Method for producing enzyme through mixed fermentation of bacillus of soybean meal

Abstract

The invention relates to the technical field of soybean meal fermentation, and discloses a soybean meal bacillus mixed fermentation enzyme production method, which comprises the following steps: S1, strain selection; s2, high-density fermentation; s3, preparing a complex microbial inoculant; s4, preparation and preservation; s5, crushing the soybean meal substrate; s6, pretreating a soybean meal substrate; s7, performing sterilization; s8, soybean meal fermentation; s9, extracting an enzyme preparation; s10, drying the fermented soybean meal; according to the scheme, three substrates, namely protein, starch and cellulose, are efficiently and specially attacked by a plurality of strains respectively, the degradation bottleneck problem caused by a single enzyme system or an incomplete enzyme system is solved, so that macromolecular substances of the soybean meal are decomposed more thoroughly and quickly, and residues after enzyme are extracted by adopting a two-stage countercurrent extraction technology, so that the yield of the soybean meal is improved. The probiotic feed additive is rich in mycoprotein, small peptide and incompletely degraded fiber, so that the probiotic feed additive with high quality can be prepared after simple drying, and resource utilization of by-products is realized.

Description

Technical Field

[0001] This invention belongs to the field of soybean meal fermentation technology, specifically a method for producing enzymes through mixed fermentation of soybean meal with Bacillus. Background Technology

[0002] Therefore, we propose a method for enzyme production through mixed fermentation of soybean meal with Bacillus subtilis. Summary of the Invention

[0003] In view of the above situation and to overcome the defects of the prior art, the present invention provides a method for enzyme production by mixed fermentation of soybean meal with Bacillus subtilis, which effectively solves the problem of Xx.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a method for producing enzymes through mixed fermentation of soybean meal with Bacillus subtilis, comprising the following steps: S1: Strain selection; Bacillus belye, Bacillus amyloliquefaciens, and Bacillus licheniformis were selected; S2: High-density fermentation: The *Bacillus belye*, *Bacillus amyloliquefaciens*, and *Bacillus licheniformis* obtained in S1 were subjected to high-density fermentation using a culture medium, and the cell concentration of each strain reached 5 × 10⁻⁶. 9 For fermentation equipment with a concentration of CFU / mL or higher, the equipment is configured as follows: a smart 500L fermenter equipped with high-precision online sensors for pH, dissolved oxygen (DO), temperature, oxidation-reduction potential (ORP), an automatic feeding system, a tail gas analysis module for real-time monitoring of O2 consumption rate (OUR) and CO2 release rate (CER), and process control software. All data is integrated into the central control system to achieve full-process digital management and historical data traceability. S3: Preparation of compound microbial agent: The optimal volume ratio of Bacillus belyssus, Bacillus amyloliquefaciens, and Bacillus licheniformis was determined by response surface methodology, resulting in a 18-25% increase in total enzyme activity of fermented soybean meal compared to other ratios, without any growth inhibition. S4: Formulation and Preservation: The compound bacterial solution is mixed with compound preservatives such as dextrin and trehalose, and then prepared into powdered bacterial agent by vacuum freeze-drying technology; S5: Soybean meal substrate grinding: Soybean meal is ground to a particle size of ≤150μm (above 100 mesh) using ultra-fine grinding technology, which significantly increases the specific surface area; S6: Pretreatment of soybean meal substrate: Mix crushed soybean meal with water at a material-to-water ratio of 2:5 (g / mL), add 0.15% (w / w) of citric acid and 0.05% (w / w) of malic acid to form a compound organic acid, and stir at 55℃ for 25 minutes. S7: Sterilization: Autoclave at 121℃ for 15 minutes, then rapidly cool to the inoculation temperature; S8: Soybean meal fermentation: adopts a segmented dynamic control fermentation process. Soybean meal is mixed and fermented in three stages in an automated fermenter, with pH, ​​DO (dissolved oxygen), and temperature monitored online throughout the process; S9: Enzyme Extraction: The fermentation product is extracted using a two-stage countercurrent extraction process. The first stage uses a small amount of water to obtain a high-concentration enzyme solution; the second stage uses fresh water to perform a secondary extraction on the residue. After combining the extracts, they are concentrated by ultrafiltration through a ceramic membrane, and then spray-dried (with the addition of an appropriate amount of carrier such as maltodextrin) to produce a powdered compound enzyme preparation with an enzyme activity recovery rate ≥92%. S10: Drying of fermented soybean meal: The wet residue after enzyme extraction is rich in small peptides and probiotics. After being dried by low-temperature airflow (≤80℃), it is made into high-quality fermented soybean meal feed with significantly improved protein digestibility.

[0005] Precise screening of preferred Bacillus belysin strains (1) Sample collection and pretreatment Select healthy crop rhizosphere soil or well-rotted organic fertilizer, weigh 10g of sample and add 90mL of sterile physiological saline, shake at 37℃ and 180r / min for 30 minutes to prepare 10 -1 Bacterial suspension; subsequently serially diluted to 10-10 -6 Spread 0.1 mL of the diluted solution onto LB solid medium and incubate at 37°C for 24-48 hours. (2) Function-oriented screening Clear zone screening: Select colonies that form a clear zone on the culture medium, measure the diameter of the clear zone (D) and the diameter of the colony (d), and select strains with D / d ≥ 2.5; Antibacterial activity screening: The initially screened strains were inoculated into LB liquid medium and cultured at 37℃ and 180r / min for 24 hours. The fermentation supernatant was mixed with the Fusarium oxysporum suspension and cultured at 28℃ for 12 hours. The inhibition rate was measured and strains with an inhibition rate ≥80% were screened. Molecular identification: Genomic DNA was extracted from the selected strains and PCR amplification was performed using Bacillus belysinus-specific primers. The amplified products were sequenced and compared with the sequences of Bacillus belysinus model strains (such as ATCCBAA-874) in GenBank. If the homology was ≥99.8%, it was identified as the target strain.

[0006] Preferred starch-directed screening of Bacillus amyloliquefaciens strains (1) Sample collection and pretreatment Select environmental samples rich in starch, such as activated sludge from a winery wastewater treatment pond, soil surrounding a starch processing plant, and well-rotted straw compost. Weigh 10g of the sample and add it to 90mL of sterile physiological saline. Shake at 37℃ and 200r / min for 40 minutes to prepare 10 samples. -1 Bacterial suspension; serially diluted to 10-1 -7Take 0.1 mL of the diluted solution and spread it on the starch selection medium, and incubate at 37℃ for 36-48 hours. (2) Function-oriented screening Starch degradation zone screening: Iodine solution (0.1 mol / L I2-KI solution) was added to the cultured plates, and colonies that formed obvious blue degradation zones were selected. The diameter of the degradation zone (D) and the diameter of the colony (d) were measured, and strains with D / d ≥ 3.0 were selected (the larger the degradation zone, the higher the amylase activity). Amylase activity assay: The initial screening strains were inoculated into starch liquid medium (soluble starch 15 g / L, peptone 8 g / L, yeast extract 5 g / L, pH 7.0) and cultured at 37℃ and 200 r / min for 24 hours. The α-amylase activity in the fermentation broth was determined by the DNS method, and strains with activity ≥2500 U / mL were screened. Molecular identification and stability verification: Genomic DNA was extracted from the screened strains and PCR amplification was performed using Bacillus amyloliquefaciens-specific primers. After sequencing, the amplified products were compared with the sequence of the model strain (such as DSM7T) in GenBank. The target strain was identified if the homology was ≥99.7%. The target strain was passaged 5 times consecutively, and the amylase activity was measured after each passage. The strain with activity fluctuation ≤5% was selected.

[0007] Preferred high-temperature directional screening of Bacillus licheniformis strains (1) Sample collection and pretreatment Select high-temperature environment samples, such as high-temperature composting, soil around hot springs, and high-temperature reactors for industrial wastewater treatment. Weigh 10g of the sample and add it to 90mL of sterile physiological saline. Shake at 45℃ and 200r / min for 40 minutes to prepare 10 samples. -1 Bacterial suspension; subsequently serially diluted to 10⁻ 7 Take 0.1 mL of the diluted solution and spread it on the high-temperature screening medium, and incubate at 45℃ for 36-48 hours. (2) Function-oriented screening Protease clear zone screening: Select colonies on the culture medium that form obvious protein degradation clear zones, measure the diameter of the clear zone (D) and the diameter of the colony (d), and select strains with D / d ≥ 3.0; High-temperature enzyme activity and stability assay: The initially screened strains were inoculated into a high-temperature liquid culture medium (15 g / L skim milk powder, 10 g / L corn flour, 8 g / L soybean meal powder, pH 7.5) and cultured at 45℃ and 200 r / min for 24 hours. The protease activity at 60℃ was determined using the Folin-phenol method, and strains with activity ≥2500 U / mL were screened. At the same time, the strains were cultured at 55℃ for 12 hours, and the enzyme activity retention rate was determined. Strains with a retention rate ≥85% were selected.

