Agricultural waste composite modified edible mushroom cultivation substrate and preparation method thereof

Abstract

This invention provides a composite modified edible fungi cultivation substrate made from agricultural waste and its preparation method, comprising the following components in parts by weight: 20-30 parts corn stalks, 15-25 parts rice husks, 10-20 parts sawdust, 25-35 parts cottonseed hulls, 5-10 parts soybean meal, 0.5-2.0 parts compound microbial agent, 3-8 parts bentonite, 1-3 parts chitosan, 1-2 parts gypsum, and 1-2 parts lime. This invention uses a compound microbial agent to modify the basic mixture through solid-state fermentation, and combines bentonite and chitosan to form an activated functional slurry. Through the synergistic effect of solid-state fermentation and the functional slurry, the granular structure and water retention and aeration of the substrate are fundamentally improved, and the efficient activation and slow release of nutrients are achieved. Ultimately, this shortens the fruiting cycle of shiitake mushrooms to less than 85 days, increases yield by more than 10%, and significantly improves the proportion of flower mushrooms, realizing the high-value utilization of agricultural waste.

Description

Technical Field

[0001] This invention relates to the field of edible fungi cultivation technology, and more specifically, to a composite modified edible fungi cultivation substrate made from agricultural waste and its preparation method. Background Technology

[0002] Edible mushroom cultivation, as a model of resource-circulating agriculture, relies heavily on the efficiency and greenness of its cultivation substrate for sustainable development. Currently, research and application of substrate formulation mainly focus on the combined utilization of various agricultural and forestry wastes. By using different proportions of raw materials such as sawdust, cottonseed hulls, corn stalks, and sugarcane bagasse, a basic carbon and nitrogen nutrient framework suitable for edible mushroom growth is constructed. To further improve substrate performance, existing technologies generally focus on two directions: first, introducing exogenous functional additives, such as directly mixing in humic acid and vermiculite to adjust the physicochemical properties of the substrate, or adding commercial nitrogen sources such as wheat bran, soybean meal, and rapeseed cake to optimize the nutrient structure; second, using microbial agents to pre-ferment the substrate, a method that has become an effective way to degrade macromolecules such as lignocellulose and promote nutrient conversion and utilization. Related bio-fermentation processes, including liquid fermentation and solid-state fermentation, have shown certain application potential in improving substrate biocompatibility and shortening the production cycle, providing a technological direction for the high-value utilization of agricultural waste.

[0003] However, existing technologies still have several obvious limitations and defects. First, in terms of raw material utilization, most technologies remain at the initial stage of physical mixing and simple fermentation, failing to achieve deep synergistic modification of the physical, chemical, and biological properties of various agricultural waste components. The functional complementarity between components is insufficient, failing to form an organically integrated functional system, thus limiting the overall performance of the substrate. Second, in terms of functional design and integration, the combination of water-retaining agents, structure modifiers, and core nutrients is often loose, mostly employing simple physical doping methods, failing to form a stable "structure-nutrient-microorganism" ternary synergistic system through chemical bonding or microstructural design. This structural defect directly leads to a difficulty in simultaneously achieving good water retention and air permeability and slow nutrient release in the substrate. Furthermore, in the mid-to-late stages of fruiting, with the metabolism of mycelium and water migration, the physical structure of the substrate is prone to collapse, affecting the yield of subsequent flushes. More importantly, existing fermentation modification processes either have cumbersome procedures and high energy consumption (such as the need for solid-liquid separation in liquid fermentation), or have limited fermentation efficiency (such as only being able to efficiently degrade cellulose but not lignin), excessively long fermentation cycles, or difficulties in precisely controlling fermentation conditions. These issues restrict their efficiency and application effects in large-scale production, ultimately limiting the further improvement of the benefits of agricultural waste matrix utilization. Summary of the Invention

[0004] The main objective of this invention is to provide a composite modified edible fungi cultivation substrate made from agricultural waste and its preparation method, in order to solve the systemic problem in the prior art where the synergy between the physical structure of the substrate, nutrient supply and microbial environment is poor, resulting in poor overall substrate performance and low cultivation efficiency.

[0005] To achieve the above objectives, the present invention provides a composite modified edible fungi cultivation substrate made from agricultural waste, comprising the following components in parts by weight: 20-30 parts corn stalks, 15-25 parts rice husks, 10-20 parts sawdust, 25-35 parts cottonseed hulls, 5-10 parts soybean meal, 0.5-2.0 parts compound microbial agent, 3-8 parts bentonite, 1-3 parts chitosan, 1-2 parts gypsum, and 1-2 parts lime.

[0006] Furthermore, the compound microbial agent is composed of Aspergillus niger, Rhizopus oryzae, and Streptococcus thermophilus in a live bacteria ratio of 2~3:1~2:1.

