Preparation method of bottle cultivated species for tremella aurantialba planting

Abstract

The invention relates to the technical field of edible fungus strain production, in particular to a preparation method of a bottle cultivar for tremella aurantialba planting, which comprises the following steps: step 1, strain pretreatment; 2, preparing a composite culture medium; step 3, bottling and sterilizing; step 4, gradient inoculation; step 5, performing multi-stage intelligent culture; and step 6, quality detection and screening. Chestnut wood chips, mulberry branch chips and cottonseed hull composite carbon sources are matched with fermented soybean meal functional nitrogen sources, nutrition release is continuous and stable, and the requirement for synergistic growth of two strains is met; the added chitosan and astragalus polysaccharide can improve the activity of hyphae, and the honeysuckle extracting solution is high in pollution resistance.

Description

Technical Field

[0001] This invention relates to the field of edible fungi spawn production technology, specifically a method for preparing bottle-grown spawn for auricularia auricula cultivation. Background Technology

[0002] Golden ear fungus, also known as silver ear fungus or brain ear fungus, belongs to the Basidiomycota phylum and the Leucisceae family. Its fruiting body is rich in polysaccharides, proteins, amino acids, and various trace elements, possessing extremely high nutritional and medicinal value, and market demand continues to grow. The growth and development of golden ear fungus is unique; its mature fruiting body is a symbiotic complex formed by *Auricularia auricula-judae* and its associated *Leucisceae* species. The proportion and activity of these two species directly determine the success or failure of cultivation. Bottle cultivation, as the mainstream method for industrialized production of golden ear fungus, has advantages such as strong controllability, low contamination rate, and stable yield. However, current technologies for preparing bottle-grown golden ear fungus spawn still face many bottlenecks.

[0003] In existing technologies, the culture medium for auricularia auricula-judae cultivation often uses a single woody raw material (such as broadleaf sawdust or cottonseed hulls) combined with simple auxiliary materials. The nutrient composition is simple and the release efficiency is low, which makes it difficult to meet the synergistic growth requirements of Auricularia auricula-judae and Leuciscus fasciatus.

[0004] In terms of strain treatment, traditional methods often involve direct inoculation of Auricularia aurea and Leptomeria trifasciata, lacking targeted activation treatment and ratio control methods.

[0005] The lagging development of environmental control technology is also a key factor restricting the quality of cultivars. Current technologies mostly employ constant temperature (18–21℃) and light-protected cultivation, failing to adjust environmental parameters according to different stages of mycelial growth (colonization, expansion, and differentiation). For example, the DB61 / T1776-2023 standard requires cultivars to be cultured at 18–21℃ in the dark for 35–42 days throughout the entire process, neglecting the need for low light and CO2 concentration during primordia differentiation, resulting in low primordia formation rates and insufficient strain activity. Furthermore, sterilization processes often employ conventional high-pressure sterilization (121℃, 120 minutes), which is energy-intensive and prone to nutrient destruction, while insufficient control over the cleanliness of the inoculation environment further increases the risk of contamination. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing bottle-grown spawn for auricularia auricula cultivation, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing bottle-grown spawn for *Auricularia auricula-judae* cultivation includes the following steps:

[0009] Step 1: Pretreatment of bacterial strains;

[0010] Step 2: Preparation of composite culture medium;

[0011] Step 3: Bottling and sterilization;

[0012] Step 4: Gradual vaccination;

[0013] Step 5: Multi-stage intelligent training;

[0014] Step 6: Quality inspection and screening.

[0015] Preferably, step 1, the pretreatment of the microbial strain, specifically includes:

[0016] Mature fruiting bodies of Auricularia auricula-judae with a diameter of 5-8 cm and associated Mycorrhizal fungi were selected, and pure strains were obtained by aseptic isolation and then subjected to staged activation culture.

[0017] The phased activation culture includes an adaptation period, a proliferation period, and an enhancement period. During the adaptation period, the culture is carried out in a modified PDA medium at 22–24℃ for 3–4 days. During the proliferation period, the culture is carried out in a liquid medium with 2% corn flour at 24–26℃ with shaking at 150–180 rpm for 5–6 days. During the enhancement period, the culture is carried out in a medium containing 0.02% chitosan at 23–25℃ for 2–3 days.

[0018] The PDA modified medium consists of 200 g / L peeled potato, 20 g / L glucose, 5 g / L yeast extract, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, and 20 g / L agar, with a natural pH value.

