Boletus aereus bag material culture medium and preparation method thereof

Abstract

The invention relates to a boletus aereus bag material culture medium and a preparation method thereof, and particularly belongs to the technical field of preparation of strain culture materials, and the boletus aereus bag material culture medium specifically comprises the following raw materials: composite strains, edible fungus residues, humus soil, dry cow dung, pine needle leaves, wheat bran, monopotassium phosphate, forchlorfenuron, quicklime and slow release components. The invention provides a coating material based on lignin and sodium alginate, a three-dimensional network structure is formed through esterification reaction and hydrogen bond and electrostatic interaction under an alkaline condition, and directional slow release of the growth promoter is realized. The synergistic effect of honeycomb powder, calcium carbonate and calcium chloride in the slow-release enhancer further optimizes the coating performance, the pH-responsive release and porous structure design concept of the slow-release enhancer can also be expanded to the field of other edible mushroom and plant root system fertilizers, and the slow-release enhancer has wide application prospects.

Description

Technical Field

[0001] This application relates to the technical field of strain culture material preparation, and more specifically, it relates to a bagged culture medium for Boletus aereus and a preparation method thereof. Background Art

[0002] As a rare edible mushroom, Boletus aereus is deeply loved by the masses. Traditional cultivation relies on mycorrhizal symbiosis or bionic cultivation models. Traditional substrates mostly use natural materials such as sawdust and red soil, which have defects such as uneven nutrient release, poor physical stability, and susceptibility to microbial contamination, and it is difficult to meet the requirements of industrialized and large-scale production.

[0003] In addition, due to the high demand of Boletus aereus for cellulose and lignin, plant-based materials are commonly used in the culture medium for Boletus aereus, but most of them contain indole-3-acetic acid (IAA), and the full concentration of IAA inhibits the development of Boletus aereus. Moreover, during the fermentation process, the substrate is not exposed to light, and IAA decomposes slowly. With the decomposition of some strains, the enrichment of IAA in the culture medium will be increased, thereby increasing the aging of the edible mushroom mycelium. Some chemical methods will increase costs and cause drug residues, affecting food safety. And artificial drying will not only increase the time cost but also lead to the loss of nutrient elements.

[0004] Based on the above statements, this application provides a bagged culture medium for Boletus aereus and a preparation method thereof. Summary of the Invention

[0005] To solve the problems raised in the background art, this application provides a bagged culture medium for Boletus aereus and a preparation method thereof.

[0006] This application provides a bagged culture medium for Boletus aereus and a preparation method thereof, adopting the following technical solutions:

[0007] A preparation method of a bagged culture medium for Boletus aereus, comprising the following steps:

[0008] S1. Prepare a slow-release material, and use the slow-release material to encapsulate a composite growth promoter to obtain a slow-release component;

[0009] S2. Weigh the formula mass of edible mushroom residue, humus soil, pine needles, wheat bran, and dry cow dung and mix them to obtain a fermentation substrate. Then use a composite strain to ferment the fermentation substrate. After fermentation, mix it with the formula mass of potassium dihydrogen phosphate, forchlorfenuron, quicklime, and the slow-release component to obtain a bagged culture medium for Boletus aereus;

[0010] Among them, the slow-release component is obtained by encapsulating the composite growth promoter with a slow-release material; the slow-release material is obtained by mixing lignin, sodium alginate, and a slow-release enhancer, coating the composite growth promoter, and then subjecting it to heat coating, rolling moisture removal, and drying.

[0011] Further, in step S1, the sustained-release component is prepared by the following steps:

[0012] S11. Add lignin into the NaOH solution, stir, then add sodium alginate and the sustained-release enhancer, raise the temperature of the system to 60 - 70 °C, continue stirring, and cool to room temperature to obtain the coating material;

[0013] S12. Thermally coat the coating material with the composite growth promoter. The mass ratio of the composite growth promoter to the inner coating material is 100:(15 - 20). Then discharge the material and dry the obtained particulate matter at 30 - 40 °C for 10 - 20 hours to obtain the sustained-release component.

[0014] Further preferably, in step S1, the sustained-release component is prepared by the following steps:

[0015] S11. Add lignin into the 0.1 mol / L NaOH solution, stir at a rate of 30 - 90 rpm for 20 - 30 minutes at room temperature, then add sodium alginate and the sustained-release enhancer, raise the temperature of the system to 60 - 70 °C, continue stirring for 20 - 30 minutes, and then cool to room temperature to obtain the coating material;

[0016] S12. Place the composite growth promoter on a disk granulator, thermally coat the coating material, roll to remove moisture to form particles. The inclination angle of the disk body is 35 - 55°, the rotation speed is 10 - 12 rpm, the mass ratio of the composite growth promoter to the inner coating material is 100:(15 - 20). Then discharge the material and dry the obtained particulate matter at 30 - 40 °C for 10 - 20 hours to obtain the sustained-release component.

[0017] In the above reaction process, using lignin and sodium alginate materials as the basis, the coating material is prepared. Lignin is partially depolymerized under alkaline conditions, its phenolic hydroxyl group reacts with the carboxyl group of sodium alginate to form an ester bond, and the phenolic hydroxyl group and the carboxyl group can also be combined by hydrogen bonds. At the same time, the cationic characteristics of lignin and the anionic group of sodium alginate form an electrostatic interaction to form a physical cross-linking network, and the coating material is prepared for coating the composite growth promoter.

