Method for regenerating red pine mushroom cultivation substrate by earthworm-diptera hybrid conversion

Abstract

The application relates to the technical field of mixtures of fertilizers and additive components without special fertilizer effect, and particularly relates to a regeneration method of earthworm-black soldier fly-collaborative conversion red pine mushroom cultivation substrate. The method comprises the following steps: constructing a substrate precursor; adding a composite prebiotic in the bionic pre-conversion of earthworms; combining light cycle, magnetic field regulation and quorum quenching agent in the directional enrichment conversion of black soldier fly; preparing signal molecule microcapsules; preparing black soldier fly sand extract; spraying the sand extract and quorum activator in sequence in the microenvironment reconstruction fermentation, and carrying out low-temperature plasma surface treatment; bionic pheromone secondary induction and maturation; gradient water replenishment. Through the time sequence coupling conversion of earthworms and black soldier fly, the bidirectional intervention of microbial quorum sensing and the time and space controllable release of signal molecules, the application constructs a substrate with bionic microporous structure and sustained induction activity, significantly improves the mycelium planting speed, the fruiting body yield and the pine mushroom alcohol content, reduces the infection rate of miscellaneous bacteria, and realizes the high-value cyclic utilization of agricultural waste.

Description

Technical Field

[0001] In the field of fertilizer and additive components without special fertilizer effects, specifically involving a method for regenerating red matsutake cultivation substrate through earthworm-black soldier fly synergistic transformation. Background Technology

[0002] As a rare edible and medicinal fungus, the cultivation of red matsutake (Agaricus blazei) requires extremely stringent conditions regarding the physicochemical properties of the substrate, the structure of the microbial community, and the signaling molecule environment. Existing technologies (such as CN114208591A) mainly rely on simple composting and fermentation of raw materials such as sawdust, rice husks, and corn cobs to prepare the substrate. While this can achieve a certain yield, it suffers from problems such as incomplete degradation of lignocellulose, lack of biomimetic microporous channels, residues of harmful metabolites (organic acids, ammonia, VOCs) during fermentation, and a lack of signaling molecules that can continuously induce mycelial growth in a wild-like manner and the accumulation of secondary metabolites (such as matsutake alcohol). These issues lead to slow mycelial colonization, unstable yields, and difficulty in improving quality. Although some studies have attempted to introduce earthworms or black soldier flies to treat agricultural waste, these have only been used as fertilizer or feed, and their temporal coupling has never been applied to substrate preparation. Furthermore, interdisciplinary technologies such as microbial quorum sensing regulation, controlled release of signaling molecules, and plasma purification have not been used to systematically reconstruct the wild-like microenvironment of red matsutake. Therefore, it is urgent to solve the technical problems of slow mycelial colonization, low yield, poor quality and high infection rate of contaminants in the existing semi-wild cultivation substrate for red matsutake mushrooms, which lack earthworm-black soldier fly temporal coupling transformation, microbial quorum sensing directional regulation and spatiotemporal controllable release of signal molecules. Summary of the Invention

[0003] The technical problem to be solved by this invention is to address the issue that existing semi-wild cultivation substrates for red matsutake mushrooms suffer from slow mycelial colonization, low yield, poor quality, and high contamination rate due to the lack of earthworm-black soldier fly temporal coupling transformation, microbial quorum sensing directional regulation, and spatiotemporally controllable release of signal molecules.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for regenerating red matsutake cultivation substrate through earthworm-black soldier fly synergistic transformation includes the following steps: Step 1: Preparing the substrate: Mix the waste residue from red matsutake mushrooms, rice husks, corn cobs, and sawdust, adjust the pH to 7.8-8.2 with quicklime water, and adjust the moisture content to 58%; Step 2: Earthworm biomimetic pre-conversion: Spread the material obtained in Step 1 evenly in the earthworm bed, inoculate Eisenia fetus, and add compound prebiotics. After conversion, earthworm castings substrate A is obtained; the compound prebiotics are composed of inulin, pectin, and chitosan oligosaccharide mixed in a weight ratio of 2:1:1. Step 3: Targeted enrichment and transformation of black soldier fly larvae: Mix substrate A with rice husks and corn cob powder and adjust the moisture content to 70%. Spread the mixture evenly in the breeding tank. Under the conditions of a photoperiod of 16h light / 8h darkness and a magnetic field strength of 0.5-0.8mT, inoculate black soldier fly larvae. During the transformation process, spray the quorum induction quencher furanone derivative C30. After transformation, substrate B is obtained, and insect sand is collected. Step 4: Preparation of signal molecule microcapsules: Using the sharp-pore-coagulation bath method, sodium alginate was used as the core material and chitosan as the wall material to encapsulate signal molecules composed of chitosan oligosaccharide, salicylic acid, and methyl jasmonate to obtain microcapsules with an encapsulation rate of ≥85%. Step 5: Preparation of black soldier fly sand extract: Soak the sand collected in step 3 in water and filter to obtain the sand extract; Step 6: Microenvironment reconstruction fermentation: Mix substrate B with nutrient soil, yeast extract, quicklime and microcapsules prepared in step 4 and build a pile. When turning the pile, spray the insect sand extract prepared in step 5 and N-acylhomoserine lactone (C6-HSL) quorum sensing activator with a concentration of 10 nM in sequence, and perform low-temperature plasma surface treatment on the pile. After fermentation, substrate C is obtained. Step 7: Secondary induction and maturation of biomimetic pheromones: Spray chitosan oligosaccharide solution into substrate C, and obtain the final cultivation substrate after maturation; Step 8: Gradual water replenishment: Water is replenished three times during the fermentation process in step 6 to control the final moisture content to 66%-68%.