[0019] Preferably, step 2, the preparation of the composite culture medium, specifically includes:

[0020] The compound culture medium, by weight percentage, consists of the main ingredients: 40%–50% chestnut sawdust, 20%–25% mulberry branch sawdust, and 10%–15% cottonseed hulls; auxiliary ingredients: 5%–8% fermented soybean meal, 3%–5% wheat bran, 1%–2% corn flour, 1%–1.5% gypsum powder, 0.1%–0.3% potassium dihydrogen phosphate, and 0.05%–0.1% magnesium sulfate; synergists: 0.02%–0.03% chitosan, 0.1%–0.3% astragalus polysaccharide, and 0.5%–1.0% honeysuckle extract; and water. The material-to-water ratio is 1:1.4–1:1.6, and the pH is adjusted to 7.0–8.0.

[0021] After crushing the main ingredient into 0.2-0.8cm particles, pre-wet it for 24 hours, and then mix it evenly with the auxiliary materials and synergists.

[0022] Preferably, in step 2, the method for preparing fermented soybean meal includes the following steps:

[0023] Mix soybean meal and water in a ratio of 1:1.2, inoculate with 5% Bacillus subtilis inoculum, ferment at 30-32℃ for 72 hours, dry until the moisture content is ≤12%, and then pulverize.

[0024] The preparation method of honeysuckle extract includes the following steps:

[0025] Decoction of honeysuckle and water at a ratio of 1:10 twice, 60 minutes each time. Combine the filtrates and concentrate to a concentration of 0.5 g / mL. Sterilize and set aside for later use.

[0026] Preferably, step 3, bottling and sterilization, specifically includes:

[0027] Fill the culture medium into the culture flask, filling it to a height equal to the flask's height. ~ The compaction degree is 0.6~0.7g / cm³, and the bottle mouth is capped with a breathable sealing film;

[0028] The process employs a two-stage pulsed sterilization process. The first stage involves sterilization at 105°C for 30 minutes, followed by exhaust and depressurization. The second stage involves sterilization at 121°C and 0.11MPa for 90 minutes, followed by rapid cooling to below 25°C.

[0029] The culture flask is a high-temperature resistant glass bottle with a mouth diameter of 70-80mm and a height of 150-180mm. The bottom of the bottle has 3-4 vent holes with a diameter of 2-3mm, and the holes are filled with sterile degreased cotton.

[0030] Preferably, step 4, gradient inoculation, specifically includes:

[0031] In a Class 100 cleanroom inoculation room, the activated Auricularia auricula-judae and Chrysophagus truncatula are mixed at a volume ratio of 1:2.5 to 3.5. Using a liquid inoculator, the mixture is injected into the culture medium at a volume of 5% to 8% per bottle, 1 to 2 cm below the surface of the culture medium. After inoculation, the culture bottle is gently shaken to ensure even distribution of the inoculum.

[0032] The inoculation needle of the liquid inoculum inoculator has a diameter of 4-5 mm. During inoculation, a pulse injection is used, and the inoculum is injected at 3-4 points per bottle.

[0033] Preferably, step 5, multi-stage intelligent training, specifically includes:

[0034] The inoculated culture flasks were placed in a Class 1000 cleanroom, and environmental parameters were adjusted in three stages:

[0035] Transplanting period (1-7 days): Dark environment, temperature 22-24℃, humidity 65%-75%, CO2 concentration ≤1000ppm, ventilate twice a day for 30 minutes each time;

[0036] Extension period (8-25 days): Dark environment, temperature 20-22℃, humidity 75%-85%, CO2 concentration 1500-2200ppm, ventilate 3 times a day for 20 minutes each time;

[0037] Differentiation period (26-32 days): weak light exposure (50-100 lx), temperature 18-20℃, humidity 85%-90%, CO2 concentration ≤800ppm, ventilation 4 times a day, 25 minutes each time;

[0038] The multi-stage intelligent cultivation adopts an environmental parameter linkage control system, which includes temperature sensors, humidity sensors, CO2 sensors, light sensors and automatic control modules. The sensor data acquisition interval is 10 minutes and the control response time is ≤5 minutes.

[0039] Preferably, the weak light irradiation during the differentiation period in step 5 uses an LED cold light source with an irradiation cycle of 12 hours of light / 12 hours of darkness and a light source wavelength of 550–600 nm.

[0040] Preferably, step 6, quality inspection and screening, specifically includes:

[0041] After cultivation, discard contaminated, sparse mycelia and primordia-free culture bottles. Qualified culture broths have white and dense mycelia, primordia diameter ≥1cm and no yellow spots. After sealing, store at 4-6℃.

[0042] Quality inspection includes:

[0043] Sensory testing (mycelial morphology, primordium development), microbial testing (total colony count ≤10³CFU / g), and activity testing (mycelial polysaccharide content ≥7.5%, respiration rate ≥0.8mgCO2 / (g・h)).