[0018] Further, in step S11, the dosage ratio of lignin, NaOH solution, sodium alginate and the sustained-release enhancer is (0.5 - 1.5) g:100 mL:(1 - 3) g:(2 - 4) g.

[0019] Further, in step S11, the sustained-release enhancer is prepared by the following steps:

[0020] Mix honeycomb powder with a plasticizer and calcium carbonate at a ratio of 1:(0.1 - 0.3):(0.1 - 0.3) at room temperature to obtain the sustained-release enhancer.

[0021] Preferably, the plasticizer is polyethylene glycol or glycerol.

[0022] Further, in step S12, the composite growth promoter is obtained by mixing vitamin B2, vitamin B6 and vitamin C in a mass ratio of (1 - 5):5:(2 - 3).

[0023] Further, in step S2, the composite strain is specifically prepared by the following steps:

[0024] S21. Prepare an activation medium according to the following components: 8 - 12 g / L glucose, 4 - 6 g / L yeast extract, 8 - 12 g / L pine needle powder, 1 - 3 g / L ammonium sulfate, 0.5 - 1.5 g / L peptone, 0.2 - 0.6 g / L magnesium sulfate heptahydrate, 0.5 - 1.5 g / L potassium dihydrogen phosphate, 0.05 - 0.2 g / L ferrous sulfate heptahydrate;

[0025] S22. Shake and culture the phagocytic bacterium and Trichoderma in the activation medium obtained in step S21, adjust the pH value of the system to 6.5 - 7.0, the culture temperature to 28 - 30 °C, the shaking speed of the shaker to 120 - 150 rpm. After culturing for 10 - 20 hours, Aspergillus niger is inoculated, the pH value of the system is adjusted to 6.0 - 6.5, the culture temperature is set to 25 - 28 °C, and after fermenting for 15 - 20 hours, the culture system is collected, centrifuged, filtered, the filter residue is washed, and dried to obtain the composite strain.

[0026] More preferably, in step S2, the composite strain is specifically prepared by the following steps:

[0027] S21. Prepare an activation medium with sterile deionized water according to the following components: 8 - 12 g / L glucose, 4 - 6 g / L yeast extract, 8 - 12 g / L pine needle powder, 1 - 3 g / L ammonium sulfate, 0.5 - 1.5 g / L peptone, 0.2 - 0.6 g / L magnesium sulfate heptahydrate, 0.5 - 1.5 g / L potassium dihydrogen phosphate, 0.05 - 0.2 g / L ferrous sulfate heptahydrate;

[0028] S22. Shake and culture the phagocytic bacterium and Trichoderma in the activation medium obtained in step S21, use 0.1 mol / L ammonia water to adjust the pH value of the system to 6.5 - 7.0, the culture temperature to 28 - 30 °C, the shaking speed of the shaker to 120 - 150 rpm. After culturing for 10 - 20 hours, Aspergillus niger is inoculated, and 0.1 mol / L hydrochloric acid solution is used to adjust the pH value of the system to 6.0 - 6.5. Set the culture temperature to 25 - 28 °C, and after fermenting for 15 - 20 hours, the culture system is collected, centrifuged at a rate of 8000 - 10000 rpm for 10 - 20 minutes, filtered, the filter residue is washed with sterile deionized water, and naturally dried until the water content is 40 - 50%, to obtain the composite strain.

[0029] Further, in step S22, the viable cell numbers ratio of phagocytic bacteria, Trichoderma, and Aspergillus niger is (3 - 5):(4 - 12):(1 - 2), and the total inoculation amount of the three strains is 6 - 10%.

[0030] Further, the specific operation of using the composite strain to ferment the fermentation substrate is as follows:

[0031] Mix the composite strain with the fermentation substrate, adjust the water content of the system to 45 - 65% using sterile deionized water, set the pH value of the system to 5.5 - 6.0, ferment at 15 - 25°C and an air humidity of 65 - 75% for 15 - 35 days, and set the stirring times to 4 - 6 times during this period.

[0032] A bagged culture medium for Boletus aereus comprises the following raw materials in parts by mass:

[0033] 5 - 10 parts of composite strain, 120 - 200 parts of edible mushroom residue, 50 - 85 parts of humus soil, 22 - 40 parts of dry cow dung, 40 - 75 parts of pine needles, 35 - 65 parts of wheat bran, 15 - 25 parts of potassium dihydrogen phosphate, 0.5 - 1.5 parts of forchlorfenuron, 35 - 85 parts of quicklime, and 10 - 20 parts of slow - release component.

[0034] In summary, the present application has the following beneficial effects:

[0035] In the technical solution of the present invention, based on lignin and sodium alginate materials, a coating material is prepared. Lignin is partially depolymerized under alkaline conditions, and its phenolic hydroxyl group reacts with the carboxyl group of sodium alginate to form an ester bond. Moreover, the phenolic hydroxyl group and the carboxyl group can also be combined through hydrogen bonds. At the same time, the cationic property of lignin forms an electrostatic interaction with the anionic group of sodium alginate to form a physical cross - linked network for coating the composite growth promoter.

