]

[0143] Table 2 is the fruiting time, yield, plant height and diameter of each test group of Stropharia rugosoannulata, from which it can be known that, compared with the control groups 1, 2, 3 and 4, the fruiting time of the example 1 is longer, the yield is higher, and the plant height and stem diameter are better, and thus it can be known that the modified bentonite, modified β-cyclodextrin, potassium dihydrogen phosphate and gypsum can control the adsorption and release of phenolic substances, inhibit the growth of Trichoderma, Aspergillus and other bacteria, create a clean substrate environment, and promote the growth of Stropharia rugosoannulata.

[0144] Finally, it should be noted that although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments or make equivalent replacements for part of the technical features, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A cultivation substrate for Stropharia rugoso-annulata prepared using highland barley straw, characterized in that, The raw materials include, by mass fraction: highland barley straw 60-100 parts, modified bentonite 8-12 parts, modified beta-cyclodextrin 5-8 parts, wheat bran 15-20 parts, dry cow manure 10-15 parts, and calcium superphosphate 1-2 parts; The cultivation substrate further comprises potassium dihydrogen phosphate and gypsum; The mass of the potassium dihydrogen phosphate is 0.3%-0.5% of the dry weight of the cultivation substrate, and the mass of the gypsum is 0.1% of the dry weight of the cultivation substrate; The preparation method of the modified bentonite comprises the following steps: S11. Take bentonite through a 200-mesh sieve and dry it in an oven at 105 DEG C to remove adsorbed water; S12. Dissolve cetyltrimethylammonium bromide in deionized water, heat in a water bath, add bentonite, and react in a constant-temperature oscillator at 60 DEG C for 2 hours. Centrifuge the reaction solution at 4000 rpm for 10 minutes, collect the precipitate, and wash the precipitate with deionized water at 60 DEG C until no foam is generated to remove free cetyltrimethylammonium bromide, to obtain a first compound; S13. Dissolve citric acid in deionized water, add the first compound, and react under ultrasonic for 30 minutes to obtain a reaction solution. Cool the reaction solution in an ice bath to below 40 DEG C, add NaOH to adjust the pH to neutral, centrifuge to collect the precipitate, and dry the precipitate in an oven at 60 DEG C to constant weight to obtain a second compound; S14. Ultrasonically disperse ZnO nano powder in anhydrous ethanol, add the second compound, constant-temperature stir at 45 DEG C, collect the solid by suction filtration, wash with ethanol, dry in a vacuum drying oven at 60 DEG C, crush and pass through a 200-mesh sieve to obtain modified bentonite; The preparation method of the modified beta-cyclodextrin comprises the following steps: S21. Dissolve beta-cyclodextrin in a NaOH solution, magnetically stir until completely transparent, dropwise add a sodium chloroacetate solution, constant-temperature oil bath at 60 DEG C, stir and react for 6 hours, adjust the pH to neutral with glacial acetic acid, purify by dialysis, and freeze-dry to obtain a third compound; S22. Add the third compound, 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide, and N-hydroxysuccinimide to a 2-morpholinoethanesulfonic acid buffer solution at pH = 6, and stir and activate for 30 minutes to obtain a fourth compound; S23. Dissolve 3-aminobenzene boronic acid in N,N-dimethylformamide, add to the fourth compound, react at constant temperature under nitrogen protection at 45 DEG C in the dark, sequentially purify by dialysis with deionized water and HCl, and freeze-dry to obtain modified beta-cyclodextrin.

2. The cultivation substrate for Stropharia rugoso-annulata prepared from highland barley straw according to claim 1, characterized in that, The masses of the cetyltrimethylammonium bromide, citric acid, and ZnO nano powder are 15%, 8%, and 3% of the mass of the bentonite, respectively.

3. The cultivation substrate for Stropharia rugoso-annulata prepared from highland barley straw according to claim 1, characterized in that, The mass ratio of the beta-cyclodextrin to the sodium chloroacetate is 5:3, and the mass ratio of the fourth compound to the 3-aminobenzene boronic acid is 4:

1.

4. A method for preparing a Stropharia rugoso-annulata cultivation substrate using highland barley straw, applied to the preparation of the Stropharia rugoso-annulata cultivation substrate using highland barley straw according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: S1. Crush highland barley straw to 3-5 cm, sieve and remove dust, soak in limewater for 24 hours, rinse with clean water until neutral, steam explosion at 1.8 MPa and 185 DEG C, crush the silica wax layer of the highland barley straw, wash with an oxalic acid solution, rinse with running water until neutral, filter by a centrifuge to obtain a first mixture; S2. The wheat bran, dry cow dung and calcium superphosphate are weighed according to the proportion, added into the first mixture, uniformly mixed, layered and laid, sprayed with water for moisture retention, and stacked for fermentation at 60-65 DEG C for 2 days; S3. The modified bentonite and modified beta-cyclodextrin are ultrasonically mixed in water according to the proportion, sodium alginate is added, stirred to form a gel, CaCl2 solution is added for solidification, the product is placed in an oven for drying, and the second mixture is obtained by crushing; S4. The first turning is carried out on the third day of fermentation, and the second mixture is uniformly layered and scattered, sprayed with water for moisture retention, and continuously fermented at 60-65 DEG C for 2 days; S5. The second turning is carried out on the fifth day of fermentation, and the potassium dihydrogen phosphate and gypsum are uniformly layered and scattered, and continuously fermented at 50-55 DEG C for 2 days; S6. After the seventh day of maturation and stabilization, the pile temperature is reduced to below 45 DEG C, the pile is spread and aired, and the moisture content is adjusted to 60-62%.

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