[0007] This invention also provides a method for preparing a composite modified edible fungus cultivation substrate made from agricultural waste, comprising the following steps:

[0008] Corn stalks, rice husks, and sawdust are crushed and then mixed evenly with cottonseed hulls and soybean meal to obtain a basic mixture.

[0009] Add compound microbial agent and water to the basic mixture, stir evenly, and then carry out solid-state fermentation to obtain modified fermented material;

[0010] Bentonite and chitosan were dissolved in an acetic acid solution and stirred until fully swollen to obtain an activated functional slurry.

[0011] The modified fermentation material, activated functional slurry, gypsum and lime are thoroughly mixed, the moisture content is adjusted to 55%~65%, and sterilization is performed to obtain the cultivation substrate.

[0012] Furthermore, the conditions for solid-state fermentation in the preparation of modified fermentation material are: temperature 35~45℃, fermentation time 3~7 days, and turning the pile 1~2 times a day during fermentation.

[0013] Furthermore, the mass concentration of the acetic acid solution in the preparation step of the activated functional slurry is 1.0%~1.5%.

[0014] Furthermore, in the preparation step of the modified fermentation material, the mass ratio of water to the basic mixture is 0.5~0.7:1.

[0015] Furthermore, in the preparation step of the activated functional slurry, the ratio of the total mass of bentonite and chitosan to the volume of acetic acid solution is 1g:8~15mL.

[0016] Furthermore, in the preparation of the cultivation substrate, sterilization is carried out using high-pressure steam sterilization at a temperature of 100-130℃ for 30-90 minutes.

[0017] This invention discloses a modified agricultural waste substrate for edible fungi cultivation, comprising the following raw materials in parts by weight: 20-30 parts corn stalks, 15-25 parts rice husks, 10-20 parts sawdust, 25-35 parts cottonseed hulls, 5-10 parts soybean meal, 0.5-2.0 parts compound microbial agent, 3-8 parts bentonite, 1-3 parts chitosan, 1-2 parts gypsum, and 1-2 parts lime. This invention uses corn stalks, rice husks, sawdust, cottonseed hulls, and soybean meal as raw materials to construct a basic nutrient system. Solid-state fermentation with a compound microbial agent of *Aspergillus niger*, *Rhizopus oryzae*, and *Streptococcus thermophilus* is used to achieve pre-digestion and activation of the raw materials. Furthermore, it innovatively employs a compounding of bentonite and chitosan under acidic conditions to form an activated functional slurry. This combination of raw materials and process design produces a triple synergistic effect: the bentonite-chitosan slurry significantly improves the substrate's granular structure and water retention and aeration; solid-state fermentation degrades macromolecules into easily absorbed small molecules, improving nutrient utilization; and the combination of soybean meal and compound microbial agents ensures a continuous and stable nitrogen source supply. The cultivation substrate and its preparation method of this invention, through the synergistic effect of each component and process, achieve the comprehensive beneficial effects of shortening the cultivation cycle, increasing yield, and significantly improving commercial quality (the proportion of shiitake mushrooms), while simultaneously realizing the high-value utilization of agricultural waste. Detailed Implementation