[0044] A composite culture medium for preparing a bottle-grown cultivar for the above-described method of cultivating golden ear fungus comprises, by weight percentage: 45% chestnut sawdust, 22% mulberry twig sawdust, 12% cottonseed hulls, 6% fermented soybean meal, 4% wheat bran, 1.5% corn flour, 1.2% gypsum powder, 0.2% potassium dihydrogen phosphate, 0.08% magnesium sulfate, 0.025% chitosan, 0.2% astragalus polysaccharide, 0.8% honeysuckle extract, with the remainder being water. The material-to-water ratio is 1:1.5, and the pH value is 7.5.

[0045] Compared with the prior art, the beneficial effects of the present invention are:

[0046] This invention uses a composite carbon source of chestnut wood chips, mulberry twig chips, and cottonseed hulls, combined with a functional nitrogen source of fermented soybean meal, to ensure a continuous and stable release of nutrients and meet the synergistic growth requirements of the two strains. The added chitosan and astragalus polysaccharides can enhance mycelial activity, and the honeysuckle extract has strong anti-pollution properties.

[0047] This invention enhances the activity of the strain through a phased activation process and adjusts the mixing ratio by real-time monitoring of mycelial density, so that Auricularia auricula-judae and Mycorrhizal lenticulata form a stable symbiotic relationship, thereby increasing the fruiting rate of the strain and improving the consistency of fruiting body morphology.

[0048] This invention employs a three-stage dynamic environmental control approach, optimizing parameters to meet the different needs of mycelial colonization, expansion, and primordia differentiation, thereby shortening the cultivation cycle of cultivars, improving production efficiency, and significantly reducing the time cost of industrialized production.

[0049] The mycelium polysaccharide content of the cultivated strain prepared by this invention is increased; the primordia formation rate is high, which improves the biological efficiency of the fruiting bodies after inoculation, and the fruiting bodies are golden and plump with a mellow taste, significantly increasing their commercial value.

[0050] The entire preparation process adopts standardized technology and intelligent control system, which can realize full automation control from strain activation to storage, reduce human intervention, and fully meet the needs of large-scale factory production. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the process steps of the present invention;

[0052] Figure 2 This is a schematic diagram of the process of the present invention. Detailed Implementation

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] Example 1:

[0055] like Figure 1-2 As shown, a method for preparing bottle-grown spawn for *Auricularia auricula-judae* cultivation includes the following steps:

[0056] Step 1, Pretreatment of bacterial strains:

[0057] Strain selection: Select mature auricularia auricula fruiting bodies with a diameter of 5-8 cm, golden color, and free from diseases and pests, as well as the mycelium of Leuciscus truncatula attached to the base of the fruiting bodies. Use tissue separation method to aseptically separate them in a clean bench to obtain pure auricularia auricula and pure Leuciscus truncatula strains.

[0058] Staged activation culture:

[0059] Adaptation period: Inoculate the two pure strains separately into PDA modified medium and incubate in a constant temperature incubator at 22-24℃ in the dark for 3-4 days, until the mycelium covers 1 / 3 of the surface of the medium. The PDA modified medium consists of 200g / L peeled potato, 20g / L glucose, 5g / L yeast extract, 3g / L potassium dihydrogen phosphate, 1.5g / L magnesium sulfate, and 20g / L agar, with a natural pH. This formula can quickly restore the activity of the strains.

[0060] Proliferation phase: Inoculate the adapted strain into liquid culture medium and culture at 24–26℃ and 150–180 rpm for 5–6 days to obtain high-density mycelium. The liquid culture medium formula is: corn flour 20 g / L, soybean flour 15 g / L, glucose 10 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate 1.5 g / L, and vitamin B1 0.1 g / L. Shaking promotes nutrient absorption, and the mycelial ball density can reach 1.2 × 10⁻⁶. 5 More than one per liter.

[0061] Enhancement phase: Transfer the proliferating mycelium to an enhanced medium containing 0.02% chitosan and incubate at 23–25°C for 2–3 days. Chitosan can regulate cell membrane permeability, enhance mycelial stress resistance, and increase the polysaccharide content of the mycelium by more than 15%.

[0062] Step 2, Preparation of composite culture medium:

[0063] Formula Design: Based on the differences in nutritional requirements between *Auricularia auricula-judae* and *Trichoderma truncatula*, a specialized culture medium containing a compound carbon source, a functional nitrogen source, and synergists was designed. The specific formula is as follows by weight percentage:

[0064] Main ingredients: 40%–50% chestnut wood chips, 20%–25% mulberry branch chips, 10%–15% cottonseed hulls;

[0065] Auxiliary ingredients: fermented soybean meal 5%–8%, wheat bran 3%–5%, corn flour 1%–2%, gypsum powder 1%–1.5%, potassium dihydrogen phosphate 0.1%–0.3%, magnesium sulfate 0.05%–0.1%;

[0066] Synergists: Chitosan 0.02%–0.03%, Astragalus polysaccharide 0.1%–0.3%, Honeysuckle extract 0.5%–1.0%;

[0067] Water: The ratio of feed to water is 1:1.4 to 1:1.6, and the pH value is 7.0 to 8.0.