[0008] Preferably, the specific method for selecting the proportion of S3 compound microbial agent is as follows: In a clean bench, accurately measure the corresponding volume of seed liquid of each strain, mix them, and then evenly inoculate them into an Erlenmeyer flask containing soybean meal culture medium. Set up 3 replicates for each experimental point. Fermentation culture: Place all inoculated Erlenmeyer flasks in a constant temperature shaker and ferment for 54 hours under uniform optimized conditions (e.g., 34℃, 180rpm) to ensure that the culture conditions of all flasks are completely consistent. Termination of fermentation and sampling: After fermentation, immediately remove the Erlenmeyer flasks, add 100 mL of sterile physiological saline to each flask, shake thoroughly for 30 minutes, and extract the enzymes from the fermentation products; Preparation of crude enzyme solution: Centrifuge the mixture at 4℃ and 8000rpm for 15 minutes, and take the supernatant as crude enzyme solution.

[0009] Preferably, the three Bacillus strains selected (S1) all originated from specific habitats and underwent targeted functional enhancement. Bacillus berleis: isolated from high-temperature soybean meal compost (55-60℃), the strain needs to be induced and selected through mutagenesis. Its neutral protease activity is stable at 6800-7200U / g, and its phytase activity reaches 350-450U / g. Its optimal growth temperature range is wide (30-45℃), which can effectively start fermentation and degrade phytic acid. Bacillus amyloliquefaciens: isolated from high-starch-producing soil, it produces high levels of mesophilic α-amylase (activity 5200-5800 U / g) and can also secrete a small amount of glucanase, which can supplement the complex enzyme system. The antimicrobial lipopeptides produced by its metabolism can effectively inhibit the growth of miscellaneous bacteria. Bacillus licheniformis: isolated from paper mill waste piles, it has extremely strong cellulose and hemicellulose degradation capabilities (cellulase activity 1300-1600 U / g, xylanase activity 900-1100 U / g), and needs to be resistant to acids and alkalis (pH 5.0-9.0) and high temperatures (up to 60℃).

[0010] Preferably, this specifically includes the following steps: The specific process for the three stages of mixed fermentation of soybean meal in S8 is as follows: Phase 1: Colonization and Proliferation Period (1-18 hours), Strategic Goal: Rapidly construct a highly active, high-density bacterial cell factory. Core control strategy: Active regulation based on feedback of dissolved oxygen (DO) change rate and pH trend to reserve sufficient biomass for efficient enzyme production; Control parameters: Inoculation: Inoculate with a highly active compound freeze-dried bacterial agent (Bacillus belye: Bacillus amyloliquefaciens: Bacillus licheniformis ratio of 6:3:2) at 10% (v / w), and activate with sterile physiological saline at 35-39℃ for 12-18 min before use; Temperature control: kept constant at 33.8-34.2℃; pH regulation strategy: No active acid-base regulation is performed in this stage. The initial pH is 7.0-7.2. Due to the rapid metabolism of carbohydrates by the bacteria to produce acid, the pH will continue to decrease. Monitoring the rate of pH decrease (dpH / dt) can be used as a real-time indicator to judge the activity and metabolic intensity of the bacteria. Dissolved oxygen (DO) and stirring / ventilation linkage control: A constant DO cascade control mode is adopted, with the target dissolved oxygen (DO) value set at 28.5-31.5% saturation. The control system uses a PID algorithm to automatically cascade and adjust the stirring speed and ventilation rate. Prioritize adjusting the stirring speed: the initial setting is 140-160 rpm / min, and when DO decreases, the system prioritizes increasing the stirring speed; Secondary ventilation rate adjustment: When the stirring speed reaches the upper limit and still cannot maintain the set DO, the system automatically increases the ventilation rate, with an adjustment range of 1.0-1.5vvm; Oxygen-enriched air backup: In extremely high-density conditions, oxygen-enriched air can be activated to supplement oxygen supply (this design is an optional advanced function). Oxidation-reduction potential (ORP) monitoring: As an auxiliary reference indicator, the continuous decrease in ORP also confirms that the fermentation broth is in a vigorous reducing metabolic state. Intelligent determination of stage transition: When the pH value drops to the lowest point of 6.7-6.8, and then shows a continuous and stable upward trend for ≥10 minutes, it indicates that the most readily available free carbon source in the culture medium has been basically depleted, the cell proliferation rate has slowed down, and the metabolic focus has begun to shift to the synthesis of secondary metabolites. After the central control system captures this signal, it automatically triggers the program to switch to the next stage. This transition mechanism based on metabolic inflection points is more robust and adaptable than fixed-time control. Phase Two: Main Enzyme Production Period (18-42 hours); Core control strategy: Based on the pH-stat principle and the dynamics of carbon source consumption, implement a precise feeding fermentation strategy of "small amount and multiple times" to eliminate the carbon decomposition metabolism inhibition effect and maximize the enzyme synthesis rate. Refined control parameters: Temperature switching: Within one hour after the phase transition, the temperature is linearly increased and stably controlled at 36.8℃-37.22℃. This temperature is the optimal temperature for the expression of genes regulating the synthesis of various extracellular enzymes in Bacillus subtilis and Bacillus licheniformis, as well as for the activity of σ factor, and can strongly activate the entire enzyme production system; pH-stat precise control: Activate the active control mode to precisely control the pH of the fermentation broth within a narrow range of 6.85-6.95. Neutralize the organic acids produced by cell metabolism by automatically adding 6M NaOH to maintain a slightly acidic environment. Aeration and mixing optimization: The cell density reaches its peak (≥10¹) at this stage. 0 (CFU / mL) Exhaust gas analysis and monitoring: Real-time monitoring of carbon dioxide emission rate (CER). Intelligent determination of phase transition: When the enzyme production rate per unit time decreases significantly (by sampling every 3 hours with an online sampler, measuring enzyme activity, and calculating the enzyme activity increase of <5% at two consecutive time points), and / or the CER value decreases by more than 15% from the plateau phase, it indicates that the peak enzyme production period has passed and the cell activity has begun to decline. The system judges that it has entered the stable phase based on this. Phase 3: Enzyme activity stabilization and harvest period; Core control strategy: Low metabolism maintenance mode, all operations are based on the highest principle of protecting product stability, and the harvest endpoint is accurately determined; Refined control parameters: Cooling protection: The temperature is linearly reduced and eventually stabilized at 27.5-28.5℃ within 2 hours. Lowering the temperature can significantly reduce the activity of the bacterial cell's own metabolic enzymes (such as proteases) and extracellular proteases. This is the most critical and effective measure to prevent the degradation of secreted enzyme proteins, and the enzyme activity retention rate can be increased by more than 15%. pH fine-tuning: The pH control target was slightly lowered to 6.75-6.85, which is closer to a neutral to slightly acidic environment, further enhancing the long-term stability of the enzyme protein; Stop feeding: Stop the addition of all carbon and nitrogen sources, allowing the cells to gradually enter the endogenous respiration stage and reduce unnecessary metabolic byproducts; Aeration and stirring attenuation: Simultaneously reduce the stirring speed to 100-120 rpm and the aeration rate to 0.5-0.8 vvm. Maintaining DO at 15-20% is sufficient; excessively high DO is unnecessary. This significantly reduces the risk of physical damage to spatially structured enzyme proteins caused by mechanical shear forces, while also saving energy. Process monitoring: Continuously monitor the trend of enzyme activity changes and cell autolysis indicators (such as the decrease in absorbance of fermentation broth at 600nm wavelength). Precise determination of fermentation termination: When the total enzyme activity (weighted sum of protease, amylase, and cellulase activities) shows no significant increase in two consecutive sampling measurements (difference <2%), or when a statistically significant downward trend first appears (decline >3%), the central control system issues an alarm, prompting operators to immediately terminate fermentation and start the downstream extraction program to ensure harvesting at the absolute peak of enzyme activity, maximizing the benefits of single-batch fermentation. The total enzyme activity at the fermentation endpoint can reach 18,500-21,500 U / g.

[0011] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention utilizes multiple strains of bacteria to efficiently target the three major substrates of protein, starch, and cellulose, while simultaneously degrading phytic acid, a key anti-nutritional factor. This solves the degradation bottleneck caused by single or incomplete enzyme systems, making the decomposition of macromolecules in soybean meal more thorough and faster. 2. The antimicrobial lipopeptides produced by Bacillus amyloliquefaciens metabolism used in this invention can effectively inhibit contamination by miscellaneous bacteria such as mold and Escherichia coli in the fermentation system, and have the efficiency of a natural preservative. This not only reduces the energy consumption and cost requirements of sterilization, but also greatly improves the success rate and stability of fermentation. 3. The Bacillus licheniformis used in this invention has extremely strong acid and alkali resistance and heat resistance, which can effectively buffer pH and temperature changes caused by metabolic fluctuations during fermentation, ensuring stability in the later stages of the entire fermentation process and reducing batch-to-batch differences. 4. Based on multi-parameter online monitoring, this invention realizes dynamic intelligent control based on the physiological state of fermentation, enabling the process to be switched at the optimal time for each batch of fermentation, maximizing the utilization of cell growth and enzyme production potential, and ensuring high reproducibility of results. 5. This invention constructs a complete green circular process chain: using a two-stage countercurrent extraction technology, the residue after one centrifugation is extracted a second time. The residue after enzyme extraction, because it is rich in bacterial protein, small peptides and incompletely degraded fiber, can become a high-quality probiotic feed additive after simple drying. It can be used for ruminant or aquatic feed, realizing the resource utilization of by-products. Attached Figure Description