[0005] Furthermore, in the above-mentioned earthworm-black soldier fly-co-transformation method for regenerating red matsutake cultivation substrate, in step 1, the raw material weight ratio is: 200-250 parts waste mushroom residue, 300-350 parts rice husk, 80-120 parts corn cob, and 150-200 parts sawdust.

[0006] Furthermore, in the above-mentioned earthworm-black soldier fly synergistic transformation method for regenerating red matsutake cultivation substrate, the earthworm inoculation density in step 2 is 1.8-2.2 kg / m³. 2 The conversion time is 18-22 days, the temperature is 20-24℃, the humidity is 70%-75%, and the amount of compound prebiotics added is 0.5%-1% of the total weight of the material.

[0007] Furthermore, in the above-mentioned earthworm-black soldier fly-co-transformation method for regenerating the cultivation substrate of red matsutake mushrooms, in step 3, the weight mixing ratio of substrate A with rice husk and corn cob powder is 5:3:2; the black soldier fly larvae are 3rd instar larvae, the inoculation density is 3.5-4.0 kg / m2, the transformation time is 10-12 days, the temperature is 28-30℃, and the humidity is 70%-75%; the concentration of the quorum sensing quencher furanone derivative C30 is 10 μM, the spraying amount is 500 mL per square meter, and it is sprayed once every 3 days.

[0008] Furthermore, in the above-mentioned earthworm-black soldier fly-co-transformation method for regenerating red matsutake cultivation substrate, the signal molecule mentioned in step 4 is composed of chitosan oligosaccharide, salicylic acid, and methyl jasmonate in a weight ratio of 10:1:1; the microcapsule preparation method is as follows: the signal molecule is mixed with sodium alginate solution, dripped into calcium chloride solution for solidification, then transferred into chitosan solution for encapsulation, and dried to obtain the microcapsule.

[0009] Furthermore, in the above-mentioned earthworm-black soldier fly-co-transformation method for regenerating the culture substrate of red matsutake mushroom, in step 5, the ratio of insect sand to water is 1:10 w / v, the soaking time is 24 h, and the insect sand extract is obtained by filtration.

[0010] Furthermore, in the above-mentioned earthworm-black soldier fly-co-transformation method for regenerating the cultivation substrate of red matsutake mushrooms, the weight ratio of substrate B, nutrient soil, yeast extract, and quicklime in step 6 is as follows: substrate B 500-600 parts, nutrient soil 400-500 parts, yeast extract 3-5 parts, and quicklime 30-35 parts; the amount of microcapsules added is 0.15% of the total dry matter; the fermentation pile is 1.2m high, 1.2m wide at the bottom, 0.8m wide at the top, and 2.9m long; for the first 3 days, the temperature is naturally raised to 58℃ and maintained for 24 hours; from the 4th to the 8th day, the pile is turned over every 48 hours, and after each turning, insect sand extract (30 L / ton of material) and 10 nM C6-HSL solution (10 L / ton of material) are sprayed; from the 9th to the 12th day, the pile is turned over every 48 hours, and after each turning, low-temperature plasma surface treatment is performed (power 50W, treatment per square meter for 10 minutes); from the 13th to the 15th day, the temperature is lowered to 28℃.

[0011] Furthermore, in the above-mentioned earthworm-black soldier fly-co-transformation method for regenerating red matsutake cultivation substrate, the concentration of chitosan oligosaccharide solution in step 7 is 0.05 g / L, the spraying amount is 20 L / ton of material; the maturation time is 3-5 days, the temperature is 20-25℃, and the relative humidity is 65%.

[0012] Furthermore, in the above-mentioned earthworm-black soldier fly-co-transformation method for regenerating red matsutake cultivation substrate, water is added three times in step 8, on the 4th, 8th and 12th days of fermentation, with each addition of water amounting to 100 kg / pile, resulting in a final moisture content of 66%-68%.

[0013] The beneficial effects of this invention are as follows: By temporally coupling earthworm gut microbiota enhancement (prebiotic-directed regulation), black soldier fly multi-physical field induction (photomagnetic synergy), and quorum sensing bidirectional intervention (quenching and activation), and combining it with signal molecule microcapsule controlled release and low-temperature plasma surface treatment, a four-in-one red matsutake mushroom semi-wild substrate regeneration system of "biopore-forming—metabolic enrichment—signal sustained release—microenvironment purification" is constructed for the first time. Comparative data show that the absence of any single key technical feature (such as prebiotics, photomagnetic induction, C6-HSL activation, or microcapsule encapsulation) leads to a prolongation of mycelial full-bag time by more than 2.0 days, a decrease in yield of more than 10%, and a reduction in matsutake alcohol content of more than 15%. In particular, the absence of microcapsules results in a yield reduction of up to 24.8% and a sharp drop in matsutake alcohol content of 37.5%. However, when all features are used simultaneously, mycelial full-bag time is only 18 days, and the yield reaches 11.3 kg / m³. 2 The matsutake alcohol content reached 0.72 mg / g, and the contamination rate of miscellaneous bacteria was only 1.0%, far superior to the comparative ratios and existing technologies. The aforementioned synergistic effect is not a simple summation of individual characteristic effects. For example, when prebiotics and C6-HSL activation are individually absent, matsutake alcohol content decreases by 25.0% and 23.6%, respectively. However, when both are present, matsutake alcohol content increases by 3.8 times compared to the simulated combination (hypothetical multiplication) when both are absent, proving that the earthworm-black soldier fly-microorganism three-level signaling network produces an unpredictable synergistic effect. This invention breaks through the technical prejudice of "improving substrate quality solely through composting fermentation," and integrates insect transformation, microbial quorum sensing engineering, and controlled release technology, realizing a paradigm shift from "passive fermentation" to "active programming." Its comprehensive performance far exceeds the conventional expectations of those skilled in the art. Detailed Implementation

[0014] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments.

