Lentinus edodes cultivation method containing heavy parasitic enzyme response type bacteriostatic microcapsules

Abstract

The present application discloses a Lentinula edodes cultivation method containing heavy parasitic enzyme response type bacteriostatic microcapsules, and belongs to the technical field of edible fungus cultivation. The method comprises the following steps in sequence: preparing slow-release nitrogen source particles, preparing bacteriostatic microcapsules, preparing composite culture medium, high-pressure sterilization inoculation, segmented temperature control culture, gradient bud induction, and graded harvesting. In the present application, the heat-induced gel film compounded by gellan gum and chitin nanofiber is used as the outer layer of the microcapsule. When the trichoderma starts to secrete lyticase, the outer layer is enzymatically degraded, and the lipopeptide bacteriostatic agent is released in situ at the pollution focus. The background enzyme activity of the normal growth of Lentinula edodes is lower than the threshold value, and the capsule wall remains intact. The outer layer does not need to be chemically cross-linked, and the 121 DEG C sterilization process serves as its final solidification. In combination with the coated slow-release nitrogen source, the nitrogen starvation window of Lentinula edodes is eliminated to ensure the specificity of the enzyme signal, so that the carbon-nitrogen ratio is autonomously reduced to 20:1-22:1; in combination with the limited speed ladder cooling bud induction, the primordium differentiation is realized synchronously. The present application can significantly reduce the pollution rate and improve the yield and quality.

Description

Technical Field

[0001] This invention relates to the field of edible fungi cultivation technology, and in particular to a method for cultivating shiitake mushrooms containing hyperparasitic enzyme-responsive antibacterial microcapsules. Background Technology

[0002] Shiitake mushrooms are one of the most produced and valuable edible fungi in my country. Unlike short-cycle varieties such as oyster mushrooms and enoki mushrooms, shiitake mycelium undergoes two unique stages after completing its vegetative growth: mycelial maturation and color change. The entire cultivation cycle typically lasts 90–120 days. This long cycle means a longer exposure time for the substrate, increasing the risk of infection with Trichoderma (…). Trichoderma spp. ), Neurospora ( Neurospora spp. ), Penicillium ( Penicillium spp. The chances of contamination by other microorganisms such as shiitake mushroom spawn are significantly increased. In large-scale production, the contamination rate of shiitake mushroom spawn is generally between 10% and 15%, which is the primary bottleneck restricting the efficiency of the industry.

[0003] First, static optimization of the carbon-nitrogen ratio in the culture medium.

[0004] Current techniques generally fix the carbon-to-nitrogen ratio of the culture medium at a certain empirical value by adjusting the ratio of sawdust to wheat bran. However, it is known in the art that the suitable carbon-to-nitrogen ratio for shiitake mushrooms is a relatively wide range of 25–40:1, and the carbon-to-nitrogen ratio requirements of shiitake mushrooms differ directionally at different growth stages: a higher carbon-to-nitrogen ratio is needed during the vegetative growth stage to promote rapid mycelial spread, while a lower carbon-to-nitrogen ratio is needed during the reproductive growth stage to break the inertia of vegetative growth and trigger primordium differentiation. Fixing the carbon-to-nitrogen ratio to a single value, regardless of its specific value, will inevitably lead to a mismatch between the ratio and the needs of the mycelium at a certain growth stage.

[0005] Second, the addition of antibacterial microbial agents.

[0006] Numerous reports have documented the use of biocontrol agents to antagonize other microorganisms. For example, CN101603009A discloses a biocontrol agent, *Sclerotinia scutellaria* ZS-1SB, for controlling sclerotinia rot, and its inoculum. However, there is a fundamental technological contradiction when introducing live biocontrol agents into edible mushroom cultivation: the culture medium must be autoclaved at 121°C to kill background microorganisms, but the live biocontrol agents are also killed simultaneously under the same conditions; if the biocontrol agents are added after sterilization, the bags must be opened, and opening the bags is itself one of the main routes for microorganism invasion.

[0007] Third, the continuous release of antibacterial active ingredients.

[0008] Existing slow-release systems mostly adopt a design logic of continuous and constant release. Firstly, during the majority of the time when the mushroom substrate is uncontaminated, the antibacterial active substances are unnecessarily consumed and degraded. Secondly, the lipopeptides produced by Bacillus (such as ituronin and fenestrant) exert their effects by forming ion channels on the cell membrane, and their targets are not limited to Bacillus and Basidiomycetes, thus significantly inhibiting the mycelium of shiitake mushrooms while inhibiting other microorganisms. The result of continuous release is often that although the contamination rate decreases, the yield decreases instead of increasing.

[0009] Fourth, the scheme of using pH as a release trigger signal is not valid in this scenario.

[0010] Stimulus-responsive microcapsules have been widely disclosed in the field of plant protection. US6500447B1 discloses a pH-sensitive microcapsule with free carboxyl groups introduced into its capsule wall, which is stable between approximately 1 and 5.5, releases its contents at pH above approximately 5.5, and is used in pesticides. CN109691444A discloses a pH-responsive controlled-release pesticide microcapsule suspension using polyurethane crosslinked with carboxyl chain extenders as the capsule material. However, such solutions cannot be applied to the edible mushroom substrate scenario: it is known in the art that during the colonization of shiitake mycelium, it continuously secretes organic acids, causing the substrate pH to decrease unidirectionally; Trichoderma, on the other hand, prefers an acidic environment of pH 4–6, and in production, lime is added to raise the pH to 7.5–8.0 to inhibit green mold. In other words, when the substrate is contaminated by other microorganisms, the substrate pH does not rise, but rather becomes further acidified.

[0011] Fifth, sudden damage during temperature-induced bud formation.

[0012] It is well known in the art that shiitake mushroom primordia induction requires a diurnal temperature range of 8℃–10℃, relative humidity of 80%–90%, and diffused light of 50lx–200lx. However, existing technologies only specify the temperature range, without limiting the cooling rate and the stepwise residence time. In practice, a sudden, one-time cooling is often used, with rates frequently exceeding 5℃ / h. The actual cooling rate differs greatly between the surface and core of the mushroom log, significantly widening the time window for primordia development and resulting in highly dispersed maturity of fruiting bodies within the same batch. This is the direct cause of the current inconsistent cap size and low graded harvesting success rate of shiitake mushrooms.

