Production method of microbial fermentation protein feed

Abstract

The invention discloses a production method of a microbial fermentation protein feed, and relates to the technical field of ferment.The production method is carried out in a micro-fluidic optomagnetic dual-mode fermentation system and comprises the following steps that a high microenvironment tolerance strain A and a high protein yield strain B which are subjected to genetic engineering modification are mixed according to the volume ratio of 1: 1-3: 1; continuously injecting into a micro-fluidic chip array at a flow rate of 0.3-0.7 uL / min; in the initial stage of fermentation, blue light of 430-450 nm is applied to each unit reaction cavity, and target protein gene expression is activated through CcaS and CcaR photosensitive regulation and control systems; in the middle and later periods of induction, an alternating magnetic field of 20-80 mT and 50 Hz is synchronously applied to each reaction unit; the fermentation wake flow is introduced into an ultrasonic-microwave coupling extraction module to realize instantaneous cracking and protein enrichment. By designing micro-fluidic optomagnetic dual-mode regulation and control, staged proliferation induction and online thallus enrichment reflux are realized, the problems of traditional large-volume mass transfer and low regulation and control precision are solved, the yield is increased, the fermentation period is shortened, and the high-efficiency cyclic utilization rate of thallus and products is increased.

Description

Technical Field

[0001] The present invention relates to the field of fermentation technology, in particular to a method for producing a microbial fermentation protein feed. Background Art

[0002] Current industrial microbial fermentation protein feed production relies primarily on large-scale batch or semi-continuous processes, which are subject to bottlenecks such as limited fermentation efficiency, high energy consumption, significant contamination risks, and high downstream separation costs. Traditional chemical and gas induction methods, particularly during the microbial proliferation and protein expression stages, have low control precision, and product separation often relies on energy-intensive centrifugation or chemical crushing, making it difficult to achieve resource recycling and a closed-loop process.

[0003] Patent CN107535671B discloses a microbial fermented rice wine lees feed and a preparation method for improving rumen protein utilization. The above patent optimizes the fermentation conditions of mixed bacteria solid-state fermentation of rice wine lees, determines the production process parameters, and improves the economic benefits of large-scale production.

[0004] The above patent provides a strain of bacteria with good synergistic effect for fermentation, which can not only significantly increase the content of small molecule proteins and amino acids in rice wine lees, but also improve the utilization and digestibility of the lees; it increases the content of essential amino acids in animal feed, especially the content of lysine and methionine, which are limiting amino acids for dairy cows, and has strong application prospects in dairy cow feed. However, product separation mostly relies on energy-consuming centrifugation or chemical crushing, which makes it difficult to achieve resource circulation and process closed loop.

[0005] To this end, the present application proposes a method for producing microbial fermentation protein feed that achieves staged proliferation induction and online bacterial enrichment and reflux. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for producing microbial fermentation protein feed to solve the technical problems raised in the above-mentioned background technology, such as low control accuracy of traditional chemical and gas induction means, product separation mostly relying on energy-consuming centrifugation or chemical crushing, and difficulty in achieving resource circulation and process closed-loop.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for producing a microbial fermentation protein feed, the production method being carried out in a microfluidic optical-magnetic dual-mode fermentation system, comprising the following steps:

[0008] The genetically engineered strain A with high microenvironment tolerance and the strain B with high protein yield were mixed at a volume ratio of 1:1-3:1 and continuously injected into the microfluidic chip array at a flow rate of 0.3-0.7 uL / min;

[0009] In the early stage of fermentation, 430-450nm blue light was applied to each unit reaction chamber, the temperature was 37±5℃, and the DO was controlled at 30%-40%;

[0010] When OD 600 When it reaches 0.6-0.8, it automatically switches to the induction stage and reduces the blue light intensity to 8-15umol / m 2 ·s, continuous irradiation for 2-4h, activating target protein gene expression through the CcaS and CcaR photosensitive regulatory systems;

[0011] In the middle and late stages of induction, a 20-80 mT, 50 Hz alternating magnetic field is applied to each reaction unit simultaneously, with a magnetic field coil density of 3-5 turns / cm 3 , realizing the rapid enrichment and online reflux of bacteria coated with polylactic acid-Fe3O4 nanocarriers;

[0012] The fermentation tail stream was introduced into an ultrasonic-microwave coupled extraction module with an ultrasonic frequency of 25kHz, a power of 100W, a microwave frequency of 2.45GHz, a power of 400W, and a processing time of 5-10s to achieve instantaneous lysis and protein enrichment.

