A thermophilic halotolerant myceliopthora grisea fungicide and a preparation method thereof

Abstract

This invention relates to the field of microbial inoculant preparation technology, specifically to a thermophilic and halophilic *Trichoderma* agent and its preparation method. The active ingredient of the *Trichoderma* MT-ST / GP agent in this invention is *Trichoderma* MT-ST / GP, deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 41587, and the agent contains 8 × 10⁸ viable cells. 6 ~1.2×10 7 The cellulase activity is 45-55 U / mL, the hemicellulase activity is 75-85 U / mL, and the salt tolerance concentration is 4-20 g / L NaCl. This invention improves the number of viable bacteria, enzyme activity, and salt tolerance of the inoculant by optimizing fermentation conditions (segmented temperature control, nutrient supplementation, and protective agent adaptation). It is suitable for scenarios such as agricultural waste degradation and biological treatment of high-salt environments.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculant preparation technology, specifically to a thermophilic and salt-tolerant mycotoxin agent and its preparation method. Background Technology

[0002] The performance of microbial inoculants (viable cell count, enzyme activity, stability) directly determines their application effect, and the preparation process is the core factor affecting the performance of the inoculant. Existing methods for preparing thermophilic and salt-tolerant *Trichoderma* inoculants have the following key problems: Monotonous fermentation conditions: Most processes use a fixed temperature (e.g., 45℃) and a single culture medium, failing to optimize fermentation conditions for the different stages of strain proliferation and enzyme production, resulting in viable cell counts generally below 5 × 10⁻⁶. 6 The CFU / mL biomass-degrading enzyme activity is less than 40 U / mL, which cannot meet the requirements for high-efficiency application.

[0003] There is a gap in the preparation of salt-tolerant microbial agents: existing preparations for salt-tolerant microbial agents mostly focus on bacteria or yeasts and fungi, and lack processes adapted to thermophilic and salt-tolerant Mycorrhizal MT-ST / GP (salt tolerance + high temperature characteristics). For example, issues such as cell osmotic pressure regulation and enzyme secretion induction under high salt conditions are not considered, resulting in rapid activity decay of the agents in high salt environments.

[0004] High post-processing loss rate: During spraying or freeze drying, the bacteria are easily inactivated due to osmotic pressure shock and temperature stress. Existing protectants (such as lactose) cannot effectively reduce the loss. The viable count of the finished bacterial agent often decreases by more than 40%, resulting in poor storage stability.

[0005] Cost and efficiency imbalance: Some processes rely on expensive commercial culture media (such as imported peptone) or have fermentation cycles of more than 72 hours, resulting in high industrialization costs and low efficiency, making them difficult to promote.

[0006] In the existing relevant patent literature, the authorized patent "A new strain of thermophilic pyriformis and its application" (patent number: ZL201110290951.3) designed a method for preparing thermophilic pyriformis using a fixed fermentation temperature of 42℃, but did not involve the preparation method of enzyme activity regulation and salt tolerance process.

[0007] The authorized patents “Salt-tolerant Bacillus, bacterial agents and their applications” (Patent No.: ZL202410967988.2), “A salt-tolerant yeast and its application” (Patent No.: ZL202211660360.5), “A salt-tolerant yellow-ochre Streptomyces and its application” (Patent No.: ZL201810869186.2), and “A salt-tolerant oxalic acid penicillin agent and its preparation method and application” (Patent No.: ZL202210106971.9) respectively designed the salt tolerance process of bacteria (Bacillus), yeast, Streptomyces and Penicillium, but did not involve the preparation method of the salt tolerance process of filomyces, which limited its application in high-salt environments.

[0008] The authorized patent, "A method and engineered strain for improving the cellulose degradation ability of thermophilic filamentous fungus" (patent number: 202411479455.6), only involves genetically engineered strains and does not cover the preparation method of the fungal agent, optimization of fermentation conditions, post-treatment protection, etc., and cannot guide the industrial production of fungal agents.

[0009] In summary, the existing technology lacks an integrated method for preparing thermophilic and salt-tolerant fungal agent MT-ST / GP, which combines "high-efficiency proliferation, high enzyme activity induction, and low-loss post-treatment". Therefore, it is urgent to develop methods to improve the performance of fungal agents and reduce application costs. Summary of the Invention

[0010] The purpose of this invention is to provide a thermophilic and salt-tolerant mycotoxin agent and its preparation method. Specifically, it involves a preparation method that improves the viable number of bacteria, enzyme activity and salt tolerance of the agent by optimizing fermentation conditions (segmented temperature control, nutrient supplementation and protective agent adaptation), which is applicable to scenarios such as agricultural waste degradation and biological treatment of high-salt environments.

[0011] The objective of this invention is achieved through the following technical solution: This invention provides a method for preparing thermophilic MT-ST / GP inoculant, comprising the following steps: (1) Seed culture: Thermophilic MT-ST / GP was inoculated into seed culture medium and cultured at 42-48℃ and 140-160r / min for 20-28h, maintaining dissolved oxygen at 20%-30%, to obtain a cell concentration of 8×10⁻⁶. 6 ~1.2×10 7 Seed culture with CFU / mL and hyphal fragment length of 100-200μm; (2) AI-assisted integrated high-density fermentation: The seed liquid was inoculated into the fermentation medium at an inoculation rate of 4%-6%. The initial fermentation parameters were pre-optimized based on the dual-module AI machine learning model. The culture was carried out using an integrated strategy of precise graded regulation of two-stage mycelial balls with AI dynamic control coupled with segmented temperature control, multi-component synergistic induction and gradient salt tolerance acclimatization. (3) Preparation of microbial agent: Add 4%-6% of a composite protective agent to the fermentation broth at the end of fermentation, and obtain the finished solid microbial agent by spray drying or freeze drying.

[0012] Furthermore, the seed culture medium mentioned in step (1) is selected from any of the following: ① Tryptone-soybean broth: tryptone 16-18 g / L, soybean peptone 2-4 g / L, glucose 2-3 g / L, NaCl 4-6 g / L, K2HPO4 2-3 g / L, pH 7.1-7.5; ② Modified LB medium: peptone 9-11 g / L, yeast extract 4-6 g / L, NaCl 9-11 g / L, glucose 4-6 g / L, pH 7.0-7.2.

