Preparation process of high-efficiency microbial degradation liquefied bacteria enzyme mixed preparation

Abstract

The application discloses a kind of high-efficiency microbial degradation liquefied bacteria enzyme mixed preparation preparation process, it is related to the field of bacterial culture, comprising the following steps: step one, parallel fermentation culture;Step two, enzyme activity triggers and membrane separation coupling operation;Step three, product flow direction control and dynamic switching;Step four, membrane pollution control;Step five, end point determination;Step six, quantitative mixed preparation.This application constructs an automatic, continuous production platform by process reconfiguration and system integration.Not only significantly improves production efficiency and process stability, but also ensures the high activity and high reliability of the final bacteria enzyme mixed preparation.

Description

Technical Field

[0001] This invention mainly relates to the technical field of bacterial culture, specifically a process for preparing a highly efficient microbial degradation liquefied bacterial enzyme mixture. Background Technology

[0002] Microbial bacterial-enzyme mixtures, due to their synergistic effects of both live bacteria and enzymes, have shown great application potential in fields such as organic waste biodegradation and environmental remediation. These preparations typically consist of specific degrading strains and their produced extracellular enzymes, and are prepared through parallel fermentation, separation, purification, and mixing, belonging to the field of microbial fermentation engineering technology.

[0003] Currently, common preparation processes typically employ a step-by-step, batch-based approach. First, enzyme-producing fermentation is carried out in a fermenter. After fermentation, the bacterial cells are separated from the fermentation broth using centrifugation and filtration. The bacterial sludge and enzyme-containing supernatant are then further processed. Finally, they are compounded in a specific ratio with the addition of a stabilizer to produce the final product. While existing technologies can produce mixed bacterial-enzyme formulations, they have significant shortcomings in efficiency and quality control: First, enzyme-producing fermentation and high-density bacterial culture are often difficult to optimize simultaneously in the same fermenter, limiting production efficiency. Second, separation is performed only after fermentation has ended, resulting in a long production cycle, and the enzyme is prone to inactivation in the later stages of fermentation, affecting yield. Third, the separation process is energy-intensive, easily damaging to the bacterial cells, and membrane separation technology faces problems such as rapid membrane fouling and frequent cleaning.

[0004] Therefore, existing preparation processes generally suffer from technical defects such as fragmented processes, low efficiency, process instability, and poor product uniformity. There is an urgent need to develop a preparation process that integrates efficient fermentation, online separation, intelligent control, and precise proportioning to fundamentally improve the production efficiency, product quality, and process economy of microbial degradation liquefied bacterial enzyme mixtures. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a highly efficient preparation process for a mixed preparation of microbial degradation liquefied bacterial enzymes, so as to solve the technical problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a high-efficiency microbial degradation liquefied bacterial enzyme mixture preparation process, comprising the following steps: Step 1, parallel fermentation culture: In the bacterial enzyme acquisition tank, the target degrading bacteria are subjected to enzyme-induced fermentation to obtain a fermentation broth containing bacterial cells and the target degrading enzyme; simultaneously, in the bacterial cell culture tank, the same or synergistic strains are subjected to high-density fermentation to obtain a high-biomass bacterial broth; Step 2, enzyme activity triggering and membrane separation coupled operation: When the enzyme activity detection device detects that the enzyme activity in the bacterial enzyme acquisition tank reaches a preset threshold, the enzyme-liquid membrane separation device is automatically started; the fermentation broth is drawn through the currently activated suction point in the multi-point suction component and enters the enzyme-liquid membrane separation device for separation; Step 3, product flow direction control and dynamic switching: The separated enzyme solution is transported to the enzyme collection tank for storage; the separated concentrated bacterial solution is transferred through the currently activated suction point in the multi-point reflux component. The activated reflux point returns to the enzyme acquisition tank, and during the return process, gas is replenished into the reflux liquid stream through the gas replenishment component; wherein, the multi-point suction component and the multi-point reflux component automatically switch the combination of the activated suction point and reflux point according to the set time cycle; Step four, membrane fouling control, during the separation process, when the permeate flux of the enzyme liquid membrane separation device drops to 70%-85% of the initial flux, the backwashing device is automatically started to backwash it online; Step five, endpoint determination, when the specific enzyme production rate of bacteria in the enzyme acquisition tank drops to below 30% of the peak value, the separation operation is stopped; Step six, quantitative mixing and formulation, through the quantitative delivery device, according to the preset enzyme activity ratio, the enzyme liquid in the enzyme collection tank and the bacterial liquid in the bacterial culture tank are delivered to the enzyme mixing formulation tank for mixing, and a stabilizer is added to prepare a liquefied enzyme mixture.