[0036] The coating material is also added with a sustained-release enhancer, which is specifically composed of honeycomb powder, plasticizer, calcium carbonate, and calcium chloride, and is used to regulate the encapsulation and release effects of the packaging material to make it more adaptable to the harvesting cycle of Boletus aereus. The high specific surface area of honeycomb powder and calcium carbonate can adsorb more growth promoters and reduce the initial burst release phenomenon. As fillers, calcium carbonate and calcium chloride can improve the hardness, wear resistance, and durability of the coating material, while adjusting the thermal expansion coefficient of the material to reduce performance fluctuations caused by environmental changes. As inorganic fillers, calcium carbonate and calcium chloride together with honeycomb powder form a porous structure. This structure can limit the diffusion rate of water and nutrients, thereby prolonging the release time. At the same time, the porosity of honeycomb powder enhances the adsorption capacity and promotes the uniform distribution of growth promoters in the coating material. Calcium carbonate gradually dissolves under acidic conditions (in the environment of organic acids secreted by plant roots), releasing calcium ions and destroying the cross-linked network of sodium alginate. This pH-responsive design enables the directional release of growth promoters near the roots, improving the nutrient utilization efficiency and avoiding drastic fluctuations in the pH value in the culture medium. The carboxyl groups of sodium alginate undergo ionic cross-linking with the free calcium ions in calcium carbonate to form a partial gel network, which can further strengthen the stability of the lignin-sodium alginate network structure, enhance the mechanical strength of the coating material, and further slow down the release rate of growth promoters through physical restriction.

[0037] In the technical solution of the present invention, phagocytic bacteria, Trichoderma viride, and Aspergillus niger are compounded to obtain a composite strain for the fermentation of the fermentation substrate. The extracellular enzymes such as protease and amylase secreted by phagocytic bacteria can decompose macromolecular substances such as cellulose, lignin, and protein in the substrate, enhance the decomposition of humus soil and pine needles, and release soluble nutrients for the utilization of Boletus aereus. At the same time, phagocytic bacteria can decompose IAA into indole and acetic acid monomers through its unique iad gene cluster, reduce the growth inhibition of IAA on Boletus aereus, and the acetic acid monomers can be used as fermentation substrates to promote the fermentation rate of synergistic bacteria. The obtained indole monomers can promote the formation of melanin in Boletus aereus. The accumulation of melanin in Boletus aereus can enhance stress resistance and improve the appearance of edible mushroom products. In addition, Trichoderma viride used in the solution mainly secretes cellulase, and Aspergillus niger strengthens the activities of xylanase and β-glucosidase after delayed inoculation to form a composite enzyme system. The three are compounded for mixed fermentation, and can transform bound substances through multiple aspects such as the cinnamic acid pathway and benzoic acid pathway, and improve the release amount of active ingredients. Specific embodiments

[0038] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

[0039] All the reagents involved in the specific implementation mode of this application are of analytical grade. In addition:

[0040] Phagocytotic bacterium: The viable count is 2×10 8 CFU / g, provided by Wuhan Warner Biotechnology Co., Ltd., with the product number WN-Je33980.

[0041] Trichoderma: Trichoderma viride, provided by Hubei Xinghengye Technology Co., Ltd., with the viable count of 5×10 9 CFU / g;

[0042] Aspergillus niger: The viable count is 2×10 8 CFU / g, provided by the China Pharmaceutical Microbial Culture Collection Management Center, with the strain number CPCC 800028;

[0043] Bacillus: Bacillus subtilis, provided by Shandong Xinzhuoyuan Biotechnology Co., Ltd., with the viable count of 1×10 11 CFU / g;

[0044] Calcium carbonate: The purity is 99%, and it is used after passing through a 50-mesh sieve.

[0045] Honeycomb powder: Provided by Anhui Kangweifu Pharmaceutical Co., Ltd.

[0046] Example 1

[0047] A bagged culture medium for Boletus aereus comprises the following raw materials in parts by mass:

[0048] 5 parts of compound bacteria, 120 parts of edible mushroom residue, 50 parts of humus soil, 22 parts of dried cow dung, 40 parts of pine needles, 35 parts of wheat bran, 15 parts of potassium dihydrogen phosphate, 0.5 part of forchlorfenuron, 35 parts of quicklime and 10 parts of slow-release component.

[0049] Its preparation method comprises the following steps:

[0050] S1. Prepare a slow-release material and use the slow-release material to encapsulate the compound growth promoter to obtain a slow-release component. The specific operation is as follows:

[0051] S11. Add lignin to 0.1 mol / L NaOH solution, stir at a rate of 90 rpm at room temperature for 25 minutes, then add sodium alginate and slow-release enhancer, raise the system temperature to 60 °C, continue to stir for 20 minutes, and then cool to room temperature to obtain a coating material; among them, the dosage ratio of lignin, NaOH solution, sodium alginate and slow-release enhancer is 0.5 g: 100 mL: 1 g: 2 g; the slow-release enhancer is obtained by mixing honeycomb powder, plasticizer and calcium carbonate in a mass ratio of 1: 0.1: 0.1 at room temperature, and the plasticizer is polyethylene glycol-2000.