[0015] Example 1 A method for regenerating red matsutake cultivation substrate through earthworm-black soldier fly synergistic transformation includes the following steps: Step 1: Base Material Precursor Construction: Take 220 kg of waste mushroom residue from the harvest of red matsutake mushrooms, 330 kg of rice husks, 100 kg of corn cobs, and 180 kg of 10-12 mm sawdust, and put them into a horizontal twin-shaft mixer (2 m³ volume). 3 Stir at 60 r / min for 15 min to ensure uniform mixing of the materials.

[0016] To prepare a 2.5% quicklime solution: Weigh 2.5 kg of quicklime (calcium oxide content ≥90%), slowly add it to 97.5 kg of tap water while stirring, continue stirring for 5 min, let stand for 30 min, and use the supernatant.

[0017] While stirring, evenly spray quicklime water onto the mixture. Use a portable pH meter (probe inserted 10 cm deep into the material) to measure and adjust the pH to 8.0. Take a sample of approximately 500 g and dry it in a 105℃ oven to constant weight. Calculate the current moisture content as 52%. Calculate the amount of pure water to be added using the following formula: The target moisture content is 58%, and the total weight of the material is 220 + 330 + 100 + 180 + 2.5 + 97.5 = 930 kg. Current water volume = 930 × 0.52 = 483.6 kg; Target water volume = 930 × 0.58 = 539.4 kg; The required amount of pure water to be added is 539.4 - 483.6 = 55.8 kg. 55.8 kg of pure water was evenly sprayed into the material, and stirring was continued for 10 min. The moisture content was retested and found to be 58.2%, and the pH was 8.0. The total mass of the base material precursor was approximately 985.8 kg.

[0018] Step 2: Earthworm biomimetic pre-conversion (combined with gut microbiota enhancement); Construct an earthworm breeding bed (5 m long, 2 m wide, and 0.3 m high), with a waterproof tarpaulin and a drainage layer (5 cm thick gravel) laid at the bottom. Spread all the materials obtained in step 1 (985.8 kg) evenly in the breeding bed to a thickness of 24 cm.

[0019] Eisenia fetida (purchased from Jiangsu Academy of Agricultural Sciences, adult earthworms 5-8 cm in length, fresh weight 0.3-0.5 g / ear) was inoculated at a density of 2.0 kg / m². 2 (Based on a breeding bed area of ​​10 m²) 2 The total inoculation amount was 20 kg (fresh weight). Earthworms were evenly scattered on the surface of the material, and they burrowed in on their own after about 30 minutes.

[0020] The compound prebiotic is prepared by uniformly mixing inulin (degree of polymerization ≥10), pectin (degree of esterification ≥70%), and chitosan oligosaccharide (degree of polymerization 3-5, degree of deacetylation 90%) in a weight ratio of 2:1:1. The total weight of the material is 985.8 kg. Based on an addition rate of 0.7%, 6.9 kg of prebiotic is required. Take 6.9 kg of prebiotic and mix it with 70 kg of material (taken from the earthworm bed) beforehand. Then, evenly spread it back onto the surface of the earthworm bed and gently turn it over to a depth of 5 cm with an iron rake to ensure even distribution of the prebiotic.

[0021] Breeding conditions: Ambient temperature 22±1℃, relative humidity 72±2%. Lightly turn the soil every 7 days with an iron rake (turning to a depth of 15 cm to avoid damaging the earthworms). After 20 days of conversion, stop feeding and let it stand for 2 days to allow the earthworms to naturally sink to the bottom. Carefully scrape off the top layer of vermicompost with a small shovel, and sieve through a 1 cm sieve to remove any remaining earthworms and large undigested pieces. Earthworm casting substrate A is obtained, weighing 680 kg. Sampling and testing: Moisture content: Take 50 g of sample, dry at 105℃ for 24 h, weigh and calculate the moisture content to be 63%.

[0022] The surface roughness Ra was measured to be 55 ± 5 nm using an atomic force microscope (Bruker Multimode 8, scanning range 10 μm × 10 μm).

[0023] The surface zeta potential was measured to be -25 mV using a zeta potential analyzer (Malvern Zetasizer Nano ZS, pH 7.0, 0.1 mM KCl dispersion).

[0024] Step 3: Targeted enrichment and transformation of black soldier fly larvae; Take 500 kg of earthworm casting substrate A obtained in step 2 (actual moisture content 63%), add 150 kg of fresh rice husks (moisture content 12%) and 100 kg of corn cob powder (passed through a 40-mesh sieve, moisture content 10%), and place them into a twin-shaft mixer (volume 1.5 m³). 3 Stir at 50 r / min for 15 min. While stirring, add pure water and monitor the moisture content in real time using a halogen moisture analyzer, adjusting to 70%.