[0018] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0019] According to an embodiment of the present invention, a composite modified edible fungi cultivation substrate made from agricultural waste comprises the following components in parts by weight: 20-30 parts corn stalks, 15-25 parts rice husks, 10-20 parts sawdust, 25-35 parts cottonseed hulls, 5-10 parts soybean meal, 0.5-2.0 parts compound microbial agent, 3-8 parts bentonite, 1-3 parts chitosan, 1-2 parts gypsum, and 1-2 parts lime. The raw material formulation of the present invention achieves functional complementarity and synergistic effect through scientific proportioning. The main function of 20-30 parts corn stalks is to provide a sustainably released carbon source and construct the macroscopic porous structure of the substrate. When its content is below 20 parts, the overall carbon-nitrogen ratio of the substrate is too low, which may lead to excessive mycelial growth in the early stages but insufficient subsequent growth; when it is above 30 parts, the lignin content is too high and difficult to completely degrade, which may instead produce benzene ring-like small molecules that inhibit mycelial growth. When combined with rice husks and sawdust, the particle size of the rice husks and sawdust forms a main skeletal structure of 2-4 mm, ensuring the initial aeration of the substrate. 15-25 parts rice husks and 10-20 parts sawdust act as "fine-tuners" for the carbon skeleton. Rice husks are rich in silica and have high hardness; their main function is to prevent excessive compaction of the substrate during sterilization and mycelial growth, maintaining long-term structural stability. Sawdust, due to its fibrous form, is easily colonized and entangled by mycelium, making it crucial for forming the "mycelium-substrate" complex. By varying the ratio of these three components from 20:15:15 to 30:25:20, the bulk density and aeration of the substrate can be flexibly adjusted to meet the growth needs of different edible fungi varieties. 25-35 parts cottonseed hulls and 5-10 parts soybean meal constitute the "nutritional duocore" of the substrate. Cottonseed hulls not only have a suitable carbon-to-nitrogen ratio, but their unique fibrous structure also facilitates water retention and mycelial penetration. Soybean meal, as a high-quality organic nitrogen source, has a protein content as high as 40%-45%. This invention discovers that when soybean meal is used in quantities of 5-10 parts, it synergistically with the compound microbial agent to achieve a "fast-slow" nitrogen supply: some nitrogen is provided by degradation in the early stages of fermentation, while the remaining portion is gradually utilized by the mycelium during the fruiting period. Excessive soybean meal (>10 parts) can easily lead to ammonia accumulation during fermentation, inhibiting the activity of the microbial agent. 3-8 parts bentonite and 1-3 parts chitosan: This compound combination is the core of the structural improvement in this invention. Bentonite and chitosan, under acidic conditions, form an organic-inorganic hybrid gel through cation exchange and hydrogen bonding, whose water-holding capacity is more than 1.8 times that of bentonite alone. When the bentonite content is below 3 parts, the gel network strength is insufficient; when it is above 8 parts, the matrix viscosity is too strong, affecting air permeability. In this system, chitosan not only acts as a gel component, but its broad-spectrum antibacterial properties can significantly reduce the incidence of miscellaneous bacteria and diseases in the matrix, and its role is particularly crucial in the middle and late stages of fruiting. Gypsum, as a structural stabilizer and buffer, can promote the formation and stability of matrix aggregate structure with its calcium ions, prevent caking, and provide essential calcium and sulfur mineral elements; while lime, as a core pH regulator, is used to precisely neutralize the acidity brought by fermentation and acidic slurry, stabilize the matrix pH in the optimal growth range of edible fungi, and also has an auxiliary antibacterial function.Preferably, the raw materials are in the following weight proportions: 25 parts corn stalks, 20 parts rice husks, 15 parts sawdust, 30 parts cottonseed hulls, 8 parts soybean meal, 1.2 parts compound microbial agent, 5 parts bentonite, 2 parts chitosan, 1.5 parts gypsum, and 1.5 parts lime.

[0020] In a preferred embodiment, the compound microbial agent is composed of *Aspergillus niger*, *Rhizopus oryzae*, and *Streptococcus thermophilus* in a viable cell ratio of 2-3:1-2:1. The compound microbial agent used in this invention, with its strain ratio of 2-3:1-2:1, is the optimal solution verified through extensive experiments. These three components form a sequentially linked and functionally complementary "microbial working chain." *Aspergillus niger* rapidly becomes the dominant microbial group within 0-48 hours of fermentation, secreting cellulase, hemicellulase, and pectinase, forming a "complex enzyme system." This system first attacks the amorphous regions of plant cell walls, breaking down the dense structure and opening channels for subsequent degradation. Its high viable cell count (2-3 parts) ensures the initiation speed and intensity of the degradation reaction. *Rhizopus oryzae* enters the logarithmic growth phase after 48 hours of fermentation. Its powerful aerial mycelial network can penetrate and physically divide material particles, greatly increasing the specific surface area of ​​the material, allowing the enzymes released by *Aspergillus niger* to more fully contact the substrate. Meanwhile, the amylase and protease secreted by *Rhizopus oryzae* can further degrade macromolecular nutrients in soybean meal and other materials, and transform its mycelium itself into microbial protein that is easily utilized by edible fungi. *Streptococcus thermophilus* activity increases in the later stages of fermentation (after 72 hours) as the pile temperature rises above 35°C. It produces L-lactic acid through homolactic fermentation, causing the pH of the fermentation system to steadily decrease to 5.0-5.5. This slightly acidic environment has three benefits: firstly, it is most suitable for the enzyme expression of *Aspergillus niger* and *Rhizopus oryzae*; secondly, it effectively inhibits the growth of common contaminants such as *Bacillus* and *Pseudomonas*; and thirdly, it creates optimal pH conditions for the subsequent gelation of chitosan. The lactic acid produced can also complex metal ions, promoting the dissolution of lignocellulose. These three factors synergistically significantly improve the degradation efficiency of lignocellulose, accelerate substrate maturation, and create a more favorable microecological environment for the growth of edible fungi mycelium. Preferably, the ratio of viable *Aspergillus niger*, *Rhizopus oryzae*, and *Streptococcus thermophilus* in the compound inoculant is 2.5:1.5:1.

