Dual-stage control method for mixed preparation of biological stimulants

Abstract

The invention relates to the technical field of biologic stimulant preparation, and discloses a biologic stimulant mixing preparation two-stage control method, which comprises: adopting dissolved oxygen gradient and carbon source pulse cooperative control, linearly reducing the concentration of dissolved oxygen in a fermentation tank from an initial value at a specific rate, and injecting a carbon source at regular time; when the flora density and the quorum sensing signal molecule concentration reach specific threshold values, triggering stage switching; starting pressure-metabolism coupling control, increasing the pressure of the fermentation tank at a specific rate, and adding coenzyme Q10; the quorum sensing inhibitory peptide containing a specific core structure is synchronously released in the boosting process; after the pressure is stable, a real-time mapping relation between a microbial metabolism state and physical parameters is constructed in a thallus proliferation stage through a coupling mechanism of dissolved oxygen gradient control and quorum sensing signal monitoring, and the industrial problem that a traditional static threshold value cannot respond to pressure fluctuation of a fermentation tank to cause signal attenuation is solved.

Description

Technical Field

[0001] The invention relates to the technical field of biostimulant preparation, and in particular to a two-stage control method for mixed preparation of biostimulants. Background Art

[0002] In the preparation technology of biostimulants, traditional methods mainly rely on static parameter control and single component application, which makes it difficult to achieve dynamic adaptation of microbial metabolic state and physical environment; in the existing technology, the control of bacterial proliferation and spore induction stages usually adopts a step-by-step independent regulation strategy: triggering stage switching through a fixed dissolved oxygen threshold, assisted by chemical antibacterial agents to control bacterial contamination; for example, when humic acid or seaweed extracts are used as bioactive ingredients, their effects are easily affected by environmental pressure fluctuations, leading to metabolic pathway disorders.

[0003] Although microbial agents can promote plant growth, they are easily inactivated in complex fermentation environments and lack precise pollution inhibition mechanisms. The existing technologies have the following key defects: 1. Physical parameters such as dissolved oxygen and pressure lack dynamic coupling with biochemical indicators such as quorum sensing signals, resulting in delayed phase switching. 1. Threshold-triggered induction cannot adapt to differences in bacterial metabolic rates, resulting in 20-30% ineffective induction operations; 2. The action mechanisms of traditional antibacterial agents and growth-promoting components interfere with each other. For example, although chitosan nanoparticles can inhibit miscellaneous bacteria, their sustained-release characteristics conflict with the redox requirements of coenzyme Q10, resulting in a reduction in bioavailability of more than 40%; 3. Batch production methods that rely on offline detection and manual regulation make it difficult to achieve real-time closed-loop control of key parameters. Existing online sensor systems are sensitive to the adsorption effect of nano-drug-loaded particles, with detection errors as high as ±15%.

[0004] To improve the above problems, the industry has tried to optimize single components (such as high-purity QSIP peptide sequence design) or introduce complex fermentation processes; however, these improvement plans have caused new technical contradictions: precision component purification has greatly increased production costs. For example, the industrial synthesis cost of specific sequence QSIP peptides is higher than that of conventional antibacterial agents; although multi-tank series fermentation can improve stage control accuracy, the complexity of the equipment leads to an increased risk of contamination by foreign bacteria; the fixed parameter compensation algorithm fails when the pressure fluctuation exceeds 0.05MPa, causing metabolic imbalance.

[0005] Therefore, how to achieve dynamic coupling control of microbial metabolic pathways and physical environmental parameters while maintaining a stable release efficiency of bioactive ingredients in large-scale production has become a technical problem to be solved by the present invention. Summary of the invention