[0013] In summary, there is an urgent need in this field for a shiitake mushroom cultivation method in which the release trigger signal is actually present in the mushroom stick and the membrane formation process of the capsule wall is compatible with the sterilization process. Summary of the Invention

[0014] To address the shortcomings of existing technologies, this invention provides a method for cultivating shiitake mushrooms containing hyperparasitic enzyme-responsive antibacterial microcapsules.

[0015] In order to achieve the objective of this invention, the following solution is proposed: A method for cultivating shiitake mushrooms containing hyperparasitic enzyme-responsive antibacterial microcapsules includes the following steps: S1. Preparation of slow-release nitrogen source particles: Soybean meal protein powder was encapsulated with calcium alginate as a gel matrix and chitosan as a coating to obtain slow-release nitrogen source particles. S2. Preparation of recombinant parasitic enzyme-responsive antibacterial microcapsules: A double-layer coating was applied to the core material using lipopeptides, with calcium alginate as the inner wall material and a composite of ketallan gum and chitin nanofibers as the outer wall material. The core material was incubated at 50℃~70℃ to form a hypostable gel, followed by drying to obtain recombinant parasitic enzyme-responsive antibacterial microcapsules. The cumulative release rate of the core material after immersion in a buffer solution with β-1,3-glucanase activity of 5 U / mL at 25℃ for 6 h was ≥75%, and the cumulative release rate after immersion in a buffer solution with β-1,3-glucanase activity of 0.60 U / mL at 25℃ for 72 h was ≤8%. S3. Preparation of modified composite culture medium: The slow-release nitrogen source particles obtained in S1 and the microcapsules obtained in S2 are added to a matrix with sawdust as the carbon source and wheat bran as the readily available nitrogen source to obtain the culture medium; the nitrogen provided by the slow-release nitrogen source particles accounts for 30% to 45% of the total nitrogen in the culture medium, and the amount of microcapsules added is 0.15% to 0.40% of the dry weight of the culture medium; the initial carbon-nitrogen ratio of the culture medium is 30:1 to 40:1, and it decreases autonomously as the culture progresses, dropping to 20:1 to 24:1 on days 35 to 45 after inoculation; S4. Autoclaving and Aseptic Inoculation: The culture medium is autoclaved at a temperature not lower than 100°C to transform the Cortex gum in the outer wall material from a low-stability gel to a heat-irreversible high-stability gel; after cooling, it is aseptically inoculated with shiitake mushroom spawn. S5. Segmented temperature-controlled culture: The culture temperature is gradually reduced from the colonization stage to the spread stage. During the color change stage, the day-night temperature difference is applied and diffused light is provided. S6. Gradient bud induction: The mushroom sticks are subjected to a step cooling treatment of no less than three levels, with a cooling rate of 1.0 to 3.0℃ / h between adjacent levels. After each level reaches the target temperature, the mushroom sticks are kept at a constant temperature for 4 to 10 hours. S7. Harvest the fruiting bodies.

[0016] Further, in S1, soybean meal protein powder is mixed with a sodium alginate aqueous solution with a mass concentration of 1.5%–2.5% at a mass ratio of 1:2–1:4, and then dripped into a calcium chloride solution with a mass concentration of 2%–4% to solidify for 20–40 minutes to obtain gel core particles; the gel core particles are immersed in a chitosan acetate solution with a mass concentration of 1.0%–2.0% to coat 2–3 times, and dried at 40℃–45℃ each time; the resulting slow-release nitrogen source particles have a particle size of 0.8 mm–1.5 mm, and the degree of deacetylation of the chitosan coating layer is 85%–95%.

[0017] Furthermore, in S2, the core material is composed of lipopeptides and chitosan oligosaccharides in a mass ratio of 1:2 to 1:4; the lipopeptides are a compound of iturin A, fenestrant and surfactant in a mass ratio of 3:2:1.

[0018] Furthermore, the microcapsules obtained by S2 have a particle size of 20 μm to 80 μm, a total capsule wall thickness of 2 μm to 5 μm, and a drug loading of 12% to 20%.

[0019] Furthermore, in S2, chitin nanofibers account for 15% to 35% of the total mass of the outer wall material, with the remainder being keratin; after sterilization in S4, the gel strength of the outer membrane is ≥400g / cm², and is more than 100% higher than before S4 sterilization.

[0020] Furthermore, the chitin nanofibers in S2 have a diameter of 10nm–50nm and an aspect ratio ≥30. They are prepared from α-chitin derived from shrimp and crab shells through dilute acid hydrolysis and high-pressure homogenization. The weight-average molecular weight of the ketallan gum is 5×10⁻⁶. 4 ~2×10 5 .

[0021] Furthermore, the enzymatic initiation threshold for the outer membrane of the microcapsules obtained by S2 is: β-1,3-glucanase activity ≥3.0 U / mL.

[0022] Furthermore, in S2, a low-stability gel is formed by incubation at 55℃~65℃ for 15min~30min, followed by spray drying to form a gel. The inlet air temperature of the spray drying is 100℃~110℃ and the outlet air temperature is 55℃~60℃. In S3, the sawdust is broadleaf tree sawdust, and its mass ratio with corn cob, wheat bran and corn flour is 60:15:18:5. The moisture content of the culture medium is 58%~62%, and the initial pH is 5.0~5.4.

[0023] Furthermore, S5 specifically includes: ① Establishment period, days 0-15, the culture temperature linearly decreases from 26℃ to 24℃ at a rate of ≤0.15℃ / d, and the environmental CO2 concentration is 2500ppm-4000ppm; ② Spreading period, days 16-35, the culture temperature decreases by 1℃ every 7 days from 24℃ to 21℃, and the environmental CO2 concentration is 1500ppm-2000ppm; ③ Color change period, days 36-55, the daytime temperature is 22℃, the nighttime temperature decreases daily from 16℃ to a diurnal temperature range of 10℃, during which 50lx-200lx diffused light is provided, and the moisture content of the substrate decreases from 60% to 55%.

[0024] Furthermore, in S6, when the color-changing area on the surface of the mushroom log accounts for ≥85% of the total surface area of ​​the mushroom log, the bud induction is initiated. The step-down cooling is in four stages, with target temperatures of 21℃, 17℃, 13℃, and 9℃ respectively. The cooling rate between adjacent stages is 1.5~2.5℃ / h, and each stage is kept at a constant temperature for 6h~8h. During the bud induction period, the ambient CO2 concentration is ≤800ppm, the relative humidity is 90%~95%, and the moisture content of the mushroom log rises to 62%±2%. After the bud induction is completed, the temperature is raised back to 15℃~18℃ for constant mushroom cultivation. In S7, the mushrooms are harvested in two grades according to the cap diameter: 4cm~6cm and 6cm~8cm.

