A fermentation system, a microbial body enrichment and purification device and a method of using the same
By using repeated filtration technology in a closed environment, the problem of cell death caused by centrifugation was solved, achieving high survival rate and high purity of cell enrichment, and improving the overall efficiency and purity of the fermentation system.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- 和度生物技术(上海)有限公司
- Filing Date
- 2022-10-24
- Publication Date
- 2026-06-16
AI Technical Summary
Existing centrifugation methods cause bacterial cell death during the enrichment and purification process, affecting the survival rate.
By employing a closed-environment, repeated circulation filtration technology is used. Through the design of the fermentation broth storage tank, filter components, and bypass pipeline, the fermentation broth is circulated and filtered between the storage chamber and the filter components, reducing the fluid pressure difference and improving the survival rate and purity of the cells.
It significantly improved the survival rate and purity of the microbial cells, reduced contamination by other microorganisms, and achieved integrated fermentation and microbial cell treatment, thereby improving processing efficiency.
Smart Images
Figure CN115637223B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bacterial cell processing technology, specifically to a fermentation system, a bacterial cell enrichment and purification device, and a method for using the same. Background Technology
[0002] Fermentation is a process by which people use the life activities of microorganisms under aerobic or anaerobic conditions to prepare the microbial cells themselves, or their direct or secondary metabolites. When it is necessary to prepare the microbial cells themselves, the cells need to be enriched and purified. Currently, the commonly used method for enriching and purifying microbial cells is centrifugation, which involves introducing the fermentation broth into a centrifuge for centrifugation, thereby separating the microbial cells from the fermentation broth supernatant. However, the centrifugal force of the centrifuge can cause some microbial cells to die, thus affecting the survival rate of the cells.
[0003] Therefore, how to provide a solution to overcome or alleviate the above-mentioned defects remains a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0004] To address the aforementioned technical problems, the present invention provides a cell enrichment and purification device, a fermentation system comprising the cell enrichment and purification device, and a method for using the cell enrichment and purification device. The cell enrichment and purification device can repeatedly circulate and filter the fermentation broth in a closed environment, thereby improving the purity and survival rate of the cells.
[0005] The specific structure of the bacterial cell enrichment and purification device includes: a fermentation broth storage tank, including a storage chamber for containing fermentation broth; a filtration component, equipped with an inlet pipe and an outlet pipe, both of which are connected to the storage chamber for circulating filtration of the fermentation broth between the storage chamber and the filtration component, the filtration component also being equipped with a filtrate outlet; and a bypass pipe, both ends of which are connected to the storage chamber, and the bypass pipe is equipped with a first switching valve.
[0006] Using the above method, the fermentation broth can be repeatedly circulated and filtered between the storage chamber and the filtration components, thereby increasing the cell concentration of the fermentation broth. This filtration method is self-circulating, and the circulation and filtration of the fermentation broth is essentially in a completely closed environment, which can greatly reduce interference from human factors and the introduction of contaminating microorganisms, thus helping to improve the purity of the cells.
[0007] In particular, the present invention also includes a bypass pipeline, which allows the fermentation broth to partially flow into the bypass pipeline when the first switching valve is partially or fully open, thereby reducing the fluid pressure in the inlet pipeline and thus reducing the pressure difference between the upstream and downstream of the filter element, thereby improving the cell survival rate during cell collection.
[0008] Optionally, the bypass pipeline and the inlet pipeline are connected to form a first connection point, and the inlet pipeline is equipped with a first pump body located upstream of the first connection point.
[0009] Optionally, both the inlet pipe and the outlet pipe are equipped with pressure detection components, and the outlet pipe is equipped with a third switching valve.
[0010] Optionally, the bacterial cell outlet pipe and the bypass pipe are connected.
[0011] Optionally, it also includes a filtrate collection component, which is equipped with a filtrate collection pipeline connected to the filtrate discharge port.
[0012] Optionally, the filtrate collection pipeline and the bypass pipeline are connected to form a second connection point. The filtrate collection pipeline is also equipped with a fourth switching valve, and the bypass pipeline is also equipped with a fifth switching valve and a sixth switching valve. The fourth switching valve is located downstream of the second connection point, and the fifth switching valve and the sixth switching valve are located upstream and downstream of the second connection point, respectively.
[0013] Optionally, the filtrate collection line and the bypass line are independent.