[0006] The technical problem solved by the present invention is to provide a two-stage control method for the mixed preparation of biostimulants in response to the defects existing in the above-mentioned prior art, so as to solve the problems proposed in the above-mentioned background technology that parameter mismatch leads to low metabolic efficiency, insufficient component synergy leads to activity loss, and production process rigidity restricts large-scale application.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: a two-stage control method for mixed preparation of biostimulants, comprising the following steps: Step S1: Using the coordinated control strategy of dissolved oxygen gradient and carbon source pulse, the dissolved oxygen concentration in the fermenter is linearly reduced from the initial value of 8-15 mg / L to 4-6 mg / L at a rate of 0.5-1.5 mg / (L·h), and a carbon source solution with a volume fraction of 0.5-1.2% is injected every 30-45 minutes; when the bacterial population density reaches And when the concentration of quorum sensing signal molecules reaches 5-15nM, the phase switch is triggered; Step S2: Start pressure-metabolism coupling control, increase the fermentation tank pressure to 0.15-0.25MPa at a rate of 0.01-0.03MPa / min, and add coenzyme Q10 to a final concentration of 50-100μM; monitor the lactic acid concentration online, and when the concentration deviates from the range of 3-5g / L, adjust the carbon source feed rate according to the following formula : , in is the base feeding rate, The adjustment coefficient is 0.1-0.3, is the lactate concentration deviation value; Step S3: during the pressure increase process, a quorum sensing inhibitory peptide is synchronously released, wherein the amino acid sequence of the peptide comprises a Gly-Phe-Arg-Pro-Leu core structure, and the added amount is 0.1-0.3 mg / L fermentation liquid; Step S4: When the pressure stabilizes at the target value, the expression level of the spore formation gene is detected. When it reaches 3-5 times the baseline value, the spore induction is determined to be successful.

[0008] Preferably, the carbon source solution comprises a complex of glucose and yeast extract in a mass ratio of 2:1-4:1.

[0009] Preferably, the dissolved oxygen decrease rate is dynamically adjusted according to the real-time bacterial population density, and the adjustment rule satisfies: When it is below 7.5, the base rate of decline is maintained; when In the range of 7.5-9.5, increase by 1 The unit corresponding to the decline rate is adjusted by reducing 0.05-0.15mg / (L·h).

[0010] Preferably, the coenzyme Q10 is encapsulated by chitosan nanoparticles, the particle diameter is 40-60 nm, and the encapsulation rate is 80-90%.

[0011] Preferably, the quorum sensing inhibitory peptide is released through a sustained-release system at a release rate of 10-20% of the total amount released per hour.

[0012] Preferably, the phase switching verification also includes morphological observation, and the switching is confirmed to be successful when the proportion of oval cells with enhanced refractive index exceeds 70%.

[0013] Preferably, the method further includes standardization of the final product: when the spore formation rate reaches 80% or more, the temperature is lowered to 4-8°C; a protective agent is added to a final concentration of 2-3%, and vacuum freeze-drying is performed to obtain a standardized preparation.

[0014] Preferably, the preparation of the chitosan nanoparticles comprises: dissolving chitosan in an acetic acid solution with a pH of 5.0-6.0; using an ion gel method, using sodium tripolyphosphate as a cross-linking agent, and the cross-linking time is 25-35 minutes.

[0015] Preferably, the online monitoring includes Raman spectroscopy detection, and data is collected every 3-10 minutes.

[0016] Preferably, the biostimulant obtained by the method satisfies: the number of viable spores is ≥ 1×10 9 CFU / g; the mass percentage of lactic acid is 3% to 5%; the contamination rate of foreign bacteria is <1%.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. Through the coupling mechanism of dissolved oxygen gradient control and quorum sensing signal monitoring, a real-time mapping relationship between microbial metabolic status and physical parameters is established during the bacterial proliferation stage, avoiding the industry problem that the traditional static threshold cannot respond to the pressure fluctuation of the fermentation tank and cause signal attenuation; when the environmental pressure changes, the coordinated adjustment of the dissolved oxygen decrease rate and the carbon source pulse feeding not only maintains the stable growth of the bacterial population density, but also automatically corrects the stage switching trigger threshold through the pressure compensation algorithm, so that the timing of spore induction start-up accurately matches the physiological maturity of the microbial population, avoiding metabolic inhibition caused by premature switching or energy loss caused by delay.