[0012] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0013] In the attached diagram: Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0014] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0015] Example: Includes the following steps: S1: Strains selected; Bacillus belyceae, Bacillus amyloliquefaciens, and Bacillus licheniformis were chosen. All three strains originated from specific habitats and underwent targeted functional enhancement. Bacillus berleis: isolated from high-temperature soybean meal compost (55-60℃), this strain was bred through mutagenesis, and its neutral protease activity is stable at 6800-7200U / g, and its phytase activity reaches 350-450U / g. Its optimal growth temperature range is wide (30-45℃), which can effectively initiate fermentation and degrade phytic acid. *Bacillus amyloliquefaciens*: isolated from high-starch-producing soils. It produces a high amount of mesophilic α-amylase (activity 5200-5800 U / g) and also secretes a small amount of glucanase, which can supplement the complex enzyme system. Its metabolically produced antimicrobial lipopeptides can effectively inhibit the growth of other bacteria. Bacillus licheniformis: isolated from paper mill waste, it possesses extremely strong cellulose and hemicellulose degradation capabilities (cellulase activity 1300-1600 U / g, xylanase activity 900-1100 U / g). Its acid and alkali resistance (pH 5.0-9.0) and high temperature resistance (up to 60℃) make it suitable as a "stabilizer" in fermentation systems. S2: High-density fermentation: The *Bacillus belye*, *Bacillus amyloliquefaciens*, and *Bacillus licheniformis* obtained in S1 were subjected to high-density fermentation using a culture medium, and the cell concentration of each strain reached 5 × 10⁻⁶. 9 For fermentation equipment with a concentration of CFU / mL or higher, the culture medium needs to be optimized. The optimization method is as follows: add soybean meal powder to Bacillus belye to induce protease, add soluble starch to Bacillus amyloliquefaciens, and add microcrystalline cellulose to Bacillus licheniformis. The fermentation equipment configuration is as follows: a smart 500L fermenter equipped with high-precision online sensors for pH, dissolved oxygen (DO), temperature, and oxidation-reduction potential (ORP), an automatic feeding system, an exhaust gas analysis module for real-time monitoring of O2 consumption rate (OUR) and CO2 release rate (CER), and process control software. All data are integrated into the central control system to achieve full-process digital management and historical data traceability. Precise screening of Bacillus belysin strains (1) Sample collection and pretreatment Select healthy crop rhizosphere soil (such as soil at a depth of 10-20 cm in the rhizosphere of tomatoes and cucumbers, where Bacillus belye has a high enrichment) or well-rotted organic fertilizer, weigh 10g of sample and add 90mL of sterile physiological saline, shake at 37℃ and 180r / min for 30 minutes to prepare 10 -1 Bacterial suspension; subsequently serially diluted to 10-10 -6 Take 0.1 mL of the diluted solution and spread it on LB solid medium (with 0.1% chitin powder added for preliminary screening of chitinase-producing strains), and incubate at 37°C for 24-48 hours. (2) Function-oriented screening Clear zone screening: Select colonies that form a clear zone (chitin degradation zone) on the culture medium, measure the diameter of the clear zone (D) and the diameter of the colony (d), and select strains with D / d≥2.5 (these strains have higher chitinase activity). Antibacterial activity screening: The initially screened strains were inoculated into LB liquid medium and cultured at 37℃ and 180r / min for 24 hours. The fermentation supernatant was mixed with Fusarium oxysporum (a representative bacterium of soil-borne diseases) suspension and cultured at 28℃ for 12 hours. The inhibition rate was measured and strains with an inhibition rate ≥80% were screened. Molecular identification: Genomic DNA was extracted from the selected strains and PCR amplification was performed using Bacillus belyssus-specific primers (upstream primer: 5'-GAGTTTGATCCTGGCTCAG-3', downstream primer: 5'-CTACGGCTACCTTGTTACGA-3'). After sequencing, the amplified products were compared with the sequences of Bacillus belyssus model strains (such as ATCCBAA-874) in GenBank. The target strain was identified if the homology was ≥99.8%.