[0013] Preferably, the production method further comprises:

[0014] Based on the pH, DO, CO2 and microflow rate data collected by online sensors, the AI control unit runs a deep neural network model to adaptively optimize blue light frequency, magnetic field intensity and flow rate;

[0015] Triggered every 48 hours by 365nm ultraviolet light to excite the self-healing polymer coating, repairing cracks on the microchannel surface and inhibiting dirt adhesion;

[0016] After the fermentation batch is completed, the temperature, time, light intensity, and magnetic field parameters are serialized and encoded into the synthetic DNA micromagnetic bead alliance chain architecture for storage, ensuring that the production data cannot be tampered with and is traceable.

[0017] Preferably, the microfluidic chip array consists of 10-100 parallel PFMS chips, each chip contains 50-200 reaction units, the unit volume is 20-40uL, the channel width is 100-200um, the depth is 50-100um, and a PTFE hydrophobic valve is provided to control the segmentation and merging of droplets.

[0018] Preferably, the blue LED array is composed of multiple 450nm light-emitting diodes with high color rendering index, equipped with a PWM dimming module to achieve 1umol / m 2 The light intensity is adjusted with s precision, and the chip surface temperature is maintained within ±0.2°C through a silicon-carbon heat sink.

[0019] Preferably, the thickness of the polylactic acid shell of the nanocarrier is controlled at 10-20 nm, the diameter of the Fe3O4 core is 80-120 nm, the surface functionalized peptide sequence is Gly-Arg-Gly-Asp, it is stably bound at pH 6.5-7.5 and can be automatically deloaded at pH 8.0.

[0020] Preferably, the alternating magnetic field is composed of a planar spiral coil and a microcurrent driving module, with 100-150 coil turns, 1mm coil spacing, and a magnetic field period repetition rate of ≤1ms, which is used for real-time separation and enrichment and supports a bacterial reflux rate of ≥90%.

[0021] Preferably, the ultrasonic-microwave coupled extraction module includes a titanium alloy ultrasonic transducer and a quartz microwave cavity not connected to the end of the microchannel, and the ultrasonic power density is 1-3W / cm 2 , the temperature rise in the microwave irradiation zone was ≤5°C to avoid thermal denaturation of the target protein.

[0022] Preferably, the deep neural network module used by the AI control unit has at least 10 6 The contribution of each process parameter to protein yield is evaluated in real time using an interpretable algorithm, and the model weights are updated through online learning for each batch.

[0023] Preferably, the self-healing polymer coating substrate is a composite material of PDMS and polypropylene, which is cross-linked by dynamic imine bonds after ultraviolet irradiation, and has a coating hardness of 1-2 MPa and a wear resistance of ≥10 5 Second-rate.

[0024] Preferably, the DNA encoding storage module adopts Fountain code error correction design, the DNA sequence verification error rate is ≤10-5, and the number of PCR amplification cycles is ≤15, so as to ensure data storage stability and reading efficiency;

[0025] The blockchain evidence storage module is based on the PoA consensus mechanism, with a ledger update delay of ≤5s. The on-chain nodes include 1 signature node, 3 verification nodes and several observation nodes. All inter-node communications are encrypted using TLS1.3 to ensure the confidentiality of production logs and traceability integrity.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention utilizes microfluidic optical-magnetic dual-mode control to achieve phased proliferation induction and online bacterial enrichment and reflux, solving the problems of traditional large-volume mass transfer and low control precision, thereby increasing yield, reducing fermentation cycle time, and improving the efficient recycling rate of bacterial cells and products.

[0028] 2. The present invention is designed with ultrasonic-microwave rapid extraction to achieve instant cell lysis and protein enrichment, solving the problems of high energy consumption of centrifugal disruption and chemical lysis pollution, shortening downstream processing time, and reducing energy and chemical reagent consumption;

[0029] 3. The present invention is designed with a self-healing nano-coating to achieve real-time repair of microchannel cracks, inhibit the adhesion of biofilm and dirt, solve the problem of easy blockage during long-term operation of the system, extend the continuous operation cycle of production, and reduce manual downtime and cleaning costs;

[0030] 4. This invention uses blockchain DNA evidence to achieve traceability of the entire production process, solve the problem of easy tampering of fermentation process data, improve production transparency and strengthen quality supervision. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the production process of the present invention. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0033] In the description of the present invention, it should be noted that the terms "upper," "lower," "inner," "outer," "front end," "rear end," "both ends," "one end," "the other end," and the like, indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0034] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "installed," "provided with," "connected," etc., should be understood in a broad sense. For example, "connected" may refer to a fixed connection, a detachable connection, or an integral connection; it may refer to a mechanical connection or an electrical connection; it may refer to a direct connection or an indirect connection through an intermediate medium; it may refer to internal communication between two components. Those skilled in the art will be able to understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0035] See also Figure 1The present invention provides an embodiment of a method for producing a microbial fermentation protein feed, wherein the method is carried out in a microfluidic optical-magnetic dual-mode fermentation system, comprising the following steps:

[0036] The genetically engineered strain A with high microenvironment tolerance and the strain B with high protein yield were mixed at a volume ratio of 1:1-3:1 and continuously injected into the microfluidic chip array at a flow rate of 0.3-0.7 uL / min;

[0037] In the early stage of fermentation, 430-450nm blue light was applied to each unit reaction chamber, the temperature was 37±5℃, and the DO was controlled at 30%-40%;

[0038] When OD 600 When it reaches 0.6-0.8, it automatically switches to the induction stage and reduces the blue light intensity to 8-15umol / m 2 ·s, continuous irradiation for 2-4h, activating target protein gene expression through the CcaS and CcaR photosensitive regulatory systems;

[0039] In the middle and late stages of induction, a 20-80 mT, 50 Hz alternating magnetic field is applied to each reaction unit simultaneously, with a magnetic field coil density of 3-5 turns / cm 3 , realizing the rapid enrichment and online reflux of bacteria coated with polylactic acid-Fe3O4 nanocarriers;

[0040] The fermentation tail stream is introduced into an ultrasonic-microwave coupled extraction module with an ultrasonic frequency of 25kHz and a power of 100W, a microwave frequency of 2.45GHz and a power of 400W, and a processing time of 5-10s to achieve instantaneous lysis and protein enrichment;

[0041] Furthermore, strain A: a modified E. coli with gene knockout and tolerance to high dissolved oxygen environment, with a vector copy number ≤10, and has shear resistance and high growth characteristics; strain B: a modified E. coli with optimized metabolic pathway, which can produce ≥2.5g / L of target protein; culture medium formula (10L scale): 20g glucose, 10g yeast extract, 15g tryptone, 2g potassium dihydrogen phosphate, 1g sodium chloride, pH adjusted to 7.0;

[0042] Strain A and strain B were mixed in a volume ratio of 1:2 from 50 mL seed tanks, with a total inoculum volume of 5% v / v. The cells were continuously injected into the chip array at a flow rate of 0.5 uL / min. The system temperature was set at 37 ± 0.5 °C, and blue light irradiation was simultaneously turned on: 450 nm, 120 μmol / m 2 ·s, DO controlled at 35%±2%, online OD 600 When the monitoring reaches 0.75, it automatically enters the induction stage; the blue light intensity is quickly reduced to 10umol / m 2·s, protein gene expression was initiated through the CcaS and CcaR systems, the DO was maintained at 25%±2%, the temperature was maintained at 37°C, and a 50mT, 50Hz alternating magnetic field was applied to each unit simultaneously, so that the bacteria coated with PLA-Fe3O4 nanocarriers were bound and refluxed to the proliferation zone; the fermentation tail flow flowed into the coupled extractor: ultrasonic 20kHz, 100W excitation and microwave 2.45GHz, 400W irradiation were started simultaneously for 8s, which instantly lyzed the bacteria and enriched the protein solution, which was collected after filtration through 0.22um.

[0043] See also Figure 1 The present invention provides an embodiment of a method for producing a microbial fermentation protein feed, the method further comprising:

[0044] Based on the pH, DO, CO2 and microflow rate data collected by online sensors, the AI control unit runs a deep neural network model to adaptively optimize blue light frequency, magnetic field intensity and flow rate;

[0045] Triggered every 48 hours by 365nm ultraviolet light to excite the self-healing polymer coating, repairing cracks on the microchannel surface and inhibiting dirt adhesion;

[0046] After the fermentation batch is completed, the temperature, time, light intensity, and magnetic field parameters are serialized and encoded into the synthetic DNA micromagnetic bead alliance chain architecture for on-chain storage, ensuring that the production data cannot be tampered with and is traceable;

[0047] Furthermore, the PLC control unit is connected to four-way pH, DO, CO2 and microflow rate sensor signals, and adopts a deep neural network DNN structure with 4 neurons in the input layer and 6 hidden layers. The number of nodes in each layer is 128, 256, 256, 138, 64, and 32 respectively, and the output layer has 3 neurons. It includes the prediction of blue light flux, magnetic field intensity, and flow rate adjustment. In the early stage, 50 batches of non-adaptive process experiments were conducted to collect about 5×10 5 Process status-yield data are used for offline model training and verification. After deployment, 1×10 4 For each new data, the model weights are fine-tuned once using the Adam optimizer with a small batch learning rate of 1e-4;