[0007] Preferably, the device includes an enzyme acquisition tank, an enzyme collection tank, a bacterial culture tank, and an enzyme mixture preparation tank. The enzyme mixture preparation tank is connected to the enzyme collection tank and the bacterial culture tank via a quantitative delivery device. The enzyme acquisition tank has a stirrer at its top, with its execution end extending into the tank. The top and bottom of the inner wall of the enzyme acquisition tank are respectively provided with a multi-point reflux component and a multi-point suction component. The device also includes a supplementary gas component with its output end connected to the multi-point reflux component. Furthermore, the device includes an enzyme liquid membrane separation device with its input end connected to the multi-point suction component and its output end connected to the multi-point reflux component. The enzyme liquid membrane separation device includes a delivery pump and a biofilm filter located on the outer wall of the enzyme acquisition tank. The input end of the delivery pump is connected to the multi-point suction component via a first pipe, and the output end of the delivery pump is connected to the biofilm filter via a second pipe. The bacterial liquid discharge end of the biofilm filter is connected to the multi-point reflux component via a third pipe, and the enzyme liquid discharge end of the biofilm filter is connected to the enzyme collection tank via a fourth pipe. In this preferred embodiment, the four core tanks and the integrated membrane separation device form an integrated platform to support continuous operation.

[0008] Preferably, the device further includes an enzyme activity detection device, which comprises a sampling tube disposed on the side wall of the enzyme acquisition tank and an exhaust pipe disposed on the top of the enzyme acquisition tank; the sampling tube is connected to an online microfluidic enzyme activity detection unit, and the exhaust pipe is connected to an online exhaust gas analyzer. In this preferred embodiment, dual-channel monitoring ensures a scientific separation triggering timing and directly optimizes enzyme harvesting efficiency.

[0009] Preferably, the multi-point reflux component includes a conveying ring disposed on the inner wall of the enzyme acquisition tank, a flared tube with its top connected to the bottom of the conveying ring, a ball valve disposed within the flared tube, and a drive motor disposed on the outer wall of the enzyme acquisition tank for driving the ball valve to rotate. The multi-point reflux component has the same structure as the multi-point suction component. In this preferred embodiment, the modular and switchable point design can prevent local dead zones, ensure uniform mixing, and mitigate contamination.

[0010] Preferably, the device further includes a pressure fluctuation elimination component disposed on the first pipeline. The pressure fluctuation elimination component includes a fluctuation elimination box disposed on the first pipeline. The fluctuation elimination box is divided into a flow channel cavity and a pressure chamber by an elastic sheet. The flow channel cavity communicates with the first pipeline. A first pressure sensor with a detection end extending into the pressure chamber is provided on the outer wall of the fluctuation elimination box. The pressure chamber communicates with an inlet pipe and an exhaust pipe. In this preferred embodiment, the pressure fluctuation elimination component can buffer pressure shocks, stabilize the separation flux, and protect the core membrane module.

[0011] Preferably, the supplementary gas component includes an auxiliary gas supply pipe, a first flow sensor sequentially disposed on the auxiliary gas supply pipe, and a first electrically controlled valve; the output end of the auxiliary gas supply pipe is connected to the conveying ring in the multi-point reflux component. In this preferred embodiment, supplementing the reflux liquid with gas can simultaneously replenish dissolved oxygen and enhance stirring, optimizing the fluid state inside the tank.

[0012] Preferably, the system further includes a flow rate control component, which comprises a second pressure sensor mounted on the second pipeline, a third pressure sensor and a back pressure valve sequentially mounted on the third pipeline along the conveying direction, and a second electrically controlled valve and a second flow sensor sequentially mounted on the fourth pipeline. In this preferred embodiment, the multi-sensor network enables precise monitoring, providing crucial data for pollution and endpoint determination.

[0013] Preferably, the biofilm filter includes an outer sleeve and a filter element disposed within the outer sleeve; the input end of the filter element is connected to a second pipe, the output end of the filter element is connected to a third pipe, and the outer wall of the outer sleeve is connected to a fourth pipe. In this preferred embodiment, the high-efficiency solid-liquid separation structure is easy to integrate and maintain, ensuring continuous clarified production of enzyme solution.

[0014] Preferably, the device further includes a backflushing apparatus, which comprises a first solenoid valve mounted on the second pipe, an inlet pipe with one end connected to the input end of the first solenoid valve, a second solenoid valve mounted on the third pipe, and a drain pipe with one end connected to the output end of the second solenoid valve; it also includes a backflushing pipe, multiple diversion pipes mounted on the outer wall of the backflushing pipe and connected to the backflushing pipe, and miniature electrically controlled valves mounted on the diversion pipes; the diversion pipes are connected to the outer sleeve. In this preferred embodiment, integrated gas-liquid multi-point backflushing can efficiently remove contaminants and restore membrane performance online.

[0015] Preferably, the quantitative delivery device includes a first quantitative delivery tube connecting an enzyme collection tank at one end and an enzyme-mixed preparation tank at the other end, and a second quantitative delivery tube connecting a bacterial culture tank at one end and an enzyme-mixed preparation tank at the other end; both the first and second quantitative delivery tubes are equipped with a third flow sensor and a third electrically controlled valve. In this preferred embodiment, high-precision quantitative delivery ensures accurate final formulation ratios and guarantees batch-to-batch consistency of the product.