[0025] Calculate the required water volume: Dry matter mass of the mixture = 500×(1-0.63)+150×(1-0.12)+100×(1-0.10)=500×0.37+150×0.88+100×0.90=185+132+90=407 kg; Initial water mass of the mixture = 500 × 0.63 + 150 × 0.12 + 100 × 0.10 = 315 + 18 + 10 = 343 kg; Target total mass = 407 / (1 - 0.70) = 1356.7 kg; The required amount of pure water is 1356.7 - 500 - 150 - 100 = 606.7 kg. 607 kg of water was added, and after thorough mixing, a mixture of 1357 kg was obtained, with a measured moisture content of 70.1%. This material was used in three aquaculture tanks (each with an area of ​​2 m²). 2 ).

[0026] The black soldier fly larvae rearing tanks are made of plastic, each measuring 2 m long, 1 m wide, and 0.15 m deep. The bottom is lined with permanent magnets (neodymium magnets, 50 mm × 50 mm × 10 mm, with a surface magnetic field strength of 0.8 mT, and a spacing of 10 cm between magnets, covering the entire bottom of the tank). Each tank is filled with 452.3 kg (1357 ÷ 3 ≈ 452.3 kg) of the aforementioned mixture, spread evenly to a thickness of approximately 11 cm.

[0027] Third-instar black soldier fly larvae (Hermetia illucens, purchased from Guangdong Institute of Entomology, average body length 1.2 cm, average weight 0.05 g / larva) were inoculated at a density of 3.8 kg / m². 2 (Based on an area of ​​2 m² per tank) 2 Each tank is inoculated with 7.6 kg of larvae. The larvae are evenly scattered on the surface of the material, and they burrow into it on their own.

[0028] An LED light strip (color temperature 6500 K, illuminance 2000 Lux) is installed 50 cm directly above each aquaculture tank, with a timer switch set: 16 hours of light (06:00-22:00) and 8 hours of darkness (22:00-06:00). A permanent magnet at the bottom generates a magnetic field of 0.8 mT in the central area of ​​the tank and 0.5 mT at the edge.

[0029] Preparation of a 10 μM furanone derivative C30 solution: Weigh 1.8 mg of furanone C30 (CAS 110812-32-9, purity ≥98%, molecular weight approximately 180), dissolve it in 2 mL of anhydrous ethanol, then add deionized water containing 0.1% Tween 80 to a final volume of 1 L and mix well. The concentration of this solution is 10 μmol / L.

[0030] Spray once every 3 days, with a dosage of 500 mL per square meter of tank area each time, that is, 1000 mL per breeding tank. Use a handheld sprayer (nozzle diameter 0.5 mm) to spray evenly on the surface of the material without stirring.

[0031] The temperature in the rearing room was controlled at 29±1℃, and the relative humidity at 72±2%. Feeding was stopped after 11 days of conversion. A photoseparation method was used: the surface of the tank was irradiated with 5000 Lux light for 30 minutes. Black soldier fly larvae, avoiding the light, burrowed to the bottom of the tank, and the upper layer of larval excrement was collected with a shovel. Approximately 700 kg of substrate B was obtained. 50 kg of this was taken out and collected separately as insect excrement, sealed and stored in a 4℃ cold storage for use in the preparation of the insect excrement extract in subsequent steps. The remaining 650 kg of substrate B was used in step 4.

[0032] Step 4: Preparation of microcapsules for spatiotemporally controlled release of signal molecules (sharp-pore coagulation bath method, encapsulation rate ≥85%). Weigh 200 g of chitosan oligosaccharide (degree of polymerization 3-5, degree of deacetylation 90%), 20 g of salicylic acid (analytical grade), and 20 g of methyl jasmonic acid (purity ≥95%), add them to 8 L of deionized water, and stir magnetically in a 50℃ water bath (300 r / min) until completely dissolved. Filter through a 0.22 μm filter membrane for sterilization, and add deionized water to a total volume of 10 L to obtain the signal molecule stock solution (chitosan oligosaccharide concentration 20 g / L, salicylic acid 2 g / L, methyl jasmonic acid 2 g / L).

[0033] Weigh 200 g of sodium alginate (viscosity 200-400 mPa·s) and add it to 10 L of deionized water. Stir magnetically for 3 h at room temperature until completely dissolved to obtain a 2% (w / v) sodium alginate solution. Mix 10 L of the above signal molecule stock solution with 10 L of the 2% sodium alginate solution, add 20 g of Tween 80, and stir for 30 min. Obtain 20 L of the mixture, in which the sodium alginate concentration is 1%, and the signal molecule concentrations are: chitosan oligosaccharide 10 g / L, salicylic acid 1 g / L, and methyl jasmonic acid 1 g / L.

[0034] A high-voltage electrostatic droplet generator (Beijing Huiyu, HGY-Ⅲ type) was used, with a voltage of 10 kV, a propulsion speed of 200 mL / h, and a needle inner diameter of 0.4 mm. The receiving solution was a 4% (w / v) calcium chloride solution (400 g of calcium chloride dissolved in 10 L of deionized water, with 10 g of Tween 80 added), with a receiving solution volume of 20 L. The solution was placed on a magnetic stirrer and stirred at 150 r / min, with the receiving solution temperature controlled at 15℃. The needle was positioned 10 cm above the receiving solution surface. The mixture was simultaneously dripped into the calcium chloride solution through 20 parallel needles (3 cm apart) and allowed to solidify for 40 min.

[0035] The gel beads were collected using a stainless steel sieve with a pore size of 300 μm and transferred to a 1% (w / v) chitosan solution (100 g of chitosan dissolved in 10 L of 1% acetic acid, pH adjusted to 5.5 with 1 M NaOH). The solution was stirred at 100 r / min for 40 min at room temperature to coat the beads. The beads were then collected again and rinsed three times with deionized water (20 L each time, soaking for 10 min).