[0014] Optionally, it also includes a bacterial cell outlet pipe, which is connected to the liquid storage chamber, and the bacterial cell outlet pipe is equipped with a second switching valve.
[0015] Optionally, the bacterial cell outlet pipe and the bypass pipe are connected.
[0016] Optionally, the fermentation broth storage tank is also equipped with an inlet pipe, which is connected to the storage chamber.
[0017] Optionally, the inlet pipeline is also equipped with a second pump body.
[0018] Optionally, the fermentation broth storage tank is also equipped with a pressure balancing component to balance the pressure inside and outside the storage chamber.
[0019] Optionally, the fermentation broth storage tank further includes a temperature control chamber isolated from the storage chamber, and the cell enrichment and purification device further includes a temperature control component connected to the temperature control chamber; it also includes a thermometer for detecting the temperature of the fermentation broth.
[0020] The present invention also provides a fermentation system, including a fermentation device and a cell enrichment and purification device, wherein the cell enrichment and purification device is the cell enrichment and purification device described above, and the fermentation device has a fermentation broth outlet, which is connected to the storage chamber.
[0021] In addition to the technical effects of the aforementioned cell enrichment and purification device, the fermentation system can also achieve the integration of fermentation and cell processing, which can improve efficiency. At the same time, it can also avoid the problem of contamination caused by the separation of the fermentation device and the cell enrichment and purification device. In other words, the fermentation system is conducive to improving the survival rate and purity of the cells.
[0022] This invention also provides a method for using a bacterial cell enrichment and purification device, applicable to the aforementioned bacterial cell enrichment and purification device. The method includes the following steps: configuring the bacterial cell enrichment and purification device; introducing a set volume of fermentation broth into the storage chamber and controlling the fermentation broth to circulate between the storage chamber and the filter component; obtaining the discharge volume of the filtrate discharged from the filtrate outlet, calculating the bacterial cell concentration using the discharge volume and the set volume; determining whether the bacterial cell concentration has reached the set concentration; if so, obtaining a fermentation broth with a bacterial cell concentration reaching the set concentration.
[0023] Optionally, after the bacterial cell concentration reaches the set concentration, the method further includes: introducing a buffer solution into the storage chamber and controlling the flow rate of the buffer solution to be consistent with the flow rate of the filtrate, wherein the buffer solution has a set parameter that characterizes the properties of the buffer solution; acquiring real-time parameters of the filtrate that characterize the properties of the filtrate; determining whether the real-time parameters and the set parameter are consistent, and if so, stopping the introduction of the buffer solution.
[0024] Optionally, the configuration of the bacterial cell enrichment and purification device includes a disinfection step and a cleaning step. The disinfection step specifically involves disinfecting the bacterial cell enrichment and purification device by setting a disinfection method, and the cleaning step specifically involves cleaning the bacterial cell enrichment and purification device with a cleaning solution.
[0025] Optionally, the disinfection method includes high-temperature dry heat sterilization, high-temperature steam sterilization, and disinfectant sterilization, wherein the disinfectant is ethanol with a purity of 75% or higher.
[0026] Optionally, the cleaning solution is sterile water, sterile saline, or sterile buffer solution.
[0027] Optionally, the configuration of the bacterial cell enrichment and purification device further includes a temperature control step for controlling the temperature of the fermentation broth. Attached Figure Description
[0028] Figure 1 This is a schematic diagram of a specific embodiment of the bacterial cell enrichment and purification device provided by the present invention;
[0029] Figure 2This is a schematic diagram of the usage method of the bacterial cell enrichment and purification device provided by the present invention.
[0030] The annotations in the attached figures are explained as follows:
[0031] 1 Fermentation broth storage tank, 11 Storage chamber, 12 Inlet pipeline, 121 Second pump body, 13 Pressure balancing component, 14 Temperature control chamber;
[0032] 2. Filtering unit, 21. Inlet pipe, 211. First pump body, 212. First pressure gauge, 22. Outlet pipe, 221. Second pressure gauge, 222. Third switch valve, 223. Thermometer, 23. Filtrate outlet;
[0033] 3. Bypass pipeline; 31. First switch valve; 32. Fifth switch valve; 33. Sixth switch valve;
[0034] 41. Bacterial cell discharge tubing; 411. Second switch valve;
[0035] 5. Filtrate collection component; 51. Filtrate collection pipeline; 511. Fourth switch valve;
[0036] 6 Temperature control components, 61 Liquid supply line, 62 Liquid return line, 63 Third pump body;
[0037] A is the first connection point, B is the second connection point, and C is the third connection point. Detailed Implementation
[0038] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
[0039] In embodiments of the present invention, the terms "first," "second," "third," "fourth," "fifth," and "sixth" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first," "second," "third," "fourth," "fifth," and "sixth" may explicitly or implicitly include one or more of that feature.