[0048] When the fermentation is carried out for 2 hours, the model starts to predict and calculate the optimal parameter adjustment value every 5 minutes. If the pH deviates from 6.8±0.1, the model recommends increasing the blue light flux by 5-10umol / m 2·s. If DO is higher than 40%, the model reduces the stirring rate by 50rpm and the ventilation volume by 0.1vvm. If CO2 drops below 0.5%, it is recommended to reduce the flow rate by 0.05uL / min to extend the buffer residence time. After receiving the adjustment command, the PLC completes the PWM dimming and magnetic field control signal update within 3s. The flow rate is automatically calibrated by the micro peristaltic pump regulating valve with an accuracy of ±0.01uL / min.

[0049] UV excitation of the self-healing polymer coating: base material: PDMS and polypropylene mixed in a mass ratio of 5:1; self-healing monomer: 10wt% dynamic imine bond precursor; nanofiller: 5wt% SiO2 to enhance mechanical strength; phototrigger: 1wt% 2,4-dihydroxybenzophenone, sensitive to 365nm UV light; when the system runs for 48 hours, the PLC triggers the UV LED cluster regularly, and the UV irradiation lasts for 8 minutes. After the exciter absorbs photons, the reversible dissociation and re-crosslinking reaction of the imine bond is triggered. After the irradiation, the crack width of the self-healing coating is reduced from an average of 5μm to <0.2μm. After online microscopic imaging acceptance, the channel resistance has recovered to 98% of the pre-irradiation level.

[0050] DNA micro-magnetic bead alliance chain evidence storage:

[0051] Preparation of DNA micromagnetic beads: Oligonucleotide design: total length 256nt, including "batch number - temperature - timestamp - illumination intensity - magnetic field parameters - CRC check - random filling"; Fountain code encoding: LT coding error correction is introduced, redundancy is 20%, and error recovery rate ≥ 99.99%; Micromagnetic bead immobilization: the 5' end of the oligonucleotide chain is coupled with biotin and captured by streptavidin micromagnetic beads, and each bead carries ≥ 10 3 DNA;

[0052] After the fermentation batch is completed, the AI control unit exports the above serialized parameters as a hexadecimal string, which is converted into a 4×10 3 DNA oligo strands are synthesized and fixed in micromagnetic beads. The micromagnetic bead information is signed by PoA node A and broadcast to verification nodes B, C, and D. The consensus time is ≤3s. A new block is generated and written into the ledger. At the same time, the block hash is returned to the AI control unit and attached to the batch report.

[0053] See also Figure 1 The present invention provides an embodiment of a method for producing microbial fermentation protein feed, wherein the microfluidic chip array is composed of 10-100 parallel PFMS chips, each chip containing 50-200 reaction units, a unit volume of 20-40uL, a channel width of 100-200um, a depth of 50-100um, and a PTFE hydrophobic valve to control droplet segmentation and merging;

[0054] The blue LED array is composed of multiple high color rendering index 450nm light emitting diodes, equipped with a PWM dimming module to achieve 1umol / m 2 ·S-precision light intensity adjustment, and the chip surface temperature is maintained within ±0.2°C through the silicon-carbon heat dissipation substrate;

[0055] The nanocarrier has a polylactic acid shell thickness of 10-20 nm, a Fe3O4 core diameter of 80-120 nm, and a surface functionalized peptide sequence of Gly-Arg-Gly-Asp. It can stably bind at pH 6.5-7.5 and automatically deload at pH 8.0.

[0056] Furthermore, the assembly and operation configuration of the microfluidic chip array are as follows: Chip collection: PDMS and glass heterogeneous integrated structure is used, microchannels are prepared by soft etching process, and the chip surface is treated with O2 plasma activation to enhance adhesion; Array scale: a total of 40 PFMS chips are connected in parallel, each containing 120 independent reaction units, arranged in a 12×10 array structure; the unit cavity volume is 30uL, the microchannel size is: width 150um, depth 75um, and a rounded rectangular cross-section is used to reduce shear damage; each chip channel Each node is equipped with two PTFE hydrophobic microvalves with an aperture of approximately 80 μm. The control strategy is based on threshold capillary pressure regulation to achieve segmented droplet control and merging. The opening and closing response time is less than 50 ms and can adapt to continuous flow rate changes of ≤ 0.7 μL / min. The chip is integrated through a customized stainless steel module fixed frame. The module adopts a stepped liquid supply structure to ensure the uniformity of parallel liquid supply. A multi-channel micro-syringe pump is used for liquid supply, and real-time flow rate feedback is adjusted. During the initial inoculation stage, each reaction unit is injected with a 0.5 μL bacterial droplet from the main channel and the reaction chamber is sealed.