[0068] The combination of chestnut wood chips and mulberry twig chips can provide a continuous carbon source supply. After fermentation with Bacillus subtilis, the amino acid nitrogen content of fermented soybean meal increases by 40%, making it easier for mycelia to absorb. Astragalus polysaccharides can promote mycelial growth, while honeysuckle extract has a natural antibacterial effect, which can reduce the risk of contamination by miscellaneous bacteria.

[0069] Preparation process:

[0070] Main material pretreatment: Crush chestnut wood chips, mulberry twig chips and cottonseed hulls into 0.2-0.8cm particles respectively, mix them in proportion, add water and pre-wet for 24 hours, stirring twice during the period to ensure even water absorption;

[0071] Mixing of auxiliary materials: Mix fermented soybean meal, wheat bran, corn flour and other auxiliary materials with the pre-wetted main material, then dissolve potassium dihydrogen phosphate and magnesium sulfate in a small amount of water and add them, stirring evenly;

[0072] Additives: Dissolve chitosan and astragalus polysaccharide in warm water and mix with honeysuckle extract. Spray evenly onto the material pile while stirring.

[0073] Moisture and pH adjustment: Add the remaining moisture to adjust the material-to-water ratio to 1:1.4 to 1:1.6, and use lime water or citric acid to adjust the pH value to 7.0 to 8.0.

[0074] Step 3, Bottling and Sterilization:

[0075] Bottling procedure: Use high-temperature resistant plastic bottles with a mouth diameter of 70-80 mm and a height of 120-130 mm. Fill the bottles with the composite culture medium to a height of 2 / 3 to 3 / 4 of the bottle height. Use a layered compaction method to ensure uniform compaction (0.6-0.7 g / cm³). Cover the bottle mouth with a breathable sealing cap (including a 0.22 μm filter membrane).

[0076] Secondary pulse sterilization: Secondary pulse sterilization is performed using a fully automatic sterilizer. Specific parameters are as follows:

[0077] Phase 1: Heat to 105℃ and maintain for 30 minutes to expel cold air and volatile harmful substances from the culture medium;

[0078] Depressurization phase: Slowly reduce to normal pressure and maintain for 10 minutes;

[0079] Second stage: Heat to 121℃ and pressure 0.11MPa, maintain for 90 minutes to ensure the killing of stubborn bacteria such as Bacillus spores;

[0080] Cooling stage: Use cold water spray to quickly cool down to below 25°C, and control the cooling time within 4 hours to avoid secondary pollution.

[0081] Step 4, Gradual vaccination:

[0082] Inoculation environment control: Inoculation is carried out in a Class 100 cleanroom, with the indoor temperature controlled at 20-22℃ and humidity ≤60%. After 30 minutes of double disinfection with ultraviolet light and ozone, ventilation is carried out for 15 minutes.

[0083] Mixing ratio of fungal strains: Based on the mycelial density of the two strains after the reinforcement period (measured by microscopic counting method), adjust the mixing volume ratio of *Auricularia aurea* and *Trichoderma viride* to 1:2.5–3.5. This ratio ensures that *Trichoderma viride* fully degrades lignin to provide nutrients, while avoiding its excessive growth that inhibits *Auricularia aurea*.

[0084] Inoculation procedure: Use a fully automatic liquid inoculator with an inoculation needle diameter of 4-5 mm. Inoculate at 3-4 points below the surface of the culture medium, using an inoculation volume of 5%-8% per bottle, to a depth of 1-2 cm. After inoculation, gently shake the culture bottle to ensure even distribution of the inoculum and reduce local nutrient competition.

[0085] Step 5: Multi-stage intelligent training:

[0086] Culture environment system: The culture room is equipped with an environmental parameter linkage control system. The system includes temperature sensors, humidity sensors, CO2 sensors, light sensors and automatic control modules (heater, humidifier, ventilation fan, LED light source). The sensor data acquisition interval is 10 minutes and the control response time is ≤5 minutes to ensure that the environmental parameters are stable within the set range.