[0025] The beneficial effects of this invention are as follows: 1. The microcapsules of this invention are released in situ at the contamination site only when Trichoderma initiates reparasitism. The lipopeptides have an inhibitory effect on the basidiomycetes themselves, and after release, they also affect the mycelium of Lentinus edodes within a few millimeters around the contamination site. This invention limits the inhibitory effect to the local area of ​​the contamination site, rather than acting on the entire substrate as in a continuously released system. Actual measurements showed that the contamination rate decreased from 12.0% to 7.6%, a reduction of 36.7%, while yield increased simultaneously.

[0026] 2. This invention uses pathogen reparasitic enzymes rather than environmental pH as a trigger, thus avoiding the defect of pH-responsive microcapsules that never release in an acidified matrix.

[0027] 3. The outer membrane of this invention is composed of thermotropic gelled gum, which is free of chemical cross-linking agents, alkali, and color. Step S2 only forms a low-stability gel, while sterilization at 121°C in S4 transforms it into a thermally irreversible, high-stability gel, increasing the gel strength from 185 g / cm² to 465 g / cm². Sterilization no longer poses a threat to the capsule wall but serves as a final curing process; after sterilization, the core material lipopeptide activity retention rate is 83.6%, and the core material retention rate is 96.2%.

[0028] 4. The carbon-nitrogen ratio of the culture medium of the present invention decreases autonomously from 37:1 to 21:1 as the culture process progresses, the yield per log increases from 0.850 kg to 1.089 kg, an increase of 28.1%, and the cultivation cycle is shortened from 105 days to 86 days, a reduction of 18.1%.

[0029] 5. The step-cooling method with a limited rate caused the core and surface of the substrate to enter the critical point of differentiation simultaneously, reducing the coefficient of variation of the fruiting body cap diameter from 21.8% to 11.6%.

[0030] 6. No living microorganisms are introduced throughout the entire process of this invention. The ketone gum, chitin, and calcium alginate are all food-grade materials. Attached Figure Description

[0031] Figure 1 This is a process flow diagram of the shiitake mushroom cultivation method in Embodiment 1 of the present invention. Detailed Implementation

[0032] Example 1 like Figure 1 As shown, this embodiment provides a method for cultivating shiitake mushrooms containing reparasitic enzyme-responsive antibacterial microcapsules, specifically including the following steps: S1. Preparation of slow-release nitrogen source particles Take 100g of soybean meal protein powder (crude protein content 46.2%) and add 300g of 2.0% sodium alginate aqueous solution (mass ratio 1:3). Stir at 500r / min for 30min until homogeneous. Use a peristaltic pump to drop the slurry into a 3.0% calcium chloride solution through a 0.6mm inner diameter needle. Solidify at room temperature for 30min, filter, and wash three times with deionized water to obtain gel core particles. Immerse the gel core particles in a 1.5% chitosan solution (deacetylation degree 90%) prepared with 1% acetic acid for 15min, remove, and dry in a forced-air dryer at 42℃ for 4h. Repeat the immersion and drying process three times in total. The resulting particles have an average particle size of 1.2mm and a nitrogen content of 6.35%.

[0033] S2. Preparation of parasitic enzyme-responsive antibacterial microcapsules (1) Core material preparation: Take Bacillus belye ( Bacillus velezensis Two L of fermentation broth (using commercially available strains, not the subject of this invention) was centrifuged at 8000 r / min for 15 min to remove bacterial cells. The supernatant was adjusted to pH 2.0 with 6 mol / L hydrochloric acid and allowed to stand overnight at 4℃ for acid precipitation. The precipitate was collected by centrifugation, extracted three times with methanol, concentrated under reduced pressure, and freeze-dried to obtain 4.86 g of crude lipopeptide. HPLC analysis and formulation showed that the mass ratio of isochoricin A, fentanyl, and surfactant was 3:2:1. This compound was mixed with chitosan oligosaccharide (degree of polymerization 2-8) at a mass ratio of 1:3 to obtain the core material.

[0034] (2) Inner layer coating: 10g of core material is dispersed in 200mL of sodium alginate solution with a mass concentration of 2.0%, and emulsified by high-speed shearing at 10000r / min for 5min. Then, 3.0% calcium chloride solution is slowly added and solidified for 20min to obtain a suspension of core particles coated with calcium alginate.

[0035] (3) Preparation of chitin nanofibers: Take α-chitin powder from shrimp shells, hydrolyze it with 3 mol / L hydrochloric acid at 95℃ for 90 min, centrifuge, wash with water until neutral, and homogenize under high pressure at 80 MPa 15 times to obtain chitin nanofiber aqueous dispersion; the fiber diameter is 20 nm to 35 nm and the aspect ratio is about 45, as determined by transmission electron microscopy.

[0036] (4) Outer coating and low-stability film formation: Take Cortex gel (weight average molecular weight 1.2×10⁻⁶) 54.2g of the above-mentioned chitin nanofibers and 1.8g of the above-mentioned chitin nanofibers (the chitin nanofibers account for 30% of the total mass of the outer wall material) were dispersed in 200mL of phosphate buffer solution with pH 6.5 and stirred at high speed for 30min until uniform. The core particle suspension obtained in (2) was added to it and re-coagulated for 30min; the temperature was raised to 60℃ and kept at 20min to make the Cordlan gum form a low-stability gel network; then it was spray dried (inlet air 105℃, outlet air 58℃, atomization pressure 0.3MPa) to form the shape. According to online infrared temperature measurement, the peak temperature of the particle surface at the end of the drying period was 71℃, which did not exceed the high-stability conversion temperature of 80℃. The pH was maintained at 6.0~7.0 throughout the process, and no chemical cross-linking agents or alkaline solvents were used.

[0037] The resulting microcapsules had an average particle size of 46 μm (distributed between 22 μm and 78 μm), a total capsule wall thickness of 3.4 μm, and a drug loading of 16.2%. At this stage, the outer membrane was a low-stability gel with a gel strength of 185 g / cm². After S4 sterilization, it transformed into a high-stability gel with a gel strength of 465 g / cm². Response characteristics are shown in Example 3.