[0036] The rinsed microcapsules were spread evenly on a stainless steel tray and dried in a 40°C hot air drying oven for 48 hours until constant weight was obtained. Approximately 1.85 kg of dry microcapsules were obtained.

[0037] Take the same volume of deionized water (10 L) as in step 2 to replace the signal molecule stock solution, mix it with 10 L of 2% sodium alginate solution, add 20 g of Tween 80, and stir for 30 min. The remaining operations are the same to obtain blank microcapsules without signal molecules.

[0038] 100 mg of dried microcapsules were ground and crushed in a mortar and pestle, then 20 mL of PBS buffer (pH 7.4) was added. The mixture was extracted by sonication for 30 min (40 kHz), centrifuged (12000 r / min, 15 min), and the supernatant was collected. The total sugar content was determined using the phenol-sulfuric acid method, and a standard curve was plotted using chitosan oligosaccharide (from the same batch) as a standard. Simultaneously, 100 mg of blank microcapsules were treated in the same manner, and background was subtracted. The calculated mass of chitosan oligosaccharide was 9.6 mg.

[0039] Theoretical chitosan oligosaccharide content: 200 g of chitosan oligosaccharide is contained in 20 L of the mixture, yielding 1850 g of dry microcapsules. The theoretical chitosan oligosaccharide mass fraction = 200 / 1850 = 10.81%. 100 mg of microcapsules contains 10.81 mg of theoretical chitosan oligosaccharide. Encapsulation efficiency = 9.6 / 10.81 = 88.8%, meeting the requirement of ≥85%.

[0040] The microcapsules are sealed in aluminum foil bags and refrigerated at 4°C for later use.

[0041] Step 5: Preparation of black soldier fly sand extract; According to the subsequent fermentation requirements: the pile is approximately 1.14 tons, and each spraying requires 30 L / ton of insect sand extract, totaling 34.2 L each time, for a total of 3 sprayings, requiring a total of 102.6 L. Take 10.3 kg of insect sand, add 103 L of deionized water, and soak at 20-25℃ for 24 hours, stirring once every 6 hours. Filter through a 200-mesh filter cloth, collect the filtrate, and obtain approximately 100-102 L of black soldier fly larvae sand extract. This extract should be prepared within 24 hours before use and stored at 4℃ for later use.

[0042] Step 6: Microenvironment reconstruction fermentation; Base material B (remaining 650 kg, moisture content 64%), dry matter = 650 × 0.36 = 234 kg. Nutrient soil (humus, sieved through a 5 mm sieve) 450 kg, moisture content 30%, dry matter = 450 × 0.7 = 315 kg. Yeast extract 4 kg, moisture content approximately 30%, dry matter ≈ 2.8 kg. Quicklime 32 kg (almost anhydrous). Signal molecule microcapsules (obtained in step 4) are added at 0.15% of the total dry matter: Total dry matter = 234 + 315 + 2.8 + 32 = 583.8 kg, 0.15% is 0.876 kg (876 g). Take 876 g of microcapsules.

[0043] Add all materials to a horizontal mixer and mix at 40 r / min for 30 min until homogeneous. The total weight of the mixed materials = 650 + 450 + 4 + 32 + 0.876 = 1136.9 kg. Take a sample to determine the moisture content: take 50 g, dry at 105℃, and calculate the moisture content as 61.8%.

[0044] Based on a total material volume of approximately 1.137 tons and a compost density of approximately 500 kg / m³, the designed pile dimensions are: bottom width 1.2 m, top width 0.8 m, height 0.8 m, length = volume / cross-sectional area, and cross-sectional area = (1.2 + 0.8) / 2 × 0.8 = 0.8 m. 2 The volume is 1137 / 500 = 2.274 m³, and the length is approximately 2.274 / 0.8 ≈ 2.84 m, so we take 2.9 m. The mixture is piled into a windrow with a base width of 1.2 m, a top width of 0.8 m, a height of 0.8 m, and a length of 2.9 m. The surface of the windrow is covered with a breathable layer of straw (5 cm thick).

[0045] Fermentation process control: Days 1-3: Natural temperature rise. Temperature was measured daily. On day 2, the core temperature reached 58°C and was maintained for 24 hours. No reactor turnover was performed.

[0046] Days 4-8: Turn the pile every 48 hours (days 4, 6, and 8). After each turning, spray in sequence: ① Black soldier fly sand extract (30 L per ton, 1.137 ton pile, 34.1 L each time); ② Quorum sensing activator – 10 nM N-acylhomoserine lactone (C6-HSL) solution (10 L per ton, 11.4 L each time). Turn the pile after spraying. Preparation method of 10 nM C6-HSL working solution: Purchase commercial C6-HSL standard (Sigma-Aldrich, catalog number K3007, 1 mM in DMSO). Before each spraying, take 114 μL of 1 mM standard, add 11.4 L of deionized water (containing 0.01% Tween 80), mix well, and you will get 11.4 L of 10 nM working solution. Calculation formula: 1 mM × (114 × 10 -6 L) / 11.4 L = 1×10 -5 mM = 10 nM.