[0013] Furthermore, the fermentation medium mentioned in step (2) is selected from any of the following: ① Corn stalk hydrolysate compound culture medium: corn stalk hydrolysate 0.7-0.9 L / L, soybean meal powder 10-13 g / L, glucose 14-16 g / L, starch 8-9.5 g / L, pH 6.5-7.5; ② Molasses-oilseed cake composite culture medium: molasses 15-25g / L, soybean meal powder 8-12g / L, rapeseed meal powder 8-12g / L, KH2PO4 0.8-1.2g / L, pH 6.5-7.5; ③ Wheat bran composite culture medium: wheat bran 48-52 g / L, ammonium sulfate 1.4-1.6 g / L, potassium dihydrogen phosphate 1.3-1.5 g / L, MgSO4 7H2O 0.4-0.6g / L, pH 6.5-7.5.

[0014] Furthermore, the dual-module AI machine learning model mentioned in step (2) includes an initial parameter pre-optimization module and a mycelial ball growth dynamic regulation module, specifically as follows: ① Initial parameter pre-optimization module: Based on the random forest algorithm, it takes the reducing sugar content of raw materials, fermenter volume, ambient temperature, and ambient humidity as input features, and the cell concentration and cellulase activity at the fermentation endpoint as output targets, and outputs the optimal initial fermentation parameters. The accuracy of the model on the test set is ≥92%. ②Mycelial ball growth dynamic control module: Based on CNN convolutional neural network, it takes dissolved oxygen, pH, stirring speed and fermentation broth turbidity data collected online during the fermentation process as input, predicts mycelial ball particle size distribution and cell growth stage in real time, and outputs dynamic adjustment parameters for stirring speed and aeration rate. The mycelial ball particle size prediction accuracy is ≥90%.

[0015] Furthermore, the AI-dynamically regulated two-stage mycelial ball precise hierarchical regulation coupled with segmented temperature control, multi-component synergistic induction, and gradient salt tolerance acclimatization integrated strategy described in step (2) is as follows: Phase 1: Based on the parameters output by the initial parameter pre-optimization module, maintain the fermentation temperature at 45-50℃, initial pH at 6.5-7.5, tank pressure at 0.03-0.08MPa, and aeration rate at 0.8-1.5L / (L). (min); The mycelial ball growth dynamic control module monitors the mycelial ball particle size in real time, and adjusts the shear force by gradually increasing the stirring speed. The stirring speed is 180 r / min from 0 to 12 h, and the speed is adjusted every 2 h from 12 to 24 h according to the particle size prediction results, gradually increasing to 200-220 r / min, so as to accurately control the mycelial ball particle size to be stable at 200-300 μm, and the particle size uniformity is ≥90%; Second stage: When the mycelial ball growth dynamic control module predicts that the mycelium has entered the late logarithmic growth phase, immediately switch the fermentation parameters: adjust the fermentation temperature to 40-45℃, reduce the stirring speed to 160-180 r / min, and increase the aeration rate to 1.2-1.5 L / (L (min); Add 0.05-0.15 g / L rhamnolipid biosurfactant, 0.1-0.3 g / L LmnSO4, 10-15 g / L microcrystalline cellulose, 15-20 g / L xylan, and 0.5-1.5 g / L glucose to the fermentation system in one go; At the same time, based on the salt tolerance threshold of the cells predicted by the model, add a total of 9-14 g / L NaCl in three gradients, with an interval of 4 h between each addition, to induce the mycelial balls to form a core-shell structure of "compact core-fluffy shell", thereby simultaneously achieving efficient enzyme production and improved salt tolerance.

[0016] Further, the post-treatment of the microbial agent in step (3) includes: adding 4-6% by mass of a protective agent to the fermentation broth, wherein the protective agent is composed of 4%-6% lactose, 2%-4% trehalose and 1%-3% glycerol; then spray drying is performed with an inlet air temperature of 120-150℃ and an outlet air temperature of 60-80℃ to obtain a solid microbial agent; or freeze drying is performed with a freezing temperature of -40 to -30℃, a vacuum degree of 10-20Pa and a drying time of 24-36h to obtain a freeze-dried microbial agent.

[0017] Furthermore, aseptic control is required during the preparation process: the culture medium is sterilized at 115-121℃ and 0.09-0.11MPa for 25-35 minutes, and inoculation and fermentation operations are carried out in an aseptic environment with an environmental cleanliness level ≥10000 to avoid contamination by other microorganisms.

[0018] This invention also provides a thermophilic and halophilic *Trichoderma tumefaciens* MT-ST / GP inoculum, which is prepared by the method described above. The active ingredient of the inoculum is *Trichoderma tumefaciens* MT-ST / GP, and it is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 41587. The inoculum contains 8 × 10⁻⁶ viable cells. 6 ~1.2×10 7 CFU / mL, cellulase activity 45~55U / mL, hemicellulase activity 75~85U / mL, salt tolerance 4~20 g / L NaCl, viable cell count decline rate ≤20% and enzyme activity retention rate ≥70% after 6 months of storage at room temperature.

[0019] The beneficial effects of this invention are as follows: This invention addresses key industry pain points in the preparation of existing thermophilic and salt-tolerant mycelial agents, such as reliance on empirically controlled fermentation conditions, irregular mycelial growth leading to low mass and oxygen transfer efficiency, antagonistic interactions between cell proliferation and enzyme production with salt tolerance, and poor batch stability. It constructs an AI-enabled, two-stage, precisely controlled mycelial ball coupled with segmented temperature control, multi-component synergistic induction, and gradient salt tolerance acclimatization—an integrated high-density fermentation technology. This technology is not simply a combination of AI parameter optimization and mycelial ball morphology control; rather, it deeply binds a dual-module AI machine learning model to the morphological and performance requirements throughout the entire cell lifecycle, targeting the growth and metabolic characteristics of thermophilic and salt-tolerant mycelial agents, forming a complete, closed-loop, and optimizable preparation system. The initial parameter pre-optimization module can accurately output the optimal initial fermentation parameters based on raw material characteristics and production conditions, avoiding the adaptability bias of empirical parameters. The prediction accuracy of the test set can reach over 92%. The mycelial ball growth dynamic control module can accurately predict the mycelial ball particle size distribution and cell growth stage based on real-time data collected online during the fermentation process, and dynamically adjust core parameters such as stirring speed and aeration rate. This provides quantitative support for the precise control of mycelial balls in both stages, and the mycelial ball particle size prediction accuracy can reach over 90%. This fundamentally solves the problems of strong lag and poor adaptability of traditional fixed parameter control.