[0021] This invention also provides a method for preparing a composite modified edible fungi cultivation substrate made from agricultural waste, comprising the following steps: crushing corn stalks, rice husks, and sawdust, and mixing them evenly with cottonseed hulls and soybean meal to obtain a basic mixture; adding a composite microbial agent and water to the basic mixture, stirring evenly, and then carrying out solid-state fermentation to obtain a modified fermented material; dissolving bentonite and chitosan in an acetic acid solution, stirring until fully swollen to obtain an activated functional slurry; thoroughly mixing the modified fermented material, the activated functional slurry, gypsum, and lime, adjusting the moisture content to 55%~65%, and sterilizing to obtain the cultivation substrate. The preparation process of this invention deeply degrades the lignocellulose in the raw materials through solid-state fermentation under the action of the composite microbial agent, releasing readily available nutrients and improving the biocompatibility of the substrate; simultaneously, the three-dimensional network structure slurry formed by bentonite and chitosan in an acidic environment effectively constructs stable substrate aggregates, significantly improving water retention and aeration. This process ultimately achieves the simultaneous completion of physical structure improvement and nutrient biological activation, giving the cultivation substrate both excellent water retention and aeration, as well as efficient nutrient supply capabilities.

[0022] In a preferred embodiment, the solid-state fermentation conditions in the modified fermentation material preparation step are: temperature 35-45℃, fermentation time 3-7 days, and turning the compost 1-2 times daily during fermentation. Solid-state fermentation is essentially a complex process of microbial ecosystem succession and biochemical reaction. The temperature is controlled at 35-45℃, which is the optimal intersection range of the enzyme activity curves of the three microbial agents: the optimal enzyme production temperature for *Aspergillus niger* is 37-40℃, for *Rhizopus oryzae* it is 30-37℃, and for *Streptococcus thermophilus* it is 40-45℃. The fermentation time is set at 3-7 days based on the lignin degradation kinetics curve in the substrate: on day 3, the rapid degradation period of cellulose is basically over; from day 5-7, hemicellulose and lignin enter a slow degradation and modification stage. At this point, the material is fully decomposed, and extending the fermentation time not only reduces efficiency but may also lead to excessive nutrient consumption, resulting in more harm than good. Turning the compost pile 1-2 times daily serves not only to supply oxygen and dissipate heat, but more importantly, to break the mycelial network through mechanical force. This encourages the inoculant to germinate new, more infectious mycelia from the aging mycelial segments, thereby achieving uniform distribution and efficient action of the inoculant throughout the material. Preferably, the fermentation temperature is 38℃, and the fermentation time is 5 days.

[0023] In a preferred embodiment, the mass ratio of water to the basic mixture in the modified fermentation feed preparation step is 0.5~0.7:1. This ratio range ensures the formation of a uniform water film on the surface of the basic mixture particles, providing the necessary liquid medium for the germination, proliferation, and secretion of hydrolytic enzymes by the compound microbial agent, thereby significantly accelerating the degradation efficiency of lignocellulose. Simultaneously, this water content maintains sufficient porosity between material particles, ensuring effective diffusion of oxygen within the fermentation pile and timely dissipation of metabolic waste heat, fundamentally preventing fermentation rancidity and the growth of miscellaneous bacteria caused by localized anaerobic conditions. Ultimately, this water-to-material ratio achieves an optimal balance between "microbial activity" and "mass transfer and permeability," which is a key guarantee for achieving efficient modification and structural stability of the fermentation feed. Preferably, the mass ratio of water to the basic mixture is 0.6:1.

[0024] In a preferred embodiment, the ratio of the total mass of bentonite and chitosan to the volume of the acetic acid solution in the preparation step of the activated functional slurry is 1 g: 8-15 mL. This ratio provides sufficient liquid medium to ensure that the bentonite sheets are fully hydrated and swollen, and that the chitosan molecular chains are fully extended and protonated under acidic conditions. This provides the necessary reaction space for the two to construct a continuous and stable "organic-inorganic" hybrid gel network through electrostatic interaction. At the same time, this ratio avoids excessive dilution of the effective component concentration caused by excessive acetic acid solution, thus ensuring that the final functional slurry has suitable viscosity and adhesion, allowing it to be uniformly dispersed and effectively coated on the surface of the material particles when mixed with the modified fermentation material, ultimately forming granules with excellent water retention, air permeability, and structural stability in the matrix. Preferably, the ratio of the total mass of bentonite and chitosan to the volume of the acetic acid solution is 1 g: 12 mL.