[0038] S3. Preparation of modified composite culture medium Weigh the following materials according to the specified mass ratio: 60 parts broadleaf tree sawdust, 15 parts corn cob (passed through a 20-mesh sieve), 18 parts wheat bran, and 5 parts corn flour, plus 1 part gypsum and 1 part light calcium carbonate. Add S1 granules to ensure that the nitrogen provided accounts for 38% of the total nitrogen; add S2 microcapsules at 0.25% of the dry weight. Mix well, add water to adjust the moisture content to 60% (wet basis), and measure the initial carbon-to-nitrogen ratio as 37.2:1 and the initial pH as 5.2. Pack into 15cm × 55cm polypropylene corner-folded bags, each bag containing 1.0 kg of dry material.

[0039] S4. Autoclaving and Aseptic Inoculation Sterilize at 121℃ for 25 minutes. This process temperature is higher than the formation temperature of the highly stable gel of Cortex gel (approximately 80℃). During this step, the outer membrane transforms from a low-stability gel to a thermally irreversible highly stable gel, completing the final curing. Within the transition window of 60℃ to 80℃ during the heating process, the low-stability gel only softens without dissolving (Cortex gel is insoluble in water), and the core material is supported by an inner layer of calcium alginate to prevent leakage. After natural cooling to 24℃, 15g / bag of shiitake mushroom spawn is inoculated under Class 100 cleanroom conditions. After sterilization, the core material lipopeptide activity retention rate is 83.6%, the core material retention rate is 96.2%, and the outer membrane gel strength increases from 185g / cm² to 465g / cm² (see Example 3).

[0040] S5, Segmented Temperature Controlled Incubation ① Days 0-15: The temperature in the mushroom house was linearly reduced from 26.0℃ to 24.0℃ at a rate of 0.13℃ / day; CO2 concentration was 3200ppm; and the entire area was kept in complete darkness.

[0041] ② Days 16-35: The temperature drops by 1℃ every 7 days, successively to 24℃ (16d-22d), 23℃ (23d-29d), and 22℃ (30d-35d), reaching 21℃ at the end of day 35; CO2 concentration is 1800ppm.

[0042] ③ Days 36–55: Daytime temperature remained constant at 22℃; nighttime temperature decreased by 0.2℃ daily from 16.0℃ on day 36, reaching 12.0℃ on day 55, with a diurnal temperature range of 10.0℃. 120 lx diffused light was provided, with a 12-hour light / 12-hour darkness cycle. Ventilation was controlled to slowly reduce the moisture content of the substrate from 60% to 55%. On day 55, the percentage of color-changing area on the substrate surface was measured to be 89%.

[0043] S6, Gradient Bud Induction The area undergoing color change accounts for ≥85%, meeting the start-up conditions. A four-stage cooling process will be implemented starting from day 56: During this period, the CO2 concentration was maintained at ≤800ppm and the relative humidity at 92%. Spraying was used to raise the moisture content of the mushroom logs back to 62%. After completing the fourth stage, the temperature was raised back to a constant 16℃ for mushroom cultivation.

[0044] S7, Harvesting Substance On day 86, the primordia generally reached the harvest standard, and were harvested in two grades: 4cm-6cm and 6cm-8cm in cap diameter.

[0045] Example 2 Except for the following parameters, the rest are the same as in Example 1: S1: Sodium alginate mass concentration 1.5%, soybean meal powder to sodium alginate solution mass ratio 1:2, calcium chloride mass concentration 2.0%, solidification for 20 min, chitosan mass concentration 1.0%, degree of deacetylation 85%, impregnation and coating twice; particle size 0.8 mm; S2: Lipopeptide to chitosan oligosaccharide mass ratio 1:2; chitin nanofibers account for 15% of the outer wall material, and ketallan gum accounts for 85%; pH of the outer wall material dispersion is 6.0; low-stability gel is kept at 55℃ for 30 min; spray drying is carried out at 100℃ inlet and 55℃ outlet; gel strength before sterilization is 168 g / cm², and after S4 sterilization it is 412 g / cm² (an increase of 145%). S3: The nitrogen provided by the slow-release nitrogen source particles accounts for 30% of the total nitrogen; the amount of microcapsules added is 0.15% of the dry weight; the measured initial carbon-nitrogen ratio is 35.4:1, the initial pH is 5.0; the measured carbon-nitrogen ratio on day 40 is 22.0:1; S4: Sterilize for 20 minutes; S6: Three-stage cooling, with target temperatures of 20℃, 14℃, and 8℃ respectively (the final stage is 14℃ lower than the daytime temperature); the cooling rate for each stage is 3.0℃ / h, and the temperature is maintained for 4 hours.

[0046] Example 3 Except for the following parameters, the rest are the same as in Example 1: S1: Sodium alginate mass concentration 2.5%, soybean meal powder to sodium alginate solution mass ratio 1:4, calcium chloride mass concentration 4.0%, curing for 40 min, chitosan mass concentration 2.0%, degree of deacetylation 95%, impregnation and coating 3 times; particle size 1.5 mm; S2: Lipopeptide to chitosan oligosaccharide mass ratio 1:4; chitin nanofibers account for 35% of the outer wall material, and ketallan gum accounts for 65%; pH of the outer wall material dispersion is 7.0; low-stability gel is kept at 65℃ for 15 min; spray drying is carried out at 110℃ for inlet air and 60℃ for outlet air; gel strength before sterilization is 214 g / cm², and after S4 sterilization it is 538 g / cm² (an increase of 151%). S3: The nitrogen provided by the slow-release nitrogen source particles accounted for 45% of the total nitrogen; the amount of microcapsules added was 0.40% of the dry weight; the measured initial carbon-nitrogen ratio was 38.0:1, and the initial pH was 5.4; the measured carbon-nitrogen ratio on day 40 was 20.2:1. S4: Sterilize for 30 minutes; S6: Five-stage cooling, with target temperatures of 21℃, 18℃, 15℃, 12℃, and 10℃ respectively (the final stage is 12℃ lower than the daytime temperature); the cooling rate for each stage is 1.0℃ / h, and the temperature is maintained for 10 hours.

[0047] Comparative Example 1 (Conventional Shiitake Mushroom Cultivation) Standard industry process: 78 parts broadleaf tree sawdust, 20 parts wheat bran, 1 part gypsum, and 1 part light calcium carbonate, with a fixed carbon-to-nitrogen ratio of 25:1. No slow-release nitrogen source particles or antibacterial microcapsules are added. Inoculation is performed after sterilization at 121℃ for 25 minutes. The entire cultivation process is carried out at a constant temperature of 23℃. After color change, the mushroom house temperature is directly lowered from 23℃ to 9℃ (actual rate approximately 5.0℃ / h) to induce bud formation. Other management procedures are the same as in Example 1.

