Method for preparing under-forest edible fungus culture medium by utilizing crop straws and application of under-forest edible fungus culture medium

Abstract

The invention discloses a method for preparing an under-forest edible fungus culture medium from crop straw and application of the under-forest edible fungus culture medium. The method comprises the steps that firstly, after the crop straw is smashed, water is added, ultrasonic cavitation treatment and optical medium splitting decomposition treatment are conducted, drying is conducted, and pretreated straw is obtained; meanwhile, chitosan and poly (3-hydroxybutyric acid) are subjected to a reaction to form a prepolymer, the prepolymer is added into the composite bacterial liquid to be cross-linked and then dried, a composite bacterial agent is obtained, then the pretreated straw, the composite bacterial agent and auxiliary materials are mixed and packaged, and the culture medium is obtained. According to the method, the compact structure of the straw surface layer and connection of lignin, cellulose and hemicellulose are effectively broken, a loose and porous matrix structure is formed, sufficient acting sites are provided for growth of edible mushrooms, the composite microbial inoculant can remarkably improve the straw degradation efficiency, sufficient nutrition is provided for growth of the edible mushrooms, and the yield of the edible mushrooms is increased. In addition, pathogenic bacteria proliferation can be inhibited, diseases are reduced, mycelial growth and sporocarp development of the edible mushrooms are effectively promoted, the nutritional quality of the edible mushrooms is optimized, and efficient resource recycling of the crop straw is achieved.

Description

Technical Field

[0001] This invention belongs to the field of forest understory edible fungi cultivation technology, specifically relating to a method for preparing a forest understory edible fungi cultivation substrate using crop straw and its application. Background Technology

[0002] Forest-grown edible fungi refer to large edible fungi that naturally occur or are cultivated in a simulated ecological manner on substrates such as humus layers, fallen leaves, and fallen logs in natural or artificial forests. They rely on extracellular enzyme systems such as ligninase, cellulase, and hemicellulase secreted by their mycelium to decompose the lignocellulose in forest litter and fallen logs, converting it into their own nutrients. This achieves efficient reuse of forest litter while simultaneously completing the rapid cycling of elements such as carbon, nitrogen, and phosphorus. Common examples include matsutake, porcini, chanterelle, rutabaga, pine lacteus, and tiger paw mushrooms. These fungi form ectomycorrhizal symbiotic relationships with specific tree species. Their fruiting bodies are mostly umbrella-shaped, trumpet-shaped, or kidney-shaped, with colors ranging from golden yellow and bright red to dark brown. They have rich and layered flavors and only require simple washing after harvesting before stir-frying, stewing, making porridge, or adding soup. This method preserves the unique rich aroma of wild fungi while providing high-quality protein, polysaccharides, vitamins, and minerals for daily diets, making them forest delicacies that combine ecological value with high-end taste.

[0003] These fungi not only possess a unique flavor but also boast rich nutritional and health benefits: they are rich in high-quality plant protein, dietary fiber, various amino acids, and minerals such as calcium, iron, and zinc, effectively supplementing the body's daily nutritional needs and compensating for deficiencies in some nutrients found in grains and vegetables. Some species, such as Ganoderma lucidum and matsutake, contain polysaccharides and triterpenoids, which also have immune-regulating and gastrointestinal-protecting effects, holding an important position in traditional dietary therapy and health preservation systems. Furthermore, the growth characteristics of edible fungi make them highly valuable in the resource utilization of agricultural and forestry waste. Many species can efficiently degrade lignocellulose, allowing for artificial cultivation using crop straws such as wheat straw, corn straw, and cotton stalks, as well as forestry logging residues and wood processing waste as substrates. This reduces cultivation costs and achieves the recycling of agricultural and forestry waste, aligning with the concept of green development.

[0004] However, current technologies for the degradation of crop straw by edible fungi still face numerous bottlenecks. Currently, straw is often simply crushed and dried before being used directly as a cultivation substrate, failing to adequately meet the growth requirements of edible fungi. This is because the cell walls of crop straw are primarily composed of cellulose, hemicellulose, and lignin tightly bound together by covalent bonds, forming a dense three-dimensional network structure. This not only significantly increases the difficulty for edible fungi to decompose and utilize the straw through their secreted enzyme systems but also hinders the absorption of nutrients by the mycelium. Furthermore, most edible fungi are extremely sensitive to environmental conditions such as temperature, humidity, and pH. If the straw substrate is poorly compatible with the fungal strain or if environmental parameters are not properly controlled, problems such as slow mycelial growth, incomplete substrate consumption, delayed fruiting time, large yield fluctuations, and inconsistent fruiting body quality are highly likely to occur. More importantly, during the open-air storage of crop straw, changes in temperature and humidity make it highly susceptible to the growth of toxin-producing fungi such as Aspergillus flavus and Fusarium. The toxins produced by these fungi are not only difficult to remove, but may also accumulate in the fruiting bodies of edible fungi, endangering human health. At the same time, straw that has not been thoroughly inactivated may also carry pathogens such as damping-off fungus and root-knot nematode eggs, as well as harmful insect eggs. These harmful organisms will compete with edible fungi for nutrients and may even cause diseases and pests, severely inhibiting the growth and reproduction of edible fungi, significantly reducing the straw degradation efficiency and the economic benefits of edible fungi cultivation, and restricting the large-scale promotion of this resource utilization model. Summary of the Invention

[0005] Technical Problem to be Solved: To address the aforementioned technical problems, the present invention discloses a method for preparing a cultivation substrate for edible fungi under forest cover using crop straw and its application. The method involves first crushing crop straw, adding water, and then subjecting it to ultrasonic cavitation and photocatalytic pyrolysis treatments, followed by drying to obtain pretreated straw. Simultaneously, chitosan and poly-3-hydroxybutyric acid are reacted to form a prepolymer, which is then added to a composite bacterial solution for cross-linking and drying to obtain a composite bacterial agent. Finally, the pretreated straw, composite bacterial agent, and auxiliary materials are mixed and packaged to obtain the cultivation substrate. This method effectively breaks down the dense surface structure of straw and the connections between lignin and cellulose / hemicellulose, forming a loose and porous substrate structure. This provides ample sites for the growth of edible fungi. The composite bacterial agent not only significantly improves the degradation efficiency of straw and provides sufficient nutrition for the growth of edible fungi, but also inhibits the proliferation of pathogens, reduces disease occurrence, effectively promotes mycelial growth and fruiting body development of edible fungi, optimizes the nutritional quality of edible fungi, and achieves efficient resource recycling of crop straw.