[0040] In this embodiment of the invention, the term "connection" includes both direct connection and indirect connection. For example, when describing the connection between A and B, it includes both a scheme in which A and B are directly connected and a scheme in which A and B are connected via other transitional components.
[0041] In the description of embodiments of the present invention, the terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.
[0042] Please refer to Figure 1 , Figure 1 This is a schematic diagram of a specific embodiment of the bacterial cell enrichment and purification device provided by the present invention.
[0043] like Figure 1 As shown, this invention provides a bacterial cell enrichment and purification device, including a fermentation broth storage tank 1, a filter component 2, a bypass pipeline 3, and a bacterial cell collection component. The fermentation broth storage tank 1 includes a storage chamber 11, which is connected to an upstream fermentation device to receive and contain the fermentation broth produced by the fermentation device. The filter component 2 can be a membrane filter, etc., and is equipped with an inlet pipeline 21 and an outlet pipeline 22, both of which are connected to the storage chamber 11. The fermentation broth in the storage chamber 11 can flow into the filter component 2 through the inlet pipeline 21 for filtration, and the filtered fermentation broth can then flow back into the storage chamber 11 through the outlet pipeline 22. The fermentation broth is circulated and filtered between the storage chamber 11 and the filter component 2. The filter component 2 is also equipped with a filtrate outlet 23 for discharging the filtrate produced by filtration. The filtrate is the supernatant in the fermentation broth and basically does not contain bacteria. The inlet and outlet of the bypass pipe 3 are connected to the storage chamber 11, and the bypass pipe 3 is equipped with a first switch valve 31. The bacteria collection component is equipped with a bacteria outlet pipe 41, which is connected to the storage chamber 11, and the bacteria outlet pipe 41 is equipped with a second switch valve 411.
[0044] Using the above scheme, the fermentation broth can be repeatedly circulated and filtered between the storage chamber 11 and the filter component 2, which can increase the cell concentration of the fermentation broth. When the cell concentration reaches the set concentration, the second switch valve 411 of the cell outlet pipe 41 can be opened to allow the fermentation broth to be passed into the cell collection component for collection, thereby meeting the technical purpose of cell collection. Furthermore, the above filtration method is self-circulating, and the circulation and filtration of the fermentation broth is basically in a completely closed environment, which can greatly reduce the interference of human factors and the introduction of contaminating bacteria, thus helping to improve the purity of the cells.
[0045] It should be understood that if the purpose of the bacterial enrichment and purification device is bacterial collection, the bacterial discharge line 41 can be connected to a bacterial collection container.
[0046] When used in the entire fermentation system, this cell enrichment and purification device can be an intermediate device. Its upstream can be directly connected to the fermentation device to receive the fermentation broth, while the downstream can be equipped with other processing devices for further processing of the enriched and purified cells, such as a freeze dryer.
[0047] In particular, the present invention is also equipped with a bypass pipe 3. When the first switch valve 31 is partially or fully opened, the fermentation broth can partially flow into the bypass pipe 3, which can reduce the fluid pressure in the inlet pipe 21, thereby reducing the pressure difference between the upstream and downstream of the filter component 2 and improving the cell survival rate during cell collection.
[0048] It should be understood that since no other components are installed in the bypass pipe 3, when the first switch valve 31 is fully open, the flow resistance in the bypass pipe 3 is much lower than that in the filter component 2, which will cause a large amount of fermentation broth to flow into the bypass pipe 3, thereby affecting the filtration efficiency. Therefore, in actual operation, the opening degree of the first switch valve 31 needs to be controlled to achieve a reasonable distribution of fermentation broth in the filter component 2 and the bypass pipe 3, so that there can be a suitable pressure difference on both sides of the filter component 2. In this way, both the cell survival rate and the filtration efficiency can be guaranteed.