[0048] Comparative Example 2 (using only dynamic carbon-nitrogen ratio) Based on Comparative Example 1, S1 and S3 from Example 1 were introduced, but without the addition of antibacterial microcapsules, and the bud-inducing rate was still rapidly decreased to 5.0℃ / h. Purpose: To investigate the effect of dynamic carbon-nitrogen ratio on yield and cycle time independently.

[0049] Comparative Example 3 (Non-responsive sustained-release capsules) Based on Comparative Example 2, non-responsive microcapsules were added: the core material and amount were the same as in Example 1, but the outer wall material was replaced by ethyl cellulose (non-enzyme-responsive, uniformly dissolved in buffer solution, with a measured cumulative release rate of 68.3% over 72 hours) instead of a Cortex gum / chitin nanofiber composite membrane. The budding induction method was still abrupt. Purpose: To demonstrate the necessity of responsive release compared to sustained release.

[0050] Comparative Example 4 (Rapid Nitrogen Medium + Enzyme Response Capsules) The S2 microcapsules of Example 1 and the S5 and S6 temperature control processes of Example 1 were used, but the culture medium did not use slow-release nitrogen source particles, but instead used wheat bran with an equal amount of nitrogen. All nitrogen sources were readily available nitrogen, and the initial carbon-nitrogen ratio was 25:1. Purpose of the setup: To verify that a slow-release nitrogen source is a necessary prerequisite for the enzyme-responsive microcapsules to respond correctly.

[0051] Comparative Example 5 (pH-responsive capsules replacing enzyme-responsive capsules) Except for the outer wall material of the S2 microcapsules, the remaining steps are exactly the same as in Example 1. The outer wall material of S2 is replaced with hydroxypropyl methylcellulose phthalate (HP-55 type, dissolution threshold pH 5.5), that is, the microcapsules are prepared according to the pH response route represented by US6500447B1 and CN109691444A. Purpose of setting: To directly verify whether the pH response route is feasible in the shiitake mushroom spawn scenario.

[0052] Comparative Example 6 (full protocol, but with a sudden drop in bud-inducing method). Except for S6, the rest is exactly the same as in Example 1. S6 is replaced by: a one-time direct drop from 21°C to 9°C at a rate of 5.0°C / h, without steps or intermediate stops, and a total stay of 28 hours after reaching 9°C (comparable to the four-stage total stay time in Example 1). Purpose of setting: to examine the effect of rate-limited stepped cooling on product uniformity (CV) separately.

[0053] Example 1: Comprehensive Comparison of Cultivation Performance Each treatment group had 300 mushroom logs, repeated 3 times, and the average value was taken. The coefficient of variation (CV) of the cap diameter was the ratio of the standard deviation to the mean of the cap diameter of 100 randomly selected mushrooms in each group.

[0054] Table 1 Comparison of cultivation performance between each embodiment and the comparative example Group Combination of technical elements Contamination rate of miscellaneous bacteria / % Single bar yield / kg Cultivation cycle / day Cap diameter CV / % Comparative Example 1 Fixed C / N ratio 25:1; capsule-free; rapid reduction in bud-inducing effect. 12.0 0.850 105 21.8 Comparative Example 2 Dynamic C / N ratio; capsule-free; sudden drop in bud-inducing effect 11.4 0.982 96 20.9 Comparative Example 3 Dynamic C / N; Non-responsive capsules; Sudden drop in bud-stimulating effect 8.9 0.901 99 21.2 Comparative Example 4 Rapidly effective nitrogen (C / N ratio 25:1); enzyme-responsive capsules; temperature-controlled process of this invention. 10.6 0.913 98 20.5 Comparative Example 5 The complete solution, but the capsules were changed to pH-responsive type (HP-55). 11.2 0.988 96 20.7 Comparative Example 6 The entire protocol was used, but the bud-inducing rate dropped sharply to 5.0℃ / h. 7.7 1.048 87 19.6 Example 1 Full protocol (median: capsules 0.25%, sustained-release nitrogen 38%, stage 4 2.0℃ / h) 7.6 1.089 86 11.6 Example 2 Full protocol (lower limit: capsule 0.15%, level 3 3.0℃ / h, residence 4h) 8.3 1.052 89 12.9 Example 3 Full protocol (upper limit: capsule 0.40%, level 5 1.0℃ / h, residence 10h) 7.4 1.071 87 12.1 Compared with Comparative Example 1, Example 1 showed a 36.7% decrease in contamination rate, a 28.1% increase in yield per log, an 18.1% reduction in cultivation cycle, and a 46.8% decrease in the coefficient of variation of cap diameter.

[0055] As shown in Table 1: (1) Compared with Comparative Example 2, Comparative Example 3 only added a non-responsive continuous-release capsule, and the contamination rate decreased from 11.4% to 8.9%. However, the yield per log decreased from 0.982 kg to 0.901 kg, and the cultivation period increased from 96 days to 99 days. The inverse relationship between contamination rate and yield directly proves that the continuously released lipopeptides act on the entire log, inhibiting shiitake mycelium with the same intensity while inhibiting other bacteria. In contrast, Example 1 had a lower contamination rate (7.6%) and a higher yield (1.089 kg), indicating that the responsive release of the parasitic enzyme limited the inhibitory effect of the lipopeptides to the local contamination site.

[0056] (2) Comparative Example 4 used the same enzyme-responsive capsules and temperature control process as Example 1, the only difference being that the nitrogen source was changed from a slow-release type to a fast-acting type. Its contamination rate only decreased to 10.6%. Tracking the matrix enzyme activity showed that the fast-acting nitrogen was exhausted around day 25, and the shiitake mycelium immediately initiated autolysis to recover nitrogen. The extracellular β-1,3-glucanase activity increased from 0.33 U / g to 5.12 U / g (equivalent to 3.41 U / mL in aqueous phase, exceeding the enzymatic hydrolysis threshold of 3.0 U / mL), and the microcapsules were mistakenly triggered. By day 30, the residual amount of core material was less than 25% when the capsules were broken, and it was unable to cope with the actual contamination during the high-incidence period after day 30. This comparative example shows that S1 / S3 and S2 are not two independent measures that can be arbitrarily separated and combined, but rather an organic whole that is a prerequisite for each other's existence.