[0006] Technical solution: A method for preparing a cultivation substrate for edible fungi under forest cover using crop straw includes the following steps: S1. Crop straw is crushed and mixed with water to prepare a straw solution. Trehalose, calcium lactate and diethyl malonate are added to the straw solution in sequence and mixed evenly. Then, ultrasonic cavitation treatment is performed to obtain a pretreated straw solution. S2. Add riboflavin to the pretreated straw solution, stir to dissolve, then perform photocatalytic pyrolysis treatment, and dry to obtain pretreated straw; S3. Chitosan and poly-3-hydroxybutyric acid are dispersed in acetic acid solution, and 1,3-diacrylate is added and reacted at 40-60℃ for 10-30 min to form a prepolymer. Then, the composite bacterial solution is added and crosslinked at 20-40℃ for 1-6 h. After washing and drying, the composite bacterial agent is prepared. S4. The pretreated straw prepared in S2 and the compound microbial agent prepared in S3 are mixed with auxiliary materials to adjust the pH to 5.5-7.5 and the moisture content to 50-70%, and then packaged to obtain the cultivation substrate for edible fungi under forest.

[0007] Preferably, in step S1, the solid-liquid ratio of crop straw to water is 1:(5-20) g / mL; the crop straw is selected from one or more of corn straw, wheat straw, rice straw, reed straw, and bean straw.

[0008] Preferably, in step S1, the amount of trehalose added is 1-5 wt%, the amount of calcium lactate added is 0.5-2%, and the amount of diethyl malonate added is 1-10%, all based on the mass of crop straw; the conditions for ultrasonic cavitation treatment are ultrasonic frequency 20-80 kHz, power density 100-300 W / L, treatment temperature 25-60℃, and treatment time 30-60 min.

[0009] Preferably, the amount of riboflavin added in step S2 is 0.1-0.5 g / L; the conditions for photocatalytic degradation treatment are light irradiation intensity of 1000-5000 lx, irradiation time of 2-8 h, light source of visible light or ultraviolet light, and light source wavelength of 365-650 nm.

[0010] Preferably, in step S3, the mass ratio of chitosan to poly(3-hydroxybutyric acid) is 1:(0.5-3), and the amount of 1,3-diacrylate added is 5-10% of the total mass of chitosan and poly(3-hydroxybutyric acid).

[0011] Preferably, in step S3, the volume ratio of the composite bacterial solution to the prepolymer is (1-5):1; the composite bacterial solution is prepared by mixing Bacillus thuringiensis bacterial solution and Bacillus tekirae bacterial solution at a volume ratio of 1:(0.5-2); the bacterial concentration of the composite bacterial solution is (1-7)×10⁻⁶. 8 CFU / mL.

[0012] Preferably, the concentration of the Bacillus thuringiensis bacterial solution is (1.5-4) × 10⁻⁶. 8The bacterial concentration of Bacillus tegmentata in the culture was (2-5.5) × 10 CFU / mL. 8 CFU / mL.

[0013] Preferably, in step S4, the amount of compound microbial agent added is 5-15% of the mass of pretreated straw, and the auxiliary material is composed of humus and wheat bran in a mass ratio of 1:(1-1.5), and the amount of auxiliary material added is 10-50% of the mass of pretreated straw. Beneficial effects

[0014] 1. This invention utilizes high-frequency ultrasound to generate a large number of microbubbles in a crop straw solution system. Under the periodic changes of ultrasound, the microbubbles undergo a stage of expansion-compression-collapse. The instantaneous high temperature and high pressure environment, strong shock wave, and micro-jet effect formed at the moment of collapse can directly destroy the dense structure of the surface layer of crop straw, break the covalent bonds such as ester bonds and ether bonds between lignin and cellulose and hemicellulose, and form a large number of microcracks and pores inside the straw. Trehalose lipids can reduce the surface tension of the crop straw solution system, stabilize the microbubbles generated by ultrasound to prolong their existence time and improve the uniformity of bursting. The fluid disturbance generated by the bursting of microbubbles can accelerate the penetration of various components into the internal pores of the straw, so that the subsequent modification treatment can extend from the surface layer to the internal matrix.

[0015] 2. This invention utilizes diethyl malonate to modify crop straw with β-dicarbonyl enol via an enolization reaction. The hydroxyl groups in diethyl malonate can undergo dehydration condensation reactions with the hydroxyl groups on the cellulose and hemicellulose molecular chains of straw, and simultaneously form hydrogen bonds with the aromatic ring hydroxyl groups of lignin and undergo substitution reactions. This introduces a large number of enol oxygen groups onto the surface of the straw, while also reacting with the Ca in the soaking solution. 2+ Stable coordination bonds are formed to expand the molecular chain spacing of straw cellulose, hemicellulose and lignin, further expanding the pores formed by ultrasonic microbubbles, breaking the lignin encapsulation barrier of cellulose and hemicellulose, providing more action sites for photo-media pyrolysis treatment, and laying the foundation for electrostatic adsorption and colonization of composite fungal hyphae.