[0057] Each chip is equipped with an independent blue LED array. The light source unit array consists of 4×8 high color rendering index LEDs with a spacing of 5mm. It uses a PWM dimming module with a frequency of ≥5kHz and a dimming resolution of 0.5umol / m 2 ·s, the minimum dimming step accuracy can be achieved to 1umol / m 2 ·s; All LED arrays are assembled on a silicon-carbon composite heat dissipation substrate with a thermal conductivity of approximately 180W / m·K. The substrate is embedded with a temperature-controlled copper water-cooling channel. Combined with a closed-loop PID control system, the measured chip surface temperature difference is controlled within the range of ±0.2℃, and the light induction consistency is good; Light operation logic: In the early stage of fermentation, the system maintains the blue light intensity at 120umol / m 2 ·s,OD 600 After reaching 0.7, the AI control unit receives sensor feedback and adjusts the blue light to 10umol / m 2·s, continuous induction for 3h, if the chip surface temperature deviates from 37℃±0.2℃, the light intensity regulation will be suspended and the water cooling flow rate will be automatically corrected;

[0058] Preparation and application of PLA-Fe3O4 nanocarriers: The magnetic core is made of spherical Fe3O4 nanoparticles with an average particle size of 100nm. The shell is coated with polylactic acid (PLA) by the oil-in-water emulsion evaporation method, and the shell thickness is controlled to 15nm. The polymer chain containing the peptide segment Gly-Arg-Gly-Asp is grafted onto the surface of the PLA shell. This sequence can bind to the α5β1 integrin receptor in the bacterial surface protein. The binding force is strongest in the pH range of 6.8-7.2. At pH ≥ 8.0, the deprotonation of the amino group causes electrostatic repulsion, achieving automatic desorption and carrier recycling. In the second hour of the induction period, the PLA-Fe3O4 nanocarrier is injected into the chip through the second channel. A 60mT alternating magnetic field is applied to the magnetron array outside the reaction chamber, and the coil layout density is 4 turns / cm. 3 , frequency 50Hz, within 10 minutes, it can be observed that the bacteria and carrier complex are enriched in the middle area of the reaction chamber. The adjustable magnetic field polarity switching is used to achieve the reflux of enriched cells to the proliferation area, thereby improving the yield. At the end of fermentation, the reaction solution is adjusted to pH 8.0, and the carrier is desorbed and regenerated by magnetic separation for the next round of reaction.

[0059] See also Figure 1 The present invention provides an embodiment of a method for producing microbial fermentation protein feed, wherein the alternating magnetic field is composed of a planar spiral coil and a microcurrent drive module, the coil has 100-150 turns, the coil spacing is 1mm, and the magnetic field period repetition rate is ≤1ms, which is used for real-time separation and enrichment and supports a bacterial cell reflux rate of ≥90%;

[0060] The ultrasonic-microwave coupled extraction module includes a titanium alloy ultrasonic transducer and a quartz microwave cavity not connected to the end of the microchannel, and the ultrasonic power density is 1-3W / cm 2 , the temperature rise in the microwave irradiation zone is ≤5°C to avoid thermal denaturation of the target protein;

[0061] Furthermore, a planar double-layer spiral coil is used, with 125 turns in both the outer and inner layers, an insulation thickness of 0.2 mm, and a turn spacing strictly controlled to 1.0 mm. This ensures that the diameter of the coil's planar uniform field area is ≥10 mm, the average diameter of a single turn is 20 mm, and the overall coil thickness is 2.5 mm. It is placed around the microfluidic chip housing to form a four-quadrant symmetrical arrangement. The microcurrent driver board outputs an adjustable square wave current with an adjustable peak current of 0-2 A, a drive frequency of 50 Hz, and a cycle repetition rate of ≤1 ms. The coil is coated with epoxy resin and covered with a heat dissipation aluminum alloy bracket. It is cooled by a 35 mL / min micro fan, and the coil operating temperature is ≤40°C.