[0057] (3) Comparative Example 5 was prepared strictly according to the pH response principle disclosed in US6500447B1 (the capsule wall is stable at pH ≤ 5.5 and releases at pH > 5.5), and the other conditions were completely consistent with those in Example 1. The result showed that its contamination rate was 11.2%, which was almost the same as that of Comparative Example 2 (11.4%) without any capsules. The pH of the substrate was monitored throughout the process (see Effect Example 4): it dropped from 5.20 at the time of inoculation to 4.47 on day 60, never exceeding the erosion threshold of 5.5; when the capsules were broken on day 60, the residual amount of core material was still as high as 92.4%. This result confirms that in the continuously acidified substrate of shiitake mushroom substrate, the physical event of pH increase simply does not occur. Although pH-responsive microcapsules are feasible in the field of pesticides, they are completely ineffective after being transplanted to this scenario.

[0058] (4) Comparative Example 6 and Example 1 differ only in the cooling method at S6. The two showed minimal differences in contamination rate (7.7% vs 7.6%), yield per log (1.048 kg vs 1.089 kg), and cultivation period (87 days vs 86 days), but the coefficient of variation for cap diameter deteriorated sharply from 11.6% to 19.6%. Under sudden cooling conditions, a significant temperature gradient formed between the surface and core of the log, widening the time window for primordia development to over 40 hours; while stepwise cooling allowed the internal and external temperatures of the log to fully balance within each stage of residence, resulting in primordia almost simultaneously entering the differentiation critical point, narrowing the development window to within 12 hours. Existing technologies only specify the diurnal temperature range without limiting the cooling rate and residence time, making this effect unpredictable. The CVs of Example 2 (Level 3, 3.0℃ / h) and Example 3 (Level 5, 1.0℃ / h) were 12.9% and 12.1%, respectively, both significantly better than Comparative Example 6, indicating that the effect can be achieved throughout the entire range defined in claim 1.

[0059] Example 2: Validation of the correlation between extracellular lysosomal enzyme activity and Trichoderma loading Take the S3 culture medium logs from Example 1, and after inoculating shiitake mushrooms, divide them into two groups on day 20: Group A is artificially inoculated with Trichoderma atroviride (a publicly available known strain, such as purchased from the China Agricultural Microbial Culture Collection Center ACCC, or a commercially available strain) spore suspension (1×10⁻⁶). 6 CFU / mL, 1mL / stick); Group B was inoculated with an equal volume of sterile water as a control. Every 12 hours, the core substrate of the mycelium was taken and extracted with sodium acetate buffer at a ratio of 1:10 (w / v) for 30 min. The activities of β-1,3-glucanase (using Cordlan gum as a substrate, reducing sugar method) and chitinase (using colloidal chitin as a substrate) were measured. Enzyme activity was expressed as U·g⁻¹ per gram of oven-dry substrate; 1 U was defined as the amount of enzyme required to release 1 μmol of product per minute. Simultaneously, the Trichoderma loading was determined using the dilution plating method.

[0060] Table 2. Dynamic changes in extracellular lysozyme activity and Trichoderma load in the matrix after inoculation (based on oven-dry matrix). Time / h Group A β-1,3-glucanase / (U·g⁻¹) Group A chitinase / (U·g⁻¹) <![CDATA[Loading of Group A Trichoderma / (10 4 CFU·g⁻¹)]]> Group B β-1,3-glucanase / (U·g⁻¹) Group B chitinase / (U·g⁻¹) 0 0.32 0.18 0.1 0.30 0.17 12 0.61 0.29 1.8 0.31 0.17 24 1.84 0.95 9.6 0.33 0.18 36 4.27 2.11 34.2 0.34 0.18 48 7.65 3.88 78.5 0.33 0.19 72 12.40 6.02 152.0 0.35 0.19 96 15.80 7.31 210.4 0.36 0.20 Conversion between solid-phase and liquid-phase enzyme activity: The substrate moisture content is 60% (wet substrate), meaning 1g of oven-dry substrate corresponds to 1.5g of aqueous substrate. Therefore, the enzyme activity (U·g⁻¹) based on oven-dry substrate is divided by 1.5 to obtain the apparent enzyme activity concentration (U·mL⁻¹) in the aqueous substrate. Accordingly: 7.65U·g⁻¹ of β-1,3-glucanase in group A after 48h corresponds to 5.10U·mL⁻¹ in the aqueous phase; 0.30–0.36U·g⁻¹ of background activity in group B corresponds to 0.20–0.24U·mL⁻¹ in the aqueous phase.

[0061] The results showed that the extracellular lysozyme activities of uncontaminated substrate (Group B) remained at a low background level for 96 h. After Trichoderma inoculation (Group A), the activities of both enzymes increased to 23-fold and 20-fold above the background value, respectively, within 48 h, and were significantly positively correlated with the logarithm of Trichoderma load (β-1,3-glucanase Pearson r = 0.976, chitinase r = 0.981, both P < 0.01). This confirms that the extracellular lysozymes secreted by Trichoderma hyperparasitism are signals of contamination that can be directly sensed by the capsule wall material, and that their increase occurs in the early expansion stage of contamination.

[0062] Example 3: Enzyme-responsive release characteristics and heat resistance of microcapsules Take 0.5 g of the microcapsules obtained in Example 1 and place them in 100 mL of each of the following: (a) Trichoderma crude enzyme solution (β-1,3-glucanase 5.0 U / mL, chitinase 2.0 U / mL), (b) shiitake mycelium extracellular crude enzyme solution (concentrated to the upper limit of background activity for the entire growth cycle, i.e. β-1,3-glucanase 0.60 U / mL, chitinase 0.30 U / mL), and (c) Trichoderma crude enzyme solution inactivated at 100 °C for 10 min (control). Shake at 25 °C and 100 r / min. Take samples at regular intervals and determine the content of lipopeptides released into the liquid phase by HPLC.

[0063] Table 3. Cumulative release rate of microcapsules core material in different media / % time (a) Crude enzyme solution of Trichoderma (b) Crude enzyme solution from shiitake mushrooms (upper limit of background) (c) Inactivated enzyme solution (control) 1h 21.3 0.4 0.2 3h 55.8 1.1 0.6 6h 82.1 1.9 1.0 12h 94.0 2.9 1.5 24h 97.5 4.2 2.1 48h 98.1 5.7 2.8 72h 98.5 7.1 3.3 The results showed that the cumulative release rate of the microcapsules in the Trichoderma crude enzyme solution reached 82.1% (≥75%) after 6 hours, while the cumulative release rate in the crude enzyme solution prepared according to the upper limit of the background activity of shiitake mushrooms throughout the entire life cycle was only 7.1% (≤8%) after 72 hours. The inactivated enzyme solution group released only 3.3% after 72 hours, indicating that the release was dominated by enzymatic hydrolysis rather than physical dissolution, and the response specificity was determined by the enzyme activity itself.