[0016] 3. In this invention, riboflavin is added to an exogenous crop straw solution system. Under light irradiation, riboflavin is excited to generate reactive oxygen species (ROS). The stable aromatic ring structure of lignin in straw can be attacked by ROS. After the conjugated structure is destroyed, it is cleaved into small molecules. At the same time, ROS can also precisely destroy the bonding bonds between lignin and cellulose, achieving efficient dissociation of the two. Through photo-mediated cleavage, cellulose, hemicellulose and lignin in straw are deeply treated to form a loose and porous matrix structure, which improves the mycelial penetration efficiency and enzymatic contact area during subsequent compound fungal fermentation, ensuring the high efficiency of fermentation degradation.

[0017] 4. In preparing the composite microbial agent, this invention utilizes chitosan and poly-3-hydroxybutyric acid to form β-hydroxy ester bonds and β-amino ester bonds through Michael addition under the cross-linking effect of acrylate molecules. The molecular chains connected by β-hydroxy ester bonds and β-amino ester bonds intertwine to obtain a three-dimensional loading network with a porous structure. The pore diameter of the network is controlled to precisely match the spore diameter of Bacillus thuringiensis and Bacillus tekirae, which can not only achieve efficient embedding and fixation of the two Bacillus species, but also provide unobstructed channels for the diffusion of nutrients and the conduction of metabolites in the straw substrate. At the same time, the ester groups of the β-hydroxy ester bonds can form hydrogen bonds with the polysaccharide molecular chains, and the amino groups of the β-amino ester bonds can form ionic bonds with the peptidoglycans of the cell walls of the two Bacillus species, further consolidating the embedding effect and improving the stability of the strains in the cultivation substrate.

[0018] 5. When inoculating edible fungi under forest cover, Bacillus thuringiensis secretes cellulase to ensure the continuous and efficient degradation of cellulose. The degradation product, glucose, provides a carbon source for the mycelium of *Matsutake*. The mycelium of *Matsutake* secretes a variety of enzyme systems to further degrade the residual components of pretreated crop straw, generating easily absorbed substances and promoting the growth of *Matsutake* fruiting body primordia. The lysin produced by Bacillus thuringiensis and the specific antimicrobial metabolites secreted by Bacillus tekirae decompose the cell wall components of root rot pathogens, destroying the cell structure and causing them to lyse and die, thus inhibiting the proliferation and infection of pathogens. The antimicrobial peptides and phenolic substances secreted by *Matsutake* act on the cell membrane of pathogens, increasing membrane permeability and inactivating them, thereby enhancing the resistance of *Matsutake* to root rot pathogens. In addition, Bacillus tekirae promotes the conversion of nitrogen, phosphorus, and potassium elements in the cultivation substrate into absorbable forms. Small molecule sugars produced by straw degradation are absorbed by the mycelium of Matsutake mushrooms and converted into energy and precursors, effectively improving the absorption and utilization rate of nitrogen, phosphorus, and potassium elements by the mycelium of Matsutake mushrooms, optimizing the nutrient content of Matsutake mushroom fruiting bodies, and improving nutritional quality. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments: The strains used in this invention, Bacillus thuringiensis (SDMCC10298) and Bacillus tekirae (HZB129373), were purchased from Wuhan Gray Algae Biotechnology Co., Ltd. Example 1

[0020] A method for preparing a compound bacterial agent using Bacillus thuringiensis and Bacillus tektina includes the following steps: Step 1. Take the Bacillus thuringiensis preserved on an agar slant, pick a single colony and inoculate it into 250 mL of LB liquid medium. Incubate at 30℃ and 180 rpm for 18 h with constant temperature shaking to obtain Bacillus thuringiensis seed culture. Then, transfer the Bacillus thuringiensis seed culture to fresh LB liquid medium at an inoculation rate of 1%, and incubate at 30℃ and 200 rpm for 12 h. Centrifuge to collect the bacterial cells and adjust the bacterial concentration to 1.5 × 10⁻⁶ with sterile physiological saline. 8 CFU / mL yields Bacillus thuringiensis bacterial suspension; Step 2. Take the *Bacillus tekirae* slant culture, pick a single colony and inoculate it into 250 mL of LB liquid medium. Incubate at 28℃ and 180 rpm for 20 h with constant temperature shaking to obtain *Bacillus tekirae* seed culture. Then, transfer the *Bacillus tekirae* seed culture to fresh LB liquid medium at a 2% inoculation rate and incubate at 28℃ and 200 rpm for 15 h. Centrifuge to collect the bacterial cells and adjust the bacterial concentration to 2 × 10⁻⁶ with sterile physiological saline. 8 CFU / mL yields the *Bacillus tekirae* bacterial suspension; 100 mL of *Bacillus thuringiensis* bacterial suspension and 100 mL of *Bacillus tekirae* bacterial suspension are mixed evenly to obtain a composite bacterial suspension (concentration 1.75 × 10⁻⁶ CFU / mL). 8 (CFU / mL) Step 3. Accurately weigh 20.0g of chitosan and 10.0g of poly-3-hydroxybutyric acid and disperse them in 500mL of 2% acetic acid solution. Stir magnetically at 500r / min at room temperature until a uniformly dispersed mixture is formed. Add 1.5g of 1,3-diacrylate and heat to 50℃. Stir magnetically at 500r / min for 20min to obtain a prepolymer solution. Slowly add 200mL of composite bacterial solution to 200mL of prepolymer solution and stir evenly. Crosslink at 30℃ for 2h. Wash the product three times with deionized water by centrifugation (each time at 8000r / min for 10min). Then dry in a vacuum drying oven at 40℃ for 6h to obtain the composite bacterial agent. Example 2

