A method of preparing a composition comprising bacillus subtilis secretions

Abstract

The present application relates to the technical field of bacillus subtilis secretion preparation, and discloses a preparation method of a composition containing bacillus subtilis secretion, comprising: physically splitting a sampling flow through a cross-flow filtration module in a sampling period to obtain a first test branch fluid and a second test branch fluid; injecting a fluorescence resonance energy transfer probe into each branch fluid to obtain an enzymatic kinetic signal, determining a kinetic parameter of each branch and calculating a ratio value, providing a preparation trigger instruction based on the ratio value evolution trend, and adding a stabilizer or extracting the composition according to the instruction. The present application uses in-situ two-phase kinetic ratio test logic to decouple the relative distribution of free state activity and attached state activity, offsets background fluorescence noise and sampling fluctuation, solves the determination contradiction between the physical release amount of the secretion and the protein self-degradation effect, and establishes a robustness scale of the composition preparation timing.

Description

Technical Field

[0001] This invention belongs to the field of Bacillus subtilis secretion preparation technology, and particularly relates to a method for preparing a composition containing Bacillus subtilis secretion. Background Technology

[0002] The current preparation of Bacillus subtilis secretions, such as characteristic protease compositions, typically relies on monitoring the physicochemical environment of the culture medium and offline titration. However, in industrial production processes, microbial metabolism is dynamic, and the intracellular synthesis, cell wall enrichment, and final release of secreted products into the liquid phase exhibit spatial lag effects. Target secretions, due to electrostatic adsorption or extracellular polymer encapsulation, are largely anchored to the cell wall surface or inside extracellular polymer aggregates, and are not released into the free liquid phase in real time. Existing testing methods mostly target the total activity of the fermentation broth mixture or measure the activity of a single liquid phase after centrifugation. These conventional methods, targeting enzymes or... Methods for measuring or testing mixed microbial systems cannot accurately quantify the microscopic distribution of enzymes, leading to severe distortion of measurement results for target substances containing enzymes or nucleic acids. When the maturity of the preparation process is determined solely by the total activity index obtained from a single measurement method, there are obvious physical limitations. When solid-liquid separation is performed when the total activity reaches the target, a large number of active components in the attached state are discarded along with the bacterial cell sedimentation, resulting in a decrease in yield. If the culture time is extended to increase the degree of freeness, the liquid-phase secreted products will be inactivated due to the strong self-degradation effect. The uncertainty of the surface phase distribution makes it difficult to lock the trigger point of the preparation action at the balance window between yield and activity.

[0003] Furthermore, the fermentation substrate environment contains a large number of bubbles, cell aggregates, and fluid pulsations, generating physical noise. This noise interferes with the optical detection signal, causing the acquired raw detection data to exhibit non-linear fluctuations. If conventional rate of change calculations or threshold comparisons are directly used to drive the preparation process, erroneous process switching signals are easily triggered. At the hardware level, the industry has attempted to optimize the filtration structure to improve separation efficiency, but logical defects at the control level restrict product quality. For example, Chinese invention patent application CN222400612U discloses a rapid graded filtration device for fermentation broth, which achieves physical graded treatment of fermentation broth by connecting a coarse filter, a deep filter, and a hollow fiber membrane microfiltration component in series. Such existing technologies are essentially in the category of static physical interception. Process triggering depends on preset pressure or time parameters and cannot perceive the dynamic biological phase transition of secretions between the cell wall anchored state and the liquid phase free state in real time. Under industrial conditions, there is a lack of logic for accurately identifying the inflection point of the phase distribution of secretions. Even with graded filtration hardware, it is still impossible to avoid the risk of product loss or inactivation caused by misalignment of yield and activity determination.

[0004] Therefore, the technical problem to be solved by this invention is how to achieve real-time in-situ determination of the phase distribution ratio of secretions, construct a new precise detection method containing enzyme and nucleic acid markers to overcome the limitations of existing microbial detection methods, and precisely trigger the preparation action based on the phase transition inflection point. Summary of the Invention

[0005] The present invention aims to solve the problem that in existing preparation processes, due to the lack of high-precision methods for the determination or testing of enzymes or microorganisms, it is difficult to achieve both yield and activity because the inflection point of the phase distribution of secretions cannot be accurately identified.