[0047] Days 9-12: Continue turning the pile every 48 hours (days 10 and 12). After each turning, perform low-temperature plasma surface treatment (remove the straw layer first). Parameters: Handheld low-temperature plasma spray gun (Nanjing Suman Electronics, CTK-1200 model, operating frequency 22 kHz, output power 50 W), nozzle distance 5 cm from the pile surface, scanning speed 10 cm / s, treatment time 10 min per square meter. Pile surface area approximately 5.57 m². 2 (Calculation: Top 2.9 × 0.8 = 2.32, Side slopes 2 × [(1.2 + 0.8) / 2 × 0.8246] = 1.6492, Ends 2 × [(1.2 + 0.8) / 2 × 0.8] = 1.6), Total 5.5692 m 2Each treatment lasted approximately 56 minutes. After treatment, the straw layer was re-covered. A total of two treatments were performed (once on day 10 and once on day 12).

[0048] Days 13-15: Reduce the stack height to 0.5 m and allow for natural cooling. By the end of day 15, the core temperature had dropped to 28°C.

[0049] Fermentation was terminated, yielding base material C, weighing approximately 1010 kg.

[0050] Step 7: Secondary induction and maturation of biomimetic pheromones; Preparation of chitosan oligosaccharide solution: For approximately 1.01 tons of base material C, 20.2 L of water is required for spraying at a rate of 20 L per ton. The chitosan oligosaccharide concentration is 0.05 g / L. Weigh out 1.01 g of chitosan oligosaccharide (degree of polymerization 3-5) and dissolve it in 20.2 L of deionized water. Spray the solution evenly onto the surface of base material C using a sprayer and stir.

[0051] Transfer to a curing warehouse (20-25℃, relative humidity 65%), pile into a long ridge 1.5 m wide and 0.5 m high, and cover with damp burlap sacks (pre-soaked in pure water and wrung out, moisture content approximately 60%). Curry for 4 days, turning once a day.

[0052] The final cultivation substrate, weighing approximately 980 kg, was obtained after maturation. Sampling and testing revealed the following: moisture content (105℃ drying method) 67%, porosity (specific gravity bottle method) 65%, Zeta potential -28.5 mV, soluble chitosan oligosaccharide content (HPLC method) 28.2 mg / kg, and total VOCs (GC-MS method) 32 μg / kg.

[0053] Step 8: Gradual hydration; During the fermentation process in step 6 (days 4, 8, and 12), water was added three times. Each time, 100 kg of pure water was added and sprayed evenly onto the surface of the pile while turning it over. After the third water addition (day 12), the measured moisture content was 67.1%. After the maturation was completed (day 19), the measured moisture content was 67.0%.

[0054] Comparative Example 1 The scheme in Example 1 is the same as in Example 1 except that no compound prebiotics are added during earthworm conversion in step 2.

[0055] Comparative Example 2 The difference from the scheme in Example 1 is that no light cycle control and bottom magnetic field are applied during the black soldier fly conversion in step 3.

[0056] Comparative Example 3 The scheme of Example 1 is the same as that of Example 1, except that furanone C30, the quorum induction quencher, is not sprayed during the black soldier fly conversion in step 3.

[0057] Comparative Example 4 The scheme of Example 1 is different in that, instead of preparing microcapsules in step 4, equal amounts of signal molecules (chitosan oligosaccharide, salicylic acid, and methyl jasmonate mixed directly in a 10:1:1 ratio) are added directly during the mixing process in step 6. The rest is the same as in Example 1.

[0058] Comparative Example 5 The scheme of Example 1 is the same as that of Example 1, except that the insect sand extract is not prepared in step 5 and the insect sand extract is not sprayed during fermentation and turning in step 6.

[0059] Comparative Example 6 The scheme of Example 1 is the same as that of Example 1, except that the quorum sensing activator C6-HSL is not sprayed during fermentation in step 6.

[0060] Comparative Example 7 The scheme of Example 1 is the same as that of Example 1, except that the low-temperature plasma surface treatment is not performed during fermentation in step 6.

[0061] Comparative Example 8 The scheme of Example 1 is different in that step 7 does not involve secondary induction of biomimetic pheromones, i.e., the chitosan oligosaccharide solution is not sprayed. Otherwise, it is the same as Example 1.

[0062] Experimental Design: Each experiment (Example 1 and 8 comparative examples) was conducted independently in 3 batches. For each batch, 100 bags were prepared and cultivated using the method described above. Within each batch, the time for mycelium to fully cover the bag was taken as the standard, and the average value of 100 bags was recorded. Yield was calculated by dividing the total yield (kg) of each batch by the fruiting area (100 bags × 0.07 m²). 2 / bag=7 m 2 The matsutake alcohol content was calculated by randomly selecting 5 bags of fruiting bodies, taking 3 representative fruiting bodies from each bag, combining them, drying and pulverizing them, and then determining them by HPLC (C18 column, mobile phase methanol:water = 55:45, detection wavelength 254 nm); the contamination rate of miscellaneous bacteria was calculated as the percentage of bags with visible contamination such as Penicillium and Trichoderma that resulted in total crop failure. The final data is the average of 3 batches.

[0063] The experimental results are shown in Table 1: Table 1 Results analysis: Comparative Example 1 compared to Example 1; Time for mycelium to fully fill the bag: increased by 3.5 days (+19.4%); Yield per unit area: decreased by 2.1 kg / m³. 2(-18.6%); Matsutake alcohol content: decreased by 0.18 mg / g (-25.0%); Contamination rate of miscellaneous bacteria: increased by 1.8 percentage points (+180%). Analysis: Prebiotics, by regulating the gut microbiota of earthworms, increased the activity of cellulase and chitinase in vermicompost, enhanced the pre-degradation of lignocellulose in the substrate, and provided a better substrate for subsequent black soldier fly larvae conversion. Without these prebiotics, the quality of vermicompost decreased, ultimately hindering mycelial growth, resulting in a significant decline in yield and quality, and an increase in contaminating bacteria.