[0021] A method for preparing a compound bacterial agent using Bacillus thuringiensis and Bacillus tektina includes the following steps: Step 1. Take the Bacillus thuringiensis preserved on an agar slant, pick a single colony and inoculate it into 250 mL of LB liquid medium. Incubate at 30℃ and 180 rpm for 18 h with constant temperature shaking to obtain Bacillus thuringiensis seed culture. Then, transfer the Bacillus thuringiensis seed culture to fresh LB liquid medium at a 1% inoculation rate and incubate at 30℃ and 200 rpm for 12 h. Centrifuge to collect the bacterial cells and adjust the bacterial concentration to 2.5 × 10⁻⁶ with sterile physiological saline. 8 CFU / mL yields Bacillus thuringiensis bacterial suspension; Step 2. Take the *Bacillus tekirae* slant culture, pick a single colony and inoculate it into 250 mL of LB liquid medium. Incubate at 28℃ and 180 rpm for 20 h with constant temperature shaking to obtain *Bacillus tekirae* seed culture. Then, transfer the *Bacillus tekirae* seed culture to fresh LB liquid medium at a 2% inoculation rate and incubate at 28℃ and 200 rpm for 15 h. Centrifuge to collect the bacterial cells and adjust the bacterial concentration to 3 × 10⁻⁶ with sterile physiological saline. 8 CFU / mL yields the *Bacillus tekirae* bacterial suspension; 100 mL of *Bacillus thuringiensis* bacterial suspension and 50 mL of *Bacillus tekirae* bacterial suspension are mixed evenly to obtain a composite bacterial suspension (concentration 2.65 × 10⁻⁶ CFU / mL). 8 (CFU / mL) Step 3. Accurately weigh 20.0 g of chitosan and 20.0 g of poly-3-hydroxybutyric acid and disperse them in 500 mL of 2% acetic acid solution. Stir magnetically at 500 r / min at room temperature until a uniformly dispersed mixture is formed. Add 2.8 g of 1,3-diacrylate and heat to 45 °C. Stir magnetically at 500 r / min for 15 min to obtain a prepolymer solution. Slowly add 100 mL of composite bacterial solution to 200 mL of prepolymer solution and stir evenly. Crosslink at 30 °C for 2 h. Wash the product three times with deionized water by centrifugation (each time at 8000 r / min for 10 min). Then dry in a vacuum drying oven at 40 °C for 6 h to obtain the composite bacterial agent. Example 3

[0022] A method for preparing a compound bacterial agent using Bacillus thuringiensis and Bacillus tektina includes the following steps: Step 1. Take the Bacillus thuringiensis preserved on the slant culture, pick a single colony and inoculate it into 250 mL of LB liquid medium. Incubate at 30℃ and 180 r / min for 18 h to obtain Bacillus thuringiensis seed culture. Then, transfer the Bacillus thuringiensis seed culture to fresh LB liquid medium at an inoculation rate of 1%, and incubate at 30℃ and 200 r / min for 12 h. Centrifuge to collect the bacterial cells and adjust the bacterial concentration to 3 × 10⁻⁶ with sterile physiological saline. 8 CFU / mL yields Bacillus thuringiensis bacterial suspension; Step 2. Take the *Bacillus tekirae* slant culture, pick a single colony and inoculate it into 250 mL of LB liquid medium. Incubate at 28℃ and 180 rpm for 20 h with constant temperature shaking to obtain *Bacillus tekirae* seed culture. Then, transfer the *Bacillus tekirae* seed culture to fresh LB liquid medium at a 2% inoculation rate and incubate at 28℃ and 200 rpm for 15 h. Centrifuge to collect the bacterial cells and adjust the bacterial concentration to 4 × 10⁻⁶ with sterile physiological saline. 8CFU / mL yields the *Bacillus tekirae* bacterial suspension; 100 mL of *Bacillus thuringiensis* bacterial suspension and 100 mL of *Bacillus tekirae* bacterial suspension are mixed evenly to obtain a composite bacterial suspension (concentration 3.5 × 10⁻⁶ CFU / mL). 8 (CFU / mL) Step 3. Accurately weigh 20.0 g of chitosan and 40.0 g of poly-3-hydroxybutyric acid and disperse them in 500 mL of 2% acetic acid solution. Stir magnetically at 500 r / min at room temperature until a uniformly dispersed mixture is formed. Add 4.2 g of 1,3-diacrylate, heat to 50 °C, and stir magnetically at 500 r / min for 25 min to obtain a prepolymer solution. Slowly add 100 mL of composite bacterial solution to 300 mL of prepolymer solution and stir evenly. Crosslink at 35 °C for 3 h. Wash the product three times with deionized water by centrifugation (each time at 8000 r / min for 10 min). Then dry in a vacuum drying oven at 45 °C for 6 h to obtain the composite bacterial agent. Example 4