[0016] In summary, the present invention has the following main beneficial effects: This solution integrates parallel fermentation, in-situ membrane separation, intelligent control, and precise proportioning into a continuous process through process design, achieving efficient and stable preparation of bacterial enzyme preparations.

[0017] The advantages of this solution stem from the deep synergy between the process and the hardware. Firstly, the process employs a continuous mode of "parallel fermentation-triggered separation-online mixing." Enzyme-induced fermentation and high-density cell culture are carried out simultaneously in two independent tanks, providing active enzymes and high-biomass cells for the formulation. When the enzyme activity in the fermentation broth reaches a threshold, the system automatically initiates membrane separation, allowing the enzyme solution to be continuously collected while the cells are returned to the tank for continued metabolism. This achieves "fermentation and separation simultaneously," significantly shortening the production cycle and improving equipment utilization.

[0018] Secondly, the hardware system provides precise assurance for the implementation of this process. The integrated membrane separation unit is directly coupled to the fermenter, ensuring efficient and low-consumption material transfer; dual-channel monitoring enables scientific triggering; the periodically switchable multi-point suction and reflux components effectively avoid dead zones inside the tank and localized contamination of the membrane surface; pressure buffering, intelligent backwashing, and a multi-sensor monitoring network jointly maintain the long-term stable operation of the separation process; finally, a high-precision quantitative delivery system ensures that the enzyme and cells are mixed according to the preset activity ratio, guaranteeing batch-to-batch consistency of the final product.

[0019] In summary, this invention constructs an automated and continuous production platform through process reconfiguration and system integration. This not only significantly improves production efficiency and process stability but also ensures the high activity and high reliability of the final bacterial-enzyme mixture. Attached Figure Description

[0020] Figure 1 This is a flowchart of the preparation process of the present invention; Figure 2 This is an isometric view of the overall structure of the preparation equipment of the present invention; Figure 3 This is an exploded view of the overall structure of the preparation equipment of the present invention; Figure 4 This is an exploded view of the bacterial enzyme acquisition tank structure of the present invention; Figure 5 This is an isometric view of the multi-point reflux component and the multi-point suction component of the present invention. Figure 6 An exploded view of the pressure fluctuation elimination component structure of the present invention; Figure 7 For the present invention Figure 6 Enlarged view of the structure at point A in the image; Figure 8 This is an exploded view of the enzyme liquid membrane separation device of the present invention; Figure 9 This is a top view of the overall structure of the preparation equipment of the present invention; Figure 10 For the present invention Figure 9 Enlarged view of the structure at point B in the image; Figure 11 This is a cross-sectional view of the bacterial enzyme acquisition tank structure of the present invention.

[0021] Figure Descriptions: 10. Enzyme Collection Tank; 11. Cell Culture Tank; 12. Bacterial Enzyme Mixture Tank; 20. Bacterial Enzyme Acquisition Tank; 21. Stirrer; 22. Multi-point Reflux Component; 221. Conveying Ring; 222. Flared Mouth Tube; 223. Ball Valve; 224. Drive Motor; 23. Multi-point Suction Component; 24. Supplementary Gas Component; 241. Auxiliary Gas Supply Pipe; 242. First Flow Sensor; 243. First Electrically Controlled Valve; 30. Quantitative Delivery Device; 31. First Quantitative Delivery Pipe; 32. Second Quantitative Delivery Pipe; 33. Third Flow Sensor; 34. Third Electrically Controlled Valve; 40. Enzyme Liquid Membrane Separation Device; 401. First Pipeline; 402. Second Pipeline; 403. Third Pipeline; 404. Fourth Pipeline; 41. Delivery Pump ; 42. Biofilm filter; 421. Outer sleeve; 422. Filter element; 43. Pressure fluctuation elimination component; 431. Fluctuation elimination box; 432. Elastic sheet; 433. Flow channel cavity; 434. Air pressure chamber; 435. First pressure sensor; 436. Air inlet pipe; 437. Exhaust pipe; 44. Separation flux adjustment component; 441. Second pressure sensor; 442. Third pressure sensor; 443. Back pressure valve; 444. Second solenoid valve; 445. Second flow sensor; 50. Backwashing device; 51. First solenoid valve; 52. Liquid inlet pipe; 53. Second solenoid valve; 54. Liquid outlet pipe; 55. Backwash pipe; 56. Diverter pipe; 57. Miniature solenoid valve; 60. Enzyme activity detection device; 61. Sampling tube; 62. Exhaust pipe. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0023] The embodiments of the present invention will now be described.