[0062] During the second hour of the induction phase, PLA-Fe3O4 nanocarriers were injected into the reaction chamber through the second channel at an initial concentration of 0.2 mg / mL. The PLC issued a command, driving the board to excite the coil with a 50 Hz square wave and a peak current of 1.5 A, generating an alternating magnetic induction intensity of 60 mT. Under the action of the magnetic field, the carrier-bacteria complex converged toward the center of the chip. The enrichment time was 2 minutes, reaching a reflux efficiency of ≥90%. The PLC then simultaneously opened the reflux valve, injecting the high-concentration bacteria + carrier complex solution from the enrichment area back into the proliferation area at a rate of 0.05 uL / min. The magnetic field-reflux action was triggered every 10 minutes, lasting 1 minute each time, and was performed 30 times in total.

[0063] Ultrasonic transducer: Titanium alloy cylinder, diameter 8mm, length 15mm, resonant frequency 25kHz, power density 2W / cm 2 Microwave cavity: A quartz tube-lined microwave cavity with an inner diameter of 10 mm and a length of 20 mm. The microwave source frequency is 2.45 GHz and the output power is 400 W. Coupling position: The outlet of the microfluidic chip is directly connected to the bottom of the ultrasonic transducer through a PEEK high-temperature-resistant tube, with the ultrasonic head immediately behind the microwave cavity inlet. A K-type thermocouple is embedded in the middle of the cavity for real-time temperature detection, with a rated allowable temperature rise of ≤5°C. The microwave cavity is covered with a lead glass shield, and a silicone elastic interface is used between the ultrasonic head and the microfluidic pipeline to prevent liquid backflow.

[0064] The fermentation tail liquid flows into the coupling module at 0.7uL / min, ensuring that the ultrasound and microwave action time is 5-10s. When the droplet enters the ultrasonic head excitation area, the amplitude is set to 10um and the power density is 2W / cm 2 The cell wall is broken, and the droplets after ultrasonic treatment enter the microwave cavity. The microwave source is turned on at the same time and lasts for 5s. The temperature rise of the cavity is controlled at ≤5℃. The intracellular proteins after rupture undergo dynamic expansion and sedimentation. At the outlet, they are intercepted by a 0.22um microfiltration membrane and refluxed for collection. The module can work continuously with a minimum idling interval of 10s until the next droplet arrives.

[0065] See also Figure 1 , an embodiment provided by the present invention: a method for producing microbial fermentation protein feed, wherein the deep neural network module used by the AI control unit has at least 10 6 Parameters, using interpretable algorithms to evaluate the contribution of each process parameter to protein yield in real time, and updating the model weights through online learning for each batch;

[0066] The self-healing polymer coating substrate is a composite material of PDMS and polypropylene, which completes dynamic imine bond cross-linking after ultraviolet light irradiation. The coating has a hardness of 1-2 MPa and a wear resistance of ≥10 5 Second-rate;

[0067] The DNA encoding storage module adopts Fountain code error correction design, and the DNA sequence verification error rate is ≤10 -5 , PCR amplification cycle number ≤ 15 to ensure data storage stability and reading efficiency;

[0068] The blockchain evidence storage module is based on the PoA consensus mechanism, with a ledger update delay of ≤5s. The on-chain nodes include 1 signing node, 3 verification nodes, and several observation nodes. All inter-node communications are encrypted using TLS1.3 to ensure the confidentiality and traceability integrity of production logs.

[0069] Further, the SHAP interpretability algorithm:

[0070] For a feature x k SHAP value Ψ k Defined as:

[0071]

[0072] N = {1, ..., n}, a total of n = 4 feature index sets;

[0073] S: feature subset without k;

[0074] F S (x S ): the expected output of the model when only the subset S is input;

[0075] Weight To ensure that the fairness criteria are met, the online calculation of each k , we can get the marginal contribution of pH, DO, CO2 and flow rate to the improvement of protein yield;

[0076] Cross-linking reaction kinetics:

[0077] Under UV excitation, the imine bonds in the coating undergo reversible cleavage and recombination:

[0078] \ceR-CHO+R'-NH2<=>[hv]R-CH=N-R'+H2O;

[0079] R-CHO: carbonyl precursor;

[0080] R'-NH2: amine cross-linker;

[0081] Reaction rate constant k fwd , k rev Linear relationship with UV intensity I: k fwd -αI,k rev =β;

[0082] α, β: empirical constants.

[0083] Performance testing:

[0084] The following is a summary of the performance test plan and test results for key items in a production method of microbial fermentation protein feed:

[0085] 1. Test item: AI model inference delay;

[0086] Test method and parameters: Input 4D sensor data and measure the delay from input to control command issuance; repeat 1000 times and take the average.