[0064] Comparative Example 2 compared to Example 1; Time to full mycelial coverage: increased by 2.0 days (+11.1%); Yield per unit area: decreased by 1.5 kg / m³. 2 (-13.3%); Matsutake alcohol content: decreased by 0.14 mg / g (-19.4%); Contamination rate of miscellaneous bacteria: increased by 1.0 percentage point (+100%). Analysis: Photoperiod and magnetic fields can induce black soldier fly larvae to secrete more antimicrobial peptides and chitinases, thereby enhancing the bioactivity of the insect sand. Their absence reduces the effective components in the insect sand, affecting subsequent fermentation and mycelial growth.

[0065] Comparative Example 3 compared to Example 1; Time for mycelium to fully fill the bag: increased by 1.5 days (+8.3%); Yield per unit area: decreased by 1.2 kg / m³. 2 (-10.6%); Matsutake alcohol content: decreased by 0.11 mg / g (-15.3%); Contamination rate of miscellaneous bacteria: increased by 2.5 percentage points (+250%). Analysis: The quencher inhibits quorum sensing of contaminating microorganisms and reduces their secretion of virulence factors. Without it, contaminating microorganisms (especially Penicillium and Trichoderma) proliferate more easily, leading to a significant increase in infection rates, which in turn affects mycelial colonization and yield.

[0066] Comparative Example 4 compared to Example 1; Time to full mycelial coverage: increased by 6.0 days (+33.3%); Yield per unit area: decreased by 2.8 kg / m³. 2 (-24.8%); Matsutake alcohol content: decreased by 0.27 mg / g (-37.5%); Contamination rate of miscellaneous bacteria: increased by 3.2 percentage points (+320%). Analysis: Microcapsules enable spatiotemporally controlled release of signaling molecules, protecting them from rapid degradation and ensuring continuous release during fermentation and cultivation. Direct addition results in significant inactivation of signaling molecules during high-temperature fermentation, preventing sustained mycelial induction and leading to a marked deterioration in various indicators. The comparative example showed the most significant decrease in performance, demonstrating that microcapsule encapsulation is a key technology.

[0067] Comparative Example 5 compared to Example 1; Time to full mycelial coverage: increased by 1.0 day (+5.6%); Yield per unit area: decreased by 0.8 kg / m³ 2 (-7.1%); Matsutake alcohol content: decreased by 0.08 mg / g (-11.1%); Contamination rate of miscellaneous bacteria: increased by 0.5 percentage points (+50%). Analysis: Chitosan oligosaccharides and antimicrobial peptides in the insect sand extract can induce beneficial microorganisms and inhibit harmful bacteria. Their absence slightly reduces the effectiveness, but has a smaller impact compared to other factors.

[0068] Comparative Example 6 compared to Example 1; Time for mycelium to fully fill the bag: increased by 2.5 days (+13.9%); Yield per unit area: decreased by 1.8 kg / m³. 2 (-15.9%); Matsutake alcohol content: decreased by 0.17 mg / g (-23.6%); Contamination rate of miscellaneous bacteria: increased by 1.2 percentage points (+120%). Analysis: C6-HSL activates quorum sensing in beneficial bacteria such as actinomycetes, promoting the secretion of antibiotics and chitinases. Its absence reduces the activity of beneficial bacteria, increases the competitiveness of other bacteria, and significantly decreases yield and quality.

[0069] Comparative Example 7 compared to Example 1; Time for mycelium to fully fill the bag: increased by 0.5 days (+2.8%); Yield per unit area: decreased by 0.5 kg / m³ 2 (-4.4%); Matsutake alcohol content: decreased by 0.04 mg / g (-5.6%); Contamination rate of miscellaneous bacteria: increased by 0.2 percentage points (+20%). Analysis: Low-temperature plasma can degrade trace amounts of VOCs accumulated on the surface of the substrate, optimizing the microenvironment. The impact of its absence is minimal, indicating that this feature primarily improves the environment and serves as a secondary optimization mechanism.

[0070] Comparative Example 8 compared to Example 1; Time to full mycelial coverage: increased by 0.5 days (+2.8%); Yield per unit area: decreased by 0.7 kg / m³. 2 (-6.2%); Matsutake alcohol content: decreased by 0.12 mg / g (-16.7%); Contamination rate of miscellaneous bacteria: increased by 0.1 percentage points (+10%). Analysis: Secondary induction with chitosan oligosaccharides provides additional signals, promoting mycelial stress resistance and the accumulation of secondary metabolites, especially affecting matsutake alcohol content. Matsutake alcohol content decreases significantly after deletion, but yield and bag filling time are less affected.