[0024] Please refer to the appendix for details. Figure 1 , Figure 2 , Figure 9As shown, in a preferred embodiment of the present invention, a process for preparing a highly efficient microbial degradation liquefied bacterial enzyme mixture includes the following steps: Step 1: Parallel fermentation culture. In the bacterial enzyme acquisition tank 20, enzyme-inducing fermentation is performed on the target degrading bacteria to obtain a fermentation broth containing bacterial cells and the target degrading enzyme; simultaneously, in the bacterial cell culture tank 11, high-density fermentation is performed on the same or synergistic strains to obtain a high-biomass bacterial broth; Step 2: Enzyme activity triggering and membrane separation coupled operation. When the enzyme activity detection device 60 detects that the enzyme activity in the bacterial enzyme acquisition tank 20 reaches a preset threshold, the enzyme liquid membrane separation device 40 is automatically started; the fermentation broth is drawn through the currently activated suction point in the multi-point suction component 23 and enters the enzyme liquid membrane separation device 40 for separation; Step 3: Product flow direction control and dynamic switching. The separated enzyme solution is transported to the enzyme collection tank 10 for storage; the separated concentrated bacterial solution is refluxed through the currently activated reflux point in the multi-point reflux component 22. The flow point returns to the enzyme acquisition tank 20, and during the return process, gas is replenished into the reflux liquid flow through the gas replenishment component 24; wherein, the multi-point suction component 23 and the multi-point reflux component 22 automatically switch the combination of the activated suction point and reflux point according to the set time cycle; Step four, membrane fouling control, during the separation process, when the permeate flux of the enzyme liquid membrane separation device 40 drops to 70%-85% of the initial flux, the backwashing device 50 is automatically started to backwash it online; Step five, endpoint determination, when the specific enzyme production rate of bacteria in the enzyme acquisition tank 20 drops to below 30% of the peak value, the separation operation is stopped; Step six, quantitative mixing and formulation, through the quantitative delivery device 30, according to the preset enzyme activity ratio, the enzyme liquid in the enzyme collection tank 10 and the bacterial liquid in the bacterial culture tank 11 are delivered to the enzyme mixing formulation tank 12 for mixing, and a stabilizer is added to prepare a liquefied enzyme mixture formulation.

[0025] It should be noted that in this embodiment, the enzyme acquisition tank 20 is a glass fermentation tank used for enzyme production fermentation. The enzyme acquisition tank 20 is equipped with pH, ​​dissolved oxygen, and temperature online probes and control systems. At the same time, the stirrer 21 can stir the solution in the enzyme acquisition tank 20. The positions of the multiple stirring rods on the stirrer 21 correspond to the positions of the multi-point reflux component 22 and the multi-point suction component 23, respectively. The bacterial culture tank 11 is used for high-density culture of the same or synergistic degrading bacteria, while the enzyme collection tank 10 and the bacterial enzyme mixture preparation tank 12 are both sterile storage tanks. Strain activation and inoculation The activated degrading bacteria were transferred to two seed bottles for expansion culture. The seed culture in the logarithmic growth phase was inoculated at a rate of 5% into the sterilized bacterial enzyme acquisition tank 20 and the bacterial cell culture tank 11, which were respectively filled with an appropriate amount of fermentation medium. Parallel fermentation culture In the enzyme acquisition tank 20, stirrer 21 is started, and the temperature is controlled at 30℃ and pH at 7.0. Dissolved oxygen is maintained above 30% by adjusting aeration and stirring. The initial culture medium uses glucose as the main carbon source to support rapid cell growth. When the OD600 reaches approximately 5.0 in the late logarithmic growth stage, an inducer is added via a feed pump to induce the cells to synthesize the degradation enzyme. This stage is called enzyme-induced fermentation.

[0026] In cell culture tank 11, a nutrient-rich medium and a fed-batch feeding strategy are employed, with the primary goal of maximizing cell biomass. Control conditions are similar to those in enzyme acquisition tank 20, but glucose remains the primary carbon source, with no or minimal addition of inducing agents later. This stage represents high-density fermentation.

[0027] Enzyme activity triggering coupled with membrane separation When fermentation in the enzyme acquisition tank 20 has been going on for about 18 hours, the online microfluidic enzyme activity detection unit continuously monitors and displays the activity through the sampling tube 61, and the online exhaust gas analyzer obtains the exhaust gas information of the enzyme acquisition tank 20 through the exhaust gas pipe 62. When the enzyme activity of the fermentation broth reaches the preset trigger threshold, for example, 50 U / L, or when the exhaust gas information reaches the trigger threshold. Upon receiving the signal, the controller automatically starts the enzyme membrane separation device 40. First, it opens the currently set set of suction points and closes the other suction points. At the same time, it opens the corresponding set of reflux points.

[0028] Product flow direction control and dynamic switching Inside the biofilm filter 42, under pressure, the enzyme protein passes through the membrane wall into the cavity of the outer sleeve 421, becoming a clear permeate, which flows into the enzyme collection tank 10 through the fourth pipe 404. The bacterial cells are completely retained and flow out with the liquid from the other end of the filter element 422, becoming a concentrated bacterial solution, which flows through the third pipe 403 to the currently activated reflux point. Before the bacterial solution enters the conveying ring 221 of the multi-point reflux component 22, the supplementary gas component 24 provides auxiliary gas supplementation.