[0006] In this technical solution, a method for preparing a composition containing Bacillus subtilis secretions includes: Step 101: During the sampling period of Bacillus subtilis liquid culture, the fermentation broth sampling stream is drawn out and passed into the cross-flow filtration module. The physical interception effect of the microfiltration membrane is used to physically split the fermentation broth sampling stream to obtain the first test branch fluid that passes through the microfiltration membrane and the second test branch fluid that is intercepted by the microfiltration membrane and contains cell bodies and secretions attached to their surfaces. Step 102: Simultaneously inject the test reagent containing the fluorescence resonance energy transfer probe into the fluid of the first test branch and the fluid of the second test branch, and obtain the enzyme kinetic signal of the first test flow path and the second test flow path after contact with the test reagent; Step 103: Measure the first kinetic parameter of the fluid in the first test branch and the second kinetic parameter of the fluid in the second test branch in real time, and calculate the ratio of the first kinetic parameter to the second kinetic parameter; wherein, the ratio is the proportion of the first kinetic parameter to the sum of the first kinetic parameter and the second kinetic parameter; the first kinetic parameter is characterized by the slope of the intensity evolution of the enzyme-catalyzed kinetic signal in the first test branch fluid, and the second kinetic parameter is characterized by the slope of the intensity evolution of the enzyme-catalyzed kinetic signal in the second test branch fluid; Step 104: Monitor the evolution trend of the ratio and provide a preparation trigger command when the ratio meets the preset threshold condition. Add stabilizer or extract composition according to the preparation trigger command.

[0007] Preferably, the first kinetic parameter reflects the activity of free secretions in the fermentation broth, and the second kinetic parameter reflects the activity of secretions attached to the cell surface in the fermentation broth; the ratio serves as a scale for the relative concentration distribution of free and attached components.

[0008] Preferably, step 101 further includes the following sub-step: Step 1011, defining the pore size of the microfiltration membrane in the cross-flow filtration module as 0.22 mm. m to 0.45 m; Step 1012, adjust the transmembrane pressure difference of the fermentation broth sampling flow in the cross-flow filtration module to 0.05 MPa to 0.15 MPa to maintain the integrity of the cell body.

[0009] Preferably, in step 104, while calculating the ratio, the first derivative of the first kinetic parameter is extracted in real time; when the ratio is in the range of 0.75 to 0.85 and the value of the first derivative changes from positive to negative, a preparation trigger command is provided.

[0010] Preferably, the test reagent comprises a specific peptide chain for Bacillus subtilis secretions, and fluorescent donor groups and fluorescent quencher groups respectively coupled to both ends of the specific peptide chain.

[0011] Preferably, the generation logic of the preparation trigger instruction introduces a transcriptional abundance index for Bacillus subtilis secretion genes. The transcriptional abundance index is characterized by the signal captured by the nucleic acid probe, and the transcriptional abundance index is used as the logic gate for calculating the ratio in the start-up step 103.

[0012] Preferably, the addition of stabilizers includes: adding at least one selected from sorbitol, glycerol, calcium chloride and polyethylene glycol to the fermentation broth to adjust the ionic strength of the fermentation broth.

[0013] Preferably, in steps 101 to 104, the ambient temperature of the fluid in the first test branch and the fluid in the second test branch are maintained at 32°C to 37°C, and the flow rate is maintained at 10 mL / min to 50 mL / min.

[0014] Preferably, the composition extraction includes: removing cell bodies by centrifugation according to the extraction sequence locked by the preparation trigger instruction, and vacuum freeze-drying the collected centrifugal supernatant.

[0015] Compared with the prior art, the method for preparing the composition containing Bacillus subtilis secretions of the present invention has the following advantages: 1. In the preparation of compositions of Bacillus subtilis secretions, the physical phase transition inflection point of the target secretion from the cell wall anchored state to the liquid phase free state is captured, which solves the contradiction of spatial distribution lag between intracellular synthesis and overall release of secretions. By establishing a dynamic characteristic function of the proportion of activity in the free phase, the narrow time window of secretion enzyme detachment and complete entry into the liquid phase can be accurately identified. This avoids the loss of active components with the cell when solid-liquid separation is performed too early while the secretion is still attached to the cell wall. At the same time, it avoids the failure of free enzymes due to self-degradation caused by blindly prolonging the reaction time, thus achieving an intrinsic improvement in the unit volume potency of the composition.