[0087] Expected performance: ≤100ms;

[0088] Actual measured results: 50ms.

[0089] 2. Test item: AI online learning convergence;

[0090] Testing method and parameters: Incremental training of 1×10 per batch 4 New data, monitoring model MAE (g / L);

[0091] Expected index: ≤0.05g / L;

[0092] Actual measured results: 0.03g / L.

[0093] 3. Test item: hardness of self-healing coating;

[0094] Test method and parameters: ASTM D2240 Shore A hardness test;

[0095] Expected index: 1-2MPa;

[0096] Actual measured results: 1.5MPa.

[0097] 4. Test items: wear resistance of self-healing coating;

[0098] Test method and parameters: Taber friction machine, 1kg load, 60rpm, until visible wear appears on the surface;

[0099] Expected index: ≥1×10 5 Second-rate;

[0100] Measured results: ≥1×10 5 Second-rate.

[0101] 5. Test items: self-healing crack healing efficiency;

[0102] Test method and parameters: After artificial scratching 5um, irradiate with 365nm UV for 8min, and measure the crack width;

[0103] Expected index: <0.5um;

[0104] Actual measured results: <0.2um.

[0105] 6. Test item: DNA storage error rate;

[0106] Test method and parameters: Sequencing and comparison with the original sequence after PCR ≤ 15 cycles;

[0107] Expected index: ≤1×10 -5 ;

[0108] Measured results: 1×10 -6 .

[0109] 7. Test items: DNA reading recovery rate;

[0110] Test method and parameters: Nanopore single read;

[0111] Expected index: ≥99.0%;

[0112] Actual measured results: 99.9%.

[0113] 8. Test item: blockchain on-chain delay;

[0114] Test method and parameters: Latency from signature request to block confirmation;

[0115] Expected indicator: ≤5s;

[0116] Actual measured results: 3s.

[0117] 9. Test items: magnetic field separation reflux efficiency;

[0118] Test method and parameters: The proportion of refluxing bacteria after enrichment was measured at 60mT and 50Hz;

[0119] Expected index: ≥90%;

[0120] Actual measured results: 92%.

[0121] 10. Test items: Chip surface temperature control accuracy;

[0122] Test method and parameters: Measure the temperature difference of each unit under LED PWM drive;

[0123] Expected index: ±0.5℃;

[0124] Actual measured results: ±0.2℃.

[0125] 11. Test items: continuous running without blocking time;

[0126] Test method and parameters: The system runs continuously until the first channel is blocked;

[0127] Expected index: ≥96h;

[0128] Actual measured results: 100h.

[0129] 12. Test item: Ultrasonic-microwave cracking efficiency;

[0130] Test method and parameters: 25kHz / 2W / cm 2 +2.45GHz / 400W, 5-10s treatment, residual cell disruption rate measured after 0.22um filtration;

[0131] Expected indicators: ≥95% wall breaking efficiency; temperature rise ≤5℃;

[0132] Measured results: 96% wall-breaking efficiency; 4.2℃.

[0133] Working Principle: Using a microfluidic array, the modified strains A and B are continuously injected into each unit reaction chamber in a set ratio. Combined with high-intensity 430-450nm blue light irradiation and high dissolved oxygen conditions, the rapid exponential growth of the bacteria is achieved.

[0134] When online OD 600 When the set threshold is reached, the system automatically reduces the blue light intensity to 8-15umol / m 2 s, initiating target protein expression through the CcaS and CcaR light-sensitive regulatory pathways; simultaneously applying a 20-80mT, 50Hz alternating magnetic field to each reaction chamber, rapidly enriching the bacteria bound to the magnetic nanocarrier surface and returning them to the proliferation section under the action of the magnetic field, achieving yield amplification and bacterial recycling;

[0135] The discharge flow passes through an ultrasonic and microwave coupling processing unit to instantly lyse the bacteria and enrich the microbial protein; the fermentation data is recorded in real time by a deep neural network model, encoded and written into DNA micromagnetic beads, and then uploaded to the chain for storage, ensuring that the production process is transparent and traceable.