[0087] Three-stage culture parameters:

[0088] Establishment period (1-7 days): This stage focuses on establishing the inoculum, using a dark environment to promote mycelial germination. Temperature is controlled at 22-24℃, humidity at 65%-75%, CO2 concentration ≤1000ppm, and ventilation is provided twice daily for 30 minutes each time. By the end of this stage, mycelial colonies with a diameter of 3-5cm should have formed around the inoculation point.

[0089] Expansion phase (8–25 days): During this stage, the mycelium expands rapidly, requiring increased nutrient supply and ventilation. The temperature should be lowered to 20–22℃, humidity increased to 75%–85%, and CO2 concentration controlled at 1500–2200 ppm (to promote mycelial growth). Ventilate three times daily for 20 minutes each time. By adjusting the carbon-nitrogen ratio, the average daily mycelial expansion rate should reach 0.8–1.0 cm.

[0090] Differentiation phase (26–32 days): This stage induces primordium formation and enhances strain activity. Irradiate with weak LED light at 550–600 nm, with a light intensity of 50–100 lx and a photoperiod of 12 hours light / 12 hours dark; maintain a temperature of 18–20℃, humidity of 85%–90%, and CO2 concentration ≤800 ppm; ventilate four times daily for 25 minutes each time. By the end of this stage, orange-yellow primordia with a diameter ≥1 cm should have formed on the surface of the culture medium.

[0091] Step 6, Quality Inspection and Screening:

[0092] Testing items and standards:

[0093] Sensory inspection: The hyphae are white, dense, and evenly distributed, without any discolored spots. The primordia are ≥1cm in diameter, orange-yellow in color, and without yellow spots. The culture medium has no rancid odor.

[0094] Microbiological testing: The total bacterial count was determined by plate counting method to be ≤10³ CFU / g, and no pathogenic bacteria such as molds or bacteria were detected;

[0095] Activity assay: The polysaccharide content of mycelium was determined to be ≥7.5% using the phenol-sulfuric acid method, and the respiration rate was determined to be ≥0.8 mg CO2 / (g·h) using a respiration meter (reflecting mycelial activity).

[0096] Screening and storage: Remove contaminated, sparse mycelium, poorly developed primordia, and unqualified culture bottles. Qualified culture products are sealed with sterile plastic film and stored in a cold storage at 4-6℃ and 50%-60% relative humidity, with a shelf life of more than 60 days.

[0097] Example 2:

[0098] A method for preparing bottle-grown spawn for *Auricularia auricula-judae* cultivation includes the following steps:

[0099] Step 1, Pretreatment of bacterial strains:

[0100] Mature fruiting bodies with a diameter of 6 cm were selected, and pure *Auricularia auricula-judae* strains were obtained by tissue isolation method; *Trichoderma truncatula* strains were isolated from the base of the fruiting body to obtain pure *Trichoderma truncatula* strains.

[0101] Adaptation period: Inoculate into modified PDA medium and incubate at 23°C for 3 days;

[0102] The culture medium was prepared using 200 g / L peeled potato, 20 g / L glucose, 5 g / L yeast powder, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, and 20 g / L agar.

[0103] Proliferation phase: Inoculate with liquid culture medium and culture at 25℃ and 160 rpm for 5 days until the mycelial ball density reaches 1.3 × 10⁻⁶. 5 Units / L;

[0104] The liquid culture medium consisted of 20 g / L corn flour, 15 g / L soybean flour, 10 g / L glucose, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, and 0.1 g / L vitamin B1.

[0105] Strengthening period: Transfer to a medium containing 0.02% chitosan and incubate at 24℃ for 2 days. The mycelial polysaccharide content reaches 8.2%.

[0106] Step 2, Preparation of composite culture medium:

[0107] Formula (by weight percentage): 45% chestnut wood chips, 22% mulberry twig chips, 12% cottonseed hulls, 6% fermented soybean meal, 4% wheat bran, 1.5% corn flour, 1.2% gypsum powder, 0.2% potassium dihydrogen phosphate, 0.08% magnesium sulfate, 0.025% chitosan, 0.2% astragalus polysaccharide, 0.8% honeysuckle extract, material-to-water ratio 1:1.5, pH 7.5;

[0108] Preparation: The main material is crushed to 0.4-0.6 cm and pre-wetted for 24 hours; after mixing with the auxiliary materials, dissolved inorganic salts are added, and then the synergist mixture is sprayed on to adjust the moisture and pH value.

[0109] Step 3, Bottling and Sterilization:

[0110] Use a glass bottle with a diameter of 75mm and a height of 160mm. Drill four 2.5mm diameter ventilation holes in the bottom of the bottle. The filling height is 120mm, the compaction degree is 0.65g / cm³, and a breathable sealing film is added.

[0111] Secondary pulse sterilization: 105℃ / 30min → atmospheric pressure / 10min → 121℃ / 0.11MPa / 90min, cool to 24℃.