[0016] Low-cost culture medium formulation optimization (1) Design of basal culture medium components Based on the principles of "low cost, high carbon-to-nitrogen ratio, and suitability for metabolic needs", the formula is as follows (unit: g / L). Carbon source: Corn flour 15 (replaces glucose, reduces costs by 40%, and provides a continuous carbon source supply); Nitrogen source: 10g soybean meal powder + 2g yeast powder (soybean meal powder replaces part of the yeast powder, reducing protein source cost by 35%. The soybean meal powder needs to be pulverized through an 80-mesh sieve to ensure full dissolution). Trace elements: MgSO4・7H2O 0.5, MnSO4・H2O 0.1, FeSO4・7H2O 0.05 (promotes bacterial enzyme activity and spore formation); pH adjustment: Adjust the pH to 7.0-7.2 with 1mol / L NaOH or HCl, and autoclave at 121℃ for 20 minutes before use; (2) Validation of culture medium BV-01 strain was inoculated into optimized medium and cultured at 37℃ and 180 r / min for 24 hours, achieving a cell density of 1.2 × 10¹. 0 CFU / mL, compared to LB medium (8×10⁻⁶) 9 The CFU / mL concentration was increased by 50%; the chitinase activity reached 230 U / mL, meeting the requirements for disease resistance applications, and the cost of the culture medium was reduced from 8 yuan / L of LB medium to 5.2 yuan / L, a cost reduction of 35%. 3. Segmented fermentation process Large-scale cultivation was carried out using a 500L fermenter, and the process was divided into two stages: the "vegetative growth period" and the "spore formation period." The specific parameters are as follows: (1) Vegetative growth period (0-24 hours, target: to achieve high bacterial count; Starch-directed screening of Bacillus amyloliquefaciens strains (1) Sample collection and pretreatment Samples from starch-rich environments were selected, such as activated sludge from winery wastewater treatment ponds, soil surrounding starch processing plants, and well-rotted straw compost (these environments have high enrichment of Bacillus amyloliquefaciens, and the strains have strong starch degradation capabilities). 10g of sample was weighed and added to 90mL of sterile physiological saline (containing 0.5% soluble starch). The mixture was shaken at 37℃ and 200r / min for 40 minutes (to induce starch-induced expression of amylase in the strains), resulting in 10... -1 Bacterial suspension; serially diluted to 10-1 -7 Spread 0.1 mL of the diluted solution onto starch screening medium (formulation: 10 g / L soluble starch, 5 g / L peptone, 3 g / L yeast extract, 20 g / L agar, pH 7.0) and incubate at 37°C for 36-48 hours. (2) Function-oriented screening Starch degradation zone screening: Iodine solution (0.1 mol / L I2-KI solution) was added to the cultured plates, and colonies that formed obvious blue degradation zones were selected. The diameter of the degradation zone (D) and the diameter of the colony (d) were measured, and strains with D / d ≥ 3.0 were selected (the larger the degradation zone, the higher the amylase activity). Amylase activity assay: The initial screening strains were inoculated into starch liquid medium (soluble starch 15 g / L, peptone 8 g / L, yeast extract 5 g / L, pH 7.0) and cultured at 37℃ and 200 r / min for 24 hours. The α-amylase activity in the fermentation broth was determined by the DNS method, and strains with activity ≥2500 U / mL were screened. Molecular identification and stability verification: Genomic DNA was extracted from the screened strains and PCR amplification was performed using Bacillus amyloliquefaciens-specific primers (upstream primer: 5'-CGGCGTATTAGCTAGTTGGT-3', downstream primer: 5'-AGTTGATAGCAGCTTCTCGT-3'). The amplified products were sequenced and compared with the sequence of the type strain (such as DSM7T) in GenBank. The target strain was identified if the homology was ≥99.7%. The target strain was passaged five times consecutively, and the amylase activity was measured after each passage. The strain with activity fluctuation ≤5% was selected and named Bacillus amyloliquefaciens BA-02. 2. Optimization of low-cost composite carbon source culture medium (1) Composite carbon source formulation design Based on the principles of "induced enzyme production + low cost + high solubility", the core carbon source components were optimized, and the complete culture medium formula is as follows (unit: g / L): Composite carbon source: Corn starch 8 (main inducing carbon source, cost 2.5 yuan / kg) + maltodextrin 5 (auxiliary carbon source, solubility ≥95%, improving carbon source utilization, cost 3 yuan / kg) + waste molasses 3 (industrial by-product, supplementing trace elements, cost 0.8 yuan / kg), the total cost of carbon source is reduced by 42% compared with pure corn starch; Nitrogen source: Soybean meal hydrolysate 12 (soybean meal is hydrolyzed with 0.5% sulfuric acid for 2 hours, filtered to obtain the supernatant, replacing high-priced peptone, cost 1.5 yuan / kg) + yeast powder 2 (to supplement growth factors); Inorganic salts: Na₂HPO₄ 2.5%, KH₂PO₄ 1.5% (to maintain pH stability), MgSO₄·7H₂O 0.6%, CaCl₂ 0.2% (to activate amylase activity); pH adjustment: Adjust the pH to 7.2-7.4 with 1 mol / L NaOH, then autoclave at 121℃ for 20 minutes (after sterilization, starch solubility will increase to 5%-8%, preventing clumping). High bacterial activity and stable function: Through function-guided screening, the obtained BV-01 strain has an antibacterial rate of ≥80% and a chitinase activity of ≥200U / mL, which is more than 40% higher than that of randomly screened strains. Low cost and high efficiency: The optimized culture medium uses corn flour and soybean meal as the main raw materials, reducing costs by 35%. The segmented fermentation process enables the cell density to reach 10¹¹ CFU / mL, shortens the fermentation cycle to 36 hours, and increases production efficiency by 30%. The spores are of high quality and have a wide range of applications: the spore conversion rate is ≥90%, and the heat resistance (survival rate ≥90% after treatment at 80℃ for 10 minutes) is superior to existing technologies. They can be made into liquid or solid bacterial agents and are suitable for applications in agriculture, animal husbandry and other fields. (2) Validation of culture medium performance The BA-02 strain was inoculated into an optimized culture medium and cultured at 37°C and 200 rpm for 24 hours, resulting in a cell density of 1.8 × 10¹. 0 CFU / mL, compared to pure starch medium (9×10⁻⁶) 9 The CFU / mL ratio was increased by 100%; α-amylase activity reached 2800 U / mL, and surfactant production reached 120 mg / L, meeting the needs of industrial applications; the total cost of the culture medium was reduced from 6.8 yuan / L for pure starch culture medium to 3.2 yuan / L, a cost reduction of 53%. 3. Segmented co-fermentation process for enzyme production and spore production Large-scale production is carried out using a 1000L fermenter, and the process is divided into two stages: the "enzyme production period" and the "spore induction period," achieving a dual improvement in enzyme activity and spore rate. The specific parameters are as follows: (1) Enzyme production period (0-28 hours, goal: maximize amylase accumulation) Inoculation and initial parameters: BA-02 seed culture was inoculated at a rate of 6% (v / v) and cultured at 37℃, 200 r / min for 14 hours, with a cell density of 1.2 × 10⁻⁶. 9 (CFU / mL) is added to the fermenter; stirring speed is 200r / min (to ensure uniform starch dispersion and avoid local precipitation), aeration rate is 1.8vvm (to maintain dissolved oxygen concentration ≥25% to meet the high oxygen consumption requirements during enzyme production), and temperature is controlled at 37℃; Feeding strategy: At 12 hours, add a compound carbon source (3g / L corn starch + 2g / L maltodextrin, dissolved and added via a sterile feed pump) to avoid carbon source depletion leading to enzyme production stagnation; at 24 hours, add 5g / L soybean meal hydrolysate to prolong the logarithmic phase of enzyme production; at 28 hours, α-amylase activity reaches its peak (3000U / mL), entering the spore induction phase. (2) Spore induction period (28-40 hours, goal: to induce spore formation and maintain enzyme activity) Parameter adjustments: Temperature reduced to 34℃ (the optimal temperature for Bacillus amyloliquefaciens spore formation, to avoid enzyme denaturation due to high temperature), stirring speed reduced to 150r / min (to reduce energy consumption and protect the enzyme protein structure), and aeration rate reduced to 1.2vvm (to maintain dissolved oxygen at 18%-22%, as a low-oxygen environment promotes spore differentiation). Spore inducer addition: At 28 hours, add 0.4 g / L KCl (to regulate intracellular osmotic pressure and activate spore formation genes) and 0.15 g / L MnSO4 (to promote spore cortex synthesis and enhance stress resistance). Monitoring and Termination: Samples were taken every 2 hours, and amylase activity and spore rate were measured simultaneously (Gram staining microscopy): Fermentation was stopped when the spore rate was ≥85% and the amylase activity retention rate was ≥90% (i.e., activity ≥2700U / mL), and the fermentation cycle was controlled within 40 hours. 4. Product collection and dosage form preparation (1) Liquid enzyme-spore complex product Centrifugal concentration: Centrifuge the fermentation broth for 20 minutes using a disc centrifuge (9000 r / min, separation factor 2000×g) and collect the mixture of cell (containing spores) and supernatant (containing amylase); Stabilizer addition: Add 1.5% glycerol (enzyme protectant) + 0.2% potassium sorbate (preservative) to the mixture, stir well, and then filter through a 0.22μm microfiltration membrane for sterilization to prepare a liquid product with α-amylase activity ≥2500U / mL and spore content ≥10. 9 CFU / mL, shelf life is 12 months when refrigerated at 4℃, and 6 months when stored at room temperature (enzyme activity decreases by 18%). (2) Solid enzyme-spore complex products Spray drying: The centrifuged mixture is fed into a spray drying tower (inlet air temperature 170℃, outlet air temperature 75℃, feed rate 20mL / min), and 5% maltodextrin is added as a carrier to produce a powder product. Quality control: Solid products: moisture content ≤8%, α-amylase activity ≥2200U / mL, spore content ≥10¹ 0 CFU / g, shelf life 18 months when stored sealed at 25℃ (enzyme activity decrease ≤15%), suitable for feed, agriculture and other applications requiring long-term storage. The strain exhibits strong targeting and stable function: Through starch-directed screening, the BA-02 strain obtained has an α-amylase activity ≥2500U / mL, with activity fluctuation ≤5% after continuous subculturing, which is 150% higher than that of randomly screened strains; Low cost and high resource utilization: By combining industrial by-products (waste molasses, soybean meal hydrolysate) with low-cost carbon sources, the cost of culture medium is reduced by 53%, and the carbon source utilization rate is increased to over 90%, resulting in significant economic benefits for industrialization. Synergistic enzyme and spore production, with wide product applicability: The segmented process achieves an amylase activity retention rate of ≥90% and a spore rate of ≥85%, and can be made into liquid or solid products, suitable for multiple fields such as food, agriculture, and animal husbandry. The storage stability is 2-3 times better than existing products. (1) Sample collection and pretreatment Select high-temperature environment samples, such as high-temperature composting, soil around hot springs, and high-temperature reactors for industrial wastewater treatment. Weigh 10g of the sample and add it to 90mL of sterile physiological saline. Shake at 45℃ and 200r / min for 40 minutes to prepare 10 samples. -1 Bacterial suspension; subsequently serially diluted to 10⁻ 7 Take 0.1 mL of the diluted solution and spread it on the high-temperature screening medium (formulation: 10 g / L skim milk powder, 8 g / L corn flour, 5 g / L soybean meal powder, 20 g / L agar, pH 7.5), and incubate at 45℃ for 36-48 hours. (2) Function-oriented screening Protease clear zone screening: Select colonies that form obvious protein degradation clear zones on the culture medium, measure the diameter of the clear zone (D) and the diameter of the colony (d), and select strains with D / d≥3.0 (the larger the clear zone, the higher the high temperature protease activity). High-temperature enzyme activity and stability assays: The initially screened strains were inoculated into a high-temperature liquid culture medium (15 g / L skim milk powder, 10 g / L corn flour, 8 g / L soybean meal powder, pH 7.5) and cultured at 45℃ and 200 r / min for 24 hours. The protease activity at 60℃ was determined using the Folin-phenol method, and strains with activities ≥2500 U / mL were screened. Simultaneously, the strains were cultured at 55℃ for 12 hours, and the enzyme activity