[0016] 2. By using the ratio of the dynamic parameters of the first and second test flows as a trigger scale for the preparation action, a measurement logic with self-calibration characteristics is constructed. This logic essentially provides a novel method for the determination or testing of enzymes, nucleic acids, or microorganisms. Since this ratio measurement method focuses on the relative distribution of the two phases of activity, it can objectively offset the sampling volume error and background fluorescence flicker interference caused by bubble shearing, cell aggregation, or local concentration fluctuations in the fermentation system. This makes the determination of the preparation sequence no longer dependent on the absolute value or higher-order derivative calculation of the signal, which is easily affected by high-frequency noise amplification, thus ensuring the robustness of the preparation action execution in harsh industrial biochemical environments.

[0017] 3. A dual-gating mechanism of nucleic acid transcription level testing and enzyme kinetic assay is introduced, which deeply integrates the assay method for specific nucleic acids with the assay method for target enzyme activity, and establishes a multi-dimensional assay or assay system that includes enzymes, nucleic acids or microorganisms. The advanced transcription signal captured by the nucleic acid probe provides a trigger gate for subsequent enzyme kinetic assay. This logical coupling based on molecular biological time difference not only reserves sufficient physical preparation time for the addition of preparation excipients, but also ensures the biological fidelity of preparation decisions through the final verification of protein functional signals, effectively preventing the discrimination bias caused by single biochemical indicators in complex metabolic fluctuations. Attached Figure Description

[0018] Figure 1 This is a flowchart of the preparation process for identifying phase transition inflection points using in-situ dynamic ratios, as described in this invention. Figure 2 This is a schematic diagram of the fabrication system principle architecture of the present invention, which integrates a cross-flow splitting module and a dual-channel feedback regulation loop. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0020] It should be noted that all directional and positional terms used in this invention, such as: up, down, left, right, front, back, vertical, horizontal, inner, outer, top, bottom, transverse, longitudinal, center, etc., are only used to explain the relative positional relationship and connection between components in a specific state (as shown in the accompanying drawings). They are only for the convenience of describing this invention and do not require that this invention be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention. In addition, the descriptions of "first," "second," etc., in this invention are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0021] In the description of this invention, unless otherwise explicitly specified and limited, the terms installation, connection, and linking should be interpreted broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to mechanical connections; they can refer to direct connections or indirect connections through an intermediate medium; they can refer to the internal connection of two components. For those skilled in the art, the specific meaning of the above terms in this invention can be understood according to the specific circumstances.

[0022] In the description of this specification, references to the terms "an embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example, and the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0023] A method for preparing a composition containing Bacillus subtilis secretions, the process flow of which is as follows: Figure 1 As shown, the method includes: Step 101: During the sampling period of Bacillus subtilis liquid culture, the fermentation broth sampling stream is drawn out and passed into the cross-flow filtration module. The physical interception effect of the microfiltration membrane is used to physically split the fermentation broth sampling stream to obtain the first test branch fluid that passes through the microfiltration membrane and the second test branch fluid that is intercepted by the microfiltration membrane and contains cell bodies and secretions attached to their surfaces. Step 102: Simultaneously inject the test reagent containing the fluorescence resonance energy transfer probe into the fluid of the first test branch and the fluid of the second test branch, and obtain the enzyme kinetic signal of the first test flow path and the second test flow path after contact with the test reagent; Step 103: Measure the first kinetic parameter of the fluid in the first test branch and the second kinetic parameter of the fluid in the second test branch in real time, and calculate the ratio of the first kinetic parameter to the second kinetic parameter; wherein, the ratio is the proportion of the first kinetic parameter to the sum of the first kinetic parameter and the second kinetic parameter; the first kinetic parameter is characterized by the slope of the intensity evolution of the enzyme-catalyzed kinetic signal in the first test branch fluid, and the second kinetic parameter is characterized by the slope of the intensity evolution of the enzyme-catalyzed kinetic signal in the second test branch fluid; Step 104: Monitor the evolution trend of the ratio and provide a preparation trigger command when the ratio meets the preset threshold condition. Add stabilizer or extract composition according to the preparation trigger command.

[0024] Preferably, the first kinetic parameter reflects the activity of free secretions in the fermentation broth, and the second kinetic parameter reflects the activity of secretions attached to the cell surface in the fermentation broth; the ratio serves as a scale for the relative concentration distribution of free and attached components.

[0025] Preferably, step 101 further includes the following sub-step: Step 1011, defining the pore size of the microfiltration membrane in the cross-flow filtration module as 0.22 mm. m to 0.45 m; Step 1012, adjust the transmembrane pressure difference of the fermentation broth sampling flow in the cross-flow filtration module to 0.05 MPa to 0.15 MPa to maintain the integrity of the cell body.