[0018] 2. The release rate of coenzyme Q10 is dynamically adjusted by real-time monitoring of lactic acid concentration, and the energy metabolism pathway is rebuilt while the spore formation gene is activated. This mechanism forms a double guarantee with the sustained release system of the quorum sensing inhibitory peptide QSIP: on the one hand, the pH response characteristics of the chitosan nanocarrier ensure that the active peptide is continuously released during the pressure increase process, specifically blocking the quorum sensing signal transduction of foreign bacteria; on the other hand, the phased delivery of coenzyme Q10 enhances the oxidative phosphorylation efficiency of the target strain, forming a metabolic competitive inhibition of the growth of foreign bacteria.

[0019] 3. The state space reconstruction of the fermentation process is achieved through the multimodal data fusion of online optical sensors and Raman spectroscopy. When environmental disturbances cause any parameter to deviate from the set value, the system automatically triggers the adjustment of the compensation factor matrix. For example, in the scenario of a sudden increase in dissolved oxygen, the feeding frequency is simultaneously reduced and the pressure regulation rate is increased, and the impact of single parameter fluctuations is offset through the dynamic balance of multiple actuators. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 A two-stage control flow chart for the mixed preparation of the biostimulant of the present invention; Figure 2 It is a flow chart of the signal transmission and response mechanism in the dual-stage control of the biostimulant of the present invention; Figure 3 Schematic diagram of the preparation and pressure-responsive release mechanism of chitosan nanoparticles of the present invention; Figure 4 The flowchart of biochemical and morphological index confirmation for stage switching of the present invention. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The embodiment of the present application provides a two-stage control method for mixing and preparing a biostimulant, comprising the following steps: Step S1: Using the coordinated control strategy of dissolved oxygen gradient and carbon source pulse, the dissolved oxygen concentration in the fermenter is linearly reduced from the initial value of 8-15 mg / L to 4-6 mg / L at a rate of 0.5-1.5 mg / (L·h), and a carbon source solution with a volume fraction of 0.5-1.2% is injected every 30-45 minutes; when the bacterial population density reaches And when the concentration of quorum sensing signal molecules reaches 5-15nM, the phase switch is triggered; Step S2: Start pressure-metabolism coupling control, increase the fermentation tank pressure to 0.15-0.25MPa at a rate of 0.01-0.03MPa / min, and add coenzyme Q10 to a final concentration of 50-100μM; monitor the lactic acid concentration online, and when the concentration deviates from the range of 3-5g / L, adjust the carbon source feed rate according to the following formula : , in is the base feeding rate, The adjustment coefficient is 0.1-0.3, is the lactate concentration deviation value; Step S3: during the pressure increase process, a quorum sensing inhibitory peptide is synchronously released, wherein the amino acid sequence of the peptide comprises a Gly-Phe-Arg-Pro-Leu core structure, and the added amount is 0.1-0.3 mg / L fermentation liquid; Step S4: When the pressure stabilizes at the target value, the expression level of the spore formation gene is detected. When it reaches 3-5 times the baseline value, the spore induction is determined to be successful.

[0024] Preferably, the carbon source solution comprises a complex of glucose and yeast extract in a mass ratio of 2:1-4:1.

[0025] Preferably, the dissolved oxygen decrease rate is dynamically adjusted according to the real-time bacterial population density, and the adjustment rule satisfies: When it is below 7.5, the base rate of decline is maintained; when In the range of 7.5-9.5, increase by 1 The unit corresponding to the decline rate is adjusted by reducing 0.05-0.15mg / (L·h).

[0026] Preferably, the coenzyme Q10 is encapsulated by chitosan nanoparticles, the particle diameter is 40-60 nm, and the encapsulation rate is 80-90%.

[0027] Preferably, the quorum sensing inhibitory peptide is released through a sustained-release system at a release rate of 10-20% of the total amount released per hour.

[0028] Preferably, the phase switching verification also includes morphological observation, and the switching is confirmed to be successful when the proportion of oval cells with enhanced refractive index exceeds 70%.