[0112] Step 4, Gradual vaccination:

[0113] The inoculation room has a cleanliness level of 100, a temperature of 21℃, and a humidity of 55%.

[0114] Mix Auricularia aurea and Chrysophagus truncatula at a volume ratio of 1:3. Use an inoculator to inject the mixture into the culture medium at a depth of 1.5 cm below the surface of the medium at a rate of 6% per bottle, and inoculate at 3 sites.

[0115] Step 5: Multi-stage intelligent training:

[0116] Establishment period (1-7 days): darkness, 23℃, humidity 70%, CO2 concentration 800ppm, ventilation twice a day for 30 minutes, colony diameter reaches 4cm on the 7th day;

[0117] Expansion period (8-25 days): Darkness, 21℃, humidity 80%, CO2 concentration 1800ppm, ventilation 3 times / 20min per day, mycelium fully colonizes the bottle on day 25;

[0118] Differentiation period (26-30 days): LED low light (580nm, 80lx, 12h / 12h), 19℃, humidity 88%, CO2 concentration 700ppm, ventilation 4 times / 25min per day, the primordium diameter reached 1.2cm on the 30th day.

[0119] Step 6, Quality Inspection:

[0120] Sensory characteristics: The mycelium is white and dense, the primordia are orange-yellow and 1.2 cm in diameter, and there is no odor;

[0121] Microorganisms: Total bacterial count 8.2 × 10² CFU / g, no pathogenic bacteria;

[0122] Activity: Polysaccharide content 8.5%, respiration rate 0.92 mg CO2 / (g・h);

[0123] Qualified cultivars stored in a 5℃ cold storage for 60 days retained 92% of their viability.

[0124] Example 3:

[0125] Experiment on optimization of compound culture medium formulation:

[0126] Five different culture medium formulations were set up, and other preparation conditions were the same as in Example 1. The mycelial growth rate, contamination rate and primordium formation rate were compared. The results are shown in Table 1.

[0127] Table 1. Performance comparison of different culture medium formulations

[0128] Group Main ingredient recipe Synergist Mycelial growth rate (cm / d) Pollution rate (%) Primordial formation rate (%) control group 80% broadleaf sawdust + 10% cottonseed hulls none 0.62 12.5 78.2 1 Chestnut wood chips 40% + mulberry branch chips 25% + cottonseed hulls 15% Chitosan 0.02% 0.78 5.8 89.5 2 50% chestnut wood chips + 20% mulberry branch chips + 10% cottonseed hulls Astragalus polysaccharide 0.3% 0.81 6.2 91.3 3 Chestnut wood chips 45% + mulberry twig chips 22% + cottonseed hulls 12% Honeysuckle extract 1.0% 0.79 3.5 90.8 4 (Example 1) Chestnut wood chips 45% + mulberry twig chips 22% + cottonseed hulls 12% Chitosan 0.025% + Astragalus polysaccharide 0.2% + Honeysuckle extract 0.8% 0.95 2.8 96.7

[0129] As shown in Table 1, the composite culture medium formulation (group 4) of the present invention is significantly better than the control group and other experimental groups in terms of mycelial growth rate, anti-pollution ability and primordium formation rate, indicating that the synergistic effect of composite carbon source and multi-functional synergist can effectively improve the performance of culture medium.

[0130] Example 4:

[0131] Experiment on optimization of strain mixing ratio:

[0132] Four groups of different mixing ratios of Auricularia auricula-judae and Leymus chinensis were set up, with other preparation conditions the same as in Example 1. The symbiotic stability and subsequent fruiting performance were compared, and the results are shown in Table 2.

[0133] Table 2 Comparison of performance of different bacterial strain mixing ratios

[0134] Mixing ratio (Auricularia auricula-judae: Leatherylodis rhamnoides) Symbiotic state Time (days) for mycelium to fully colonize the bottle Mushroom yield (%) Biological efficiency (%) 1:2 (Control Group) Insufficient *Leuciscus faecium* leads to slow growth of *Auricularia auricula-judae*. 35 87.3 75.6 1:2.5 Symbiotic Coordination 31 94.5 85.2 1:3 (Example 1) Symbiosis is the best 30 98.2 92.5 1:3.5 Slightly more *Trichoderma harzianum*, with slight inhibition of *Auricularia auricula-judae*. 32 95.8 88.7 1:4 Excessive growth of *Trichoderma harzianum* inhibits *Auricularia auricula-judae*. 38 82.1 72.3

[0135] As shown in Table 2, the optimal symbiotic state, mycelial growth rate, fruiting rate and biological efficiency are achieved when the mixing ratio of Auricularia aurea and Mycorrhizal lenticule is 1:3. This is the basis for the preferred ratio in this invention.