[0023] A method for preparing a compound bacterial agent using Bacillus thuringiensis and Bacillus tektina includes the following steps: Step 1. Take the Bacillus thuringiensis preserved on an agar slant, pick a single colony and inoculate it into 250 mL of LB liquid medium. Incubate at 30℃ and 180 rpm for 18 h with constant temperature shaking to obtain Bacillus thuringiensis seed culture. Then, transfer the Bacillus thuringiensis seed culture to fresh LB liquid medium at a 1% inoculation rate and incubate at 30℃ and 200 rpm for 12 h. Centrifuge to collect the bacterial cells and adjust the bacterial concentration to 2.5 × 10⁻⁶ with sterile physiological saline. 8 CFU / mL yields Bacillus thuringiensis bacterial suspension; Step 2. Take the *Bacillus tekirae* slant culture, pick a single colony and inoculate it into 250 mL of LB liquid medium. Incubate at 28℃ and 180 rpm for 20 h with constant temperature shaking to obtain *Bacillus tekirae* seed culture. Then, transfer the *Bacillus tekirae* seed culture to fresh LB liquid medium at a 2% inoculation rate and incubate at 28℃ and 200 rpm for 15 h. Centrifuge to collect the bacterial cells and adjust the bacterial concentration to 2 × 10⁻⁶ with sterile physiological saline. 8 CFU / mL yields the *Bacillus tekirae* bacterial suspension; 100 mL of *Bacillus thuringiensis* bacterial suspension and 200 mL of *Bacillus tekirae* bacterial suspension are mixed evenly to obtain a composite bacterial suspension (concentration 1.5 × 10⁻⁶ CFU / mL). 8 (CFU / mL) Step 3. Accurately weigh 10.0 g of chitosan and 30.0 g of poly-3-hydroxybutyric acid and disperse them in 500 mL of 2% acetic acid solution. Stir magnetically at 500 r / min at room temperature until a uniformly dispersed mixture is formed. Add 4.0 g of 1,3-diacrylate and heat to 45 °C. Stir magnetically at 500 r / min for 25 min to obtain a prepolymer solution. Slowly add 60 mL of composite bacterial solution to 300 mL of prepolymer solution and stir evenly. Crosslink at 40 °C for 3.5 h. Wash the product three times with deionized water by centrifugation (each time at 8000 r / min for 10 min). Then dry in a vacuum drying oven at 45 °C for 5 h to obtain the composite bacterial agent. Example 5

[0024] A method for preparing a compound bacterial agent using Bacillus thuringiensis and Bacillus tektina includes the following steps: Step 1. Take the Bacillus thuringiensis preserved on the slant culture, pick a single colony and inoculate it into 250 mL of LB liquid medium. Incubate at 30℃ and 180 r / min for 18 h to obtain Bacillus thuringiensis seed culture. Then, transfer the Bacillus thuringiensis seed culture to fresh LB liquid medium at an inoculation rate of 1%, and incubate at 30℃ and 200 r / min for 12 h. Centrifuge to collect the bacterial cells and adjust the bacterial concentration to 3 × 10⁻⁶ with sterile physiological saline. 8 CFU / mL yields Bacillus thuringiensis bacterial suspension; Step 2. Take the *Bacillus tekirae* slant culture, pick a single colony and inoculate it into 250 mL of LB liquid medium. Incubate at 28℃ and 180 rpm for 20 h with constant temperature shaking to obtain *Bacillus tekirae* seed culture. Then, transfer the *Bacillus tekirae* seed culture to fresh LB liquid medium at a 2% inoculation rate and incubate at 28℃ and 200 rpm for 15 h. Centrifuge to collect the bacterial cells and adjust the bacterial concentration to 3.5 × 10⁻⁶ with sterile physiological saline. 8 CFU / mL yields the *Bacillus tekirae* bacterial suspension; 100 mL of *Bacillus thuringiensis* bacterial suspension and 150 mL of *Bacillus tekirae* bacterial suspension are mixed evenly to obtain a composite bacterial suspension (concentration 3.3 × 10⁻⁶ CFU / mL). 8 (CFU / mL) Step 3. Accurately weigh 20.0 g of chitosan and 50.0 g of poly-3-hydroxybutyric acid and disperse them in 500 mL of 2% acetic acid solution. Stir magnetically at 500 r / min at room temperature until a uniformly dispersed mixture is formed. Add 6.3 g of 1,3-diacrylate, heat to 40 °C, and stir magnetically at 500 r / min for 20 min to obtain a prepolymer solution. Slowly add 100 mL of composite bacterial solution to 400 mL of prepolymer solution and stir evenly. Crosslink at 40 °C for 4.5 h. Wash the product three times with deionized water by centrifugation (each time at 8000 r / min for 10 min). Then dry in a vacuum drying oven at 45 °C for 5 h to obtain the composite bacterial agent. Comparative Example 1

[0025] The difference between this comparative example and Example 3 is that poly-3-hydroxybutyric acid is not added; the remaining operations are the same as in Example 3. Comparative Example 2

[0026] The difference between this comparative example and Example 3 is that chitosan is not added; the remaining operations are the same as in Example 3. Comparative Example 3

[0027] The difference between this comparative example and Example 3 is that 1,3-diacrylate is not added; the remaining operations are the same as in Example 3. Physicochemical indicators

[0028] (1) Average particle size Accurately weigh 0.05g of the compound bacterial agent sample and place it in 5mL of sterile physiological saline. Add 1 drop of Tween-80 and ultrasonically disperse it for 10min at 200W in an ultrasonic cell disruptor. Turn on the laser particle size analyzer and calibrate the instrument with standard particle size calibration solution. After successful calibration, set the measurement parameters as follows: measurement temperature 25℃, dispersion medium sterile physiological saline, and stirring speed 200r / min. Slowly inject the pretreated sample suspension into the sample cell of the laser particle size analyzer. After the instrument shows that the particle dispersion is stable, start the measurement program. Each measurement time is 30s.

[0029] (2) Embedding efficiency Accurately weigh 0.1g of the compound bacterial agent sample and place it in 9mL of sterile physiological saline. Dilute it serially to an appropriate concentration (10). -6 -10 -8(1) Take 100 μL of the diluted solution and spread it evenly on LB agar plates. Set up 3 replicates for each dilution gradient. Incubate at 30℃ for 24 h. Count the number of colonies on the plates and calculate the total viable count of the compound bacterial agent according to the dilution ratio. Take 9 mL of the suspension corresponding to the above 0.1 g compound bacterial agent sample and centrifuge at 8000 r / min for 10 min. Collect the supernatant and dilute it to the appropriate concentration. Take 100 μL of the diluted solution and spread it on LB agar plates. Set up 3 replicates for each dilution gradient. Incubate at 30℃ for 24 h and count the number of free viable bacteria. Calculate the encapsulation efficiency: Encapsulation efficiency (%) = (total viable bacteria - free viable bacteria) / total viable bacteria × 100%.