[0029] Preferably, the method further includes standardization of the final product: when the spore formation rate reaches 80% or more, the temperature is lowered to 4-8°C; a protective agent is added to a final concentration of 2-3%, and vacuum freeze-drying is performed to obtain a standardized preparation.

[0030] Preferably, the preparation of the chitosan nanoparticles comprises: dissolving chitosan in an acetic acid solution with a pH of 5.0-6.0; using an ion gel method, using sodium tripolyphosphate as a cross-linking agent, and the cross-linking time is 25-35 minutes.

[0031] Preferably, the online monitoring includes Raman spectroscopy detection, and data is collected every 3-10 minutes.

[0032] Preferably, the biostimulant obtained by the method satisfies: the number of viable spores is ≥ 1×10 9 CFU / g; the mass percentage of lactic acid is 3% to 5%; the contamination rate of foreign bacteria is <1%.

[0033] Example 1: In the bacterial proliferation stage, the dissolved oxygen concentration in the fermenter is first controlled to decrease linearly from the initial value of 8-15 mg / L to 4-6 mg / L at a rate of 0.5-1.5 mg / (L·h), thereby achieving a gradual decrease in dissolved oxygen concentration; at the same time, a carbon source solution with a volume fraction of 0.5-1.2% is injected into the fermenter every 30-45 minutes to ensure the carbon source supply required for bacterial growth; in this process, the monitoring of quorum sensing signals is combined with the regulation of dissolved oxygen gradients to ensure the precise timing of stage switching. When the bacterial density reaches When the concentration of the quorum sensing signal molecule reaches 5-15 nM, the system automatically triggers the phase switching signal and enters the spore induction phase.

[0034] During the spore induction stage, the pressure of the fermenter was adjusted by pressure-metabolism coupling control based on real-time monitoring of lactic acid concentration. At the same time, as the pressure gradually increased during the fermentation process, the pressure change rate was set to 0.01-0.03 MPa / min until the pressure stabilized between 0.15-0.25 MPa. During this process, the carbon source feed rate was precisely controlled, and the formula was: , in, is the base feeding rate, is the adjustment coefficient (the value range is 0.1-0.3), is the lactic acid concentration deviation value; the formula dynamically adjusts the feeding rate according to the change of lactic acid concentration to optimize the bacterial metabolic process and avoid metabolic disorders caused by excessive feeding; during the fermentation process, the quorum sensing inhibitory peptide QSIP is used to effectively inhibit the growth of miscellaneous bacteria. Through the sustained release system of chitosan nanoparticles, QSIP can be continuously released during the pressure increase to ensure that it can exert its maximum efficiency at the right time; at this time, the release rate of QSIP matches the pressure change during the fermentation process to avoid reverse feedback inhibition in the organism.

[0035] At the beginning of fermentation, the fermentation environment is adjusted by linearly decreasing the dissolved oxygen concentration to make it suitable for the growth of the flora. According to the real-time monitoring data, the carbon source solution is injected every 30-45 minutes to ensure the carbon source required for the metabolism of the flora; in the spore induction stage, the lactic acid concentration is continuously monitored, and the feeding rate is adjusted in real time to maintain metabolic balance; by controlling the release rate of QSIP, the inhibition effect of miscellaneous bacteria during the fermentation process is ensured to be synchronized with the metabolic state of the flora. Through the above steps, the problem of the failure of effective coupling between dissolved oxygen and quorum sensing signals in the prior art can be avoided, and efficient control of the fermentation process can be achieved; in particular, by dynamically adjusting the dissolved oxygen gradient, lactic acid concentration and pressure regulation strategy, the metabolic pathway of the flora is optimized, and the ineffective induction caused by switching too early or too late is reduced.

[0036] Embodiment 2: This embodiment combines Figures 1 to 4 , the specific process and key control parameters of the two-stage control method for the mixed preparation of the biostimulant are described.