[0052] S12. Place the composite growth promoter on a disk granulator for hot coating with the coating material, and roll to remove moisture to form granules. The inclination angle of the disk body is 45°, the rotation speed is 12 rpm, and the mass ratio of the composite growth promoter to the inner-layer coating material is 100:15. Then discharge the material and dry the obtained particulate matter at 35°C for 15 hours to obtain the sustained-release component; wherein, the composite growth promoter is obtained by mixing riboflavin, pyridoxine and ascorbic acid in a mass ratio of 1:5:2.

[0053] S2. Weigh the edible mushroom residue, humus soil, pine needles, wheat bran, and dried cow dung in the formula mass to obtain the fermentation substrate, and then ferment the fermentation substrate with the composite strain: Stir and mix the formula mass of the composite strain with the fermentation substrate, use sterile deionized water to adjust the water content of the system to 55%, set the pH value of the system to 5.5, and ferment for 30 days at 20°C and an air humidity of 70%. During this period, set the stirring times to 5 times; after fermentation, mix with the formula mass of potassium dihydrogen phosphate, forchlorfenuron, quicklime and the sustained-release component obtained in step S1 to obtain a black bolete bagged culture substrate.

[0054] Among them, the composite strain is specifically prepared by the following steps:

[0055] S21. Prepare an activation medium with sterile deionized water according to the following components: 8 g / L glucose, 4 g / L yeast extract, 8 g / L pine needle powder, 1 g / L ammonium sulfate, 0.5 g / L peptone, 0.2 g / L magnesium sulfate heptahydrate, 0.5 g / L potassium dihydrogen phosphate, 0.05 g / L ferrous sulfate heptahydrate;

[0056] S22. Place the Variovorax paradoxus and Trichoderma viride in the activation medium obtained in step S21, and culture them with shaking. Use 0.1 mol / L ammonia water to adjust the pH value of the system to 6.5, the culture temperature is 28°C, the shaking rate is 120 rpm. After culturing for 10 hours, Aspergillus niger is inoculated, and 0.1 mol / L hydrochloric acid solution is used to adjust the pH value of the system to 6.0. Set the culture temperature to 25°C, and after fermentation for 15 hours, collect the culture system, centrifuge at a rate of 8000 rpm for 10 minutes, filter, wash the filtrate with sterile deionized water, and naturally dry it to a moisture content of 40% to obtain the composite strain; the viable cell numbers ratio of Variovorax paradoxus, Trichoderma viride, and Aspergillus niger is 3:4:1, and the total inoculation amount of the three strains is 6%.

[0057] Example 2

[0058] A black bolete bagged culture substrate, comprising the following raw materials in parts by mass:

[0059] 8 parts of compound bacteria, 160 parts of edible mushroom residue, 65 parts of humus soil, 30 parts of dry cow dung, 55 parts of pine needles, 45 parts of wheat bran, 20 parts of potassium dihydrogen phosphate, 1 part of forchlorfenuron, 60 parts of quicklime and 15 parts of slow-release component.

[0060] Its preparation method includes the following steps:

[0061] S1. Prepare the slow-release material and use the slow-release material to encapsulate the compound growth promoter to obtain the slow-release component. The specific operation is as follows:

[0062] S11. Add lignin to 0.1mol / L NaOH solution, stir at a rate of 90rpm at room temperature for 25 minutes, then add sodium alginate and slow-release enhancer, raise the system temperature to 65°C, continue to stir for 25 minutes, and then cool to room temperature to obtain the coating material; among them, the dosage ratio of lignin, NaOH solution, sodium alginate and slow-release enhancer is 1g:100mL:2g:3g; the slow-release enhancer is obtained by mixing honeycomb powder, plasticizer and calcium carbonate in a mass ratio of 1:0.2:0.2 at room temperature, and the plasticizer is polyethylene glycol-2000.

[0063] S12. Place the compound growth promoter on a disk granulator for hot coating with the coating material, roll to remove moisture to form particles, the inclination angle of the disk body is 45°, the rotation speed is 12rpm, and the mass ratio of the compound growth promoter to the inner coating material is 100:18. Then discharge the material and dry the obtained particulate matter at 35°C for 15 hours to obtain the slow-release component; among them, the compound growth promoter is obtained by mixing vitamin B2, vitamin B6 and vitamin C in a mass ratio of 3:5:2.