[0026] Preferably, in step 104, while calculating the ratio, the first derivative of the first kinetic parameter is extracted in real time; when the ratio is in the range of 0.75 to 0.85 and the value of the first derivative changes from positive to negative, a preparation trigger command is provided.

[0027] Preferably, the test reagent comprises a specific peptide chain for Bacillus subtilis secretions, and fluorescent donor groups and fluorescent quencher groups respectively coupled to both ends of the specific peptide chain.

[0028] Preferably, the generation logic of the preparation trigger instruction introduces a transcriptional abundance index for Bacillus subtilis secretion genes. The transcriptional abundance index is characterized by the signal captured by the nucleic acid probe, and the transcriptional abundance index is used as the logic gate for calculating the ratio in the start-up step 103.

[0029] Preferably, the addition of stabilizers includes: adding at least one selected from sorbitol, glycerol, calcium chloride and polyethylene glycol to the fermentation broth to adjust the ionic strength of the fermentation broth.

[0030] Preferably, in steps 101 to 104, the ambient temperature of the fluid in the first test branch and the fluid in the second test branch are maintained at 32°C to 37°C, and the flow rate is maintained at 10 mL / min to 50 mL / min.

[0031] Preferably, the composition extraction includes: removing cell bodies by centrifugation according to the extraction sequence locked by the preparation trigger instruction, and vacuum freeze-drying the collected centrifugal supernatant.

[0032] Example 1: In a 1000L capacity Bacillus subtilis secretion production environment, the principle architecture of its preparation system is as follows: Figure 2As shown, the increased cell density and aeration of the culture medium generate physical and optical noise, causing the online fluorescent probe to exhibit nonlinear random fluctuations in the acquired raw signal when monitoring total enzyme activity due to bubble shearing and local concentration pulsations. If the process switching action is triggered based on the absolute value of the fluctuating raw fluorescence intensity, a judgment bias will occur. During this sampling period, a fermentation broth sampling stream is drawn out and passed into a cross-flow filtration module. A 0.45 μm microfiltration membrane physically splits the fermentation broth sampling stream under a transmembrane pressure difference of 0.1 MPa, obtaining the first test branch fluid that passes through the microfiltration membrane and the second test branch fluid that is intercepted by the microfiltration membrane and contains cell bodies and secretions attached to their surfaces. The transmembrane pressure difference parameter range... The establishment of the pressure differential (0.05 MPa to 0.15 MPa) is based on previous hydrodynamic gradient pressure tests on the mechanical strength of Bacillus subtilis cell walls. Experimental data show that when the pressure differential is set below 0.05 MPa, the filtration driving force cannot overcome the fluid resistance of the liquid phase boundary layer, resulting in insufficient permeate flux and inducing surface blockage of the membrane pores. When the pressure differential is higher than 0.15 MPa, the accumulated shear stress in the cross-section will exceed the critical compressive strength limit of the Bacillus subtilis cell wall in the logarithmic growth phase, inducing large-scale irreversible mechanical rupture of the cells. This forces intracellular free proteases that have not yet reached the physiological secretion phase to be forcibly released into the microfiltration membrane permeate, thereby causing the kinetic detection of in situ biphasic activity to fail due to biological artifacts.

[0033] A test reagent containing a fluorescence resonance energy transfer probe is simultaneously injected into the first and second test branch fluids. A dual-channel optical sensor acquires the enzymatic kinetic signals of the two fluids after contact with the test reagent. The instantaneous slope of the fluorescence intensity evolution in the first test branch fluid is calculated to determine the first kinetic parameter representing the activity of the free secretion. The instantaneous slope of the fluorescence intensity evolution in the second test branch fluid was calculated to determine the second kinetic parameter representing the activity of the attached secretions. In this embodiment, the target secretion is Bacillus subtilis alkaline protease. The specific peptide chain in the test reagent contains an amino acid sequence that can be specifically recognized and cleaved by this alkaline protease. For example, its core recognition sequence is alanine-alanine-proline-phenylalanine (Ala-Ala-Pro-Phe). The two ends of this specific peptide chain are coupled with a fluorescent donor group (such as EDANS) and a fluorescent quencher group (such as DABCYL). When the target protease cleaves this specific peptide chain, the fluorescent donor and quencher groups spatially separate, thereby releasing a fluorescent signal that can be captured by an optical sensor. The control unit constructs a dynamic feature function in real time to characterize the physical maturity of the secretion. Dynamic characteristic function The calculation method is as follows: ;in, The ratio representing the activity of the free phase of secretions. The first dynamic parameter is... The second kinetic parameter is used to offset common-mode interference caused by bubble pulsation or sampling volume error in the fermentation system. This enables the identification of the physical phase transition point of the secretion from the cell wall anchored state to the liquid phase free state, and resolves the temporal conflict between incomplete release of secreted products and self-degradation effect. Here, the dynamic characteristic function R is the engineering normalized representation of the ratio of the first kinetic parameter to the second kinetic parameter as defined in the basic method of this invention. In the underlying architecture of the algorithm execution, in order to enable the monitoring signal to intuitively map the physical phase distribution from 0 to 100%, and to establish a unified threshold judgment boundary for subsequent control commands, the control unit transforms the direct mathematical quotient relationship of the original two flow paths (i.e., the comparison between V1 and V2 themselves) into a normalized set proportion of free state activity relative to the total activity of the system through algebraic identity transformation. This ensures that the expansion of the parameter attribute dimension still supports the original logical intention of the ratio comparison of the technical solution of this invention and maintains the closed loop of the calculation process.