[0025] Furthermore, the mass concentration of the acetic acid solution in the preparation step of the activated functional slurry is 1.0%~1.5%. The preparation of the activated functional slurry is a dynamic process from physical swelling to chemical cross-linking. An acetic acid solution with a mass concentration of 1.0%~1.5% is used because at this concentration, the H+ in the solution... + The concentration is sufficient to fully protonate the amino groups (-NH2) on the chitosan molecular chains into cations (-NH3⁺), allowing the molecular chains to fully extend and hydrate, but not to cause acidic degradation of chitosan. In this acidic environment, the Na+ in the interlayer of bentonite... + Ca 2+ Isocations and H +Ion exchange occurs, leading to increased interlayer spacing and easier penetration of water molecules, thus achieving full swelling. When bentonite and chitosan come into contact under stirring, the negatively charged bentonite sheets and positively charged chitosan molecular chains rapidly form a three-dimensional network gel structure through strong electrostatic interactions. Stirring until full swelling is achieved is crucial to ensuring a uniform and stable gel structure free of undispersed particles. This functional slurry is no longer a simple mixture but a viscoplastic smart material that can uniformly coat the surface of fermentation particles within a matrix, forming a robust yet elastic microcapsule membrane after solidification. Preferably, the acetic acid solution has a mass concentration of 1.2%.

[0026] In a preferred embodiment, sterilization in the cultivation substrate preparation step is performed using high-pressure steam sterilization at a temperature of 100-130°C for 30-90 minutes. High-temperature saturated steam at 100°C and above can efficiently penetrate the interior of substrate particles, thoroughly inactivating various potential contaminants and pathogens, including bacterial vegetative cells, spores, fungal spores, and insect eggs, creating a sterile colonization environment for the pure culture of edible fungi mycelia. The sterilization temperature combined with a sterilization time of 30-90 minutes ensures thorough sterilization while effectively avoiding damage to the activated fast-acting nutrients, compound microbial agent metabolites, and functional slurry gel structure in the substrate caused by prolonged and excessive heat-pressing, thereby maximizing the preservation of the substrate's biological activity and nutritional value. Preferably, the sterilization temperature is 121°C and the sterilization time is 60 minutes.

[0027] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0028] Preparation of the compound microbial agent: Aspergillus niger, Rhizopus oryzae, and Streptococcus thermophilus were mixed at a viable count ratio of 2.5:1.5:1 to obtain the compound microbial agent. The microbial strains used in this invention were purchased from the China Industrial Microbial Culture Collection Center. Example 1

[0029] (1) Weigh the raw materials: 25 parts corn stalks, 20 parts rice husks, 15 parts sawdust, 30 parts cottonseed hulls, 8 parts soybean meal, 1.2 parts compound microbial agent, 5 parts bentonite, 2 parts chitosan, 1.5 parts gypsum, and 1.5 parts lime.

[0030] (2) Crush corn stalks, rice husks, and sawdust into 2-4 mm, mix them evenly with cottonseed hulls and soybean meal to obtain the basic mixture.

[0031] (3) Add compound microbial agent and water (water to basic mixture mass ratio is 0.6:1) to the basic mixture, stir evenly, and carry out solid fermentation at 38℃ for 5 days, turning the pile twice a day during the period to obtain modified fermented material.

[0032] (4) Dissolve bentonite and chitosan in a 1.2% acetic acid solution (solid-liquid mass-volume ratio of 1g:12mL), stir for 40 minutes until fully swollen, and obtain the activated functional slurry.

[0033] (5) Thoroughly mix the modified fermentation material, activated functional slurry, gypsum and lime, spray a small amount of water to adjust the overall moisture content to 60%, and sterilize with high pressure steam at 121℃ for 60 minutes to obtain the cultivation substrate. Example 2

[0034] (1) Weigh the raw materials: 20 parts corn stalks, 15 parts rice husks, 10 parts sawdust, 25 parts cottonseed hulls, 5 parts soybean meal, 0.5 parts compound microbial agent, 3 parts bentonite, 1 part chitosan, 1 part gypsum, and 1 part lime.

[0035] (2) Crush corn stalks, rice husks, and sawdust into 2-4 mm, mix them evenly with cottonseed hulls and soybean meal to obtain the basic mixture.

[0036] (3) Add compound microbial agent and water (water to basic mixture mass ratio is 0.6:1) to the basic mixture, stir evenly, and carry out solid fermentation at 35℃ for 7 days, turning the pile twice a day during the period to obtain modified fermented material.

[0037] (4) Dissolve bentonite and chitosan in a 1.0% acetic acid solution (solid-liquid mass-volume ratio of 1g:15mL), stir for 40 minutes until fully swollen, and obtain the activated functional slurry.