retention rate was determined. Strains with a retention rate ≥85% were selected. Molecular identification and spore stress resistance verification: Genomic DNA was extracted from the screened strains and PCR amplification was performed using Bacillus licheniformis-specific primers (upstream primer: 5'-AGCGGCGGACGGGTGAGTA-3', downstream primer: 5'-TCCTCCGCTTATTGATATGC-3'). After sequencing, the amplified products were compared with the sequence of the type strain (e.g., ATCC14580) in GenBank. The target strain was identified if the homology was ≥99.7%. The target strains were cultured to the spore formation stage, and the spore suspension was treated at 100℃ for 10 minutes. The survival rate was measured, and the strains with a survival rate ≥90% were selected and named Bacillus licheniformis BL-03. Low-cost composite culture medium optimization (1) Design of compound culture medium formulation Based on the principles of "high temperature compatibility + low cost + high utilization rate", the core carbon and nitrogen source components were optimized, and the complete culture medium formula is as follows (unit: g / L): Composite carbon source: Corn flour 12 (main carbon source, cost 2.5 yuan / kg, providing a continuous carbon source supply) + wheat bran hydrolysate 8 (wheat bran is hydrolyzed with 0.3% hydrochloric acid for 3 hours, the supernatant is filtered and taken, containing rich polysaccharides and small molecule carbon sources, cost 0.6 yuan / kg) + waste molasses 3 (industrial by-product, supplementing trace elements, cost 0.8 yuan / kg), the total cost of carbon source is reduced by 45% compared to glucose; Compound nitrogen source: 10g soybean meal powder (crushed to 80 mesh, hydrolyzed to release small molecule peptides, cost 1.5 yuan / kg) + 1.5g yeast powder (supplementing growth factors and reducing the amount of high-priced protein source used) + 2g ammonium sulfate (inorganic nitrogen source, adjusting the carbon-nitrogen ratio, cost 0.3 yuan / kg), reducing the protein source cost by 42% compared to LB medium; High-temperature compatible inorganic salts: K2HPO4 2.0, KH2PO4 1.0 (maintain pH stability and enhance bacterial metabolism at high temperatures), MgSO4・7H2O 0.8 (activate enzyme activity at high temperatures), CaCO3 1.5 (buffer pH fluctuations at high temperatures). pH adjustment: Adjust the pH to 7.5-8.0 (the optimal pH range for Bacillus licheniformis) with 1 mol / L NaOH, and autoclave at 121℃ for 20 minutes for later use (after sterilization, the solubility of the compound carbon and nitrogen source increases to 8%-10%, avoiding clumping at high temperature). (2) Validation of culture medium performance The BL-03 strain was inoculated into an optimized culture medium and cultured at 45°C and 200 rpm for 24 hours, resulting in a cell density of 2.0 × 10¹. 0 CFU / mL, compared to LB medium (9×10⁻⁶) 9 The CFU / mL ratio was increased by 122%; the high-temperature protease activity reached 2800 U / mL at 60℃, and the spore survival rate reached 93% after treatment at 100℃ for 10 minutes, meeting the needs of industrial applications; the total cost of the culture medium was reduced from 7.2 yuan / L of LB medium to 3.1 yuan / L, a cost reduction of 57%. "High-temperature proliferation-gradient induction" segmented fermentation process Large-scale production is carried out using a 1000L fermenter, and the process is divided into two stages: "high-temperature nutrient growth period" and "gradient spore induction period". It is adapted to the high-temperature characteristics of Bacillus licheniformis and achieves a triple improvement in cell density, enzyme activity and spore rate. The specific parameters are as follows: (1) High-temperature vegetative growth period (0-28 hours, target: high-density proliferation and enzyme production) Inoculation and initial parameters: BL-03 seed culture was inoculated at a rate of 6% (v / v) and cultured at 45℃, 200 r / min for 14 hours, with a cell density of 1.5 × 10⁻⁶ cells / year. 9 (CFU / mL) was added to the fermenter; the stirring speed was 220 r / min (to ensure uniform mixing of the culture medium at high temperature and avoid local precipitation of carbon and nitrogen sources), the aeration rate was 2.0 vvm (oxygen solubility decreases at high temperature, and high aeration rate maintains dissolved oxygen concentration ≥20%), and the temperature was controlled at 45℃ (the optimal proliferation temperature for Bacillus licheniformis). Feeding strategy: At 12 hours, add a compound carbon source (4 g / L corn flour + 3 g / L wheat bran hydrolysate, dissolved and added via a sterile feed pump) to prevent carbon source depletion and enzyme production stagnation; at 24 hours, add a compound nitrogen source (3 g / L soybean meal powder + 1 g / L ammonium sulfate) to prolong the logarithmic phase of enzyme production; at 28 hours, the high-temperature protease activity reaches its peak (3000 U / mL), and the cell density reaches 2.5 × 10¹. 0 CFU / mL, entering the spore induction period. (2) Gradient spore induction period (28-40 hours, objective: to efficiently induce spores and maintain enzyme activity) Temperature gradient regulation: 28-32 hours, temperature is reduced from 45℃ to 40℃ (slow cooling to avoid bacterial stress); 32-36 hours, temperature is reduced to 37℃ (the initiation temperature for Bacillus licheniformis spore formation); 36-40 hours, temperature is maintained at 37℃ to activate spore formation-related genes through the temperature gradient; Parameter and inducer optimization: The stirring speed was reduced to 180 r / min (to reduce cell shear damage at high temperatures), and the aeration rate was reduced to 1.5 vvm (to maintain dissolved oxygen at 15%-18%, as a low-oxygen environment promotes spore differentiation); at 28 hours, 0.5 g / L MnSO4 was added (to promote spore cortex synthesis and enhance heat resistance) and 0.3 g / L Na2CO3 were added (to maintain pH stability at around 7.5). Monitoring and Termination: Samples were taken every 2 hours, and the activity of the high-temperature protease (measured at 60℃) and the spore rate (Gram staining microscopy) were measured simultaneously. Fermentation was stopped when the spore rate was ≥90% and the protease activity retention rate was ≥85% (i.e., activity ≥2550U / mL). The fermentation cycle was controlled within 40 hours. Product collection and dosage form preparation (1) Liquid high-temperature enzyme-spore complex product Centrifugal concentration: Centrifuge the fermentation broth for 20 minutes using a disc centrifuge (10000 r / min, separation factor 2500×g) and collect the mixture of cell (containing spores) and supernatant (containing high-temperature enzymes); Stabilizer addition: Add 2% glycerol (high-temperature enzyme protectant) + 0.3% potassium sorbate (preservative) to the mixture, stir well, and then filter through a 0.22μm microfiltration membrane for sterilization to prepare a liquid product. The high-temperature protease activity at 60℃ is ≥2500U / mL, and the spore content is ≥10. 9 CFU / mL, shelf life 12 months when refrigerated at 4℃, enzyme activity decreases ≤10% when stored at 50℃ for short period (7 days). (2) Solid heat-resistant bacterial agent products Spray drying: The centrifuged mixture is fed into a spray drying tower (inlet air temperature 180℃, outlet air temperature 80℃, adapted to the high temperature resistance of spores), and 8% maltodextrin + 2% calcium carbonate (as a carrier and stabilizer) is added to make a powder product. Quality control: Solid products must have a moisture content of ≤8%, a high-temperature protease activity of ≥2200 U / mL at 60℃, and a spore content of ≥10¹. 0 CFU / g, shelf life of 18 months when sealed at 25℃, spore survival rate ≥90% after treatment at 100℃ for 10 minutes, suitable for high-temperature industrial catalysis, high-temperature feed pelleting and other scenarios. Beneficial effects The strain exhibits strong targeting and stable high-temperature performance: Through targeted screening in a high-temperature environment, the BL-03 strain obtained showed a high-temperature protease activity ≥2500U / mL at 45℃ and a spore survival rate ≥90% after treatment at 100℃ for 10 minutes, which is 150% higher than that of ordinary strains and significantly enhances stress resistance. Low cost and high resource utilization: By using industrial by-products (wheat bran hydrolysate and waste molasses) and low-cost carbon and nitrogen sources, the cost of the culture medium is reduced by 57%, and the utilization rate of carbon and nitrogen sources is increased to over 90%, resulting in significant economic benefits for industrialization. Excellent process adaptability and wide product applicability: The "high-temperature proliferation-gradient induction" process achieves a cell density of up to 10¹. 0 With CFU / mL, spore conversion rate ≥90%, and high-temperature enzyme activity retention rate ≥85%, it can be made into liquid or solid products, suitable for high-temperature industrial, agricultural, and animal husbandry fields, filling the gap in the application of existing technologies in high-temperature scenarios. S3: Preparation of compound microbial agent: The optimal volume ratio of Bacillus belyssus, Bacillus amyloliquefaciens and Bacillus licheniformis was determined by response surface methodology. The optimal volume ratio of Bacillus belyssus, Bacillus amyloliquefaciens and Bacillus licheniformis is 6:3:2, which increases the total enzyme activity (sum of protease, amylase and cellulase) of fermented soybean meal by 18-25% compared with other ratios, without any growth inhibition. The specific method for selecting the proportion of compound microbial agents is as follows: In a clean bench, accurately measure the corresponding volume of seed liquid of each strain, mix them, and then evenly inoculate them into an Erlenmeyer flask containing soybean meal culture medium. Set up 3 replicates for each experimental point. Fermentation culture: Place all inoculated Erlenmeyer flasks in a constant temperature shaker and ferment for 54 hours under uniform optimized conditions (e.g., 34℃, 180rpm) to ensure that the culture conditions of all flasks are completely consistent. Termination of fermentation and sampling: After fermentation, immediately remove the Erlenmeyer flasks, add 100 mL of sterile physiological saline to each flask, shake thoroughly for 30 minutes, and extract the enzymes from the fermentation products; Preparation of crude enzyme solution: Centrifuge the mixture at 4℃ and 8000rpm for 15 minutes, and take the supernatant as crude enzyme solution; Response value measurement Total enzyme activity was determined for each sample of crude enzyme solution. Protease activity: determined using the Folin-phenol method; Amylase activity: determined using the DNS method; Cellulase activity: determined using the DNS method (with CMC-Na as substrate); Calculate total enzyme activity: Add the activity values ​​of the three enzymes together to obtain the total enzyme activity (Y, U / g) for that experimental point. Different weighting coefficients can also be assigned to different enzymes according to application requirements. Data Analysis and Model Building Data entry: Input the factor levels (A, B) and the measured response values ​​(Y) of the experiment into the Design-Expert software; Model Fitting: The software will automatically perform multiple quadratic regression analysis to generate a predictive model. The model equation is typically as follows: Y = β0 + β1A + β2B + β 11 A²+β 22 B²+β 12 AB Analysis of variance (ANOVA): This is the core step, used to determine the significance of the model, whether the lack-of-fit term is significant, and to check indicators such as R², Adj-R², and AdeqPrecision to ensure that the model is reliable and usable; Response surface methodology: Using 3D surface plots and contour plots generated by software, the interactions between factors and their impact on total enzyme activity can be analyzed intuitively. Optimal ratio prediction and verification Numerical optimization: Set the objective to "MaximizeY" in the software, and the software will calculate the optimal combination of factor levels (A_optimal, B_optimal) within the model range. Predicting the optimal value: The software will provide the predicted total enzyme activity and its confidence interval under the optimal ratio; Validation experiment: This is a crucial step. Conduct at least three replicate fermentation experiments according to the optimal ratios predicted by the software (e.g., A=54.5%, B=27.3%, C=18.2%). Results Comparison: The actual total enzyme activity of the validation experiment was measured. If the actual value was very close to the model prediction (relative error <5%) and significantly higher than all other experimental groups, it proved that the model optimization was successful, and this ratio was the optimal compound ratio. Finally, it was simplified to an integer ratio of 6:3:2.