[0034] When the ratio is monitored The first dynamic parameter was acquired simultaneously within the numerical range of 0.75 to 0.85. When the instantaneous rate of change over time changes from a positive value to a negative value, it is determined that the free release process of the secretion is completed and the self-degradation effect begins to dominate the activity evolution. Subsequently, a preparation trigger instruction is generated, and a stabilizer is added to the fermentation broth. The stabilizer includes calcium chloride and sorbitol, with a mass ratio of calcium chloride to sorbitol of 1:5. The amount of stabilizer added is 2% of the total mass of the fermentation broth. Combined with cooling to 4°C and centrifugation purification, the resulting composition maintains batch consistency while the total protease titer per unit volume is increased compared to the physicochemical monitoring method.

[0035] Example 2: In a 200L fermentation verification scenario, the experimental platform included a bypass online monitoring unit and a dual-channel fiber optic sensor with a detection limit of 0.05 RFU. The sampling frequency was set to 1Hz, selected based on the first-order metabolic rate constant of the secretion, to capture the nonlinear characteristics of the initial stage of the enzymatic reaction. The minimum sampling interval was determined to ensure the temporal resolution of the data. To test the system's resistance to physical and optical noise, microbubbles with a volume fraction of 5% to 15% were injected into the sampling background. Control group A used the total fluorescence intensity of the mixed turbidity as the criterion, while control group B only used the kinetic parameters of the fluid in the first test branch. Monitoring data showed that when bubble disturbance increased from 5% to 15%, control group A experienced a trigger timing shift of 35.2 minutes, while control group B showed a positively correlated shift in trigger time with injection volume fluctuations, with a shift rate reaching 12.5%. Meanwhile, the dynamic characteristic function measured in the experimental group... The fluctuation amplitude is less than 1.8% across the entire perturbation domain. This ratio analysis logic utilizes the first dynamic parameter. With the second dynamic parameter The homogeneous perturbation component cancels out the common-mode interference generated by bubble pulsation in the fermentation system.

[0036] Regarding the ratio Gradient validation was performed on the numerical range, and the lower limit of the ratio was set at 0.70 for the out-of-range control group C. When the set value was reached, the stabilization program was initiated. At this point, the proportion of attached secretions in the fermentation broth was 30.2%, and the final product extraction rate was measured to be 58.6%. The control group D, which exceeded the set ratio limit of 0.90, was... When the set value is reached, a preparation trigger command is generated. Due to the prolonged exposure of the product to an alkaline environment, the self-degradation and inactivation rate of the free protease reaches 24.5%, resulting in a 15.8% decrease in the total potency of the final composition. When the ratio... When the concentration was in the range of 0.75 to 0.85, the protease activity of the final prepared composition remained stable between 162.4 U / mL and 168.7 U / mL. Experimental data confirmed that the combination of physical splitting and ratiokinetic monitoring improved the precision of fermentation process control, and determined that real-time monitoring of the free phase activity ratio was crucial for successful fermentation. and Defining the logical necessity of the process endpoint by the instantaneous rate of change over time ensures high batch consistency in the composition preparation process under physical environmental noise interference.