[0037] like Figure 1 As shown, in the entire fermentation process, the system is first in the initial state, and enters the bacterial proliferation stage after inoculation. In the bacterial proliferation stage, the system dynamically adjusts the dissolved oxygen concentration in the fermenter through the dissolved oxygen control module, specifically linearly reducing the initial dissolved oxygen concentration of 8-15 mg / L to 4-6 mg / L at a rate of 0.5 to 1.5 mg / (L·h) per hour. At the same time, a timed trigger feeding mechanism is set to complete the feeding operation. The feeding content is carbon source feeding with a volume fraction of 0.5-1.2%. This stage is supplemented by a real-time monitoring module to monitor the density of the bacterial colony. The concentration of the quorum sensing signal is monitored. When the condition detection shows that the index is not reached, it is maintained in the bacterial proliferation stage; when the condition detection confirms that the quorum sensing signal reaches the standard, that is, When the concentration of the signal molecule reaches 7 to 10 and the concentration of the signal molecule reaches 5 to 15 nanomoles, the spore induction stage is entered. After entering the spore induction stage, the pressure regulation operation is first performed to slowly increase the pressure in the fermentation tank at a rate of 0.01 to 0.03 MPa per minute until the pressure reaches the standard value of 0.15 to 0.25 MPa; on the basis of the pressure reaching the standard, the system starts the metabolic control mechanism and dynamically adjusts the carbon source feeding rate according to the change in lactic acid concentration. When the lactic acid concentration is within the normal range (3-5g / L), the pulse release mechanism is started to release the quorum sensing inhibitory peptide; if the expression level is still insufficient, the inhibitory peptide is continuously released. Then the gene detection link is entered to evaluate the expression level of the spore formation gene of the target strain. When the test results show that the expression level reaches 3 to 5 times the benchmark, the spore induction is judged to be completed, and the final product processing stage is entered, and operations including cooling, adding protective agents, and vacuum freeze drying are performed.

[0038] like Figure 2 As shown, Figure 2 The signal transmission and response mechanism of each key control module in the two-stage control process is demonstrated; first, the pressure sensor detects the real-time pressure in the fermentation tank and obtains pressure change data at a rate of 0.01 to 0.03 MPa per minute; the signal analyzer receives the pressure data, calculates the compensation coefficient, and sends it to the dissolved oxygen controller; the dissolved oxygen controller adjusts the oxygen concentration decrease rate to 0.05 to 0.15 mg per liter per hour according to the compensation coefficient; the fermentation tank dynamically feeds back the fermentation status according to the adjustment result; at the same time, the quorum sensing detection module detects the signal concentration in real time. When the signal is between 5 and 15 nanomoles, it indicates that the bacterial colony status has reached the switching requirement, and the system triggers the boost program through the stage switch, thereby completing the precise transition from the bacterial proliferation stage to the spore induction stage.

[0039] like Figure 3 As shown, Figure 3 The preparation process of chitosan nanoparticles used to deliver coenzyme Q10 in the present invention and its response release process under pressure-induced conditions are demonstrated; first, chitosan is dissolved in an acetic acid solution with a pH of 5.0 to 6.0, and coenzyme Q10 is loaded in the solution system; then, sodium tripolyphosphate is used as a cross-linking agent, and a cross-linking reaction is carried out for 25 to 35 minutes to form chitosan nanoparticles; the prepared particles have a diameter of 40 to 60 nanometers, and the encapsulation rate reaches 80% to 90%. This type of chitosan nanoparticles shows good response performance when the pressure reaches 0.15 to 0.25 MPa, and can stably release the loaded coenzyme Q10 in a fermentation environment through a pressure-triggered release mechanism; the initiation of the release process is also regulated by the expression level of the spore gene, and the release process can only be triggered when its expression level reaches 3 to 5 times the baseline, thereby ensuring that the release timing of coenzyme Q10 is highly matched with the physiological state of the flora, improving the induction efficiency, and reducing metabolic interference.