[0020] The integrated process of this invention achieves precise particle size control of mycelial balls (200-300 μm) through gradient shear force regulation in the first stage, ensuring mass and oxygen transfer efficiency during the cell proliferation stage and realizing high-density cell growth. In the second stage, through the coupling of biosurfactants and gradient salt tolerance, the mycelial balls are induced to form a special core-shell structure of "compact core-fluffy shell". This not only opens up the secretion channels of extracellular enzymes, but also forms a natural osmotic pressure buffer barrier through the fluffy shell. This achieves the simultaneous completion of cell proliferation, efficient enzyme production and salt tolerance improvement, breaking the technical prejudice that the three cannot be achieved simultaneously in the prior art. Parallel controlled experiments verified that, compared with the traditional uncontrolled basic fermentation process, the integrated process of this invention achieves a 78% increase in cell concentration at the fermentation endpoint, a 28% increase in cellulase activity, a 24.4% increase in hemicellulase activity, a 25% increase in maximum salt tolerance, and a 60.3% increase in average mycelial pellet size, representing a significant leap in core performance indicators. Furthermore, compared with single processes using AI-assisted fermentation or two-stage mycelial pellet control alone, the integrated process of this invention still shows significant improvements in core indicators such as cell concentration, enzyme activity, and salt tolerance. Specifically, the cell concentration is 22.9% higher than the average of the two single processes, and the cellulase activity is 10.3% higher, demonstrating an unexpected synergistic effect between AI dynamic control and precise mycelial pellet classification control—a technical solution that cannot be easily obtained by those skilled in the art through conventional technical combinations.

[0021] The integrated process of this invention not only achieves breakthroughs in core performance, but also significantly improves the batch stability and industrial scale-up feasibility of fungal agent production. Through AI-standardized parameter control and precise control of mycelial ball morphology, the relative standard deviation between batches in industrial production can be reduced to 2.1%, which is 81.2% lower than that of traditional processes. This completely solves the industry problem of large batch fluctuations and unstable product quality in the traditional filamentous fungal fermentation process. During the industrial-scale pilot-scale production in a 100L fermenter, three batches of microbial agents produced continuously using the integrated process of this invention showed highly consistent core performance indicators with the laboratory small-scale test results, with no significant scale-up effect. Moreover, the entire process requires no additional dedicated production equipment and can be achieved solely through online data acquisition and model calculations using existing fermenters. The amount of rhamnolipin added for core regulation is only 0.05-0.15 g / L, resulting in a raw material cost increase of less than 1%. Furthermore, this process is adaptable to various low-cost culture media based on agricultural waste, such as corn stalk hydrolysate, molasses-cake meal, and wheat bran. Compared to traditional processes that rely on expensive commercial peptones and yeast extracts, the overall industrial production cost can be reduced by 30%-50%, demonstrating strong adaptability for large-scale industrial production.

[0022] This invention provides a thermophilic and salt-tolerant mycotoxin agent prepared through an integrated fermentation process. Combined with a suitable ternary composite protectant and an optimized drying process, the final liquid inoculum can achieve a viable cell count of 8 × 10⁻⁶. 6 ~1.2×10 7 CFU / mL, the highest viable count of solid bacterial agents can reach 8.5 × 10⁻⁶. 10 With a CFU / g concentration, the viable cell count at room temperature for 6 months exhibits a decline rate of ≤18% and an enzyme activity retention rate of ≥75%, far superior to bacterial agents prepared using traditional processes. This solves the problems of high loss rates and poor storage stability during the drying process of existing bacterial agents. Furthermore, this bacterial agent maintains stable cell activity and enzymatic hydrolysis performance across a wide salt concentration range of 4–25 g / L NaCl. Even under high salt stress at 25 g / L NaCl, the cell survival rate can still reach over 85%, completely resolving the issues of rapid activity decay and limited application range of existing thermophilic fungal agents in high-salt environments. It is widely adaptable to various complex application scenarios, such as high-temperature and high-efficiency degradation of agricultural straw and other organic waste, biological treatment of high-salt organic wastewater, and improvement and enhancement of organic matter in saline-alkali soils, demonstrating extremely high practical application value and promising market prospects. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] The present invention will be described in detail below. This embodiment is only used to explain the present invention and is not intended to limit the scope of protection of the present invention.

[0029] The strain used in this embodiment is *Myceliophthorathermophila* MT-ST / GP, which was deposited on October 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 41587 and classified as *Myceliophthorathermophila*. The collection center confirmed the viability of the strain on October 30, 2024, and the preservation period is 30 years from the date of deposit (extendable for 5 years upon expiration), which meets the requirements for biological material preservation in patent procedures.

[0030] Example 1 Seed culture 1. Experimental Materials and Equipment Strain source: Thermophilic pyriformis MT-ST / GP (CGMCC No. 41587), obtained from slant culture preserved in glycerol at -80℃; Culture medium formulation: Modified LB medium, specific components: peptone 10g / L (analytical grade from Sinopharm Group), yeast extract 5g / L (Angel Yeast industrial grade), NaCl 10g / L (food grade), glucose 5g / L (analytical grade), adjust pH to 7.1 with 1mol / L NaOH, add deionized water to each 1000mL of medium to make up the volume, and dispense into 250mL Erlenmeyer flasks (100mL of medium per flask). Equipment: Constant temperature shaking incubator (model THZ-300, temperature control accuracy ±1℃, speed range 0-300r / min), ultra-clean workbench (model SW-CJ-2FD, cleanliness level 100), high pressure steam sterilizer (model YXQ-LS-50SII), dissolved oxygen meter (model JPB-607A, accuracy 0.1mg / L), colony counter (model JJ-2).

[0031] 2. Detailed operating steps Culture medium sterilization: Seal the dispensed Erlenmeyer flasks and place them in an autoclave. Sterilize at 121℃ and 0.1MPa for 30 minutes. After sterilization, remove the flasks and cool them to below 45℃ (to avoid scalding the culture medium). Inoculation: In a clean bench, use a sterile inoculation loop to pick up 1 cm² of slant culture (mycelium + spores) and inoculate it into the cooled modified LB medium (1 cm² of slant culture corresponds to 100 mL of medium). Shaking culture: Place the inoculated Erlenmeyer flasks into a constant temperature shaking incubator and set the parameters as follows: temperature 45℃, rotation speed 150r / min. During the culture process, monitor the dissolved oxygen content of the culture medium in real time using a dissolved oxygen meter. When the dissolved oxygen content is lower than 20%, maintain the dissolved oxygen content ≥25% by increasing the shaking speed (maximum 160r / min) or opening the vent membrane of the Erlenmeyer flask (aseptic operation). The total culture time is 24 hours. Seed culture sampling: After the culture is completed, use a sterile pipette to take 10 mL of seed culture and place it in a sterile centrifuge tube for subsequent cell concentration detection.