[0049] Specifically, pressure detection components, which can be pressure gauges, can be installed in both the inlet pipe 21 and the outlet pipe 22. For ease of description, the pressure detection component in the inlet pipe 21 can be referred to as the first pressure gauge 212, which is used to detect the inlet pressure, and the pressure detection component in the outlet pipe 22 can be referred to as the second pressure gauge 221, which is used to detect the outlet pressure. The difference between the inlet pressure and the outlet pressure is the pressure difference across the filter component 2, and the opening degree of the first switching valve 31 can be adjusted by this pressure difference.
[0050] The first switching valve 31 can be adjusted manually, meaning that the opening of the first switching valve 31 can be manually adjusted by the operator based on the measured pressure difference. Alternatively, the first switching valve 31 can also be adjusted automatically. In this case, a controller can be configured. The controller can be connected to the first pressure gauge 212, the second pressure gauge 221, and the first switching valve 31. The controller can receive the inlet pressure measured by the first pressure gauge 212 and the outlet pressure measured by the second pressure gauge 221, calculate the pressure difference based on the inlet and outlet pressures, and then determine the opening of the first switching valve 31 based on the pressure difference and a predetermined mapping relationship, thus enabling automatic adjustment of the first switching valve 31.
[0051] It should be noted that, unless otherwise specified, the adjustment methods of each switching valve involved in the following embodiments include both manual and automatic adjustment.
[0052] The outflow pipe 22 can also be equipped with a third switching valve 222. By adjusting the opening degree of the third switching valve 222, the outflow pressure of the outflow pipe 22 can be regulated.
[0053] The two ends of the bypass pipe 3 can be directly connected to the liquid storage chamber 11, or the bypass pipe 3 can be indirectly connected to the liquid storage chamber 11 through other pipes.
[0054] In the embodiments shown in the accompanying drawings, as Figure 1 As shown, the inlet end of the bypass pipe 3 can be connected to the inlet pipe 21, so that the fermentation broth in the storage chamber 11 can be indirectly introduced into the bypass pipe 3 through the inlet pipe 21. In this way, the bypass pipe 3 and the inlet pipe 21 can establish a structural connection, which can improve the structural strength of each pipe, and also helps to reduce the length of the pipes on the outside of the fermentation broth storage tank 1, thereby improving the compactness and integration of the pipes on the outside of the fermentation broth storage tank 1. The outlet end of the bypass pipe 3 can be directly connected to the storage chamber 11 to avoid affecting the outflow pressure in the outflow pipe 22.
[0055] The connection point between the bypass pipe 3 and the inlet pipe 21 can be referred to as the first connection point A. The inlet pipe 21 can be equipped with a first pump body 211, which can be located upstream of the first connection point A to provide unified driving force for both the inlet pipe 21 and the bypass pipe 3. The first pressure gauge 212 can be located downstream of the first connection point A to directly detect the inlet pressure of the fermentation broth entering the filter element 2.
[0056] Furthermore, the cell outlet pipe 41 and the bypass pipe 3 can also be connected. This can improve the structural strength of the pipes and further simplify the pipe structure outside the fermentation broth storage tank 1, improving the compactness and integration of the pipes; a third connection point C can be formed between the cell outlet pipe 41 and the bypass pipe 3.
[0057] It should be understood that the cell outlet pipe 41 can also be connected to other pipes, such as the inlet pipe 21, the outlet pipe 22, etc.; or, the cell outlet pipe 41 can also be directly connected to the storage chamber 11, as long as the fermentation broth in the cell enrichment and purification device can be exported.
[0058] Please continue to refer to this. Figure 1The cell enrichment and purification device provided by the present invention may further include a filtrate collection component 5, which may be equipped with a filtrate collection pipe 51. The filtrate collection pipe 51 may be connected to the filtrate discharge port 23 for collecting the filtrate discharged by the filtration component 2. By collecting the filtrate, the discharged volume of the filtrate can be easily counted. Combined with the volume of the fermentation broth initially injected into the storage chamber 11, the cell concentration of the current fermentation broth can be calculated, so as to control the concentration of the cells to be collected.
[0059] It should be understood that the method of obtaining the volume of filtrate discharge is not limited to the filtrate collection component 5 mentioned above. For example, a flow meter can also be configured at the filtrate discharge outlet 23, which can also achieve the statistical acquisition of the filtrate discharge volume.