[0029] Dynamic switching: The controller automatically shuts down the currently used suction point and reflux point combination every 30 minutes, while starting the next preset combination.

[0030] Membrane Fouling Control During the separation process, the separation flux regulating component 44 continuously monitors the flow and transmits the information to the controller. When the flow information is abnormal, the controller triggers the backwashing device 50 to perform a backwashing operation. End point determination During decoupled operation, the controller synchronously executes the endpoint determination logic.

[0031] The specific enzyme production rate (SP) is calculated every 15 minutes: SP = ΔE / (X * Δt). Where ΔE is the increase in total enzyme activity collected in the permeate within 15 minutes, obtained by integrating online enzyme activity detection data with permeate flow rate, and X is the average bacterial concentration in the tank during this time period, converted from OD600 by offline sampling or estimated by an online turbidity sensor.

[0032] The system records the peak value of SP (SP_max) during operation. When the calculated SP value is lower than 30% of SP_max for three consecutive times, the controller determines that the enzyme production efficiency has decreased significantly and continued operation is uneconomical. It then issues a command to stop the delivery pump 41, ending the membrane separation coupling operation phase. Quantitative Mixed Formulation The central controller calculates the volume to be transported based on the preset optimal bacterial-enzyme synergy ratio, the measured total activity of the enzyme solution in enzyme collection tank 10, and the biomass of the bacterial solution in bacterial culture tank 11.

[0033] The controller automatically activates the quantitative delivery device 30, which simultaneously delivers the calculated volumes of enzyme solution and bacterial solution to the bacterial-enzyme mixture preparation tank 12.

[0034] In the bacterial enzyme mixture preparation tank 12, start stirring and add the composite stabilizer through the feeding port, and mix evenly.

[0035] Finally, aseptic filtration and dispensing are performed to obtain the target product.

[0036] Please refer to the appendix for details. Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 10 , Figure 11As shown, in another preferred embodiment of the present invention, the system includes a bacterial enzyme acquisition tank 20, an enzyme collection tank 10, a bacterial culture tank 11, and a bacterial enzyme mixture preparation tank 12. The bacterial enzyme mixture preparation tank 12 is connected to the enzyme collection tank 10 and the bacterial culture tank 11 via a quantitative delivery device 30. The top of the bacterial enzyme acquisition tank 20 is provided with a stirrer 21 whose execution end extends into the bacterial enzyme acquisition tank 20. The top and bottom of the inner wall of the bacterial enzyme acquisition tank 20 are respectively provided with a multi-point reflux component 22 and a multi-point suction component 23. It also includes a supplementary gas component 24 whose output end is connected to a multi-point reflux component 22; it also includes an enzyme activity detection device 60, which includes a sampling tube 61 disposed on the side wall of the enzyme acquisition tank 20 and a tail gas pipe 62 disposed on the top of the enzyme acquisition tank 20; the sampling tube 61 is connected to an online microfluidic enzyme activity detection unit, and the tail gas pipe 62 is connected to an online tail gas analyzer; the multi-point reflux component 22 includes a conveying ring 221 disposed on the inner wall of the enzyme acquisition tank 20, and a top... The part is connected to the bell-shaped pipe 222 at the bottom of the conveying ring 221, a ball valve 223 is provided in the bell-shaped pipe 222, and a drive motor 224 is provided on the outer wall of the enzyme acquisition tank 20 for driving the ball valve 223 to rotate. The multi-point reflux component 22 has the same structure as the multi-point suction component 23. The supplementary gas component 24 includes an auxiliary gas supply pipe 241, a first flow sensor 242 and a first electric control valve 243 sequentially provided on the auxiliary gas supply pipe 241. The output end of the auxiliary gas supply pipe 241 is connected to the conveying ring 221 in the multi-point reflux component 22. The quantitative conveying device 30 includes a first quantitative conveying pipe 31 with one end connected to the enzyme collection tank 10 and the other end connected to the bacterial enzyme mixed preparation tank 12, and a second quantitative conveying pipe 32 with one end connected to the bacterial culture tank 11 and the other end connected to the bacterial enzyme mixed preparation tank 12. The first quantitative conveying pipe 31 and the second quantitative conveying pipe 32 are each equipped with a third flow sensor 33 and a third electric control valve 34.