[0032] 3. Key Control Points Dissolved oxygen maintenance: Thermophilic MT-ST / GP is an aerobic fungus. Dissolved oxygen levels below 20% will cause the fungal growth to stop. It is necessary to ensure sufficient dissolved oxygen by adjusting the rotation speed or controlling the aeration. This operation is based on the "Dissolved Oxygen Management" requirements in "Recommended Culture Medium Formulation 3.docx". pH stability: The addition of NaCl (10 g / L) to the modified LB medium can simulate a low-salt environment, preventing the strain from becoming inactive due to sudden changes in osmotic pressure. At the same time, the buffering effect of yeast extract and peptone can maintain the pH at 7.0-7.2 without the need for additional adjustment.

[0033] 4. Detection methods and results Cell concentration detection: Plate count method was used, and the seed culture was serially diluted to 10⁻⁶ with sterile physiological saline. -6 Take 0.1 mL of the diluted solution and spread it evenly on a PDA solid plate (formulation: potato 200 g / L, glucose 20 g / L, agar 15 g / L, pH natural). Incubate at 50℃ for 48 h, and then count the colonies using a colony counter. Actual testing showed that the bacterial cell concentration in the seed culture reached 1×10⁻⁶. 7 CFU / mL, relative standard deviation (RSD) of 3 parallel experiments ≤5%.

[0034] Activity verification: 1 mL of seed culture was inoculated into cellulase liquid culture medium (formulation: sodium carboxymethyl cellulose 10 g / L, peptone 5 g / L, yeast extract 3 g / L, NaCl 5 g / L, sodium bicarbonate 2 g / L, pH 7.0), and cultured at 50℃ and 180 r / min for 72 h. The cellulase activity was measured to be 50 U / mL, proving that the seed culture cells have normal metabolic activity.

[0035] 5. Results Analysis and Verification The modified LB medium in this embodiment increases the bacterial concentration by 30% compared to the traditional LB medium (without glucose) (the bacterial concentration of traditional LB medium after 24 hours of culture is approximately 7.7 × 10⁻⁶). 6 The addition of glucose (CFU / mL) is because it provides the strain with a readily available carbon source, accelerating logarithmic growth; at the same time, the addition of 10 g / L NaCl allows the strain to adapt to salt tolerance in advance, avoiding osmotic pressure shocks during subsequent fermentation.

[0036] Example 2 High-density fermentation 1. Experimental Materials and Equipment Seed culture: Logarithmic growth phase seed culture prepared in Example 1 (cell concentration 1×10⁻⁶) 7 (CFU / mL) Fermentation medium: Corn straw hydrolysate composite culture medium, specific preparation steps: Corn stalk pretreatment: Take fresh corn stalks and crush them to 20 mesh. Add 1% sulfuric acid solution at a solid-liquid ratio of 1:10. Hydrolyze at 121℃ for 30 min. After cooling, neutralize with 10% NaOH to pH 6.5. Filter to remove residue and obtain corn stalk hydrolysate (reducing sugar content ≥15g / L). Culture medium preparation: Add corn straw hydrolysate at a volume ratio of 0.8 L / L, then add soybean meal powder 12 g / L (crushed to 40 mesh), glucose 15 g / L, starch 9 g / L, and supplement with KH2PO4 1.0 g / L (to adjust buffer capacity). Adjust the pH to 7.0 with 1 mol / L HCl or NaOH, and dispense into 5L fermenters (3L of liquid per tank). Equipment: 5L mechanically stirred fermenter (model BLBIO-5GJ, with online monitoring of pH, dissolved oxygen, and temperature), autoclave, DNS reagent (for enzyme activity detection), UV-Vis spectrophotometer (model UV-1800).

[0037] 2. Detailed operating steps Culture medium sterilization: After the fermenter is sealed, it is sterilized with the following parameters: 115℃, 0.11MPa for 35min (to avoid high temperature from destroying the reducing sugars in the corn straw hydrolysate). After sterilization, sterile air is introduced to cool it to 48℃. Seed culture inoculation: Under aseptic conditions, add 5% volume of seed culture (150 mL of seed culture to 3 L of culture medium) through the inoculation port of the fermenter. After inoculation, close the inoculation port and turn on stirring and aeration. Initial fermentation stage (0-24h, cell proliferation period): Fermentation parameters were set as follows: temperature 48℃, initial pH 7.0 (maintained by automatic addition of 1mol / L HCl / NaOH), tank pressure 0.05MPa, and aeration rate 1.2L / (L). The stirring speed was 200 r / min, and samples were taken every 4 hours to test the bacterial concentration. Late fermentation stage (24-40h, enzyme activity induction period): After 24h of fermentation, when the cell concentration reaches 5×10⁻⁶... 8 At CFU / mL, segmented regulation is initiated: Temperature control: Lower the fermentation temperature to 42℃; Nutrient supplementation: Add 0.2 g / L MnSO4 (analytical grade, dissolved in sterile water), 10-15 g / L microcrystalline cellulose (cellulase-inducing substrate), 15-20 g / L xylan (hemicellulase-inducing substrate), and 1 g / L glucose (sterile aqueous solution) through the feeding port. The supplementation time is 1 hour to avoid excessive local concentrations that inhibit the cells. Parameter adjustment: Ventilation rate increased to 1.5 L / (L) (min), reduce the stirring speed to 180r / min (to reduce mycelial breakage), and continue fermentation for 16h; Fermentation endpoint determination: When the cell concentration no longer increases and the cellulase activity stabilizes, stop fermentation (total fermentation time 40h).

[0038] 3. Key Control Points Quality control of corn stalk hydrolysate: The sulfuric acid concentration must be strictly controlled at 1% during the hydrolysis process. Too high a concentration will lead to furfural formation (inhibiting cell growth), while too low a concentration will result in insufficient reducing sugar production. Segmented temperature control logic: In the early stage, the strains proliferate rapidly at 48℃, and in the later stage, enzyme secretion is induced at 42℃—because cellulase is an inducible enzyme, and a low temperature environment can promote enzyme gene expression. Mn 2+ Effect of supplementation: The addition of MnSO4 can activate the active site of cellulase in the strain. Experiments have shown that without Mn supplementation... 2+ In the control group, cellulase activity was only 60% of that in the supplemented group.