[0064] S2. Weigh the edible mushroom residue, humus soil, pine needles, wheat bran, and dry cow dung in the formula parts by mass and mix them to obtain the fermentation substrate. Then use the compound bacteria to ferment the fermentation substrate: stir and mix the compound bacteria in the formula parts by mass with the fermentation substrate, use sterile deionized water to adjust the water content of the system to 55%, set the pH value of the system to 5.8, and ferment at 20°C and an air humidity of 70% for 30 days. During this period, set the stirring times to 5 times; after fermentation, mix with the potassium dihydrogen phosphate, forchlorfenuron, quicklime and the slow-release component obtained in step S1 in the formula parts by mass to obtain a bagged culture substrate for Boletus aereus;

[0065] Among them, the compound bacteria are specifically prepared by the following steps:

[0066] S21. Prepare an activation medium with sterile deionized water according to the following components: 10g / L glucose, 5g / L yeast extract, 10g / L pine needle powder, 2g / L ammonium sulfate, 1.5g / L peptone, 0.4g / L magnesium sulfate heptahydrate, 1.0g / L potassium dihydrogen phosphate, 0.1g / L ferrous sulfate heptahydrate;

[0067] S22. Shake the bacterium Devorans and Trichoderma viride in the activated culture medium obtained in step S21 on a shaker, adjust the pH value of the system to 6.8 with 0.1 mol / L ammonia water, set the culture temperature at 29 °C, the shaker speed at 130 rpm, and inoculate Aspergillus niger after culturing for 15 hours. Then adjust the pH value of the system to 6.5 with 0.1 mol / L hydrochloric acid solution, set the culture temperature at 26 °C, and after fermenting for 20 hours, collect the culture system, centrifuge at a speed of 9000 rpm for 20 minutes, filter, wash the filter residue with sterile deionized water, and naturally dry it until the water content is 45%, thus obtaining the composite strain; the viable cell count ratio of the bacterium Devorans, Trichoderma viride, and Aspergillus niger is 4:8:2, and the total inoculation amount of the three strains is 8%.

[0068] Example 3

[0069] A substrate for bagged culture of Boletus aereus comprises the following raw materials in parts by mass:

[0070] 10 parts of composite strain, 200 parts of edible mushroom residue, 85 parts of humus soil, 40 parts of dried cow dung, 75 parts of pine needles, 65 parts of wheat bran, 25 parts of potassium dihydrogen phosphate, 1.5 parts of forchlorfenuron, 85 parts of quicklime, and 20 parts of slow-release component.

[0071] Its preparation method comprises the following steps:

[0072] S1. Prepare a slow-release material and encapsulate the composite growth promoter with the slow-release material to obtain a slow-release component. The specific operation is as follows:

[0073] S11. Add lignin to 0.1 mol / L NaOH solution, stir at a speed of 90 rpm at room temperature for 25 minutes, then add sodium alginate and a slow-release enhancer, raise the temperature of the system to 70 °C, continue to stir for 30 minutes, and then cool to room temperature to obtain a coating material; wherein, the dosage ratio of lignin, NaOH solution, sodium alginate, and slow-release enhancer is 1.5 g:100 mL:3 g:4 g; the slow-release enhancer is obtained by mixing honeycomb powder, a plasticizer, and calcium carbonate in a mass ratio of 1:0.3:0.3 at room temperature, and the plasticizer is glycerol.

[0074] S12. Place the composite growth promoter on a disk granulator for hot coating with the coating material, roll to remove moisture to form particles, the inclination angle of the disk is 45°, the rotation speed is 12 rpm, the mass ratio of the composite growth promoter to the inner coating material is 100:20, then discharge the material and dry the obtained particulate matter at 35 °C for 15 hours to obtain the slow-release component; wherein, the composite growth promoter is obtained by mixing vitamin B2, vitamin B6, and vitamin C in a mass ratio of 5:5:3.

[0075] S2. Weigh the formula amounts of edible mushroom residues, humus soil, pine needles, wheat bran, and dry cow dung, mix them to obtain a fermentation substrate, and then ferment the fermentation substrate using a composite strain: Stir and mix the formula amount of the composite strain with the fermentation substrate, adjust the water content of the system to 55% using sterile deionized water, set the pH value of the system to 6.0, and ferment for 30 days at 20 °C and an air humidity of 70%. During this period, set the number of turning and mixing times to 5 times; after fermentation, mix with the formula amounts of potassium dihydrogen phosphate, forchlorfenuron, quicklime, and the slow-release component obtained in step S1 to obtain a bagged culture substrate for Boletus aereus;

[0076] Among them, the composite strain is specifically prepared by the following steps:

[0077] S21. Prepare an activation medium with sterile deionized water according to the following components: 12 g / L glucose, 6 g / L yeast extract, 12 g / L pine needle powder, 3 g / L ammonium sulfate, 1.5 g / L peptone, 0.6 g / L magnesium sulfate heptahydrate, 1.5 g / L potassium dihydrogen phosphate, 0.2 g / L ferrous sulfate heptahydrate;

[0078] S22. Shake and culture the phagocytic bacterium and Trichoderma viride in the activation medium obtained in step S21. Use 0.1 mol / L ammonia water to adjust the pH value of the system to 7.0, the culture temperature to 30 °C, and the shaker speed to 150 rpm. After culturing for 20 hours, Aspergillus niger is inoculated. Use 0.1 mol / L hydrochloric acid solution to adjust the pH value of the system to 6.5, set the culture temperature to 28 °C, and after fermentation for 20 hours, collect the culture system, centrifuge at a speed of 0000 rpm for 20 minutes, filter, wash the filter residue with sterile deionized water, and naturally dry it to a moisture content of 45% to obtain the composite strain; the viable cell ratio of the phagocytic bacterium, Trichoderma viride, and Aspergillus niger is 5:12:2, and the total inoculation amount of the three strains is 9%.