[0064] Heat resistance and gel transition testing: After autoclaving at 121℃ for 25 min, the microcapsules were ruptured with methanol, and ituronic acid A was extracted and quantified by HPLC. The contents before and after sterilization were 162.0 mg / g and 135.4 mg / g, respectively, with a lipopeptide activity retention rate of 83.6%. The retention rate of unencapsulated free lipopeptides was only 51.2% under the same conditions. The core material retention rate (based on core material that did not leak into the matrix) was 96.2%, indicating that no substantial leakage occurred within the transition window of 60℃ to 80℃ during the heating process.

[0065] Table 3-1 Changes in the gel properties of the outer membrane before and after sterilization in step S4 project Before S4 sterilization (low-stability type) After S4 sterilization (high stability type) change Gel strength / (g·cm⁻²) 185 465 +151% Does it melt after reheating to 90℃? Yes (thermally reversible) No (heat is irreversible) — Solubility loss rate after soaking in water at 25℃ for 24 hours / % 1.8 0.4 −78% Core material retention rate / % 100.0 96.2 −3.8% The gel strength jumped from 185 g / cm² to 465 g / cm² (an increase of 151%), and the outer film, after sterilization, no longer melted upon reheating to 90°C. This directly confirms that the 121°C sterilization process transformed the Cortex gel from a thermally reversible, low-stability gel to a thermally irreversible, high-stability gel, meaning the sterilization process also served as the final curing process for the outer film. In contrast, if the holding temperature was increased to 90°C in S2, the gel strength before and after sterilization was 458 g / cm² and 471 g / cm², respectively, a change of only 2.8%. Sterilization no longer had a curing function, and the loss of lipopeptide activity during the high-temperature holding stage reduced the final retention rate to 74.1%.

[0066] Example 4: Temporal changes in substrate pH For the mycelium sticks of Example 1 and Comparative Example 5, the pH of the core substrate was measured every 10 days from the time of inoculation.

[0067] Table 4. Changes in pH of the substrate over culture time Culture time / d Example 1 Matrix pH Comparative Example 5: Matrix pH Comparative Example 5: Capsule Core Material Residual Rate / % 0 5.20 5.20 100.0 10 5.02 5.03 99.5 20 4.86 4.85 98.9 30 4.71 4.70 97.6 40 4.60 4.59 95.8 50 4.52 4.51 93.7 60 4.47 4.46 92.4 The substrate pH decreased unidirectionally from 5.20 at inoculation to 4.47 on day 60, without any rebound or exceeding the HP-55 dissolution threshold of pH 5.5. In Comparative Example 5, the microcapsules still retained 92.4% of their core material on day 60 (the slight loss was due to physical wear rather than dissolution). This data positively demonstrates that the physical event of pH increase does not exist in shiitake mushroom spawn, and that the use of a reparasitic enzyme as a trigger signal in this invention is irreplaceable.

[0068] Example 5: Background activity of extracellular lysing enzymes throughout the entire growth cycle of shiitake mushrooms To rule out the possibility that the capsules would open spontaneously due to autolysis of shiitake mycelium in the later stages of mycelial growth, the extracellular β-1,3-glucanase and chitinase activities of the uncontaminated substrate in Example 1 (confirmed by multiplex PCR to be free of Trichoderma, Neurospora, and Penicillium) were measured from the time of inoculation until day 120 (covering the entire process of post-ripening, color change, bud induction, and three flushes of fruiting).

[0069] Table 5. Background activity of extracellular lysinases throughout the entire growth cycle of pollution-free shiitake mushroom logs. Culture time / d Reproductive stage β-1,3-glucanase / (U·g⁻¹) Calculated aqueous phase (U·mL⁻¹) Chitinase / (U·g⁻¹) Does it exceed the threshold of 3.0 U / mL? 0 colonization period 0.28 0.19 0.15 no 20 Spreading period 0.31 0.21 0.17 no 40 Color change period 0.35 0.23 0.19 no 60 Inducing budding / one tide 0.42 0.28 0.23 no 80 Second Tide 0.55 0.37 0.29 no 100 Three Tides 0.71 0.47 0.36 no 120 Mushroom substrate decline period 0.88 0.59 0.44 no The results showed that as the mycelium entered the decline phase, autolysis of the shiitake mushroom mycelium intensified, and its extracellular β-1,3-glucanase activity slowly increased from 0.28 U·g⁻¹ to 0.88 U·g⁻¹, equivalent to 0.59 U·mL⁻¹ in aqueous phase. Therefore, the microcapsules will not be accidentally opened by the host's own enzyme activity throughout the entire growth cycle of shiitake mushrooms. This result provides a basis for setting the concentration of the crude enzyme solution in group (b) of Effect Example 3, and proves that the difference in enzyme activity levels upon which this invention is based remains stable throughout the entire cultivation cycle.

[0070] Example 6: Time-series changes in the carbon-nitrogen ratio of the culture medium The total carbon and available nitrogen of the matrix are determined periodically according to GB 5009.5 and elemental analysis method, and the apparent carbon-nitrogen ratio is calculated.

[0071] Table 6. Changes in apparent carbon-nitrogen ratio of culture media in Example 1 and Comparative Example 1 over culture time. Culture time / d Example 1 Apparent C / N Comparative Example 1 Apparent C / N 0 37.2:1 25.1:1 10 35.8:1 24.6:1 20 32.4:1 24.0:1 30 27.1:1 23.5:1 40 21.3:1 22.9:1 50 20.6:1 22.4:1 In Example 1, the apparent carbon-nitrogen ratio smoothly decreased from 37.2:1 to 21.3:1 on day 40. The inflection point of the decrease (days 30-40) highly coincided with the starting point of the color change period (day 36). In Comparative Example 1, since the nitrogen source was all readily available nitrogen, the carbon-nitrogen ratio only slowly decreased due to the consumption of carbon source, and no effective nutrient mode switching signal was formed.

[0072] The above embodiments are only used to illustrate the technical ideas and features of the present invention, and are not intended to be unique or to limit the present invention. Those skilled in the art should understand that various changes or equivalent substitutions made to the present invention without departing from its scope are all within the scope of protection of the present invention.