[0037] It should be noted that in this embodiment, the working principle of the multi-point suction component 23 and the multi-point reflux component 22 is the same. Taking the multi-point reflux component 22 as an example, the drive motor 224 drives the ball valve 223 to rotate, so as to open or close the bell mouth tube 222. The refluxed bacterial liquid can be returned to the bacterial enzyme acquisition tank 20 through the opened bell mouth tube 222. Furthermore, when the supplementary gas component 24 is working, the auxiliary gas supply pipe 241 is connected to the sterile air supply system. The controller automatically adjusts the first electronic control valve 243 according to the signal of the first flow sensor 242 and the dissolved oxygen value in the tank, injecting a certain flow of sterile air into the conveying ring 221. After the bacterial liquid and gas are initially mixed in the conveying ring 221, they are sprayed into the tank through the opened bell pipe 222. This effectively replenishes the dissolved oxygen in the reflux bacterial liquid and plays a certain role in stirring. Furthermore, when the quantitative delivery device 30 is working, the controller receives the flow information measured by the third flow sensor 33, and closes the third electric control valve 34 after the product of the flow information and time reaches a set value, so as to realize the delivery of quantitative liquid. Furthermore, when the enzyme activity detection device 60 is in operation, the sampling tube 61 is connected to the online microfluidic enzyme activity detection unit to measure the activity concentration of the target degrading enzyme in the fermentation broth in real time.

[0038] The exhaust pipe 62 is connected to an online exhaust gas analyzer for indirect and continuous monitoring of the overall metabolic state of the bacteria, serving as an auxiliary judgment and process monitoring signal for the enzyme production period.

[0039] Please refer to the appendix for details. Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 11As shown, in another preferred embodiment of the present invention, an enzyme liquid membrane separation device 40 is further included, with its input end connected to a multi-point suction component 23 and its output end connected to a multi-point reflux component 22. The enzyme liquid membrane separation device 40 includes a delivery pump 41 disposed on the outer wall of the bacterial enzyme acquisition tank 20 and a biofilm filter 42. The input end of the delivery pump 41 is connected to the multi-point suction component 23 via a first pipe 401, and the output end of the delivery pump 41 is connected to the biofilm filter 42 via a second pipe 402. The bacterial liquid discharge end of the biofilm filter 42 is connected to the multi-point reflux component 22 via a third pipe 403. The enzyme solution discharge end of 42 is connected to the enzyme collection tank 10 via a fourth pipe 404. It also includes a pressure fluctuation elimination component 43 disposed on the first pipe 401. The pressure fluctuation elimination component 43 includes a fluctuation elimination box 431 disposed on the first pipe 401. The fluctuation elimination box 431 is divided into a flow channel cavity 433 and a pressure chamber 434 by an elastic sheet 432. The flow channel cavity 433 communicates with the first pipe 401. A first pressure sensor 435 with its detection end extending into the pressure chamber 434 is provided on the outer wall of the fluctuation elimination box 431. The pressure chamber 434 is connected to an air inlet pipe 436 and an exhaust pipe 437. The system also includes a flow rate regulating component 44, which comprises a second pressure sensor 441 mounted on the second pipe 402, a third pressure sensor 442 and a back pressure valve 443 sequentially mounted on the third pipe 403 along the conveying direction, and a second electrically controlled valve 444 and a second flow sensor 445 sequentially mounted on the fourth pipe 404. The biofilm filter 42 includes an outer sleeve 421 and a filter element 422 disposed within the outer sleeve 421; the input end of the filter element 422 is connected to the second pipe 402, and the output end of the filter element 422 is connected to the third pipe 403. The outer wall of the outer sleeve 421 is connected to the fourth pipe 404, and also includes a backflushing device 50. The backflushing device 50 includes a first solenoid valve 51 disposed on the second pipe 402, an inlet pipe 52 with one end connected to the input end of the first solenoid valve 51, a second solenoid valve 53 disposed on the third pipe 403, and a drain pipe 54 with one end connected to the output end of the second solenoid valve 53; it also includes a backflushing pipe 55, a plurality of diversion pipes 56 disposed on the outer wall of the backflushing pipe 55 and connected to the backflushing pipe 55, and a miniature electronically controlled valve 57 disposed on the diversion pipes 56; the diversion pipes 56 are connected to the outer sleeve 421.

[0040] It should be noted that in this embodiment, when the enzyme liquid membrane separation device 40 is working, the delivery pump 41 is turned on, and the bacterial enzyme mixture enters the biofilm filter 42 through the first pipe 401 and the second pipe 402. After the filter element 422 filters the bacterial enzyme mixture, the enzyme liquid is discharged into the enzyme collection tank 10 through the fourth pipe 404, and the bacterial liquid is returned to the enzyme collection tank 10 through the third pipe 403. Furthermore, when the separation flux regulating component 44 is working, the back pressure valve 443 increases the liquid flow pressure, thereby increasing the pressure inside the filter element 422, which facilitates the seepage of the enzyme solution through the filter element 422; The second flow sensor 445 monitors the permeate flow rate in real time and calculates the instantaneous permeate flux J(t) based on the known membrane area. The second pressure sensor 441 and the third pressure sensor 442 monitor the pressure, and the controller performs calculations.

[0041] The initial flux J0 is recorded as the average value after the system has been running stably for 5 minutes. The controller continuously compares J(t) / J0, and when J(t) / J0 drops to 80%, it is determined that backwashing is required.