[0038] (5) Thoroughly mix the modified fermentation material, activated functional slurry, gypsum and lime, spray a small amount of water to adjust the overall moisture content to 60%, and sterilize with high-pressure steam at 115℃ for 90 minutes to obtain the cultivation substrate. Example 3

[0039] (1) Weigh the raw materials: 30 parts corn stalks, 25 parts rice husks, 20 parts sawdust, 35 parts cottonseed hulls, 10 parts soybean meal, 2.0 parts compound microbial agent, 8 parts bentonite, 3 parts chitosan, 2 parts gypsum, and 2 parts lime.

[0040] (2) Crush corn stalks, rice husks, and sawdust into 2-4 mm, mix them evenly with cottonseed hulls and soybean meal to obtain the basic mixture.

[0041] (3) Add compound microbial agent and water (water to basic mixture mass ratio is 0.6:1) to the basic mixture, stir evenly, and carry out solid fermentation at 40℃ for 3 days. During the period, turn the pile twice a day to obtain modified fermented material.

[0042] (4) Dissolve bentonite and chitosan in a 1.5% acetic acid solution (solid-liquid mass-volume ratio of 1g:8mL), stir for 40 minutes until fully swollen, and obtain the activated functional slurry.

[0043] (5) Thoroughly mix the modified fermentation material, activated functional slurry, gypsum and lime, spray a small amount of water to adjust the overall moisture content to 60%, and sterilize with high pressure steam at 124℃ for 30 minutes to obtain the cultivation substrate.

[0044] Comparative Example 1

[0045] The same raw materials and steps as in Example 1 were used, the only difference being that solid-state fermentation modification was not performed. Instead, the basic mixture was mixed with water and then directly mixed with the activated functional slurry, gypsum, and lime, and then sterilized.

[0046] Comparative Example 2

[0047] The same raw materials and steps as in Example 1 were used, the only difference being that no activated functional slurry was used, i.e., no bentonite and chitosan were added.

[0048] Comparative Example 3

[0049] The same raw materials and steps as in Example 1 were used, the only difference being the composition of the compound microbial agent. An equal amount of Bacillus subtilis and Bacillus licheniformis in a 1:1 ratio was used to replace the specific compound microbial agent of this invention.

[0050] Comparative Example 4

[0051] The same raw materials and steps as in Example 1 were used, the only difference being the preparation parameters of the activated slurry. A 0.5% acetic acid solution was used, and the solid-liquid ratio was 1g:20mL.

[0052] Experimental Example: Verification of Cultivation Effects

[0053] Within a standard mushroom shed, the production area was divided into multiple plots. The cultivation substrates prepared in Examples 1-3 and Comparative Examples 1-4 were used in each plot. A commonly used commercial substrate for local shiitake mushroom cultivation (commercially available, mainly composed of sawdust, cottonseed hulls, and wheat bran) served as the control group (CK). The cultivar was "Zhexiang No. 6" shiitake. The fruiting time, yield, and proportion of flower-shaped mushrooms in the first flush were recorded. The results are shown in Table 1.

[0054] Table 1. Effects of different cultivation substrates on fruiting and quality of shiitake mushrooms

[0055]

[0056] As shown in Table 1, firstly, regarding fruiting efficiency, the fruiting time of Examples 1-3 was concentrated in 82-84 days, which was more than 14 days shorter than the control group (98 days). This significant improvement was not due to a single factor, but rather the result of the synergistic effect of solid-state fermentation modification and the activation of the functional slurry. Solid-state fermentation pre-degraded lignocellulose, enabling faster "start-up" of mycelial colonization and nutrient absorption; while the stable granular structure constructed by the functional slurry created an optimal environment of balanced water and air for the mycelium, resulting in stronger growth vitality, which together led to a significant shortening of the growth cycle. Secondly, regarding yield composition, the yield increase rate of Examples 1-3 remained consistently high at 12.6%-14.5%. This directly confirms the highly efficient activation and slow-release effect of nutrients. The yield increase rate of Comparative Example 1 (without fermentation) was only 5.8%, demonstrating that the nutrient utilization rate of undegraded raw materials is extremely low. In contrast, the yield increase rate of Comparative Example 3 (using alternative inoculants) was only 3.4%, highlighting the irreplaceable role of the specific compound inoculant (Aspergillus niger-Rhizopus oryzae-Streptococcus thermophilus) in degrading lignocellulose. This invention converts "potential nutrients" into "quick-acting nutrients" through fermentation and achieves "slow release" through the functional slurry portion, meeting the continuous and balanced nutritional needs of the mycelium throughout its growth cycle. Finally, in terms of commercial quality, the proportion of shiitake mushrooms with flower patterns (29.8%~32.5%) cultivated using the substrate of this invention is more than 1.6 times that of the control group. The formation of flower patterns requires specific environmental conditions, especially during the bud development stage, requiring the substrate to maintain a stable and low moisture content and high air humidity. The excellent pore structure and water retention and air permeability of the substrate of this invention can more precisely control the rate of water evaporation from the mushroom surface, thus making it easier to form ideal patterns. The lowest proportion of shiitake mushrooms was found in Comparative Example 2 (non-functional slurry) (20.5%), which, conversely, demonstrates the decisive role of bentonite-chitosan slurry in optimizing the mushroom-growing microenvironment and enhancing commercial value.