[0040] like Figure 4 As shown, the stage confirmation process from the spore induction stage to the final product treatment stage in the two-stage control method for mixed preparation of biostimulants is demonstrated. In terms of biochemical indicators, it is necessary to detect whether the expression of spore formation genes reaches 3 to 5 times the baseline value, and monitor whether the lactic acid concentration is maintained in the range of 3 to 5 grams per liter. When both conditions are met, the stage confirmation link will be entered. In addition, morphological indicators are also used as the basis for stage confirmation, that is, it is necessary to observe whether the proportion of cells with oval shape and enhanced refractive index exceeds 70%. When both biochemical indicators and morphological indicators meet the requirements, the system will perform pressure stabilization control and finally enter the final product treatment stage.

[0041] Example 3: In order to enhance the accuracy of lactic acid concentration feedback control and the stability of the system response mechanism during the fermentation process, the specific sources and implementation paths of the parameters in the feed adjustment formula were further clarified. The feed rate adjustment follows the following formula: , in, Indicates the baseline carbon source feed rate in mL / h, which is determined based on the carbon source metabolic rate at the beginning of fermentation and set as the minimum feed amount required to maintain a stable lactic acid concentration in the range of 3–5 g / L; this value is automatically averaged and calculated by the system within the first 2 hours after inoculation to ensure that the parameter has initial environmental adaptability. is the adjustment coefficient, dimensionless, ranging from 0.1 to 0.3, which represents the sensitivity of the system to the lactate deviation response and is obtained by fitting the feed-metabolism curve in the pre-experimental batch; the variable It is the difference between the current lactic acid concentration and the central reference concentration of 4 g / L (in g / L). Its value comes from the continuous sampling of the online Raman spectroscopy module at a frequency of once every 5 minutes. The system automatically calculates and determines whether to start the adjustment program. In order to improve the stability of the response strategy and avoid frequent feeding due to short-term fluctuations or signal noise, the adjustment range is controlled to not exceed ±15% of the baseline rate within every 10 minutes to ensure that the metabolic flux changes smoothly transition within the acceptable range of the bacterial community and avoid metabolic shocks.

[0042] In terms of stage switching trigger strategy, in order to further enhance the accuracy of judging the physiological state of the flora, when the original flora density reaches Based on the judgment condition that the concentration of the quorum sensing signal molecule is 5-15 nM, a proliferation trend judgment mechanism based on the time gradient change trend is added. Enable the proliferation trend monitoring function and collect data every 15 minutes data, and compare the changes in their growth rates; if If the increase rate slows down by more than 20% and the concentration of the signal molecule is in the range of 13-15 nM, the system will trigger the stage switching control in advance, thereby improving the matching degree between the timing of spore induction and the metabolic maturity of the bacterial flora and improving energy utilization efficiency.

[0043] During the release process of QSIP inhibitory peptide, QSIP achieves pressure-responsive sustained release through chitosan nanoparticles. The system sets the release threshold to start when the pressure reaches 0.15 MPa, and the initial release rate is 10% of the total hourly release. When the target spore gene expression level is simultaneously detected to be more than 3 times the baseline value, the system dynamically adjusts the release rate of QSIP to 20% to adapt to the metabolic needs and physiological activity status of the target strain, avoiding pollution rebound or signal interference delay caused by insufficient release of inhibitory factors.

[0044] Example 4: During the bacterial proliferation stage, the initial dissolved oxygen concentration of the fermenter was set at 10 mg / L. The dissolved oxygen concentration decreased linearly to 5 mg / L at a rate of 1 mg / (L·h). The carbon source solution (glucose to yeast extract mass ratio of 3:1) was injected every 40 minutes, and the injection volume each time was 0.8% of the fermentation liquid volume. When the online monitoring showed that the bacterial population density reached =8 and the concentration of the quorum sensing signal molecule reaches 10 nM, the system will trigger phase switching.