[0071] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for regenerating red matsutake mushroom cultivation substrate through earthworm-black soldier fly synergistic transformation, characterized in that, Includes the following steps: Step 1: Preparing the substrate: Mix the waste residue from red matsutake mushrooms, rice husks, corn cobs, and sawdust, adjust the pH to 7.8-8.2 with quicklime water, and adjust the moisture content to 58%; Step 2: Earthworm biomimetic pre-conversion: Spread the material obtained in Step 1 evenly in the earthworm bed, inoculate Eisenia fetus, and add compound prebiotics. After conversion, earthworm castings substrate A is obtained; the compound prebiotics are composed of inulin, pectin, and chitosan oligosaccharide mixed in a weight ratio of 2:1:

1. Step 3: Targeted enrichment and transformation of black soldier fly larvae: Mix substrate A with rice husks and corn cob powder and adjust the moisture content to 70%. Spread the mixture evenly in the breeding tank. Under the conditions of a photoperiod of 16h light / 8h darkness and a magnetic field strength of 0.5-0.8mT, inoculate black soldier fly larvae. During the transformation process, spray the quorum induction quencher furanone derivative C30. After transformation, substrate B is obtained, and insect sand is collected. Step 4: Preparation of signal molecule microcapsules: Using the sharp-pore-coagulation bath method, sodium alginate was used as the core material and chitosan as the wall material to encapsulate signal molecules composed of chitosan oligosaccharide, salicylic acid, and methyl jasmonate to obtain microcapsules with an encapsulation rate of ≥85%. Step 5: Preparation of black soldier fly sand extract: Soak the sand collected in step 3 in water and filter to obtain the sand extract; Step 6: Microenvironment reconstruction fermentation: Mix substrate B with nutrient soil, yeast extract, quicklime and microcapsules prepared in step 4 and build a pile. When turning the pile, spray the insect sand extract prepared in step 5 and N-acylhomoserine lactone (C6-HSL) quorum sensing activator with a concentration of 10 nM in sequence, and perform low-temperature plasma surface treatment on the pile. After fermentation, substrate C is obtained. Step 7: Secondary induction and maturation of biomimetic pheromones: Spray chitosan oligosaccharide solution into substrate C, and obtain the final cultivation substrate after maturation; Step 8: Gradual water replenishment: Water is replenished three times during the fermentation process in step 6 to control the final moisture content to 66%-68%.

2. The method for regenerating red matsutake cultivation substrate through earthworm-black soldier fly synergistic transformation according to claim 1, characterized in that, In step 1, the raw materials are proportioned as follows by weight: 200-250 parts waste mushroom residue, 300-350 parts rice husk, 80-120 parts corn cob, and 150-200 parts sawdust.

3. The method for regenerating red matsutake cultivation substrate through earthworm-black soldier fly synergistic transformation according to claim 1, characterized in that, In step 2, the earthworm inoculation density is 1.8-2.2 kg / m². 2 The conversion time is 18-22 days, the temperature is 20-24℃, the humidity is 70%-75%, and the amount of compound prebiotics added is 0.5%-1% of the total weight of the material.

4. The method for regenerating red matsutake cultivation substrate through earthworm-black soldier fly synergistic transformation according to claim 1, characterized in that, In step 3, the weight ratio of substrate A to rice husks and corn cob powder is 5:3:2; the black soldier fly larvae are third instar larvae, and the inoculation density is 3.5-4.0 kg / m². 2 The conversion time is 10-12 days, the temperature is 28-30℃, and the humidity is 70%-75%. The concentration of furanone derivative C30, the quorum sensing quencher, is 10 μM, the spraying amount is 500 mL per square meter, and it is sprayed once every 3 days.

5. The method for regenerating red matsutake cultivation substrate through earthworm-black soldier fly synergistic transformation according to claim 1, characterized in that, The signal molecule mentioned in step 4 is composed of chitosan oligosaccharide, salicylic acid, and methyl jasmonate in a weight ratio of 10:1:

1. The microcapsule is prepared by mixing the signal molecule with sodium alginate solution, adding it to calcium chloride solution for solidification, then transferring it into chitosan solution for encapsulation, and drying it.

6. The method for regenerating red matsutake cultivation substrate through earthworm-black soldier fly synergistic transformation according to claim 1, characterized in that, In step 5, the ratio of insect sand to water is 1:10 w / v, the soaking time is 24 h, and the insect sand extract is obtained by filtration.

7. The method for regenerating red matsutake cultivation substrate through earthworm-black soldier fly synergistic transformation according to claim 1, characterized in that: In step 6, the weight ratio of base material B, nutrient soil, yeast paste, and quicklime is as follows: base material B 500-600 parts, nutrient soil 400-500 parts, yeast paste 3-5 parts, and quicklime 30-35 parts; the amount of microcapsules added is 0.15% of the total dry matter; the fermentation pile is 1.2m high, 1.2m wide at the bottom, 0.8m wide at the top, and 2.9m long; for the first 3 days, the temperature is naturally raised to 58℃ and maintained for 24 hours; from the 4th to the 8th day, the pile is turned over every 48 hours, and after each turning, insect sand extract (30 L / ton of material) and 10 nM C6-HSL solution (10 L / ton of material) are sprayed; from the 9th to the 12th day, the pile is turned over every 48 hours, and after each turning, low-temperature plasma surface treatment is performed (power 50W, treatment per square meter for 10 minutes); from the 13th to the 15th day, the temperature is lowered to 28℃.

8. The method for regenerating red matsutake cultivation substrate through earthworm-black soldier fly synergistic transformation according to claim 1, characterized in that: In step 7, the concentration of the chitosan oligosaccharide solution is 0.05 g / L, and the spraying amount is 20 L / ton of material; the maturation time is 3-5 days, the temperature is 20-25℃, and the relative humidity is 65%.

9. The method for regenerating red matsutake cultivation substrate through earthworm-black soldier fly synergistic transformation according to claim 1, characterized in that: In step 8, water is added three times, on the 4th, 8th and 12th day of fermentation, with each addition being 100 kg / pile, resulting in a final moisture content of 66%-68%.

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