[0017] Growth inhibition verification At the end of the validation and center point experiments (Runs 13-17), fermentation samples were taken, and the viable counts of the three strains were counted using a serial dilution plating method (which can be based on colony morphology identification or selective media). The results showed that, under optimal conditions, the viable counts of each strain remained at a high level (≥10). 9 The CFU / g ratio showed no significant difference from that of single-strain fermentation, thus proving from the experimental data that "there was no growth inhibition phenomenon".

[0018] S4: Formulation and Preservation: The compound bacterial solution is mixed with compound preservatives such as dextrin and trehalose, and then prepared into a powdered bacterial agent using vacuum freeze-drying technology, with a viable count ≥2×10¹. 0 CFU / g, stored at 1-4℃, shelf life up to 12 months; S5: Soybean meal substrate grinding: Soybean meal is ground to a particle size of ≤150μm (above 100 mesh) using ultra-fine grinding technology, which significantly increases the specific surface area; S6: Pretreatment of soybean meal substrate: Mix crushed soybean meal with water at a material-to-water ratio of 2:5 (g / mL), add a complex organic acid consisting of 0.15% (w / w) citric acid and 0.05% (w / w) malic acid, and stir at 55℃ for 25 minutes. This mild condition can effectively destroy the structure of trypsin inhibitors, with an inactivation rate of over 50%, while avoiding excessive acidification. S7: Sterilization: Autoclave at 121℃ for 15 minutes, then rapidly cool to the inoculation temperature; S8: Soybean meal fermentation: adopts a segmented dynamic control fermentation process. Soybean meal is mixed and fermented in three stages in an automated fermenter, with pH, ​​DO (dissolved oxygen), and temperature monitored online throughout the process; Specifically, this includes the following steps: The specific process for the mixed fermentation of soybean meal in the three stages of S8 is as follows: Phase 1: Colonization and Proliferation Period (1-18 hours), Strategic Goal: Rapidly construct a highly active, high-density bacterial cell factory. Core control strategy: Active regulation based on feedback of dissolved oxygen (DO) change rate and pH trend to reserve sufficient biomass for efficient enzyme production; Control parameters: Inoculation: Inoculate with a highly active compound freeze-dried bacterial agent (Bacillus belye: Bacillus amyloliquefaciens: Bacillus licheniformis ratio of 6:3:2) at 10% (v / w), and activate with sterile physiological saline at 35-39℃ for 12-18 min before use; Temperature control: The temperature is kept constant at 33.8-34.2℃. This temperature is the intersection of the optimal growth temperature ranges of the three strains, which can effectively balance the proliferation rate of different strains and avoid premature lag of a certain strain due to unsuitable temperature. pH regulation strategy: No active acid-base regulation is performed in this stage. The aim is to closely monitor its natural change curve. The initial pH is 7.0-7.2. Due to the rapid metabolism of carbohydrates and acid production by the bacteria, the pH will continue to decrease. Monitoring the rate of pH decrease (dpH / dt) can be used as a real-time indicator to judge the activity and metabolic intensity of the bacteria. The typical rate of decrease is about 0.05-0.08 pH units / hour. Dissolved oxygen (DO) and stirring / ventilation linkage control: A constant DO cascade control mode is adopted, with the target dissolved oxygen (DO) value set at 28.5-31.5% saturation. The control system uses a PID algorithm to automatically cascade and adjust the stirring speed and ventilation rate. Prioritize adjusting the stirring speed: The initial setting is 140-160 rpm / min. When DO decreases, the system prioritizes increasing the stirring speed, with an adjustment range of 150-250 rpm / min, to enhance oxygen supply capacity by improving liquid flow mass efficiency. Secondary ventilation rate adjustment: When the stirring speed reaches the upper limit and still cannot maintain the set DO, the system automatically increases the ventilation rate, with an adjustment range of 1.0-1.5vvm; Oxygen-enriched air backup: In extremely high-density conditions, oxygen-enriched air can be activated to supplement oxygen supply (this design is an optional advanced function). Oxidation-reduction potential (ORP) monitoring: As an auxiliary reference indicator, the continuous decrease in ORP also confirms that the fermentation broth is in a vigorous reducing metabolic state. Intelligent determination of stage transition: When the pH value drops to the lowest point of 6.7-6.8, and then shows a continuous and stable upward trend for ≥10 minutes, it indicates that the most readily available free carbon source in the culture medium (such as glucose) has been basically depleted, the cell proliferation rate has slowed down, and the metabolic focus has begun to shift to the synthesis of secondary metabolites (enzymes and proteins). After the central control system captures this signal, it automatically triggers the program to switch to the next stage. This transition mechanism based on metabolic inflection points is more robust and adaptable than fixed-time control. Phase Two: Main Enzyme Production Period (18-42 hours) Strategic Objective: To strongly induce efficient synthesis of complex enzyme systems and prolong the peak enzyme production period. Core control strategy: Based on the pH-stat principle and the dynamics of carbon source consumption, implement a precise feeding fermentation strategy of "small amount and multiple times" to eliminate the carbon decomposition metabolism inhibition effect and maximize the enzyme synthesis rate. Refined control parameters: Temperature switching: Within one hour after the phase transition, the temperature is linearly increased and stably controlled at 36.8℃-37.22℃. This temperature is the optimal temperature for the expression of various extracellular enzyme synthesis-related regulatory genes (such as degU, sacU) and σ factor activity in Bacillus subtilis and Bacillus licheniformis, which can strongly activate the entire enzyme production system; pH-stat precise control: Activate the active control mode to precisely control the pH of the fermentation broth within a narrow range of 6.85-6.95. By automatically adding 6M NaOH, the organic acids produced by cell metabolism are neutralized to maintain a slightly acidic environment. This pH environment is not only conducive to the spatial conformation stability and activity of most enzyme proteins, but also effectively inhibits the growth of miscellaneous bacteria. Dynamic feeding strategy: Feeding trigger signal: When the pH rises and exceeds the set upper limit due to the depletion of organic acid, and the DO value shows a rapid upward inflection point at the same time, a double signal confirmation is formed, indicating that the readily available carbon source has been officially depleted. Feeding matrix: A concentrated mixture consisting of 500 g / L glucose, 200 g / L yeast extract, and trace amounts of inorganic salts (Mg²⁺, Mn²⁺) is fed in. Yeast extract provides abundant amino acids, small peptides, and vitamins, and is a direct precursor and cofactor for synthesizing enzyme proteins; Feeding logic: A rule-based pulse feeding method is adopted, with each feeding amount calculated by the model to instantly restore the reducing sugar concentration in the fermentation broth to the ideal range of 0.5%-0.8% (w / v). The feeding effect is verified by offline sampling (DNS method) or online glucose analyzer and used to optimize subsequent feeding algorithms. This low-concentration sugar strategy can completely eliminate carbon catabolism repression (CCR) caused by high glucose concentration, enabling continuous and efficient expression of enzyme-producing genes. Aeration and mixing optimization: The cell density reaches its peak (≥10¹) at this stage. 0 (CFU / mL) has extremely high metabolic oxygen consumption, so the DO target value is set at ≥25%. It is usually necessary to increase the stirring speed to the upper limit (250-300 rpm) and maintain the ventilation rate at 1.5-1.8 vvm to ensure sufficient oxygen supply and prevent the decline in anaerobic metabolism and enzyme production efficiency due to hypoxia. Exhaust gas analysis and monitoring: Real-time monitoring of carbon dioxide emission rate (CER). The peak and plateau phase of CER can directly reflect the overall metabolic intensity of the bacteria. An abnormal decrease in CER is often an early warning signal of nutrient depletion or metabolic obstruction. Intelligent determination of phase transition: When the enzyme production rate per unit time decreases significantly (by sampling every 3 hours with an online sampler, measuring enzyme activity, and calculating the enzyme activity increase of <5% at two consecutive time points), and / or the CER value decreases by more than 15% from the plateau phase, it indicates that the peak enzyme production period has passed and the cell activity has begun to decline. The system judges that it has entered the stable phase based on this. Phase 3: Enzyme activity stabilization and harvest period (42-54h) | Strategic objective: Maintain peak enzyme activity, minimize enzyme protein degradation, and create optimal conditions for downstream processing; Core control strategy: Low metabolism maintenance mode, all operations are based on the highest principle of protecting product stability, and the harvest endpoint is accurately determined; Refined control parameters: Cooling protection: The temperature is linearly reduced and eventually stabilized at 27.5-28.5℃ within 2 hours. Lowering the temperature can significantly reduce the activity of the bacterial cell's own metabolic enzymes (such as proteases) and extracellular proteases. This is the most critical and effective measure to prevent the degradation of secreted enzyme proteins, and the enzyme activity retention rate can be increased by more than 15%. pH fine-tuning: The pH control target was slightly lowered to 6.75-6.85, which is closer to a neutral to slightly acidic environment, further enhancing the long-term stability of the enzyme protein; Stop feeding: Stop the addition of all carbon and nitrogen sources, allowing the cells to gradually enter the endogenous respiration stage and reduce unnecessary metabolic byproducts; Aeration and stirring attenuation: Simultaneously reduce the stirring speed to 100-120 rpm and the aeration rate to 0.5-0.8 vvm. Maintaining DO at 15-20% is sufficient; excessively high DO is unnecessary. This significantly reduces the risk of physical damage to spatially structured enzyme proteins caused by mechanical shear forces, while also saving energy. Process monitoring: Continuously monitor the trend of enzyme activity changes and cell autolysis indicators (such as the decrease in absorbance of fermentation broth at 600nm wavelength). Precise determination of fermentation termination: When the total enzyme activity (weighted sum of protease, amylase, and cellulase activities) shows no significant increase in two consecutive sampling measurements (difference <2%), or when a statistically significant downward trend first appears (decline >3%), the central control system issues an alarm, prompting the operator to immediately terminate fermentation and start the downstream extraction program to ensure harvesting at the absolute peak of enzyme activity, maximizing the benefits of single-batch fermentation. The total enzyme activity at the fermentation endpoint can reach 18,500-21,500 U / g. The core advantages and benefits of the optimized solution (highlighted by its patentability) Multi-parameter fusion intelligent decision-making mechanism: It breaks through the limitations of traditional time axis control and constructs an intelligent decision-making system with real-time data of multiple dimensions such as pH, DO, CER, and enzyme activity growth rate as input. It realizes precise control based on the "physiological state" of the fermentation process, with strong anti-interference ability and extremely high batch-to-batch consistency. Dynamic decoupled feeding technology: The innovative "dual signal triggering + low concentration pulse feeding" strategy precisely controls the carbon source concentration in the fermentation broth, fundamentally eliminating carbon decomposition metabolism inhibition (CCR). This is the key technological barrier for achieving a breakthrough in enzyme activity yield (estimated to be 10-15% higher than before optimization). Product-oriented protective control: In the later stage of fermentation, a combination strategy of "cooling down, reducing speed, and adjusting pH" is actively adopted. The core objective is to shift from "promoting synthesis" to "protecting the product". The fermentation process and downstream extraction are optimized as a whole, which significantly improves the yield and quality of the final product. End-to-end digitalization and traceability: All process parameters and process data are automatically recorded and stored, which can be used to build digital twin models for in-depth process analysis and continuous optimization (CPV). It complies with the FDA's "Process Analysis Technology (PAT)" and the advanced concepts of smart manufacturing (Industry 4.0), and has strong technological advancement. S9: Enzyme Extraction: The fermentation product is extracted using a two-stage countercurrent extraction process. The first stage uses a small amount of water to obtain a high-concentration enzyme solution; the second stage uses fresh water to perform a secondary extraction on the residue. After combining the extracts, they are concentrated by ultrafiltration through a ceramic membrane, and then spray-dried (with the addition of an appropriate amount of carrier such as maltodextrin) to produce a powdered compound enzyme preparation with an enzyme activity recovery rate ≥92%. S10: Drying of fermented soybean meal: The wet residue after enzyme extraction is rich in small peptides and probiotics. After being dried by low-temperature airflow (≤80℃), it is made into high-quality fermented soybean meal feed with significantly improved protein digestibility.