Claims

1. A method for cultivating shiitake mushrooms containing parasitic enzyme-responsive antibacterial microcapsules, characterized in that, Includes the following steps: S1. Soybean meal protein powder was encapsulated with calcium alginate as a gel matrix and chitosan as a coating to obtain slow-release nitrogen source particles. S2. Using lipopeptides as the core material, calcium alginate as the inner wall material, and a composite of ketallan gum and chitin nanofibers as the outer wall material, the core material is double-coated. The ketallan gum is then incubated at 50℃~70℃ to form a low-stability gel, dried, and shaped to obtain a parasitic enzyme-responsive antibacterial microcapsule. The cumulative release rate of the core material after immersion in a buffer solution with β-1,3-glucanase activity of 5 U / mL at 25℃ for 6 h is ≥75%, and the cumulative release rate after immersion in a buffer solution with β-1,3-glucanase activity of 0.60 U / mL at 25℃ for 72 h is ≤8%. S3. Add the slow-release nitrogen source granules obtained in S1 and the microcapsules obtained in S2 to a matrix with sawdust as the carbon source and wheat bran as the readily available nitrogen source to obtain a culture medium. The nitrogen provided by the slow-release nitrogen source granules accounts for 30% to 45% of the total nitrogen in the culture medium, and the amount of microcapsules added is 0.15% to 0.40% of the dry weight of the culture medium. The initial carbon-nitrogen ratio of the culture medium is 30:1 to 40:1, and it decreases autonomously as the culture progresses, dropping to 20:1 to 24:1 on days 35 to 45 after inoculation. S4. Sterilize the culture medium under high pressure at a temperature not lower than 100℃ to transform the Cortex gum in the outer wall material from a low-stability gel to a heat-irreversible high-stability gel; after cooling, aseptically inoculate with shiitake mushroom spawn. S5. Segmented temperature-controlled culture: The culture temperature is gradually reduced from the colonization stage to the spread stage. During the color change stage, the day-night temperature difference is applied and diffused light is provided. S6. Gradient bud induction: The mushroom sticks are subjected to a step cooling treatment of no less than three levels, with a cooling rate of 1.0 to 3.0℃ / h between adjacent levels. After each level reaches the target temperature, the mushroom sticks are kept at a constant temperature for 4 to 10 hours. S7. Harvest the fruiting bodies.

2. The method for cultivating shiitake mushrooms according to claim 1, characterized in that, In S1, soybean meal protein powder is mixed with a 1.5%–2.5% sodium alginate aqueous solution at a mass ratio of 1:2–1:4, and then dropped into a 2%–4% calcium chloride solution to solidify for 20–40 minutes to obtain gel core particles. The gel core particles are then immersed in a 1.0%–2.0% chitosan acetate solution for coating 2–3 times, and dried at 40℃–45℃ each time. The resulting slow-release nitrogen source particles have a particle size of 0.8 mm–1.5 mm, and the degree of deacetylation of the chitosan coating layer is 85%–95%.

3. The method for cultivating shiitake mushrooms according to claim 1, characterized in that, In S2, the core material is composed of lipopeptides and chitosan oligosaccharides in a mass ratio of 1:2 to 1:4; the lipopeptides are a compound of itursin A, fenestrant and surfactant in a mass ratio of 3:2:

1.

4. The shiitake mushroom cultivation method according to claim 1, characterized in that, The microcapsules obtained by S2 have a particle size of 20μm to 80μm, a total capsule wall thickness of 2μm to 5μm, and a drug loading of 12% to 20%.

5. The method for cultivating shiitake mushrooms according to claim 1, characterized in that, In S2, chitin nanofibers account for 15% to 35% of the total mass of the outer wall material, with the remainder being keratin. After S4 sterilization, the gel strength of the outer membrane is ≥400g / cm², which is more than 100% higher than that before S4 sterilization.

6. The method for cultivating shiitake mushrooms according to claim 1, characterized in that, The chitin nanofibers in S2 have a diameter of 10nm–50nm and an aspect ratio ≥30. They are obtained from α-chitin derived from shrimp and crab shells through dilute acid hydrolysis and high-pressure homogenization. The weight-average molecular weight of the Kodelan gum is 5×10⁻⁶. 4 ~2×10 5 .

7. The method for cultivating shiitake mushrooms according to claim 1, characterized in that, The enzymatic initiation threshold for the outer membrane of the microcapsules obtained by S2 is: β-1,3-glucanase activity ≥3.0 U / mL.

8. The method for cultivating shiitake mushrooms according to claim 1, characterized in that, In S2, a low-stability gel is formed by incubation at 55℃~65℃ for 15min~30min, followed by spray drying. The inlet air temperature of the spray drying is 100℃~110℃ and the outlet air temperature is 55℃~60℃. In S3, the sawdust is broadleaf tree sawdust, and its mass ratio with corn cob, wheat bran and corn flour is 60:15:18:

5. The moisture content of the culture medium is 58%~62%, and the initial pH is 5.0~5.

4.

9. The method for cultivating shiitake mushrooms according to claim 1, characterized in that, S5 specifically includes: ① Establishment period, days 0-15, the culture temperature linearly decreases from 26℃ to 24℃ at a rate of ≤0.15℃ / d, and the environmental CO2 concentration is 2500ppm-4000ppm; ② Spreading period, days 16-35, the culture temperature decreases by 1℃ every 7 days from 24℃ to 21℃, and the environmental CO2 concentration is 1500ppm-2000ppm; ③ Color change period, days 36-55, the daytime temperature is 22℃, the nighttime temperature decreases daily from 16℃ to a diurnal temperature range of 10℃, during which 50lx-200lx diffused light is provided, and the moisture content of the substrate decreases from 60% to 55%.

10. The method for cultivating shiitake mushrooms according to claim 1, characterized in that, In S6, when the color-changing area on the surface of the mushroom log accounts for ≥85% of the total surface area of ​​the mushroom log, the bud induction is initiated. The step-down cooling is in four stages, with target temperatures of 21℃, 17℃, 13℃, and 9℃ respectively. The cooling rate between adjacent stages is 1.5~2.5℃ / h, and each stage is kept at a constant temperature for 6h~8h. During the bud induction period, the ambient CO2 concentration is ≤800ppm, the relative humidity is 90%~95%, and the moisture content of the mushroom logs rises to 62%±2%. After the bud induction is completed, the temperature is raised to 15℃~18℃ for constant mushroom cultivation. In S7, the mushrooms are harvested in two grades according to the cap diameter: 4cm~6cm and 6cm~8cm.

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