[0042] Furthermore, when the pressure fluctuation elimination component 43 is working, the air inlet pipe 436 is connected to the air source system. The controller receives the air pressure information in the air pressure chamber 434 measured by the first pressure sensor 435 and triggers the air source system until the air pressure information reaches the set value. When it is necessary to reduce the air pressure in the air pressure chamber 434, the valve on the exhaust pipe 437 can be opened. When the suction point and the return point are switched, the pressure in the flow channel cavity 433 connected to the first quantitative delivery pipe 31 changes, and the elastic sheet 432 deforms, smoothing the pressure fluctuation in the first pipe 401. Furthermore, when the backwashing device 50 is working, the first solenoid valve 51 switches to connect to the inlet pipe 52, and the second solenoid valve 53 switches to connect to the outlet pipe 54. The backwashing liquid supply system connected to the inlet pipe 52 supplies sterile buffer solution, which rinses the membrane core of the filter element 422. The backwashing liquid is discharged through the outlet pipe 54. The backflush fluid supply system stops supplying, and the sterile gas supply system connected to the backflush pipe 55 supplies sterile gas. The micro-electrically controlled valves 57 on the multiple diversion pipes 56 open sequentially at set times to backflush the filter element 422 with airflow. After the airflow backwash, the backwash liquid supply system is restarted to re-flush the membrane element of filter element 422 to complete the backwashing process.

[0043] Although embodiments of the present invention have been shown and described, these specific embodiments are merely explanations of the invention and are not intended to limit it. The specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. After reading this specification, those skilled in the art may make modifications, substitutions, and variations to the embodiments as needed without departing from the principles and spirit of the invention, but such modifications, substitutions, and variations are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A process for preparing a highly efficient microbial degradation liquefied bacterial enzyme mixture, characterized in that, Includes the following steps: Step 1: Parallel fermentation culture. In the enzyme acquisition tank (20), the target degrading bacteria are induced to produce enzymes and fermentation to obtain a fermentation broth containing bacterial cells and the target degrading enzyme. At the same time, in the bacterial cell culture tank (11), the same or synergistic strains are fermented at high density to obtain a high biomass bacterial broth. Step 2: Enzyme activity triggering and membrane separation coupled operation. When the enzyme activity detection device (60) detects that the enzyme activity in the bacterial enzyme acquisition tank (20) reaches the preset threshold, the enzyme liquid membrane separation device (40) is automatically started. The fermentation broth is drawn through the currently activated suction point in the multi-point suction component (23) and enters the enzyme liquid membrane separation device (40) for separation. Step 3: Product flow direction control and dynamic switching. The separated enzyme solution is transported to the enzyme collection tank (10) for storage. The separated concentrated bacterial solution is returned to the bacterial enzyme acquisition tank (20) through the currently activated reflux point in the multi-point reflux component (22). During the return process, gas is added to the reflux liquid flow through the gas replenishment component (24). The multi-point suction component (23) and the multi-point reflux component (22) automatically switch the combination of activated suction points and reflux points according to the set time period. Step 4: Membrane fouling control. During the separation process, when the permeate flux of the enzyme liquid membrane separation device (40) drops to 70%-85% of the initial flux, the backwashing device (50) is automatically started to backwash it online. Step 5, endpoint determination: When the specific enzyme production rate of bacteria in the bacterial enzyme acquisition tank (20) drops to below 30% of the peak value, the separation operation is stopped. Step 6: Quantitative mixing and formulation. Using a quantitative delivery device (30), the enzyme solution in the enzyme collection tank (10) and the bacterial solution in the bacterial culture tank (11) are transported to the bacterial enzyme mixing preparation tank (12) according to the preset bacterial enzyme activity ratio, and a stabilizer is added to prepare a liquefied bacterial enzyme mixture.

2. The preparation process of a highly efficient microbial degradation liquefied bacterial enzyme mixture according to claim 1, characterized in that, It includes a bacterial enzyme acquisition tank (20), an enzyme collection tank (10), a bacterial culture tank (11), and a bacterial enzyme mixed preparation tank (12). The bacterial enzyme mixed preparation tank (12) is connected to the enzyme collection tank (10) and the bacterial culture tank (11) through a quantitative delivery device (30). The top of the bacterial enzyme acquisition tank (20) is provided with a stirrer (21) whose execution end extends into the bacterial enzyme acquisition tank (20). The top and bottom of the inner wall of the bacterial enzyme acquisition tank (20) are respectively provided with a multi-point reflux component (22) and a multi-point suction component (23). It also includes a supplementary gas component (24) whose output end is connected to the multi-point reflux component (22). It also includes an enzyme liquid membrane separation device (40) with an input end connected to a multi-point suction component (23) and an output end connected to a multi-point reflux component (22). The enzyme liquid membrane separation device (40) includes a delivery pump (41) and a biofilm filter (42) disposed on the outer wall of the bacterial enzyme acquisition tank (20). The input end of the delivery pump (41) is connected to the multi-point suction component (23) through a first pipe (401). The output end of the delivery pump (41) is connected to the biofilm filter (42) through a second pipe (402). The bacterial liquid discharge end of the biofilm filter (42) is connected to the multi-point reflux component (22) through a third pipe (403). The enzyme liquid discharge end of the biofilm filter (42) is connected to the enzyme collection tank (10) through a fourth pipe (404).