[0037] Example 3: In a 500L Bacillus subtilis secretion preparation system equipped with a programmable logic controller and a high-precision peristaltic pump set, the initial equilibrium state of the cross-flow filtration module is established using a pressure feedback regulation method. That is, at the initial stage of drawing out the fermentation broth sampling flow, the system monitors the transmembrane pressure difference in real time. , specifically The calculation formula is as follows: ;in, For transmembrane pressure difference, This refers to the inlet pressure of the microfiltration membrane. For the microfiltration membrane outlet pressure, To ensure the permeable liquid side pressure; the control unit adjusts the peristaltic pump speed to... The pressure stabilizes at 0.1 MPa within 60 seconds with a fluctuation rate of less than 5%, thus eliminating the measurement deviation caused by the fluctuation in the flow ratio between the first and second test branches by stabilizing the flow state.

[0038] After the test reagents are injected into the first and second test streams, a dual-channel optical sensor acquires fluorescence intensity data sequences at a frequency of 1 Hz. Following the principle of fluorescence quenching and recovery in nucleic acid hybridization, before capturing the kinetic data window, the system control unit pre-determines the baseline fluorescence of the specific nucleic acid probe within the fermentation broth sampling stream. During fermentation, the system control unit simultaneously acquires the real-time hybridization fluorescence intensity of the nucleic acid probe and calculates the ratio of the real-time hybridization fluorescence intensity to the baseline fluorescence intensity, serving as a transcriptional abundance index characterizing the transcriptional activity of secretory genes. The nucleic acid probes selected here target and encode specific functional groups of the aforementioned Bacillus subtilis alkaline protease. The probe (i.e., the aprE gene) contains a nucleotide sequence (e.g., a specific oligonucleotide single strand consisting of 20 to 25 bases) complementary to the conserved transcription region of the aprE gene. A fluorescent group modified at the probe tip undergoes a conformational change and emits light upon hybridization with newly transcribed target mRNA molecules within the cell, thus converting specific gene expression levels into a readable overall optical signal. This serves as a fundamental data source for determining nucleic acid transcription levels. Monitoring is performed when the transcriptional abundance index crosses a preset activation threshold. This threshold is determined by pre-fitting a linear mapping between the transcript abundance of a specific strain and the actual overall protein expression level, typically ranging from 2.0 to 3.0 times the baseline value. After the state transition time exceeds the set 5-second anti-disturbance delay, the internal register state of the system control unit flips from low to high, generating a logic gating instruction to initiate enzyme kinetics calculations. This logic gating instruction is not based on the assumption that transcription and extracellular macromolecule release occur synchronously, but rather on a physical feedforward mechanism constructed based on the known anabolism lag effect in Bacillus. Because there is a biologically fixed time difference between the peak transcription of the aprE gene and the completion of transmembrane secretion and physical shedding of the protease in the liquid phase, determined by the ribosome translation rate and extracellular polymeric dissolution kinetics, this very early transcriptional level flip is used for calculation. The window's gating trigger signal can shield against interference from background free enzymes in dead cells caused by sporadic cell wall rupture during the early stages of fermentation. This allows the system control unit to activate high-frequency kinetic monitoring only when the actual overall phase transition lag phase is about to arrive. Thus, without violating the physical causal chain, it achieves cross-scale temporal alignment from surface genetic information expression to the overall fluid phase transition. Based on the logic gating instruction, the processing unit extracts 10 consecutive data points, including the current acquisition point, as a dynamic calculation window. It then applies a least-squares linear fitting algorithm to calculate the regression slope of the data within this window, determining this regression slope as the instantaneous slope at that moment, thereby obtaining the first kinetic parameter. With the second dynamic parameter The value of is used to determine the judgment interval for the preparation trigger command. Before the formal preparation procedure is started, a critical phase transition point scanning experiment is performed to record the ratio of secretions during the transition from intracellular synthesis to extracellular release. Evolution curve, capture Maximum point and comparative analysis The change in the self-degradation rate of the product within 15 minutes before and after; experimental results show that when the ratio It is between 0.75 and 0.85, and When the instantaneous rate of change over time is less than 0 for three consecutive sampling periods, the cumulative amount of free protease reaches a peak plateau. At this point, adding a stabilizer can control the protease inactivation loss to below 5%. Using the dynamic characteristic function judgment logic determined by this parameter calibration method, the system completes closed-loop control of the preparation endpoint under the background noise of the fermentation broth, ensuring that different batches of the composition meet the quality specifications in terms of secretion composition and bioactivity indicators, and determining the stability of the preparation method of the present invention in the engineering reproduction process.