[0136] 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 invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. A method for producing a microbial fermented protein feed, characterized in that: The production method is carried out in a microfluidic optical-magnetic dual-mode fermentation system, comprising the following steps: The genetically engineered strain A with high microenvironment tolerance and the strain B with high protein yield were mixed at a volume ratio of 1:1-3:1 and continuously injected into the microfluidic chip array at a flow rate of 0.3-0.7 uL / min; In the early stage of fermentation, 430-450nm blue light was applied to each unit reaction chamber, the temperature was 37±5℃, and the DO was controlled at 30%-40%; When OD 600 When it reaches 0.6-0.8, it automatically switches to the induction stage and reduces the blue light intensity to 8-15umol / m 2 ·s, continuous irradiation for 2-4h, activating target protein gene expression through the CcaS and CcaR photosensitive regulatory systems; In the middle and late stages of induction, a 20-80 mT, 50 Hz alternating magnetic field is applied to each reaction unit simultaneously, with a magnetic field coil density of 3-5 turns / cm 3 , realizing the rapid enrichment and online reflux of bacteria coated with polylactic acid-Fe3O4 nanocarriers; The fermentation tail stream was introduced into an ultrasonic-microwave coupled extraction module with an ultrasonic frequency of 25kHz, a power of 100W, a microwave frequency of 2.45GHz, a power of 400W, and a processing time of 5-10s to achieve instantaneous lysis and protein enrichment.

2. The method for producing a microbial fermented protein feed according to claim 1, wherein: The production method further comprises: Based on the pH, DO, CO2 and microflow rate data collected by online sensors, the AI control unit runs a deep neural network model to adaptively optimize blue light frequency, magnetic field intensity and flow rate; Triggered every 48 hours by 365nm ultraviolet light to excite the self-healing polymer coating, repairing cracks on the microchannel surface and inhibiting dirt adhesion; After the fermentation batch is completed, the temperature, time, light intensity, and magnetic field parameters are serialized and encoded into the synthetic DNA micromagnetic bead alliance chain architecture for storage, ensuring that the production data cannot be tampered with and is traceable.

3. The method for producing a microbial fermented protein feed according to claim 1, wherein: The microfluidic chip array consists of 10-100 parallel PFMS chips, each chip contains 50-200 reaction units, the unit volume is 20-40uL, the channel width is 100-200um, the depth is 50-100um, and a PTFE hydrophobic valve is provided to control the segmentation and merging of droplets.

4. The method for producing a microbial fermented protein feed according to claim 1, wherein: The blue LED array is composed of multiple high color rendering index 450nm light emitting diodes, equipped with a PWM dimming module to achieve 1umol / m 2 The light intensity is adjusted with s precision, and the chip surface temperature is maintained within ±0.2°C through a silicon-carbon heat sink.

5. The method for producing a microbial fermented protein feed according to claim 1, wherein: The thickness of the polylactic acid shell of the nanocarrier is controlled at 10-20nm, the diameter of the Fe3O4 core is 80-120nm, the surface functionalized peptide sequence is Gly-Arg-Gly-Asp, it is stably bound at pH 6.5-7.5 and can be automatically deloaded at pH 8.

0.

6. The method for producing a microbial fermented protein feed according to claim 1, wherein: The alternating magnetic field is composed of a planar spiral coil and a microcurrent driving module. The coil has 100-150 turns, a coil spacing of 1mm, and a magnetic field period repetition rate of ≤1ms. It is used for real-time separation and enrichment and supports a bacterial reflux rate of ≥90%.

7. The method for producing a microbial fermented protein feed according to claim 1, wherein: The ultrasonic-microwave coupled extraction module includes a titanium alloy ultrasonic transducer and a quartz microwave cavity not connected to the end of the microchannel, and the ultrasonic power density is 1-3W / cm 2 , the temperature rise in the microwave irradiation zone was ≤5°C to avoid thermal denaturation of the target protein.

8. The method for producing a microbial fermented protein feed according to claim 2, wherein: The deep neural network module used by the AI control unit has at least 10 6 The contribution of each process parameter to protein yield is evaluated in real time using an interpretable algorithm, and the model weights are updated through online learning for each batch.

9. The method for producing a microbial fermented protein feed according to claim 2, wherein: The self-healing polymer coating substrate is a composite material of PDMS and polypropylene, which completes dynamic imine bond cross-linking after ultraviolet light irradiation. The coating hardness is 1-2MPa and the number of wear resistance is ≥10 5 Second-rate.

10. The method for producing a microbial fermented protein feed according to claim 2, characterized in that: The DNA encoding storage module adopts Fountain code error correction design, the DNA sequence verification error rate is ≤10-5, and the number of PCR amplification cycles is ≤15 to ensure data storage stability and reading efficiency; The blockchain evidence storage module is based on the PoA consensus mechanism, with a ledger update delay of ≤5s. The on-chain nodes include 1 signature node, 3 verification nodes and several observation nodes. All inter-node communications are encrypted using TLS1.3 to ensure the confidentiality of production logs and traceability integrity.

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