[0079] Comparative Example 1

[0080] The difference between this comparative example and Example 1 is that in step S2 of this comparative example, the phagocytic bacterium in the composite strain is replaced by Aspergillus niger. The specific preparation steps of the used composite strain are as follows:

[0081] S21. Prepare an activation medium with sterile deionized water according to the following components: 8 g / L glucose, 4 g / L yeast extract, 8 g / L pine needle powder, 1 g / L ammonium sulfate, 0.5 g / L peptone, 0.2 g / L magnesium sulfate heptahydrate, 0.5 g / L potassium dihydrogen phosphate, 0.05 g / L ferrous sulfate heptahydrate;

[0082] S22. Shake and culture the Trichoderma viride in the activated medium obtained in step S21 on a shaker. Adjust the pH value of the system to 6.5 with 0.1 mol / L ammonia water, set the culture temperature at 28 °C, the shaker speed at 120 rpm. After culturing for 10 hours, inoculate Aspergillus niger, adjust the pH value of the system to 6.0 with 0.1 mol / L hydrochloric acid solution, set the culture temperature at 25 °C, and after fermentation for 15 hours, collect the culture system, centrifuge at a speed of 8000 rpm for 10 minutes, filter, wash the filtered product with sterile deionized water, and naturally dry it to a moisture content of 40% to obtain the composite strain; the viable count ratio of Trichoderma viride to Aspergillus niger is 4:4, and the total inoculation amount of the two strains is 6%.

[0083] Comparative Example 2

[0084] The difference between this comparative example and Example 1 is that in step S2 of this comparative example, Bacillus is used instead of Trichoderma viride for the composite strain, and the specific composite strain is prepared by the following steps:

[0085] S21. Prepare an activated medium with sterile deionized water according to the following components: 8 g / L glucose, 4 g / L yeast extract, 8 g / L pine needle powder, 1 g / L ammonium sulfate, 0.5 g / L peptone, 0.2 g / L magnesium sulfate heptahydrate, 0.5 g / L potassium dihydrogen phosphate, 0.05 g / L ferrous sulfate heptahydrate;

[0086] S22. Shake and culture the Variovorax paradoxus and Bacillus in the activated medium obtained in step S21 on a shaker. Adjust the pH value of the system to 6.5 with 0.1 mol / L ammonia water, set the culture temperature at 28 °C, the shaker speed at 120 rpm. After culturing for 10 hours, inoculate Aspergillus niger, adjust the pH value of the system to 6.0 with 0.1 mol / L hydrochloric acid solution, set the culture temperature at 25 °C, and after fermentation for 15 hours, collect the culture system, centrifuge at a speed of 8000 rpm for 10 minutes, filter, wash the filtered product with sterile deionized water, and naturally dry it to a moisture content of 40% to obtain the composite strain; the viable count ratio of Variovorax paradoxus, Bacillus, and Aspergillus niger is 3:4:1, and the total inoculation amount of the three strains is 6%.

[0087] Comparative Example 3

[0088] The difference between this comparative example and Example 1 is that in the slow-release enhancer, instead of adding calcium carbonate, honeycomb powder is used, and the slow-release enhancer is specifically obtained by mixing honeycomb powder and a plasticizer at a mass ratio of 1:0.2 at room temperature.

[0089] Comparative Example 4

[0090] The difference between this comparative example and Example 1 is that in step S22, different strains are co-activated, and the specific operation is as follows:

[0091] The phagocytic bacterium, Aspergillus niger, and Trichoderma were cultured in a shaker in the activated culture medium obtained in step S21. The pH value of the system was adjusted to 6.5 using 0.1 mol / L ammonia water, the culture temperature was 28 °C, the shaker speed was 120 rpm. After culturing for 10 hours, the pH value of the system was adjusted to 6.0 using 0.1 mol / L hydrochloric acid solution, the culture temperature was set to 25 °C, and after fermenting for 15 hours, the culture system was collected, centrifuged at a speed of 8000 rpm for 10 minutes, filtered, the filter residue was washed with sterile deionized water, and naturally dried to a moisture content of 40%, thus obtaining the composite strain; the viable bacteria count ratio of the phagocytic bacterium, Trichoderma, and Aspergillus niger was 3:4:1, and the total inoculation amount of the three strains was 6%.

[0092] Comparative Example 5

[0093] The difference between this comparative example and Example 1 is that in step S22, no temperature gradient activation is set, and the specific operation is as follows:

[0094] The phagocytic bacterium and Trichoderma were cultured in a shaker in the activated culture medium obtained in step S21. The pH value of the system was adjusted to 6.5 using 0.1 mol / L ammonia water, the culture temperature was 28 °C, the shaker speed was 120 rpm. After culturing for 10 hours, Aspergillus niger was inoculated, and fermentation continued for 15 hours under the same fermentation conditions. Subsequently, the culture system was collected, centrifuged at a speed of 8000 rpm for 10 minutes, filtered, the filter residue was washed with sterile deionized water, and naturally dried to a moisture content of 40%, thus obtaining the composite strain; the viable bacteria count ratio of the phagocytic bacterium, Trichoderma, and Aspergillus niger was 3:4:1, and the total inoculation amount of the three strains was 6%.