[0057] To further explore the underlying reasons for the improved performance of the matrix of the present invention, the key physicochemical properties and microbial communities of the matrices of Example 1, Comparative Examples 1-4 and the control group were measured, and the results are shown in Table 2.

[0058] 1. Determination of water holding capacity

[0059] Weigh a certain weight (W1) of the dried cultivation substrate sample to constant weight, and completely immerse it in deionized water for 24 hours to ensure full water absorption. Then remove it, let it drain on a sieve until no water drips, and weigh its saturated wet weight (W2). The water holding capacity is calculated using the following formula:

[0060] Water holding capacity (%) = [(W2-W1) / W1] × 100%.

[0061] Each sample group was set up with 3 replicates, and the results were averaged.

[0062] 2. Porosity determination

[0063] The ring cutter method was used for determination. A undisturbed matrix sample was taken using a ring cutter of known volume (V), and its natural wet weight (Wwet) was measured. The matrix sample was then dried at 105°C to constant weight, and its dry weight (Wdry) was measured. The bulk density (ρb) of the matrix was calculated as ρb = Wdry / V. Simultaneously, the particle density (ρs) of the matrix was determined using the pyridine method. The total porosity (Pt) was calculated using the following formula:

[0064] Porosity (%) = [1 - (ρb / ρs)] × 100%.

[0065] 3. Determination of carbon-nitrogen ratio (C / N)

[0066] The cultivation substrate sample was dried, pulverized, and passed through a 100-mesh sieve. The percentage content of total carbon (TC) and total nitrogen (TN) in the sample was directly determined using an elemental analyzer. The carbon-to-nitrogen ratio (C / N) was calculated.

[0067] C / N = TC% / TN.

[0068] 4. Determination of cellulose degradation rate

[0069] Referring to the "Determination of Chemical Composition of Lignocellulosic Biomass Raw Materials", the Panthen washing method was used for determination. The cellulose content in the basic mixture (unfermented) and the modified fermented mixture were determined separately.

[0070] The formula for calculating cellulose degradation rate is:

[0071] Cellulose degradation rate (%) = [(cellulose content before fermentation - cellulose content after fermentation) / cellulose content before fermentation] × 100%.

[0072] 5. Microbial community analysis (bacterial / fungal ratio and Shannon index)

[0073] Total DNA was extracted from culture substrate samples using a microbial genomic DNA kit. PCR amplification was performed using primers targeting the V3-V4 variable region of the bacterial 16S rRNA gene (e.g., 338F / 806R) and primers targeting the ITS1 region of fungi (e.g., ITS5-1737F / ITS2-2043R). After purification, the amplified products were sequenced on high-throughput sequencing platforms such as Illumina MiSeq. After quality control of the raw sequencing data, operational taxonomic unit (OTU) clustering and species taxonomy analysis were performed using bioinformatics software such as QIIME2 and USEARCH. Based on the standardized OTU table, the bacterial to fungal sequence ratio and the Shannon-Wiener index, reflecting community diversity, were calculated.

[0074] Table 2. Physicochemical and biological properties of different cultivation substrates

[0075]

[0076] As shown in Table 2, the water holding capacity (78.2%) and porosity (62.5%) of Example 1 were significantly higher than those of the other groups. This is not a simple numerical summation, but rather demonstrates that the bentonite-chitosan activated slurry successfully constructed an ideal structure with a "high water-to-air ratio". Comparative Example 2 (without slurry) had the lowest values ​​for both of these indicators and poor cultivation results, indicating that traditional physical mixing cannot achieve this structural improvement. High water holding capacity ensures a continuous water supply for mycelial growth, while high porosity guarantees the oxygen required for mycelial respiration. The synergy of these two factors is the physical basis for shortening the cycle and increasing yield. The carbon-to-nitrogen ratio (C / N = 28.3) of Example 1 is most suitable for shiitake mushroom growth, and its cellulose degradation rate (45.6%) is much higher than that of Comparative Example 1 (18.4%, no fermentation) and Comparative Example 3 (25.7%, with substitute inoculant). This eloquently demonstrates that the solid-state fermentation process of the composite inoculant of this invention efficiently completes the "pre-digestion" of the substrate, transforming complex carbon sources into easily usable forms and adjusting the C / N ratio to the ideal range. This provides the chemical basis for improved nutrient utilization and sufficient mycelial nutrition. Example 1 exhibited the lowest bacteria / fungus ratio (0.85) and the highest bacterial Shannon index (3.21). This combination of data has profound ecological significance: the low bacteria / fungus ratio indicates a shift from bacterial to fungal dominance in the substrate environment, which is extremely beneficial for eukaryotic edible fungi mycelia to occupy a niche advantage and competitively suppress other microorganisms; the high Shannon index indicates higher bacterial community diversity and a more stable structure. A diverse bacterial community usually means a more balanced micro-ecosystem with complementary functions, strong resistance to disturbance, and less susceptibility to monopolization by a few pathogens, thus providing a healthy, stable, and low-disease-risk growth environment for edible fungi mycelia.