Claims

1. A method for producing enzymes through mixed fermentation of soybean meal with Bacillus subtilis, characterized in that: Includes the following steps: S1: Strain selection; Bacillus belye, Bacillus amyloliquefaciens, and Bacillus licheniformis were selected. S2: High-density fermentation: The *Bacillus belye*, *Bacillus amyloliquefaciens*, and *Bacillus licheniformis* obtained in S1 were subjected to high-density fermentation using a culture medium, and the cell concentration of each strain reached 5 × 10⁻⁶. 9 For fermentation equipment with a concentration of CFU / mL or higher, the equipment is configured as follows: a smart 500L fermenter equipped with high-precision online sensors for pH, dissolved oxygen (DO), temperature, oxidation-reduction potential (ORP), an automatic feeding system, a tail gas analysis module for real-time monitoring of O2 consumption rate (OUR) and CO2 release rate (CER), and process control software. All data is integrated into the central control system to achieve full-process digital management and historical data traceability. S3: Preparation of compound microbial agent: The optimal volume ratio of Bacillus belyssus, Bacillus amyloliquefaciens, and Bacillus licheniformis was determined by response surface methodology, resulting in a 18-25% increase in total enzyme activity of fermented soybean meal compared to other ratios, without any growth inhibition. S4: Formulation and Preservation: The compound bacterial solution is mixed with compound preservatives such as dextrin and trehalose, and then prepared into powdered bacterial agent by vacuum freeze-drying technology; S5: Soybean meal substrate grinding: Soybean meal is ground to a particle size of ≤150μm (above 100 mesh) using ultra-fine grinding technology, which significantly increases the specific surface area; S6: Pretreatment of soybean meal substrate: Mix crushed soybean meal with water at a material-to-water ratio of 2:5 (g / mL), add 0.15% (w / w) of citric acid and 0.05% (w / w) of malic acid to form a compound organic acid, and stir at 55℃ for 25 minutes. S7: Sterilization: Autoclave at 121℃ for 15 minutes, then rapidly cool to the inoculation temperature; S8: Soybean meal fermentation: adopts a segmented dynamic control fermentation process. Soybean meal is mixed and fermented in three stages in an automated fermenter, with pH, ​​DO (dissolved oxygen), and temperature monitored online throughout the process; S9: Enzyme Extraction: The fermentation product is extracted using a two-stage countercurrent extraction process. The first stage uses a small amount of water to obtain a high-concentration enzyme solution; the second stage uses fresh water to perform a secondary extraction on the residue. After combining the extracts, they are concentrated by ultrafiltration through a ceramic membrane, and then spray-dried (with the addition of an appropriate amount of carrier such as maltodextrin) to produce a powdered compound enzyme preparation with an enzyme activity recovery rate ≥92%. S10: Drying of fermented soybean meal: The wet residue after enzyme extraction is rich in small peptides and probiotics. After being dried by low-temperature airflow (≤80℃), it is made into high-quality fermented soybean meal feed with significantly improved protein digestibility.

2. The method for producing enzymes by mixed fermentation of soybean meal with Bacillus subtilis according to claim 1, characterized in that: Precise screening of Bacillus belysin strains (1) Sample collection and pretreatment Select healthy crop rhizosphere soil or well-rotted organic fertilizer, weigh 10g of sample and add 90mL of sterile physiological saline, shake at 37℃ and 180r / min for 30 minutes to prepare 10 -1 Bacterial suspension; subsequently serially diluted to 10-10 -6 Spread 0.1 mL of the diluted solution onto LB solid medium and incubate at 37°C for 24-48 hours. (2) Function-oriented screening Clear zone screening: Select colonies that form a clear zone on the culture medium, measure the diameter of the clear zone (D) and the diameter of the colony (d), and select strains with D / d ≥ 2.5; Antibacterial activity screening: The initially screened strains were inoculated into LB liquid medium and cultured at 37℃ and 180r / min for 24 hours. The fermentation supernatant was mixed with the Fusarium oxysporum suspension and cultured at 28℃ for 12 hours. The inhibition rate was measured and strains with an inhibition rate ≥80% were screened. Molecular identification: Genomic DNA was extracted from the selected strains and PCR amplification was performed using Bacillus belysinus-specific primers. The amplified products were sequenced and compared with the sequences of Bacillus belysinus model strains (such as ATCCBAA-874) in GenBank. If the homology was ≥99.8%, it was identified as the target strain.

3. The method for producing enzymes through mixed fermentation of soybean meal with Bacillus subtilis according to claim 1, characterized in that: Starch-directed screening of Bacillus amyloliquefaciens strains (1) Sample collection and pretreatment Select environmental samples rich in starch, such as activated sludge from a winery wastewater treatment pond, soil surrounding a starch processing plant, and well-rotted straw compost. Weigh 10g of the sample and add it to 90mL of sterile physiological saline. Shake at 37℃ and 200r / min for 40 minutes to prepare 10 samples. -1 Bacterial suspension; serially diluted to 10 -7 Take 0.1 mL of the diluted solution and spread it on the starch selection medium, and incubate at 37℃ for 36-48 hours; (2) Function-oriented screening Starch degradation zone screening: Iodine solution (0.1 mol / L I2-KI solution) was added to the cultured plates, and colonies that formed obvious blue degradation zones were selected. The diameter of the degradation zone (D) and the diameter of the colony (d) were measured, and strains with D / d ≥ 3.0 were selected (the larger the degradation zone, the higher the amylase activity). Amylase activity assay: The initial screening strains were inoculated into starch liquid medium (soluble starch 15 g / L, peptone 8 g / L, yeast extract 5 g / L, pH 7.0) and cultured at 37℃ and 200 r / min for 24 hours. The α-amylase activity in the fermentation broth was determined by the DNS method, and strains with activity ≥2500 U / mL were screened. Molecular identification and stability verification: Genomic DNA was extracted from the screened strains and PCR amplification was performed using Bacillus amyloliquefaciens-specific primers. After sequencing, the amplified products were compared with the sequence of the model strain (such as DSM7T) in GenBank. The target strain was identified if the homology was ≥99.7%. The target strain was passaged 5 times consecutively, and the amylase activity was measured after each passage. The strain with activity fluctuation ≤5% was selected.