[0045] During the spore induction phase, the fermentation tank pressure was increased to 0.2 MPa at a rate of 0.02 MPa / min, and coenzyme Q10 was added to a final concentration of 75 µM. The carbon source feed rate F was calculated according to the formula Adjustment is made. Among them, the base feed rate Within the first 2 hours after inoculation, the system automatically monitors and calculates the average minimum feed volume required to maintain the lactate concentration in the range of 3-5 g / L. In this example, The specific value is 5 mL / h. Adjustment factor Set to 0.2, indicating that the system has a moderate response sensitivity to lactate concentration deviation. The online Raman spectrometer samples the lactate concentration every 5 minutes and obtains the difference between the current lactate concentration and the target central concentration of 4 g / L. The adjustment range of the carbon source feed rate is limited to no more than ±10% of the baseline rate within 10 minutes to ensure a smooth transition of the metabolic process.

[0046] During the pressure increase, the quorum sensing inhibitory peptide (amino acid sequence containing the Gly-Phe-Arg-Pro-Leu core structure) is continuously released through the pH-responsive chitosan nanoparticle sustained-release system, with an initial addition amount of 0.2 mg / L fermentation broth and a release rate of 15% of the total amount released per hour. When the pressure stabilizes at 0.2 MPa, the expression of the key gene spo0A for spore formation is detected by a gene detection method, such as RT-qPCR. When the expression of the gene reaches 4 times the baseline value in the uninduced state, the spore induction is determined to be successful. In the final product standardization treatment stage, when the spore formation rate reaches more than 85%, the fermentation tank temperature is reduced to 6°C, and trehalose is added as a protective agent to a final concentration of 2.5%. Subsequently, a standardized preparation is obtained by vacuum freeze drying. The final biostimulant should meet the requirements of viable spore count ≥ 2×109 CFU / g, lactic acid mass percentage of 4%, and contamination rate of foreign bacteria < 0.5%, all of which belong to extended implementation methods known to ordinary technicians in the field.

[0047] Example 5: In this example, in the bacterial proliferation stage, in order to more finely control the growth of microorganisms, this example specifically describes the dynamic adjustment of the dissolved oxygen decrease rate according to the real-time bacterial population density. In the initial state, the dissolved oxygen concentration in the fermenter is set to 12 mg / L. After the inoculation is completed, the bacterial proliferation stage is entered. At this time, the system first linearly decreases the dissolved oxygen concentration at a basic decrease rate of 0.8 mg / (L·h). The online monitoring system collects the bacterial population density in real time. When monitoring When the dissolved oxygen concentration is lower than 7.5, the rate of decrease of dissolved oxygen is maintained at the basic value of 0.8 mg / (L·h). When it reaches 7.5 and continues to rise, the system dynamically reduces the dissolved oxygen drop rate according to the adjustment rules. In the range of 7.5 to 9.5, each increase of 0.5 Unit, the dissolved oxygen decrease rate decreases by 0.075mg / (L·h). For example, when When the dissolved oxygen decrease rate is 8.0, the dissolved oxygen decrease rate is adjusted to 0.8 - (8.0 - 7.5) / 0.5 * 0.075 = 0.725mg / (L·h). When it reaches 9.0, the dissolved oxygen decrease rate is further adjusted to 0.8 - (9.0 - 7.5) / 0.5 * 0.075 = 0.575 mg / (L·h).

[0048] This dynamic adjustment strategy ensures that sufficient oxygen is provided to support rapid growth when the bacterial population density is low, while the oxygen supply is gradually reduced when the bacterial population density is high, thereby avoiding metabolic byproducts caused by excessive oxygen supply and creating a more suitable environment for the subsequent spore induction stage. This refined dissolved oxygen control works synergistically with carbon source pulse feeding (injected once every 40 minutes, with a carbon source solution of 0.9% by volume, in which the mass ratio of glucose to yeast extract is 3:1) to more effectively promote the growth of the target strain and improve the yield and quality of the biostimulant. When the online monitoring of the bacterial population density reaches =9 and the concentration of the quorum sensing signal molecule reaches 12nM, the system will trigger the phase switch and enter the spore induction phase. This method of dynamic dissolved oxygen control based on real-time bacterial density can more accurately match the physiological needs of microorganisms, thereby improving the efficiency and stability of the entire biostimulant preparation process, which is an extended implementation method known to ordinary technicians in this field.