[0030] Table 1. Average particle size and encapsulation efficiency of the composite microbial agents prepared in Examples 1-5 and Comparative Examples 1-3.

[0031] As shown in Table 1, the composite bacterial agents prepared in Examples 1-5 exhibit excellent overall performance, uniform particle dispersion, and good carrier encapsulation effect. This is because the interaction between chitosan, poly-3-hydroxybutyric acid, and 1,3-diacrylate forms a stable and dense carrier structure, effectively achieving efficient encapsulation of Bacillus thuringiensis and Bacillus tekirae. In contrast, Comparative Examples 1-3 lacked one of these three components, resulting in the inability of the carrier to form a stable cross-linked structure, thus affecting the encapsulation performance. Consequently, the composite bacterial agents prepared in Comparative Examples 1-3 were prone to particle agglomeration, leading to a significant decrease in encapsulation effect. Example 6

[0032] A method for preparing a cultivation substrate for edible fungi under forest cover using crop straw includes the following steps: S1. Weigh 7 kg of corn stalks, 6 kg of wheat stalks, and 7 kg of rice stalks, crush them, and mix them with 100 L of water to prepare a straw solution. Add 200 g of trehalose, 100 g of calcium lactate, and 200 g of diethyl malonate to the straw solution in sequence. After mixing evenly, perform ultrasonic cavitation treatment for 30 min under the conditions of ultrasonic frequency 20 kHz, power density 100 W / L, and treatment temperature 25 ℃ to obtain a pretreated straw solution. S2. Add 10g of riboflavin to 100L of pretreated straw solution, stir to dissolve, and then perform photocatalytic degradation treatment for 2h under visible light with an intensity of 1000lx and a wavelength of 650nm. After treatment, dry for 8h at a drying temperature of 50℃ and a wind speed of 1.0m / s to obtain pretreated straw. S3. Take 17 kg of the pretreated straw prepared in S2, add 0.85 kg of the compound microbial agent prepared in Example 3, and then add 1.7 kg of auxiliary material made from 0.85 kg of humus and 0.85 kg of wheat bran. After mixing evenly, adjust the pH to 5.5 and the moisture content to 50%. After packaging, the substrate for cultivating edible fungi under forests is obtained. Example 7

[0033] A method for preparing a cultivation substrate for edible fungi under forest cover using crop straw includes the following steps: S1. Weigh 6 kg of wheat straw, 7 kg of reed straw, and 7 kg of soybean straw, crush them, and mix them with 200 L of water to prepare a straw solution. Add 400 g of trehalose lipids, 200 g of calcium lactate, and 500 g of diethyl malonate to the straw solution in sequence. After mixing evenly, perform ultrasonic cavitation treatment for 40 min under the conditions of ultrasonic frequency 40 kHz, power density 150 W / L, and treatment temperature 35 ℃ to obtain a pretreated straw solution. S2. Add 40g of riboflavin to 200L of pretreated straw solution, stir to dissolve, and then perform photocatalytic degradation treatment under ultraviolet light with an intensity of 2000lx and a wavelength of 365nm for 2h. After treatment, dry for 8h at a drying temperature of 50℃ and a wind speed of 1.0m / s to obtain pretreated straw. S3. Take 16 kg of the pretreated straw prepared in S2, add 1.6 kg of the compound microbial agent prepared in Example 3, then add 4.1 kg of the auxiliary material made from 1.85 kg of humus and 2.25 kg of wheat bran, mix evenly, adjust the pH to 6.0 and the moisture content to 55%, and package to obtain the forest edible fungus cultivation substrate. Example 8

[0034] A method for preparing a cultivation substrate for edible fungi under forest cover using crop straw includes the following steps: S1. Weigh 4 kg of corn stalks, 4 kg of rice stalks, 4 kg of reed stalks, 4 kg of soybean stalks, and 4 kg of wheat stalks, crush them, and mix them with 160 L of water to prepare a straw solution. Add 400 g of trehalose, 200 g of calcium lactate, and 500 g of diethyl malonate to the straw solution in sequence. After mixing evenly, perform ultrasonic cavitation treatment for 35 min under the conditions of ultrasonic frequency 30 kHz, power density 180 W / L, and treatment temperature 30 ℃ to obtain a pretreated straw solution. S2. Add 64g of riboflavin to 160L of pretreated straw solution, stir to dissolve, and then perform photocatalytic degradation treatment for 3h under visible light with an intensity of 2500lx and a wavelength of 500nm. After treatment, dry for 6h at a drying temperature of 60℃ and a wind speed of 2.0m / s to obtain pretreated straw. S3. Take 20 kg of pretreated straw prepared in S2, add 1.6 kg of compound microbial agent prepared in Example 3, then add 4 kg of auxiliary material made of 2 kg of humus and 2 kg of wheat bran, mix evenly, adjust the pH to 6.0 and the moisture content to 55%, and package to obtain the forest edible fungus cultivation substrate. Example 9