[0039] 4. Detection methods and results Cell concentration detection: Take 1 mL of fermentation broth, serially dilute it and spread it on a PDA plate, incubate at 50℃ for 48 h and count the cells, using the same method as in Example 1; Cellulase activity assay: The DNS method was used: 0.5 mL of fermentation broth supernatant was centrifuged at 4000 rpm for 10 min, 1 mL of 1% sodium carboxymethyl cellulose solution (prepared with pH 4.8 citrate buffer) was added, the mixture was reacted at 50 °C for 30 min, 1.5 mL of DNS reagent was added, the mixture was boiled in a water bath for 5 min, and after cooling, the absorbance was measured at 540 nm using a spectrophotometer. The enzyme activity was calculated using the glucose standard curve. Hemicellulase activity assay: The substrate was replaced with 1% xylan solution, and the other steps were the same as for cellulase assay; The results are shown in Table 1: Table 1

[0040] It can be seen that the RSD of the three parallel experiments is ≤8%, which fully meets the design goal of "high viable bacteria + high enzyme activity", and the fermentation cycle of 40h is 44% shorter than that of the traditional process (72h).

[0041] 5. Results Analysis and Verification In this embodiment, corn straw hydrolysate (agricultural waste) is used as the fermentation medium instead of traditional glucose medium, reducing raw material costs by 40% (the cost of traditional medium is about 2.5 yuan / L, while that of this medium is about 1.5 yuan / L). At the same time, the segmented temperature control strategy increases enzyme activity by 87.5% compared to fermentation at a fixed temperature (45℃), demonstrating the dual advantages of this process in terms of efficiency and cost.

[0042] Example 3: Preparation of bacterial agent by spray drying 1. Experimental Materials and Equipment Fermentation broth: Fermentation broth at the end of fermentation in Example 2 (viable cell count 5 × 10⁻⁶) 9 CFU / mL, cellulase activity 150 U / mL); Protectant: A compound protectant composed of 5% lactose (food grade), 3% trehalose (biochemical reagent) and 2% glycerol (analytical grade), which is dissolved in sterile water and then sterilized (sterilized at 121℃ and 0.1MPa for 20 min). Equipment: Centrifugal spray dryer (model LPG-5, inlet air temperature range 80-200℃, outlet air temperature range 40-100℃), sterile collection tank, moisture analyzer (model SFY-60), vacuum drying oven (for auxiliary testing).

[0043] 2. Detailed operating steps Protectant mixing: Under aseptic conditions, add the protectant to the fermentation broth at a mass fraction of 5% (e.g., add 50 mL of protectant to 1000 mL of fermentation broth), and mix with a sterile stirrer (500 r / min) for 30 min to ensure that the protectant is evenly dispersed; Spray drying parameter settings: inlet air temperature 135℃, outlet air temperature 70℃ (error ±2℃), feed rate 500mL / h, atomization pressure 0.2MPa; Drying process control: Start the spray dryer, first introduce hot air to stabilize the inlet air temperature to 135℃, then pump the fermentation liquid mixed with the protective agent into the atomizer, and the dried solid microbial agent falls into the sterile collection tank. During the collection process, maintain a slight positive pressure in the collection tank (introduce sterile air) to avoid external contamination. Post-treatment of microbial agents: The collected solid microbial agents are sealed in sterile aluminum foil bags (10g per bag) and stored in a dry environment at room temperature (25℃).

[0044] 3. Key Control Points The rationale behind the formulation of the preservatives is as follows: lactose can form a glassy matrix to encapsulate the bacteria, trehalose maintains the integrity of the cell membrane, and glycerol reduces moisture loss during the drying process. The combined use of these three preservatives, compared to lactose alone, increases the viable cell retention rate by 50%. Air inlet / outlet temperature control: If the air inlet temperature is too high (>150℃), it will cause denaturation of bacterial proteins; if it is too low (<120℃), the drying will not be complete (the moisture content of the bacterial agent is >10%). The air outlet temperature needs to be controlled at 60-80℃ to ensure that the moisture content of the bacterial agent is ≤5% (to avoid clumping during storage). In this embodiment, the final moisture content of the bacterial agent was 3.8%, which meets the requirements.

[0045] 4. Detection methods and results Viable bacteria count: Take 1g of solid bacterial agent, dissolve and serially dilute it with sterile physiological saline, spread it on PDA plates for counting (method as in Example 1), and calculate CFU / g; Enzyme activity assay: Take 1g of bacterial agent, add 10mL of sterile water, shake for 30min, centrifuge and collect the supernatant, and detect enzyme activity according to the DNS method in Example 2; Storage stability test: The sealed bacterial agent was placed in an environment of 25°C and 60% relative humidity, and samples were taken at 0 days (tested immediately after drying), 1 month, 3 months and 6 months to test the number of viable bacteria and enzyme activity; The results are shown in Table 2: Table 2

[0046] The enzyme activity retention rate was ≥87.5%, meeting the application requirements.

[0047] 5. Results Analysis and Verification The retention rate of live bacteria by spray drying is significantly higher than that of traditional hot air drying (retention rate <20%). This is due to the synergistic effect of composite protective agent and precise temperature control. At the same time, this process can produce 1 kg of solid bacterial agent per hour, which is suitable for large-scale industrial production.

[0048] Example 4: Preparation of bacterial agent by freeze-drying 1. Experimental Materials and Equipment Fermentation broth and protectant: Same as in Example 3; Equipment: Freeze dryer (model LGJ-10, freezing temperature range -50℃ to 0℃, vacuum range 1-100Pa), sterile freeze-drying bottles (10mL), ultra-low temperature freezer (-80℃, for auxiliary pre-freezing).

[0049] 2. Detailed operating steps Pre-freezing treatment: Pour the fermentation broth mixed with the protectant (10mL / bottle) into a sterile freeze-drying bottle and place it in an ultra-low temperature freezer at -80℃ for 4 hours to ensure that the fermentation broth is completely frozen (to avoid damage to the bacteria due to ice crystals during the freeze-drying process). Freeze-drying parameter settings: Primary drying: Freezing temperature -35℃, vacuum degree 15Pa, drying time 24h (to remove free water); Secondary drying: freezing temperature -20℃, vacuum degree 10Pa, drying time 6h (to remove bound water). Freeze-drying process control: Place the pre-frozen freeze-drying bottle into the freeze dryer, start the vacuum pump and refrigeration system, and monitor the vacuum level and temperature in real time to avoid the sample melting due to a sudden increase in vacuum level; Sealing of bacterial agent: After freeze-drying, seal the freeze-dried bottle with a sterile rubber stopper under vacuum and store at room temperature.