3. The preparation process of a highly efficient microbial degradation liquefied bacterial enzyme mixture according to claim 2, characterized in that, It also includes an enzyme activity detection device (60), which includes a sampling tube (61) disposed on the side wall of the bacterial enzyme acquisition tank (20) and a tail gas pipe (62) disposed on the top of the bacterial enzyme acquisition tank (20). The sampling tube (61) is connected to the online microfluidic enzyme activity detection unit, and the exhaust pipe (62) is connected to the online exhaust gas analyzer.

4. The preparation process of a highly efficient microbial degradation liquefied bacterial enzyme mixture according to claim 2, characterized in that, The multi-point reflux component (22) includes a conveying ring (221) disposed on the inner wall of the enzyme acquisition tank (20), a bell-shaped pipe (222) with its top connected to the bottom of the conveying ring (221), a ball valve (223) disposed in the bell-shaped pipe (222), and a drive motor (224) disposed on the outer wall of the enzyme acquisition tank (20) for driving the ball valve (223) to rotate. The multi-point reflux component (22) has the same structure as the multi-point suction component (23).

5. The preparation process of a highly efficient microbial degradation liquefied bacterial enzyme mixture according to claim 2, characterized in that, It also includes a pressure fluctuation elimination component (43) disposed on the first pipe (401). The pressure fluctuation elimination component (43) includes a fluctuation elimination box (431) disposed on the first pipe (401). The fluctuation elimination box (431) is divided into a flow channel cavity (433) and a pressure cavity (434) by an elastic sheet (432). The flow channel cavity (433) is connected to the first pipe (401), and the outer wall of the fluctuation elimination box (431) is provided with a first pressure sensor (435) whose detection end extends into the air pressure cavity (434). The air pressure cavity (434) is connected to the air inlet pipe (436) and the exhaust pipe (437).

6. The preparation process of a highly efficient microbial degradation liquefied bacterial enzyme mixture according to claim 4, characterized in that, The supplementary gas component (24) includes an auxiliary gas supply pipe (241), a first flow sensor (242) and a first electronically controlled valve (243) sequentially disposed on the auxiliary gas supply pipe (241). The output end of the auxiliary gas supply pipe (241) is connected to the conveying ring (221) in the multi-point reflux component (22).

7. The preparation process of a highly efficient microbial degradation liquefied bacterial enzyme mixture according to claim 2, characterized in that, It also includes a separation flow rate regulating component (44), which includes a second pressure sensor (441) disposed on the second pipeline (402), a third pressure sensor (442) and a back pressure valve (443) disposed sequentially on the third pipeline (403) along the conveying direction, and a second electrically controlled valve (444) and a second flow sensor (445) disposed sequentially on the fourth pipeline (404).

8. The preparation process of a highly efficient microbial degradation liquefied bacterial enzyme mixture according to claim 2, characterized in that, The biofilm filter (42) includes an outer sleeve (421) and a filter element (422) disposed inside the outer sleeve (421). The input end of the filter element (422) is connected to the second pipe (402), the output end of the filter element (422) is connected to the third pipe (403), and the outer wall of the outer sleeve (421) is connected to the fourth pipe (404).

9. The preparation process of a highly efficient microbial degradation liquefied bacterial enzyme mixture according to claim 8, characterized in that, It also includes a backwashing device (50), which includes a first solenoid valve (51) disposed on the second pipe (402), an inlet pipe (52) with one end connected to the input end of the first solenoid valve (51), a second solenoid valve (53) disposed on the third pipe (403), and a drain pipe (54) with one end connected to the output end of the second solenoid valve (53). It also includes a backflush tube (55), a plurality of shunt tubes (56) disposed on the outer wall of the backflush tube (55) and connected to the backflush tube (55), and a miniature electronically controlled valve (57) disposed on the shunt tube (56). The shunt pipe (56) is connected to the outer sleeve (421).

10. The preparation process of a highly efficient microbial degradation liquefied bacterial enzyme mixture according to claim 2, characterized in that, The quantitative delivery device (30) includes a first quantitative delivery tube (31) with one end connected to an enzyme collection tank (10) and the other end connected to a bacterial enzyme mixture preparation tank (12), and a second quantitative delivery tube (32) with one end connected to a bacterial culture tank (11) and the other end connected to a bacterial enzyme mixture preparation tank (12). Both the first quantitative delivery pipe (31) and the second quantitative delivery pipe (32) are equipped with a third flow sensor (33) and a third electrically controlled valve (34).

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