[0035] A method for preparing a cultivation substrate for edible fungi under forest cover using crop straw includes the following steps: S1. Weigh 6 kg of soybean straw, 4 kg of corn straw, 5 kg of rice straw, and 5 kg of reed straw, crush them, and mix them with 400 L of water to prepare a straw solution. Add 1 kg of trehalose lipid, 0.4 kg of calcium lactate, and 1 kg of diethyl malonate to the straw solution in sequence. After mixing evenly, perform ultrasonic cavitation treatment for 60 min under the conditions of ultrasonic frequency 50 kHz, power density 300 W / L, and treatment temperature 60 ℃ to obtain a pretreated straw solution. S2. Add 120g of riboflavin to 400L of pretreated straw solution, stir to dissolve, and then perform photocatalytic degradation treatment for 6h under visible light with an intensity of 3000lx and a wavelength of 400nm. After treatment, dry for 9h at a drying temperature of 65℃ and a wind speed of 1.5m / s to obtain pretreated straw. S3. Take 16.2 kg of pretreated straw prepared in S2, add 2.43 kg of compound microbial agent prepared in Example 3, then add 1.62 kg of auxiliary material made from 0.85 kg of humus and 0.85 kg of wheat bran, mix evenly, adjust the pH to 7.0 and the moisture content to 60%, and then package to obtain the understory edible fungus cultivation substrate. Example 10

[0036] A method for preparing a cultivation substrate for edible fungi under forest cover using crop straw includes the following steps: S1. Weigh 4 kg of reed straw, 6 kg of rice straw, 5 kg of wheat straw, and 5 kg of corn straw, crush them, and mix them with 300 L of water to prepare a straw solution. Add 0.8 kg of trehalose lipids, 0.3 kg of calcium lactate, and 0.8 kg of diethyl malonate to the straw solution in sequence. After mixing evenly, perform ultrasonic cavitation treatment for 45 min under the conditions of ultrasonic frequency 35 kHz, power density 200 W / L, and treatment temperature 50 ℃ to obtain a pretreated straw solution. S2. Add 90g of riboflavin to 300L of pretreated straw solution, stir to dissolve, and then perform photocatalytic degradation treatment under ultraviolet light with an intensity of 2500lx and a wavelength of 380nm for 5.5h. After treatment, dry for 7h at a drying temperature of 60℃ and a wind speed of 1.0m / s to obtain pretreated straw. S3. Take 16 kg of the pretreated straw prepared in S2, add 2.15 kg of the compound microbial agent prepared in Example 3, then add 2.5 kg of auxiliary material made from 1 kg of humus and 1.5 kg of wheat bran, mix evenly, adjust the pH to 6.5 and the moisture content to 70%, and package to obtain the understory edible fungus cultivation substrate. Comparative Example 4

[0037] The difference between this comparative example and Example 9 is that the composite bacterial agent prepared in Comparative Example 1 is used; the remaining operations are the same as in Example 9. Comparative Example 5

[0038] The difference between this comparative example and Example 9 is that the composite bacterial agent prepared in Comparative Example 2 is used; the remaining operations are the same as in Example 9. Comparative Example 6

[0039] The difference between this comparative example and Example 9 is that the composite bacterial agent prepared in Comparative Example 3 is used; the remaining operations are the same as in Example 9. Comparative Example 7

[0040] The difference between this comparative example and Example 9 is that ultrasonic cavitation treatment is not used; the remaining operations are the same as in Example 9. Comparative Example 8

[0041] The difference between this comparative example and Example 9 is that riboflavin is not added and photocatalytic degradation is not used; the remaining operations are the same as in Example 9. Comparative Example 9

[0042] The difference between this comparative example and Example 9 is that diethyl malonate is not added; the remaining operations are the same as in Example 9. Comparative Example 10

[0043] The difference between this comparative example and Example 9 is that the compound bacterial agent prepared in Example 3 is not added; the remaining operations are the same as in Example 9. Verification Experiment on the Cultivation Effect of Edible Fungi under Forest

[0044] A cultivation experiment was conducted using *Matsutake*, an edible fungus grown under forest cover, as an example. The experiment was conducted in broad-leaved forest areas with flat terrain, good ventilation and light penetration, soil pH of 6.0-7.0, and organic matter content ≥2.0%. The experimental plots were uniformly divided into four groups: Example 6, Example 7, Example 8, Example 9, Example 10, Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 7, Comparative Example 8, Comparative Example 9, and Comparative Example 10. The cultivation substrate for each experimental group was moistened by spraying water 12 hours in advance. Sowing was carried out using a spot-sowing method. The inoculum amount of *Matsutake* was uniformly 5% of the dry weight of the cultivation substrate, and the sowing density was 20cm × 20cm. After sowing, a 5cm layer of sterile humus soil was covered and gently compacted. A small amount of sterile water was sprayed to keep the soil moist. During the experiment, the forest environment was uniformly controlled, with the temperature maintained at 25℃ and the relative humidity at 85-90%. Ventilation was carried out twice a day for 30 minutes each time (9-10 am and 4-5 pm). Weeds and bacteria in the plots were regularly removed to ensure the normal growth of red matsutake mushrooms. The management conditions of each group were completely consistent. When the fruiting bodies of *Matsutake* matured, the material conversion rates of each group's cultivation substrate were measured (including cellulose, hemicellulose, and lignin, referring to "NY / T 3494-2019 Determination of Cellulose, Hemicellulose, and Lignin in Agricultural Biomass Raw Materials"). The agronomical traits of *Matsutake* were recorded. The cap diameter, cap thickness, stipe diameter, and stipe length were measured using vernier calipers, and the weight of a single mushroom was measured using an electronic scale. The biological efficiency was defined as the ratio of the total fresh weight of the fruiting bodies to the dry weight of the cultivation substrate. Nutritional indicators of the *Matsutake* fruiting bodies were also measured: protein was determined according to "GB 5009.5-2025 National Food Safety Standard - Determination of Protein in Food"; polysaccharides were determined according to "NY / T 1676-2023 Determination of Crude Polysaccharides in Edible Fungi - Spectrophotometric Method"; flavonoids were determined using the sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method; polyphenols were determined using the Folin-Ciocalteu method; and sterols were determined according to "NY / T 4724-2025". The determination of ergosterol and ergosterol peroxide in edible fungi and their products.