[0050] 3. Key Control Points Effect of pre-freezing rate: Pre-freezing at -80℃ (rapid freezing) can form small ice crystals, reduce the rupture of bacterial cell membranes, and increase the viable cell retention rate by 30% compared with slow pre-freezing (-20℃); Vacuum maintenance: The vacuum level needs to be kept stable at around 15Pa during the initial drying stage. If it is too low (<10Pa), the drying rate will slow down. If it is too high (>20Pa), the ice crystals will not sublimate completely, and the moisture content of the bacterial agent will exceed the standard.

[0051] 4. Detection methods and results Detection items and methods: Same as in Example 3 (live bacteria count, enzyme activity, storage stability); The results are shown in Table 3: Table 3

[0052] Freeze-dried bacterial agents have higher viable bacterial counts and enzyme activities than spray-dried bacterial agents, and their 6-month decay rate is only 16.7%, making them suitable for high-value-added scenarios (such as deep treatment of high-salt wastewater and precision agriculture). However, the equipment cost is relatively high (the unit price of a freeze dryer is about 500,000 yuan, and that of a spray dryer is about 200,000 yuan), so the process needs to be selected according to the application scenario.

[0053] 5. Results Analysis and Verification The viable cell retention rate of freeze-dried bacteria (approximately 60%) is significantly higher than that of spray-dried bacteria. This is because the low-temperature environment prevents the bacteria from being inactivated due to high-temperature stress. At the same time, the enzyme activity retention rate of freeze-dried bacteria in a high-salt environment (NaCl 10g / L) reaches 90%, while that of spray-dried bacteria is 80%, proving that it is more suitable for high-salt environments.

[0054] Example 5: Comparative Experiment of Synergistic Induction Effects of Multi-Components Experimental design: A control group (no feed), a single-component feed group (with MnSO4, microcrystalline cellulose, xylan, and NaCl added respectively), and a quaternary feed group (process of this invention) were set up. Each group was tested in 3 parallel experiments, and the fermentation conditions were the same as in Example 2.

[0055] Test indicators: cell concentration, cellulase activity, hemicellulase activity, and salt tolerance concentration after 40 hours of fermentation.

[0056] The experimental results are shown in Table 4: Table 4 Experimental Results

[0057] Results analysis: The cellulase activity of the quaternary combination group was 1.76 times that of the highest value of single-component feeding, the hemicellulase activity was 1.61 times, and the salt tolerance concentration was 1.67 times that of the NaCl single group. This proves that there is a significant synergistic effect among the four components, which is not a simple superposition of existing technologies and produces unexpected technical effects.

[0058] Example 6: Detection of Transcription Levels of Key Functional Genes Experimental methods: Fermented cells were collected for 24 h (proliferation phase) and 40 h (induction phase), respectively. Total RNA was extracted using the Trizol method and reverse transcribed into cDNA. The transcription levels of cellulase gene (cbh1), hemicellulase gene (xyn1), and salt tolerance-related gene (hog1) were detected by qPCR, with actin gene as an internal control.

[0059] The experimental results are shown in Table 5: Table 5 Experimental Results

[0060] Results analysis: The segmented regulation + multi-component feeding strategy of the present invention can specifically upregulate the transcriptional levels of cellulase, hemicellulase and salt tolerance-related genes, proving the mechanism of action of the method from the molecular level, rather than simply adjusting process parameters.

[0061] Example 7 Single-factor optimization experiment of drying process 7.1 Optimization of spray drying process Using viable bacteria retention rate and bacterial agent moisture content as evaluation indicators, single-factor optimization was performed on inlet air temperature, outlet air temperature, feed rate, and atomization pressure. The results showed that the optimal process window was: inlet air temperature 135℃, outlet air temperature 70℃, feed rate 500mL / h, and atomization pressure 0.2MPa. At this point, the viable bacteria retention rate reached 40%, and the bacterial agent moisture content was 3.8%, achieving the optimal balance between drying efficiency and activity retention.

[0062] 7.2 Optimization of freeze-drying process Using the viable cell retention rate as the evaluation index, single-factor optimization was performed on the pre-freezing temperature, pre-freezing time, primary drying temperature, and vacuum degree. The results showed that the optimal process parameters were: pre-freezing at -80℃ for 4 hours, primary drying at -35℃ / 15Pa for 24 hours, and secondary drying at -20℃ / 10Pa for 6 hours. At this time, the viable cell retention rate reached 60%, which was significantly higher than that of the traditional freeze-drying process (retention rate of about 35%).

[0063] The comparison results of the industrial adaptability of the two drying processes are shown in Table 6: Table 6 Comparison of Industrial Adaptability of Two Drying Processes

[0064] Example 8: Comparison Experiment with Existing Technologies The closest existing technologies (ZL201110290951.3 fixed-temperature fermentation process and conventional thermophilic fungal liquid fermentation process) were selected and compared with the process of this invention. The results are shown in Table 7: Table 7 Comparison with Existing Technologies

[0065] Example 9: AI-assisted integrated high-density fermentation Experimental materials and equipment: Seed culture: Logarithmic phase seed culture prepared in Example 1 (cell concentration 1×10⁻⁶) 7 CFU / mL, mycelial fragment length 150μm±20μm; Fermentation medium: corn straw hydrolysate composite medium (0.8L / L corn straw hydrolysate, 12g / L soybean meal powder, 15g / L glucose, 9g / L starch, 1.0g / L KH2PO4, pH 7.0); Reagents: rhamnolipid (purity ≥95%, food grade), other reagents as in Example 2; Equipment: 5L mechanically stirred fermenter (with online dissolved oxygen, pH, and turbidity acquisition functions), laser particle size analyzer (Malvin Mastersizer 3000), scanning electron microscope (SEM, Hitachi SU8010), high-performance computer (equipped with Python 3.9, Scikit-learn, TensorFlow framework), other equipment as in Example 2.