[0095] Performance test

[0096] The bagged culture media prepared in Examples 1 - 3 and Comparative Examples 1 - 5 of this application were subjected to performance tests.

[0097] The bagged culture media prepared in different groups were filled into polyethylene plastic mushroom bags with a length of 60 cm, a width of 15 cm, and a thickness of 0.05 mm. 20 bags were prepared for each sample, and they were respectively inoculated with Boletus aereus solid strain, the inoculation amount was 5%, the cultivation temperature was set to 29 °C, the environmental humidity was 90%, and they were placed in a ventilated environment for cultivation, and all the mushrooms that had opened umbrellas were harvested within 150 days.

[0098] The fresh weight of the fresh mushrooms harvested in different groups was weighed, and the average harvest amount per bag of the culture medium was calculated; 10 Boletus aereus were randomly selected from each group of samples for appearance evaluation; 10 Boletus aereus were randomly selected from each group of samples, and the melanin content of the Boletus aereus products was measured.

[0099] The sensory evaluation criteria are as follows:

[0100] 96 - 100 points: The mycelium is intact without damage, the flesh is thick and nearly white, the color change at the injured part is normal (light purple or light yellow), the stipe is thick and not hollow, the surface has no mildew or impurities, and the smell is almond - like;

[0101] 90 - 95 points: The flesh is relatively thick but slightly thinned locally, the hardness of the stipe is moderate, there are occasionally a small amount of impurities or slight scratches on the surface, the smell is normal without peculiar smell, and there is no obvious swelling or abnormal reticulation on the stipe;

[0102] 80 - 89 points: The flesh thickness is medium, the hardness of the stipe is soft or slightly hollow, there are obvious scratches or a small amount of mildew spots on the surface, the smell is normal but slightly earthy, and the stipe has obvious swelling or disordered reticulation;

[0103] 60 - 79 points: The flesh is thin and soft, the stipe is significantly softened or hollow, there are obvious mildew spots or residual impurities on the surface, there are abnormal smells such as sour smell, and the stipe is thin and weak with water stains on the surface;

[0104] Below 60 points: The mycelium is severely damaged or fractured, worm - eaten, the cap is fully opened and the flesh is shriveled, the color is abnormal (such as blackening, turning green), the stipe is completely softened or rotten, there are mildew, peculiar smells or characteristics of poisonous mushrooms such as slime, annulus, etc.

[0105] The test method for melanin content is as follows:

[0106] Chop 5 Boletus aereus in different groups, freeze - dry, grind into powder, sieve through 80 - mesh sieve, then add to 1 mol / L NaOH solution according to the mass - volume ratio of 1 g:10 mL, ultrasonically treat at 200 W for 20 minutes at room temperature, then water - bath treat the system at 60 °C for 40 minutes, centrifuge at 10000×g for 10 minutes, collect the supernatant, adjust the pH value of the system to 2 with 6 mol / L HCl, water - bath at 80 °C for 12 hours, after natural cooling, centrifuge at 10000×g for 10 minutes, wash the precipitate with deionized water until colorless, freeze - dry, then add the product to 7 mol / L HCl solution according to the mass - volume ratio of 1 g:5 mL, water - bath at 80 °C for 12 hours, collect the precipitate and wash it 3 times with ultrapure water, dissolve the precipitate with 1 mmol / L KOH solution, add 10 mL of chloroform:isoamyl alcohol (1:5) and mix well, centrifuge at 10000×g for 10 minutes, repeat 3 times. Adjust the pH value to 2.0 with 1 mol / L HCl solution, centrifuge at 10000×g for 10 minutes again, wash the precipitate with absolute ethanol, centrifuge at 10000×g for 10 minutes, wash the precipitate 4 times with ultrapure water, collect the precipitate, freeze - dry, measure the mass of the product, and judge the melanin content in Boletus aereus.

[0107] The specific performance test results are shown in Table 1 below:

[0108] Table 1

[0109]

[0110] As can be seen from the results shown in Table 1 above:

[0111] The comprehensive performance of the black bolete bag culture medium prepared in Examples 1-3 of this application is significantly better than that of the products prepared in Comparative Examples 1-5. That is, within the limited technical solutions of this application, the comprehensive performance of the prepared culture medium product is excellent.

[0112] As can be seen from the results in Comparative Example 1, reducing the use of Phagocytella results in a lower production effect of promoting black bolete than the three-bacteria synergy. As can be seen from the results in Comparative Example 2, compared with the examples, the Bacillus in Comparative Example 2 mainly secretes protease, has a lower decomposition of lignin and cellulose substrates, and causes the accumulation of indole acetic acid in the system, relatively inhibiting the growth of black bolete. As can be seen from the results in Comparative Example 3, the absence of the calcium carbonate component weakens the pH responsiveness and mechanical strength of the coating material and cannot control the nutrient release rate. As can be seen from the results in Comparative Examples 4 and 5, staged activation and staged temperature activation can reduce the nutritional competition of the strains and improve their promoting effect on black bolete production.