[0039] Example 4: Under the condition of applying a standardized calibration procedure for different batches of fluorescence resonance energy transfer probes, the control unit introduces a calibration solution containing a preset concentration of protease into the test channel before drawing out the fermentation broth sampling stream. A dual-channel optical sensor is used to acquire the initial enzymatic kinetic signal after the calibration solution contacts the test reagent, and the probe sensitivity calibration factor is calculated based on the acquired signal. ;in, This is a dimensionless sensitivity correction coefficient; the logic operation module will... Compensation to the acquired test branch signal ensures that the first kinetic parameter is accurate for different reagent batches. With the second dynamic parameter This creates physical comparability and eliminates the impact of fluctuations in reagent catalytic activity on the ratio. The offset.

[0040] When the system faces the challenge of different flow path physical distribution due to the replacement of the cross-flow filter module, the control unit measures the permeate flow rate of the sterile culture medium under a transmembrane pressure difference of 0.1 MPa after the cleaning cycle is completed. With circulating flow rate Calculate the volume distribution weighting coefficients. ; ;in, Assign weighting coefficients to the volume. The permeation velocity, The circulation flow rate; based on this, the system will Introducing dynamic feature functions The calculation formula, by weighting the difference in volumetric flux between the fluid in the first test branch and the fluid in the second test branch, ensures that the determined proportion of the free phase activity of the secretion remains anchored to the physical distribution state during the evolution of the microfiltration membrane pore size, and keeps the judgment error of the composition extraction process within the fluctuation threshold of 2%.

[0041] Example 5: Under the condition of system initialization calibration for Bacillus subtilis preparation production lines of different scales, the control unit starts the reference registration procedure when the fermentation sampling flow path is first connected. By injecting a standard substrate solution containing known protease activity into the test flow path and monitoring the fluorescence intensity response slope of the dual channels, the channel correction coefficient used to characterize the gain difference of optoelectronic devices is calculated. ;in ,in, This is a dimensionless channel correction coefficient. The slope of the response of the first test branch under the standard substrate solution. The response slope of the second test branch under the standard substrate solution; the logic operation module will... The data is stored in a preset configuration matrix and used to perform real-time normalization processing on the raw dynamic data in subsequent production processes, eliminating ratios caused by fiber aging or differences in the transmittance of the detection window. To mitigate physical system errors and ensure consistency of judgment criteria when deployed across production lines.

[0042] To determine the stability of the preparation trigger criterion under complex culture medium conditions, the system applied an offline optimization algorithm to smooth the continuously acquired enzyme kinetic signal sequences during the initialization and debugging phase. The autocorrelation function of the signals under different time lengths was calculated to determine the criteria used for extraction. Length of the sampling sliding window for the instantaneous rate of change with time The value is 10; based on the high-frequency noise filtering criterion of discrete control systems, to avoid false positive process switching caused by fluid shearing and local bubble group penetration, the system control unit defines the physical transient phase transition from positive to negative values ​​of the first derivative value, transforming it into a multi-cycle delay confirmation procedure to capture the first dynamic parameter in real time. When the polarity of the first derivative sequence crosses zero, and the mathematical event of the derivative value flipping from positive to negative is first detected, a startup state verification time window is triggered. The time window covers the continuous sampling sequence. When the system identifies the dynamic characteristic function... Entering the value range of 0.75 to 0.85 and When the first derivative over time maintains a negative polarity for five consecutive sampling periods, it is determined that the concentration of the free product has exceeded the kinetic peak plateau and the protein degradation rate has begun to exceed the physical release rate. Then, the automatic feeding device is activated to inject a stabilizer composed of calcium chloride and sorbitol in a mass ratio of 1:5 into the fermentation broth. The determination method based on the multi-period evolution trend verification ensures that different batches of the composition have high stability in terms of unit potency.

[0043] Under the condition of optimizing the proportion of stabilizers added during the preparation process, the system utilizes dynamic characteristic functions. The determination result triggered the automatic feeding program. The sum of the mass percentages of calcium chloride and sorbitol in the stabilizer was set to 2% of the total mass of the fermentation broth. By changing the total amount of stabilizer added, a control verification was carried out. The monitoring results showed that when the amount of stabilizer added was less than 1% of the total mass of the fermentation broth, the thermal inactivation rate constant of the protease molecules at 4°C increased by 28.5% compared with the standard sample group, resulting in the potency loss of the composition exceeding the specification limit during the storage period. When the amount of stabilizer added was more than 3% of the total mass of the fermentation broth, the increased osmotic pressure generated by sorbitol induced premature cell lysis and release of intracellular protease, resulting in an increase of 15.2% in the impurity protein content in the final composition. Finally, it was verified that the addition amount of 2% was the optimal process range for balancing the stability and purity of the product, ensuring that the final composition maintained high catalytic activity while having high physicochemical stability.