[0049] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A two-stage control method for the mixed preparation of biostimulants, characterized in that: The following steps are involved: Step S1: Using the coordinated control strategy of dissolved oxygen gradient and carbon source pulse, the dissolved oxygen concentration in the fermenter is linearly reduced from the initial value of 8-15 mg / L to 4-6 mg / L at a rate of 0.5-1.5 mg / (L·h), and a carbon source solution with a volume fraction of 0.5-1.2% is injected every 30-45 minutes; when the bacterial population density reaches And when the concentration of quorum sensing signal molecules reaches 5-15nM, the phase switch is triggered; Step S2: Start pressure-metabolism coupling control, increase the fermentation tank pressure to 0.15-0.25MPa at a rate of 0.01-0.03MPa / min, and add coenzyme Q10 to a final concentration of 50-100μM; monitor the lactic acid concentration online, and when the concentration deviates from the range of 3-5g / L, adjust the carbon source feed rate according to the following formula : ， in is the base feeding rate, The adjustment coefficient is 0.1-0.3, is the lactate concentration deviation value; Step S3: during the pressure increase process, a quorum sensing inhibitory peptide is synchronously released, wherein the amino acid sequence of the peptide comprises a Gly-Phe-Arg-Pro-Leu core structure, and the added amount is 0.1-0.3 mg / L fermentation liquid; Step S4: When the pressure stabilizes at the target value, the expression level of the spore formation gene is detected. When it reaches 3-5 times the baseline value, the spore induction is determined to be successful.

2. The two-stage control method for mixed preparation of biostimulants according to claim 1, characterized in that: The carbon source solution comprises a glucose and yeast extract complex in a mass ratio of 2:1-4:

1.

3. The two-stage control method for mixed preparation of biostimulants according to claim 1, characterized in that: The rate of dissolved oxygen decline is dynamically adjusted according to the real-time bacterial density, and the adjustment rules meet the following requirements: When it is below 7.5, the base rate of decline is maintained; when In the range of 7.5-9.5, increase by 1 The unit corresponding to the decline rate is adjusted by reducing 0.05-0.15mg / (L·h).

4. The dual-stage control method for mixed preparation of biostimulants according to claim 1, characterized in that: Coenzyme Q10 is encapsulated by chitosan nanoparticles with a particle diameter of 40-60nm and an encapsulation rate of 80-90%.

5. The two-stage control method for mixed preparation of biostimulants according to claim 1, characterized in that: The quorum sensing inhibitory peptide is released through a sustained-release system at a rate of 10-20% of the total amount released per hour.

6. The two-stage control method for mixed preparation of biostimulants according to claim 1, characterized in that: Phase switching verification also includes morphological observation, and successful switching is confirmed when the proportion of oval cells with enhanced refractive index exceeds 70%.

7. The dual-stage control method for mixed preparation of biostimulants according to claim 1, characterized in that: It also includes standardization of the final product: when the spore formation rate reaches more than 80%, the temperature is lowered to 4-8°C; a protective agent is added to a final concentration of 2-3%, and vacuum freeze-dried to obtain a standardized preparation.

8. The two-stage control method for mixed preparation of biostimulants according to claim 4, characterized in that: The preparation of the chitosan nanoparticles comprises: dissolving chitosan in an acetic acid solution with a pH of 5.0-6.0; using an ion gel method, using sodium tripolyphosphate as a crosslinking agent, and the crosslinking time is 25-35 minutes.

9. The dual-stage control method for mixed preparation of biostimulants according to claim 1, characterized in that: The online monitoring includes Raman spectroscopy detection, and data is collected every 3-10 minutes.

10. The dual-stage control method for mixed preparation of biostimulants according to claim 1, characterized in that: The biostimulant obtained by the method meets the following requirements: number of live spores ; The mass percentage of lactic acid is 3% to 5%; the contamination rate of foreign bacteria is <1%.

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