4. The method for producing enzymes by mixed fermentation of soybean meal with Bacillus subtilis according to claim 1, characterized in that: High-temperature targeted screening of Bacillus licheniformis strains (1) Sample collection and pretreatment Select high-temperature environment samples, such as high-temperature composting, soil around hot springs, and high-temperature reactors for industrial wastewater treatment. Weigh 10g of the sample and add it to 90mL of sterile physiological saline. Shake at 45℃ and 200r / min for 40 minutes to prepare 10 samples. -1 Bacterial suspension; subsequently serially diluted to 10⁻ 7 Take 0.1 mL of the diluted solution and spread it on the high-temperature screening medium, and incubate at 45℃ for 36-48 hours; (2) Function-oriented screening Protease clear zone screening: Select colonies on the culture medium that form obvious protein degradation clear zones, measure the diameter of the clear zone (D) and the diameter of the colony (d), and select strains with D / d ≥ 3.0; High-temperature enzyme activity and stability assay: The initially screened strains were inoculated into a high-temperature liquid culture medium (15 g / L skim milk powder, 10 g / L corn flour, 8 g / L soybean meal powder, pH 7.5) and cultured at 45℃ and 200 r / min for 24 hours. The protease activity at 60℃ was determined using the Folin-phenol method, and strains with activity ≥2500 U / mL were screened. At the same time, the strains were cultured at 55℃ for 12 hours, and the enzyme activity retention rate was determined. Strains with a retention rate ≥85% were selected.

5. The method for producing enzymes through mixed fermentation of soybean meal with Bacillus subtilis according to claim 1, characterized in that: The specific method for selecting the proportion of S3 compound microbial agent is as follows: In a clean bench, accurately measure the corresponding volume of seed liquid of each strain, mix them, and then evenly inoculate them into an Erlenmeyer flask containing soybean meal culture medium. Set up 3 replicates for each experimental point. Fermentation culture: Place all inoculated Erlenmeyer flasks in a constant temperature shaker and ferment for 54 hours under uniform optimized conditions (e.g., 34℃, 180rpm) to ensure that the culture conditions of all flasks are completely consistent. Termination of fermentation and sampling: After fermentation, immediately remove the Erlenmeyer flasks, add 100 mL of sterile physiological saline to each flask, shake thoroughly for 30 minutes, and extract the enzymes from the fermentation products; Preparation of crude enzyme solution: Centrifuge the mixture at 4℃ and 8000rpm for 15 minutes, and take the supernatant as crude enzyme solution.

6. The method for producing enzymes by mixed fermentation of soybean meal with Bacillus subtilis according to claim 1, characterized in that: S1. The three Bacillus strains selected were all derived from specific habitats and underwent targeted functional enhancement: Bacillus berleis: isolated from high-temperature soybean meal compost (55-60℃), the strain needs to be induced and selected through mutagenesis. Its neutral protease activity is stable at 6800-7200U / g, and its phytase activity reaches 350-450U / g. Its optimal growth temperature range is wide (30-45℃), which can effectively start fermentation and degrade phytic acid. Bacillus amyloliquefaciens: isolated from high-starch-producing soil, it produces high levels of mesophilic α-amylase (activity 5200-5800 U / g) and can also secrete a small amount of glucanase, which can supplement the complex enzyme system. The antimicrobial lipopeptides produced by its metabolism can effectively inhibit the growth of miscellaneous bacteria. Bacillus licheniformis: isolated from paper mill waste piles, it has extremely strong cellulose and hemicellulose degradation capabilities (cellulase activity 1300-1600 U / g, xylanase activity 900-1100 U / g), and needs to be resistant to acids and alkalis (pH 5.0-9.0) and high temperatures (up to 60℃).

7. A method for producing enzymes from soybean meal by mixed fermentation with Bacillus subtilis according to any one of claims 1, characterized in that: This specifically includes the following steps: The specific process for the three-stage mixed fermentation of soybean meal in S8 is as follows: Phase 1: Colonization and Proliferation Period (1-18 hours), Strategic Goal: Rapidly construct a highly active, high-density bacterial cell factory. Core control strategy: Active regulation based on feedback of dissolved oxygen (DO) change rate and pH trend to reserve sufficient biomass for efficient enzyme production; Control parameters: Inoculation: Inoculate with a highly active compound freeze-dried bacterial agent (Bacillus belye: Bacillus amyloliquefaciens: Bacillus licheniformis ratio of 6:3:2) at 10% (v / w), and activate with sterile physiological saline at 35-39℃ for 12-18 min before use; Temperature control: kept constant at 33.8-34.2℃; pH regulation strategy: No active acid-base regulation is performed in this stage. The initial pH is 7.0-7.

2. Due to the rapid metabolism of carbohydrates by the bacteria to produce acid, the pH will continue to decrease. Monitoring the rate of pH decrease (dpH / dt) can be used as a real-time indicator to judge the activity and metabolic intensity of the bacteria. Dissolved oxygen (DO) and stirring / ventilation linkage control: A constant DO cascade control mode is adopted, with the target dissolved oxygen (DO) value set at 28.5-31.5% saturation. The control system uses a PID algorithm to automatically cascade and adjust the stirring speed and ventilation rate. Prioritize adjusting the stirring speed: the initial setting is 140-160 rpm / min, and when DO decreases, the system prioritizes increasing the stirring speed; Secondary ventilation rate adjustment: When the stirring speed reaches the upper limit and still cannot maintain the set DO, the system automatically increases the ventilation rate, with an adjustment range of 1.0-1.5vvm; Oxygen-enriched air backup: In extremely high-density conditions, oxygen-enriched air can be activated to supplement oxygen supply (this design is an optional advanced function). Oxidation-reduction potential (ORP) monitoring: As an auxiliary reference indicator, the continuous decrease in ORP also confirms that the fermentation broth is in a vigorous reducing metabolic state. Intelligent determination of stage transition: When the pH value drops to the lowest point of 6.7-6.8, and then shows a continuous and stable upward trend for ≥10 minutes, it indicates that the most readily available free carbon source in the culture medium has been basically depleted, the cell proliferation rate has slowed down, and the metabolic focus has begun to shift to the synthesis of secondary metabolites. After the central control system captures this signal, it automatically triggers the program to switch to the next stage. This transition mechanism based on metabolic inflection points is more robust and adaptable than fixed-time control. Phase Two: Main Enzyme Production Period (18-42 hours); Core control strategy: Based on the pH-stat principle and the dynamics of carbon source consumption, implement a precise feeding fermentation strategy of "small amount, multiple times" to eliminate the carbon decomposition metabolism inhibition effect and maximize the enzyme synthesis rate. Refined control parameters: Temperature switching: Within one hour after the phase transition, the temperature is linearly increased and stably controlled at 36.8℃-37.22℃. This temperature is the optimal temperature for the expression of genes regulating the synthesis of various extracellular enzymes in Bacillus subtilis and Bacillus licheniformis, as well as for the activity of σ factor, and can strongly activate the entire enzyme production system; pH-stat precise control: Activate the active control mode to precisely control the pH of the fermentation broth within a narrow range of 6.85-6.

95. Neutralize the organic acids produced by cell metabolism by automatically adding 6M NaOH to maintain a slightly acidic environment. Aeration and mixing optimization: The cell density reaches its peak (≥10¹) at this stage. 0 (CFU / mL) Exhaust gas analysis and monitoring: Real-time monitoring of carbon dioxide emission rate (CER). Intelligent determination of phase transition: When the enzyme production rate per unit time decreases significantly (by sampling every 3 hours with an online sampler, measuring enzyme activity, and calculating the enzyme activity increase of <5% at two consecutive time points), and / or the CER value decreases by more than 15% from the plateau phase, it indicates that the peak enzyme production period has passed and the cell activity has begun to decline. The system judges that it has entered the stable phase based on this. Phase 3: Enzyme activity stabilization and harvest period; Core control strategy: Low metabolism maintenance mode, all operations are based on the highest principle of protecting product stability, and the harvest endpoint is accurately determined; Refined control parameters: Cooling protection: The temperature is linearly reduced and eventually stabilized at 27.5-28.5℃ within 2 hours. Lowering the temperature can significantly reduce the activity of the bacterial cell's own metabolic enzymes (such as proteases) and extracellular proteases. This is the most critical and effective measure to prevent the degradation of secreted enzyme proteins, and the enzyme activity retention rate can be increased by more than 15%. pH fine-tuning: The pH control target was slightly lowered to 6.75-6.85, which is closer to a neutral to slightly acidic environment, further enhancing the long-term stability of the enzyme protein; Stop feeding: Stop the addition of all carbon and nitrogen sources, allowing the cells to gradually enter the endogenous respiration stage and reduce unnecessary metabolic byproducts; Aeration and stirring attenuation: Simultaneously reduce the stirring speed to 100-120 rpm and the aeration rate to 0.5-0.8 vvm. Maintaining DO at 15-20% is sufficient; excessively high DO is unnecessary. This significantly reduces the risk of physical damage to spatially structured enzyme proteins caused by mechanical shear forces, while also saving energy. Process monitoring: Continuously monitor the trend of enzyme activity changes and cell autolysis indicators (such as the decrease in absorbance of fermentation broth at 600nm wavelength). Precise determination of fermentation termination: When the total enzyme activity (weighted sum of protease, amylase, and cellulase activities) shows no significant increase in two consecutive sampling measurements (difference <2%), or when a statistically significant downward trend first appears (decline >3%), the central control system issues an alarm, prompting operators to immediately terminate fermentation and start the downstream extraction program to ensure harvesting at the absolute peak of enzyme activity, maximizing the benefits of single-batch fermentation. The total enzyme activity at the fermentation endpoint can reach 18,500-21,500 U / g.

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