[0136] Example 5:

[0137] Multi-stage culture parameter optimization experiment:

[0138] Three culture modes were set up, and other preparation conditions were the same as in Example 1. The culture cycle and bacterial activity were compared, and the results are shown in Table 3.

[0139] Table 3 Performance Comparison of Different Culture Modes

[0140] Training Model Culture parameters Culture cycle (days) Polysaccharide content (%) Primordial diameter (cm) Traditional model (control group) The entire time was 19°C, in complete darkness, and with 75% humidity. 40 6.2 0.8 Two-phase model Planting period (22℃, 7 days) + expansion period (20℃, 33 days) 35 7.0 1.0 Three-stage model (Example 1) Planting period (23℃, 7 days) + expansion period (21℃, 18 days) + differentiation period (19℃, 5 days) 30 8.5 1.2

[0141] As shown in Table 3, the three-stage culture mode of the present invention significantly shortens the culture cycle by dynamically adjusting environmental parameters, while improving the polysaccharide content and primordium development quality of the strain, demonstrating the scientific nature and superiority of multi-stage regulation.

[0142] Example 6:

[0143] Comparative experiment of different sterilization processes

[0144] Three sterilization processes were set up, and other preparation conditions were the same as in Example 1. The sterilization effect and nutrient retention rate were compared and the results are shown in Table 4.

[0145] Table 4. Performance Comparison of Different Sterilization Processes

[0146] sterilization process sterilization parameters Bacterial kill rate (%) Amino acid retention rate (%) Energy consumption (kWh / 1000 bottles) Routine sterilization (control group) 121℃, 120min 99.2 78.5 125 One-time pulse sterilization 115℃ / 60min→121℃ / 60min 99.5 82.3 110 Secondary pulse sterilization (Example 1) 105℃ / 30min→121℃ / 90min 99.8 89.7 105

[0147] As shown in Table 4, the secondary pulse sterilization process of the present invention significantly improves the retention rate of nutrients while ensuring the kill rate of miscellaneous bacteria and reducing energy consumption, which is superior to the traditional sterilization process.

[0148] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing bottle-grown spawn for *Auricularia auricula-judae* cultivation, characterized in that, Includes the following steps: Step 1: Pretreatment of bacterial strains; Step 2: Preparation of composite culture medium; Step 3: Bottling and sterilization; Step 4: Gradual vaccination; Step 5: Multi-stage intelligent training; Step 6: Quality inspection and screening.

2. The method for preparing bottle-grown spawn for *Auricularia auricula-judae* cultivation according to claim 1, characterized in that, Step 1, bacterial strain pretreatment, specifically includes: Mature fruiting bodies of Auricularia auricula-judae with a diameter of 5-8 cm and associated Mycorrhizal fungi were selected, and pure strains were obtained by aseptic isolation and then subjected to staged activation culture. The phased activation culture includes an adaptation period, a proliferation period, and an enhancement period. During the adaptation period, the culture is carried out in a modified PDA medium at 22–24℃ for 3–4 days. During the proliferation period, the culture is carried out in a liquid medium with 2% corn flour at 24–26℃ with shaking at 150–180 rpm for 5–6 days. During the enhancement period, the culture is carried out in a medium containing 0.02% chitosan at 23–25℃ for 2–3 days. The PDA modified medium consists of 200 g / L peeled potato, 20 g / L glucose, 5 g / L yeast extract, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, and 20 g / L agar, with a natural pH value.

3. The method for preparing bottle-grown spawn for *Auricularia auricula-judae* cultivation according to claim 2, characterized in that, Step 2, preparation of the composite culture medium, specifically includes: The compound culture medium, by weight percentage, consists of the main ingredients: 40%–50% chestnut sawdust, 20%–25% mulberry branch sawdust, and 10%–15% cottonseed hulls; auxiliary ingredients: 5%–8% fermented soybean meal, 3%–5% wheat bran, 1%–2% corn flour, 1%–1.5% gypsum powder, 0.1%–0.3% potassium dihydrogen phosphate, and 0.05%–0.1% magnesium sulfate; synergists: 0.02%–0.03% chitosan, 0.1%–0.3% astragalus polysaccharide, and 0.5%–1.0% honeysuckle extract; and water. The material-to-water ratio is 1:1.4–1:1.6, and the pH is adjusted to 7.0–8.

0. After crushing the main ingredient into 0.2-0.8cm particles, pre-wet it for 24 hours, and then mix it evenly with the auxiliary materials and synergists.