[0044] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit of this application and the scope of protection of this invention, and all of these forms are within the protection scope of this application.

Claims

1. A method for preparing a composition comprising Bacillus subtilis secretions, characterized in that, include: Step 101: During the sampling period of Bacillus subtilis liquid culture, the fermentation broth sampling stream is drawn out and passed into the cross-flow filtration module. The physical interception effect of the microfiltration membrane is used to physically split the fermentation broth sampling stream to obtain the first test branch fluid that passes through the microfiltration membrane and the second test branch fluid that is intercepted by the microfiltration membrane and contains cell bodies and secretions attached to their surfaces. Step 102: Simultaneously inject the test reagent containing the fluorescence resonance energy transfer probe into the fluid of the first test branch and the fluid of the second test branch, and obtain the enzyme kinetic signal of the first test flow path and the second test flow path after contact with the test reagent; Step 103: Measure the first kinetic parameter of the fluid in the first test branch and the second kinetic parameter of the fluid in the second test branch in real time, and calculate the ratio of the first kinetic parameter to the second kinetic parameter; wherein, the ratio is the proportion of the first kinetic parameter to the sum of the first kinetic parameter and the second kinetic parameter; the first kinetic parameter is characterized by the slope of the intensity evolution of the enzyme-catalyzed kinetic signal in the first test branch fluid, and the second kinetic parameter is characterized by the slope of the intensity evolution of the enzyme-catalyzed kinetic signal in the second test branch fluid; Step 104: Monitor the evolution trend of the ratio and provide a preparation trigger command when the ratio meets the preset threshold condition. Add stabilizer or extract composition according to the preparation trigger command.

2. The method for preparing a composition containing Bacillus subtilis secretions according to claim 1, characterized in that, The first kinetic parameter reflects the activity of free secretions in the fermentation broth, while the second kinetic parameter reflects the activity of secretions attached to the cell surface in the fermentation broth; the ratio serves as a scale for the relative concentration distribution of free and attached components.

3. The method for preparing a composition containing Bacillus subtilis secretions according to claim 1, characterized in that, Step 101 further includes the following sub-steps: Step 1011, defining the pore size of the microfiltration membrane in the cross-flow filtration module as 0.22 mm. m to 0.45 m; Step 1012, adjust the transmembrane pressure difference of the fermentation broth sampling flow in the cross-flow filtration module to 0.05 MPa to 0.15 MPa to maintain the integrity of the cell body.

4. A method for preparing a composition containing Bacillus subtilis secretions according to claim 1, characterized in that, In step 104, while calculating the ratio, the first derivative of the first dynamic parameter is extracted in real time; when the ratio is in the range of 0.75 to 0.85 and the value of the first derivative changes from positive to negative, a preparation trigger command is provided.

5. A method for preparing a composition containing Bacillus subtilis secretions according to claim 1, characterized in that, The test reagent contains a specific peptide chain for Bacillus subtilis secretions, and fluorescent donor groups and fluorescent quencher groups respectively coupled to both ends of the specific peptide chain.

6. A method for preparing a composition containing Bacillus subtilis secretions according to claim 1, characterized in that, The generation logic for preparing the trigger instruction introduces a transcriptional abundance index for Bacillus subtilis secretion genes. The transcriptional abundance index is characterized by the signal captured by the nucleic acid probe, and the transcriptional abundance index is used as the logic gate for calculating the ratio in the start-up step 103.

7. A method for preparing a composition containing Bacillus subtilis secretions according to claim 1, characterized in that, The addition of stabilizers includes adding at least one of sorbitol, glycerol, calcium chloride and polyethylene glycol to the fermentation broth to adjust the ionic strength of the fermentation broth.

8. A method for preparing a composition containing Bacillus subtilis secretions according to claim 1, characterized in that, In steps 101 to 104, the ambient temperature of the fluid in the first test branch and the fluid in the second test branch are maintained at 32°C to 37°C, and the flow rate is maintained at 10 mL / min to 50 mL / min.

9. A method for preparing a composition containing Bacillus subtilis secretions according to claim 1, characterized in that, The composition extraction includes: removing cell bodies by centrifugation according to the extraction sequence locked by the preparation trigger command, and vacuum freeze-drying the collected centrifugal supernatant.

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