[0066] Operating steps: (1) Pre-run of the dual-module AI model: Input the parameters of the raw materials for this fermentation (reducing sugar content of corn straw hydrolysate is 16.2 g / L), fermentation tank volume is 5 L, ambient temperature is 25 ℃, and ambient humidity is 55%. The optimal initial fermentation parameters are output through the initial parameter pre-optimization module: fermentation temperature is 48 ℃, initial pH is 7.0, tank pressure is 0.05 MPa, and aeration rate is 1.2 L / (L (min), initial stirring speed 180 r / min; (2) Sterilization and inoculation of culture medium: Fermentation culture medium was dispensed into 5L fermenters (3L of liquid), sterilized at 115℃ and 0.11MPa for 35min, cooled to 48℃, and then inoculated into seed liquid at a rate of 5% under sterile conditions; (3) First stage (0-24h, precise control of mycelial ball particle size): Fermentation was started according to the initial parameters output by AI. Online data was collected in real time through the dynamic control module for mycelial ball growth, and the mycelial ball particle size prediction result was output every 2h. From 0-12h, the stirring speed was maintained at 180r / min. From 12-24h, the stirring speed was increased by 10r / min every 2h according to the particle size prediction result, and finally increased to 220r / min. After 24h, the mycelial ball particle size was tested and found to be stable at 220-280μm, with a particle size uniformity of 93.2%. (4) Second stage (24-40h, core-shell structure induction and synergistic acclimatization): At 24h, the AI ​​model predicts that the cells have entered the late logarithmic growth phase, and automatically switches fermentation parameters: the temperature is reduced to 42℃, the stirring speed is reduced to 180r / min, and the aeration rate is increased to 1.5L / (L (min); add 0.1 g / L rhamnolipin, 0.2 g / L MnSO4, 12 g / L microcrystalline cellulose, 18 g / L xylan, and 1 g / L glucose at one time; at the same time, based on the salt tolerance threshold of the cells predicted by the model, add NaCl in three gradients, 4 g / L each time, at 4 h intervals, for a total of 12 g / L; continue fermentation for 40 h to terminate; (5) Preparation of microbial agent: Add 5% by mass of compound protective agent (5% lactose + 3% trehalose + 2% glycerol) to the fermentation broth, mix evenly, and spray dry at an inlet air temperature of 135℃, an outlet air temperature of 70℃, a feed rate of 500mL / h, and an atomization pressure of 0.2MPa to obtain the finished solid microbial agent.

[0067] The test results showed that the final fermentation broth concentration reached 8.9 × 10⁻⁶ cells / day. 9 CFU / mL, cellulase activity 192 U / mL, hemicellulase activity 224 U / mL, salt tolerance up to 25 g / L NaCl; finished solid inoculum: viable count up to 3.6 × 10⁻⁶ 10CFU / g, cellulase activity 154 U / g, hemicellulase activity 179 U / g, viable cell decline rate 15.2% after 6 months of storage at room temperature, enzyme activity retention rate 80.2%.

[0068] Example 10: Construction and Validation of a Dual-Module AI Machine Learning Model The dataset was constructed by collecting 150 sets of historical fermentation data of thermophilic and salt-tolerant Mycorrhizal pyridostigma, covering different raw material batches, fermenter specifications, environmental conditions, operating parameters and corresponding fermentation results. The dataset was divided into a training set (120 sets) and a test set (30 sets) in an 8:2 ratio.

[0069] Model building and training: (1) Initial parameter pre-optimization module: Based on the random forest regression algorithm, the number of decision trees is set to 100 and the maximum depth is 15. The input features are the reducing sugar content of raw materials, the volume of fermenter, the ambient temperature and the ambient humidity. The output targets are the cell concentration at the end of fermentation and the cellulase activity. The model is trained by five-fold cross-validation. (2) Dynamic regulation module for mycelial ball growth: It is constructed based on CNN convolutional neural network. The input layer is the time series data of four features collected online during the fermentation process: dissolved oxygen, pH, stirring speed and turbidity. The convolutional layer is set with 2 convolutional blocks, the fully connected layer is set with 2 hidden layers, and the output layer is the predicted results of mycelial ball size and mycelial growth stage. The Adam optimizer and mean squared error loss function are used to train the model.

[0070] Model validation results: Initial parameter pre-optimization module: training set determination coefficient R²=0.962, test set R²=0.927, parameter prediction accuracy 93.1%; Mycelial ball growth dynamic regulation module: mycelial ball particle size prediction average absolute error ≤25μm, prediction accuracy 91.4%, cell growth stage identification accuracy 95.2%, can accurately determine the switching node between the proliferation period and the enzyme production period.

[0071] Example 11 Technical Comparison and Verification The experimental design included four parallel experiments, with three replicates per group. The basic fermentation medium, inoculum size, fermentation cycle, and post-treatment process were completely identical, with only the core fermentation strategy being adjusted to verify the synergistic effect of the integrated technology. Control group 1: Original basic process (no AI assistance, no mycelium ball control, fixed rotation speed 200 r / min, fixed temperature 48℃, original process of Example 2); Control group 2: AI-assisted fermentation process only (initial parameters were optimized using AI only, without two-stage mycelial ball regulation). Control group 3: Two-stage mycelial ball control process only (no AI assistance, fixed gradient rotation speed and switching time); Experimental group: The integrated AI + mycelial ball regulation process of this invention (process of Example 14).

[0072] The test results are shown in Table 8: Table 8 Test Results

[0073] Results analysis: The core performance indicators of the experimental group were not only significantly better than those of the single-technology control group, but also better than the average performance of control groups 2 and 3. This proves that AI assistance and precise control of mycelial balls are not simply superimposed, but produce an unexpected synergistic effect: AI dynamic control solves the lag problem of fixed rotation speed / fixed time control, further improving the accuracy of mycelial ball control; while the precise control of mycelial ball morphology provides more stable training samples for the AI ​​model, improves the model prediction accuracy, and forms a positive closed loop, fundamentally breaking through the performance ceiling of a single technology.

[0074] Example 12: Pilot-scale industrialization verification of integrated technology Pilot-scale conditions: A 100L mechanically stirred fermenter was used, the fermentation medium was molasses-cake composite medium (low-cost industrial formula), the inoculum size was 5%, the fermentation cycle was 40 hours, and three batches of pilot-scale production were carried out continuously using the integrated process of this invention to verify the feasibility of industrial scale-up.

[0075] Pilot-scale results: The average cell concentration at the endpoint of the three pilot-scale fermentations reached 8.5 × 10⁻⁶. 9 The average cellulase activity was 187 U / mL, the average hemicellulase activity was 218 U / mL, and the batch-to-batch relative standard deviation was ≤2.7%. The finished microbial agent prepared by spray drying had an average viable count of 3.4 × 10⁻⁶ cells / mL. 10 The CFU / g viable cell attenuation rate after 6 months of storage at room temperature is ≤17%, which is highly consistent with the results of laboratory small-scale tests, proving that the process has excellent industrial scale-up stability and no scale-up effect.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a thermophilic mitochondrial MT-ST / GP inoculant, characterized in that, Includes the following steps: (1) Seed culture: Thermophilic MT-ST / GP was inoculated into seed culture medium and cultured at 42-48℃ and 140-160r / min for 20-28h, maintaining dissolved oxygen at 20%-30%, to obtain a cell concentration of 8×10⁻⁶. 6 ~1.2×10 7 Seed culture with CFU / mL and hyphal fragment length of 100-200μm; (2) AI-assisted integrated high-density fermentation: The seed liquid was inoculated into the fermentation medium at an inoculation rate of 4%-6%. The initial fermentation parameters were pre-optimized based on the dual-module AI machine learning model. The culture was carried out using an integrated strategy of precise graded regulation of two-stage mycelial balls with AI dynamic control coupled with segmented temperature control, multi-component synergistic induction and gradient salt tolerance acclimatization. (3) Preparation of microbial agent: Add 4%-6% of a composite protective agent to the fermentation broth at the end of fermentation, and obtain the finished solid microbial agent by spray drying or freeze drying.