[0113] In the description of the specification, the description referring to terms such as "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0114] The above content is only an example and explanation of the present invention. Those skilled in the art of this technology can make various modifications or supplements to the specific embodiments described or use similar methods to replace them. As long as they do not deviate from the invention or exceed the scope defined by this claim book, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing a bag culture matrix for Boletus edulis, characterized in that: The following steps are involved: S1. preparing a sustained-release material, and encapsulating the composite growth promoter using the sustained-release material to prepare a sustained-release component; S2. Weigh the formula weight of edible fungi residue, humus, pine needles, wheat bran, dry cow dung mixed to obtain a fermentation matrix, and then use the composite bacteria to ferment the fermentation matrix, and after fermentation, mix with the formula weight of potassium dihydrogen phosphate, chlorfenapyr, quicklime and a slow-release component to obtain a black boletus bag culture matrix; The slow-release component is obtained by encapsulating the composite growth promoter with the slow-release material; the slow-release material is obtained by mixing lignin, sodium alginate and a slow-release enhancer, and then coating the composite growth promoter, and then performing heat coating, rolling dehumidification and drying.

2. The method for preparing a bag culture matrix of Boletus edulis according to claim 1, characterized in that: In step S1, the sustained-release component is prepared by the following steps: S11. Add lignin to the NaOH solution, add sodium alginate and a sustained-release enhancer after stirring, raise the system temperature to 60-70°C, continue stirring, and cool to room temperature to obtain a coating material; S12. The composite growth promoter is thermally coated on the coating material, the mass ratio of the composite growth promoter to the inner coating material is 100:(15-20), and then the material is discharged and the obtained particles are dried at 30-40° C. for 10-20 hours to obtain a sustained-release component.

3. The method for preparing a bag culture matrix of Boletus edulis according to claim 2, characterized in that: In step S11, the dosage ratio of lignin, NaOH solution, sodium alginate and sustained-release enhancer is (0.5-1.5) g:100 mL:(1-3) g:(2-4) g.

4. The method for preparing a bag culture matrix of Boletus edulis according to claim 2, characterized in that: In step S11, the sustained-release enhancer is prepared by the following steps: The honeycomb powder is mixed with a plasticizer and calcium carbonate in a ratio of 1:(0.1-0.3):(0.1-0.3) at room temperature to obtain a sustained-release enhancer.

5. The method for preparing a bag culture matrix of Boletus edulis according to claim 2, characterized in that: In step S12, the composite growth promoter is obtained by mixing vitamin B2, vitamin B6 and vitamin C in a mass ratio of (1-5):5:(2-3).

6. The method for preparing a bag culture matrix of Boletus edulis according to claim 1, characterized in that: In step S2, the composite bacteria are prepared by the following steps: S21, prepare activation medium according to the following components: 8-12 g / L glucose, 4-6 g / L yeast extract, 8-12 g / L pine needle powder, 1-3 g / L ammonium sulfate, 0.5-1.5 g / L peptone, 0.2-0.6 g / L magnesium sulfate heptahydrate, 0.5-1.5 g / L potassium dihydrogen phosphate, 0.05-0.2 g / L ferrous sulfate heptahydrate; S22. In the activated culture medium obtained in step S21, the phagocytes and Trichoderma are cultured on a shaking table, the pH value of the system is adjusted to 6.5-7.0, the culture temperature is 28-30°C, the shaking rate is 120-150rpm, and after culturing for 10-20 hours, Aspergillus niger is inoculated, the pH value of the system is adjusted to 6.0-6.5, the culture temperature is set to 25-28°C, and after fermentation for 15-20 hours, the culture system is collected, centrifuged, filtered, the filtrate is washed, and dried to obtain a composite strain.

7. The method for preparing a bag culture matrix of Boletus edulis according to claim 6, characterized in that: In step S22, the ratio of the number of live bacteria of Bacillus thuringiensis, Trichoderma spp., and Aspergillus niger is (3-5):(4-12):(1-2).

8. The method for preparing a bag culture matrix of Boletus edulis according to claim 6, characterized in that: In step S22, the total inoculation amount of the three species of Bacillus phagnum, Trichoderma and Aspergillus niger is 6-10%.

9. The method for preparing a bag culture matrix of Boletus edulis according to claim 1, characterized in that: In step S2, the specific operation of fermenting the fermentation substrate using the composite strain is as follows: The composite bacteria and the fermentation matrix are stirred and mixed, the water content of the system is adjusted to 45-65%, the pH value of the system is set to 5.5-6.0, and ferment for 15-35 days at 15-25° C. and 65-75% air humidity, during which the stirring frequency is set to 4-6 times.

10. A culture matrix obtained by the method for preparing a bag culture matrix of Boletus edulis according to any one of claims 1 to 9, characterized in that: Including the following raw materials by weight: 5-10 parts of composite bacteria, 120-200 parts of edible fungus residues, 50-85 parts of humus soil, 22-40 parts of dry cow dung, 40-75 parts of pine needles, 35-65 parts of wheat bran, 15-25 parts of potassium dihydrogen phosphate, 0.5-1.5 parts of chlorpyrifos, 35-85 parts of quicklime and 10-20 parts of slow-release components.

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