[0077] In summary, the technical solution of this invention achieves systematic optimization and reconstruction of the physicochemical properties and microecological structure of the cultivation substrate through the synergistic effect of "compound microbial agent solid-state fermentation" and "bentonite-chitosan functional slurry". The activated functional slurry effectively constructs stable substrate aggregates, giving them both high water holding capacity and high porosity, creating an excellent water-air balance environment for mycelial growth. This is the physical basis for shortening the fruiting cycle and increasing yield. This invention successfully creates a microecological system dominated by beneficial fungi, with high bacterial community diversity and strong system stability. This environment is extremely conducive to the ecological colonization and healthy growth of edible fungi mycelium, and is the biological basis for ensuring high yield, stability, and a significant increase in the proportion of marketable mushrooms (flower mushroom rate). The cultivation substrate and its preparation method provided by this invention, through the above-mentioned multi-dimensional and systematic improvements, fundamentally solve the key technical bottlenecks of traditional substrates, such as poor structural stability, low nutrient utilization rate, and easy imbalance of micro-ecology. Ultimately, it achieves comprehensive beneficial effects in edible fungi cultivation, including significantly shortened fruiting cycle, steadily improved biological efficiency, and excellent product quality.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composite modified edible fungus cultivation substrate made from agricultural waste, characterized in that, The components include the following parts by mass: 20-30 parts corn stalks, 15-25 parts rice husks, 10-20 parts sawdust, 25-35 parts cottonseed hulls, 5-10 parts soybean meal, 0.5-2.0 parts compound microbial agent, 3-8 parts bentonite, 1-3 parts chitosan, 1-2 parts gypsum, and 1-2 parts lime; The compound microbial agent is composed of Aspergillus niger, Rhizopus oryzae, and Streptococcus thermophilus in a live bacteria ratio of 2-3:1-2:

1.

2. The method for preparing a composite modified edible fungus cultivation substrate from agricultural waste according to claim 1, characterized in that, Includes the following steps: Corn stalks, rice husks, and sawdust are crushed and then mixed evenly with cottonseed hulls and soybean meal to obtain a basic mixture. Add compound microbial agent and water to the basic mixture, stir evenly, and then carry out solid-state fermentation to obtain modified fermented material; Bentonite and chitosan were dissolved in an acetic acid solution and stirred until fully swollen to obtain an activated functional slurry. The modified fermentation material, activated functional slurry, gypsum and lime are thoroughly mixed, the moisture content is adjusted to 55%~65%, and sterilization is performed to obtain the cultivation substrate.

3. The method for preparing a composite modified edible fungus cultivation substrate from agricultural waste according to claim 2, characterized in that, The solid-state fermentation conditions in the preparation steps of the modified fermentation material are: temperature 35~45℃, fermentation time 3~7 days, and turning the pile 1~2 times a day during the fermentation period.

4. The method for preparing a composite modified edible fungus cultivation substrate from agricultural waste according to claim 2, characterized in that, In the modified fermentation material preparation step, the mass ratio of water to basic mixture is 0.5~0.7:

1.

5. The method for preparing a composite modified edible fungus cultivation substrate from agricultural waste according to claim 2, characterized in that, In the preparation step of the activated functional slurry, the ratio of the total mass of bentonite and chitosan to the volume of acetic acid solution is 1g:8~15mL.

6. The method for preparing a composite modified edible fungus cultivation substrate from agricultural waste according to claim 5, characterized in that, The mass concentration of the acetic acid solution in the preparation step of the activated functional slurry is 1.0%~1.5%.

7. The method for preparing a composite modified edible fungus cultivation substrate from agricultural waste according to claim 2, characterized in that, The sterilization process in the cultivation substrate preparation step involves high-pressure steam sterilization at a temperature of 100-130°C for 30-90 minutes.

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