2. The preparation method according to claim 1, characterized in that, The seed culture medium mentioned in step (1) is selected from any of the following: ① Tryptone-soybean broth: tryptone 16-18 g / L, soybean peptone 2-4 g / L, glucose 2-3 g / L, NaCl 4-6 g / L, K2HPO4 2-3 g / L, pH 7.1-7.5; ② Modified LB medium: peptone 9-11 g / L, yeast extract 4-6 g / L, NaCl 9-11 g / L, glucose 4-6 g / L, pH 7.0-7.

2.

3. The preparation method according to claim 1, characterized in that, The fermentation medium mentioned in step (2) is selected from any of the following: ① Corn stalk hydrolysate compound culture medium: corn stalk hydrolysate 0.7-0.9 L / L, soybean meal powder 10-13 g / L, glucose 14-16 g / L, starch 8-9.5 g / L, pH 6.5-7.5; ② Molasses-oilseed cake composite culture medium: molasses 15-25g / L, soybean meal powder 8-12g / L, rapeseed meal powder 8-12g / L, KH2PO4 0.8-1.2g / L, pH 6.5-7.5; ③ Wheat bran composite culture medium: wheat bran 48-52 g / L, ammonium sulfate 1.4-1.6 g / L, potassium dihydrogen phosphate 1.3-1.5 g / L, MgSO4 7H2O 0.4-0.6g / L, pH 6.5-7.

5.

4. The preparation method according to claim 1, characterized in that, The dual-module AI machine learning model mentioned in step (2) includes an initial parameter pre-optimization module and a mycelial ball growth dynamic regulation module, specifically: ① Initial parameter pre-optimization module: Based on the random forest algorithm, it takes the reducing sugar content of raw materials, fermenter volume, ambient temperature, and ambient humidity as input features, and the cell concentration and cellulase activity at the fermentation endpoint as output targets, and outputs the optimal initial fermentation parameters. The accuracy of the model on the test set is ≥92%. ②Mycelial ball growth dynamic control module: Based on CNN convolutional neural network, it takes dissolved oxygen, pH, stirring speed and fermentation broth turbidity data collected online during the fermentation process as input, predicts mycelial ball particle size distribution and cell growth stage in real time, and outputs dynamic adjustment parameters for stirring speed and aeration rate. The mycelial ball particle size prediction accuracy is ≥90%.

5. The preparation method according to claim 1, characterized in that, The AI-dynamically regulated two-stage mycelial ball precise hierarchical regulation coupled with segmented temperature control, multi-component synergistic induction, and gradient salt tolerance acclimatization integrated strategy described in step (2) is as follows: Phase 1: Based on the parameters output by the initial parameter pre-optimization module, maintain the fermentation temperature at 45-50℃, initial pH at 6.5-7.5, tank pressure at 0.03-0.08MPa, and aeration rate at 0.8-1.5L / (L). (min); The mycelial ball growth dynamic control module monitors the mycelial ball particle size in real time, and adjusts the shear force by gradually increasing the stirring speed. The stirring speed is 180 r / min from 0 to 12 h, and the speed is adjusted every 2 h from 12 to 24 h according to the particle size prediction results, gradually increasing to 200-220 r / min, so as to accurately control the mycelial ball particle size to be stable at 200-300 μm, and the particle size uniformity is ≥90%; Second stage: When the mycelial ball growth dynamic control module predicts that the mycelium has entered the late logarithmic growth phase, immediately switch the fermentation parameters: adjust the fermentation temperature to 40-45℃, reduce the stirring speed to 160-180 r / min, and increase the aeration rate to 1.2-1.5 L / (L (min); Add 0.05-0.15 g / L rhamnolipid biosurfactant, 0.1-0.3 g / L LmnSO4, 10-15 g / L microcrystalline cellulose, 15-20 g / L xylan, and 0.5-1.5 g / L glucose to the fermentation system in one go; At the same time, based on the salt tolerance threshold of the cells predicted by the model, add a total of 9-14 g / L NaCl in three gradients, with an interval of 4 h between each addition, to induce the mycelial balls to form a core-shell structure of "compact core-fluffy shell", thereby simultaneously achieving efficient enzyme production and improved salt tolerance.

6. The preparation method according to claim 1, characterized in that, The post-treatment of the microbial agent in step (3) includes: adding 4-6% by mass of a protectant to the fermentation broth, wherein the protectant is composed of 4%-6% lactose, 2%-4% trehalose and 1%-3% glycerol; then spray drying is performed with an inlet air temperature of 120-150℃ and an outlet air temperature of 60-80℃ to obtain a solid microbial agent; or freeze drying is performed with a freezing temperature of -40 to -30℃, a vacuum degree of 10-20Pa and a drying time of 24-36h to obtain a freeze-dried microbial agent.

7. The preparation method according to claim 1, characterized in that, Aseptic control is required during the preparation process: the culture medium is sterilized at 115-121℃ and 0.09-0.11MPa for 25-35 minutes. Inoculation and fermentation operations are carried out in an aseptic environment with a cleanliness level ≥10000 to avoid contamination by other microorganisms.

8. A thermophilic and salt-tolerant fungal agent of *Hypericum tumefaciens* MT-ST / GP, characterized in that... The thermophilic and salt-tolerant *Trichoderma tumefaciens* MT-ST / GP inoculum is prepared by the method according to any one of claims 1-7, wherein the active ingredient of the inoculum is *Trichoderma tumefaciens* MT-ST / GP, and it is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41587. The viable count in the inoculum is 8 × 10⁻⁶. 6 ~1.2×10 7 CFU / mL, cellulase activity 45~55U / mL, hemicellulase activity 75~85U / mL, salt tolerance 4~20 g / L NaCl, viable cell count decline rate ≤20% and enzyme activity retention rate ≥70% after 6 months of storage at room temperature.

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