[0045] Table 2. Material conversion rate of *Matsutake* mushrooms in cultivation substrate for Examples 6-10 and Comparative Examples 4-10.

[0046] Table 3. Agronomic traits of red matsutake mushrooms in Examples 6-10 and Comparative Examples 4-10

[0047] Table 4. Nutritional indicators of red matsutake fruiting bodies in Examples 6-10 and Comparative Examples 4-10

[0048] As shown in Tables 2, 3, and 4, the conversion rates of cellulose, hemicellulose, and lignin in the forest understory edible fungi cultivation substrate of Examples 6-10 were all superior to those of Comparative Examples 4-10. This indicates that the compound microbial agent in the forest understory edible fungi cultivation substrate can work synergistically with the mycelium of *Matsutake* to efficiently degrade and utilize cellulose, hemicellulose, and lignin. Furthermore, the agronomic traits, single mushroom weight, biological efficiency, and nutritional indicators of the fruiting bodies of *Matsutake* in Examples 6-10 were all superior to those in Comparative Examples 4-10. This demonstrates that the method described in this invention, through a three-stage synergistic treatment system of ultrasonic cavitation, photocatalytic lysis, and compound microbial agent, significantly improves the degradation efficiency of lignocellulose in the cultivation substrate, promotes the conversion and utilization of nutrients in the cultivation substrate by *Matsutake*, thereby significantly improving the agronomic traits of the fruiting bodies and enriching them with functional nutrients such as proteins, polysaccharides, and flavonoids. This invention provides an effective way to achieve high-value utilization of crop straw and promotes the development of the forest understory edible fungi industry towards high efficiency, high quality, and ecological sustainability.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for preparing a cultivation substrate for edible fungi under forest cover using crop straw, characterized in that, Includes the following steps: S1. Crop straw is crushed and mixed with water to prepare a straw solution. Trehalose, calcium lactate and diethyl malonate are added to the straw solution in sequence and mixed evenly. Then, ultrasonic cavitation treatment is performed to obtain a pretreated straw solution. S2. Add riboflavin to the pretreated straw solution, stir to dissolve, then perform photocatalytic pyrolysis treatment, and dry to obtain pretreated straw; S3. Chitosan and poly-3-hydroxybutyric acid are dispersed in acetic acid solution, and 1,3-diacrylate is added and reacted at 40-60℃ for 10-30 min to form a prepolymer. Then, the composite bacterial solution is added and crosslinked at 20-40℃ for 1-6 h. After washing and drying, the composite bacterial agent is prepared. S4. The pretreated straw prepared in S2 and the compound microbial agent prepared in S3 are mixed with auxiliary materials to adjust the pH to 5.5-7.5 and the moisture content to 50-70%, and then packaged to obtain the cultivation substrate for edible fungi under forest.

2. The method for preparing a cultivation substrate for edible fungi under forest cover using crop straw according to claim 1, characterized in that: In step S1, the solid-liquid ratio of crop straw to water is 1:(5-20)g / mL; the crop straw is selected from one or more of corn straw, wheat straw, rice straw, reed straw, and bean straw.

3. The method for preparing a cultivation substrate for edible fungi under forest cover using crop straw according to claim 1, characterized in that: In step S1, the amount of trehalose added is 1-5 wt%, the amount of calcium lactate added is 0.5-2%, and the amount of diethyl malonate added is 1-10%, all based on the mass of crop straw; the conditions for ultrasonic cavitation treatment are ultrasonic frequency 20-80 kHz, power density 100-300 W / L, treatment temperature 25-60℃, and treatment time 30-60 min.

4. The method for preparing a cultivation substrate for edible fungi under forest cover using crop straw according to claim 1, characterized in that: In step S2, the amount of riboflavin added is 0.1-0.5 g / L; the conditions for photocatalytic degradation treatment are: light intensity of 1000-5000 lx, irradiation time of 2-8 h, light source of visible or ultraviolet light, and light source wavelength of 365-650 nm.

5. The method for preparing a cultivation substrate for edible fungi under forest cover using crop straw according to claim 1, characterized in that: In step S3, the mass ratio of chitosan to poly(3-hydroxybutyric acid) is 1:(0.5-3), and the amount of 1,3-diacrylate added is 5-10% of the total mass of chitosan and poly(3-hydroxybutyric acid).

6. The method for preparing a cultivation substrate for edible fungi under forest cover using crop straw according to claim 1, characterized in that: In step S3, the volume ratio of the composite bacterial solution to the prepolymer is (1-5):1; the composite bacterial solution is prepared by mixing Bacillus thuringiensis bacterial solution and Bacillus tekirae bacterial solution at a volume ratio of 1:(0.5-2); the bacterial concentration of the composite bacterial solution is (1-7)×10⁻⁶. 8 CFU / mL.

7. The method for preparing a cultivation substrate for edible fungi under forest cover using crop straw according to claim 6, characterized in that: The concentration of the Bacillus thuringiensis bacterial suspension was (1.5-4)×10⁻⁶. 8 The bacterial concentration of Bacillus tegmentata in the culture was (2-5.5) × 10 CFU / mL. 8 CFU / mL.

8. The method for preparing a cultivation substrate for edible fungi under forest cover using crop straw according to claim 1, characterized in that: In step S4, the amount of compound microbial agent added is 5-15% of the mass of pretreated straw, and the auxiliary material is composed of humus and wheat bran in a mass ratio of 1:(1-1.5), and the amount of auxiliary material added is 10-50% of the mass of pretreated straw.

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