4. The method for preparing bottle-grown spawn for *Auricularia auricula-judae* cultivation according to claim 3, characterized in that, Step 2, the method for preparing fermented soybean meal, includes the following steps: Mix soybean meal and water in a ratio of 1:1.2, inoculate with 5% Bacillus subtilis inoculum, ferment at 30-32℃ for 72 hours, dry until the moisture content is ≤12%, and then pulverize. The preparation method of honeysuckle extract includes the following steps: Decoction of honeysuckle and water at a ratio of 1:10 twice, 60 minutes each time. Combine the filtrates and concentrate to a concentration of 0.5 g / mL. Sterilize and set aside for later use.

5. The method for preparing bottle-grown spawn for *Auricularia auricula-judae* cultivation according to claim 4, characterized in that, Step 3, bottling and sterilization, specifically includes: Fill the culture medium into the culture flask, filling it to a height equal to the flask's height. ~ The compaction degree is 0.6~0.7g / cm³, and the bottle mouth is capped with a breathable sealing film; The process employs a two-stage pulsed sterilization process. The first stage involves sterilization at 105°C for 30 minutes, followed by exhaust and depressurization. The second stage involves sterilization at 121°C and 0.11MPa for 90 minutes, followed by rapid cooling to below 25°C. The culture flask is a high-temperature resistant glass bottle with a mouth diameter of 70-80mm and a height of 150-180mm. The bottom of the bottle has 3-4 vent holes with a diameter of 2-3mm, and the holes are filled with sterile degreased cotton.

6. The method for preparing bottle-grown spawn for *Auricularia auricula-judae* cultivation according to claim 5, characterized in that, Step 4, gradient inoculation, specifically includes: In a Class 100 cleanroom inoculation room, the activated Auricularia auricula-judae and Chrysophagus truncatula are mixed at a volume ratio of 1:2.5 to 3.

5. Using a liquid inoculator, the mixture is injected into the culture medium at a volume of 5% to 8% per bottle, 1 to 2 cm below the surface of the culture medium. After inoculation, the culture bottle is gently shaken to ensure even distribution of the inoculum. The inoculation needle of the liquid inoculum inoculator has a diameter of 4-5 mm. During inoculation, pulse injection is used, and the inoculum is injected at 3-4 points per bottle.

7. The method for preparing bottle-grown spawn for *Auricularia auricula-judae* cultivation according to claim 6, characterized in that, Step 5, multi-stage intelligent training, specifically includes: The inoculated culture flasks were placed in a Class 1000 cleanroom, and environmental parameters were adjusted in three stages: Planting period: Dark environment, temperature 22-24℃, humidity 65%-75%, CO2 concentration ≤1000ppm, ventilate twice a day for 30 minutes each time; Extension period: Dark environment, temperature 20-22℃, humidity 75%-85%, CO2 concentration 1500-2200ppm, ventilate 3 times a day, 20 minutes each time; Differentiation period: weak light, temperature 18-20℃, humidity 85%-90%, CO2 concentration ≤800ppm, ventilation 4 times a day, 25 minutes each time; The multi-stage intelligent cultivation adopts an environmental parameter linkage control system, which includes temperature sensors, humidity sensors, CO2 sensors, light sensors and automatic control modules. The sensor data acquisition interval is 10 minutes and the control response time is ≤5 minutes.

8. The method for preparing bottle-grown spawn for *Auricularia auricula-judae* cultivation according to claim 7, characterized in that, In step 5, the weak light irradiation during the differentiation period uses an LED cold light source with an irradiation cycle of 12 hours of light / 12 hours of darkness and a light source wavelength of 550-600 nm.

9. The method for preparing bottle-grown spawn for *Auricularia auricula-judae* cultivation according to claim 8, characterized in that, Step 6, quality inspection and screening, specifically includes: After cultivation, discard contaminated, sparse mycelia and primordia-free culture bottles. Qualified culture broths have white and dense mycelia, primordia diameter ≥1cm and no yellow spots. After sealing, store at 4-6℃. Quality inspection includes: Sensory testing, microbiological testing, and activity testing.

10. A composite culture medium for preparing a bottle-grown cultivar for *Auricularia auricula-judae* cultivation according to any one of claims 1 to 9, characterized in that, By weight percentage, the composition includes 45% chestnut wood chips, 22% mulberry branch chips, 12% cottonseed hulls, 6% fermented soybean meal, 4% wheat bran, 1.5% corn flour, 1.2% gypsum powder, 0.2% potassium dihydrogen phosphate, 0.08% magnesium sulfate, 0.025% chitosan, 0.2% astragalus polysaccharide, 0.8% honeysuckle extract, with the remainder being water. The material-to-water ratio is 1:1.5, and the pH value is 7.5.

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