[0060] In some alternative embodiments, when the filtrate collection component 5 is configured, the filtrate collection pipe 51 and the bypass pipe 3 can also be connected. In this way, the filtrate collection pipe 51 and the bypass pipe 3 can also establish a structural connection, which can improve the structural strength of each pipe and reduce the pipe length, thereby improving the integration and compactness of the pipe system.
[0061] Combination Figure 1 A second connection point B can be formed between the filtrate collection line 51 and the bypass line 3. The filtrate collection line 51 can also be equipped with a fourth switching valve 511, and the bypass line 3 can also be equipped with a fifth switching valve 32 and a sixth switching valve 33. The fourth switching valve 511 can be located downstream of the second connection point B, and the fifth switching valve 32 and the sixth switching valve 33 can be located upstream and downstream of the second connection point B, respectively. When the fourth switching valve 511 is closed, the filtrate cannot flow into the filtrate collection component 5. When the fifth switching valve 32 or the sixth switching valve 33 is closed, the bypass line 3 is in an open state.
[0062] Furthermore, when the first switch valve 31, the second switch valve 411, the third switch valve 222, the fourth switch valve 511, the fifth switch valve 32, and the sixth switch valve 33 are all in the open state, each pipeline can be directly or indirectly connected, which can conveniently disinfect and clean the entire bacterial enrichment and purification device provided in the embodiment of the present invention.
[0063] It should be understood that the filtrate collection line 51 and the bypass line 3 can also be independent, so that the discharge of the filtrate and the flow of the fermentation broth in the bypass line 3 can be independent of each other.
[0064] In some alternative embodiments, the fermentation broth storage tank 1 may also be equipped with an inlet pipe 12, which can be connected to the storage chamber 11, and the fermentation broth can enter the storage chamber 11 through the inlet pipe 12.
[0065] It should be noted that the function of the inlet pipe 12 is not limited to feeding fermentation broth into the fermentation broth storage tank 1. It can also be used to feed buffer into the fermentation broth storage tank 1 after the cell concentration reaches the set concentration, in order to wash and resuspend the cells. This can remove residual culture medium components, cell debris, and other impurities, thereby improving cell survival rate and cell purity. The composition of the buffer is not limited here.
[0066] The inlet pipe 12 may also be equipped with a second pump 121 to provide power for the flow of fermentation broth or buffer solution within the inlet pipe 12. It should be understood that in the scenario where the inlet pipe 12 is used to introduce fermentation broth, the second pump 121 may also be located in the upstream fermentation device, which can also provide power for the flow of fermentation broth from the fermentation device to the fermentation broth storage tank 1.
[0067] In some optional embodiments, the fermentation broth storage tank 1 may also be equipped with a pressure balancing component 13, which may specifically be a breathable membrane, a balancing valve, etc., to balance the pressure inside and outside the storage chamber 11 and to prevent external bacteria from entering.
[0068] In some optional embodiments, the fermentation broth storage tank 1 may further include a temperature control chamber 14 isolated from the storage chamber 11. The temperature control chamber 14 may be arranged around the storage chamber 11 and may be filled with a temperature control liquid to control the temperature inside the storage chamber 11, thereby controlling the temperature of the fermentation broth and improving the survival rate of the cells.
[0069] The relative positional relationship between the temperature control cavity 14 and the liquid storage cavity 11 is not limited to the temperature control cavity 14 surrounding the liquid storage cavity 11. It can also be that the liquid storage cavity 11 surrounds the temperature control cavity 14. Alternatively, the temperature control cavity 14 and the liquid storage cavity 11 can be arranged in a non-surrounding manner, as long as the temperature control liquid in the temperature control cavity 14 can regulate the temperature in the liquid storage cavity 11.
[0070] Based on the temperature control chamber 14, the bacterial cell enrichment and purification device provided by the present invention may further include a temperature control component 6, which contains a temperature control liquid. The temperature control component 6 and the temperature control chamber 14 can be connected to circulate the temperature control liquid. Specifically, the temperature control liquid can be water, and the temperature control component 6 can be an electric heater or an electric refrigerator, capable of heating or cooling the temperature control liquid.
[0071] In detail, the temperature control component 6 is also equipped with a liquid supply line 61 and a liquid return line 62. Both the liquid supply line 61 and the liquid return line 62 can be connected to the temperature control cavity 14. The liquid supply line 61 can be equipped with a third pump body 63. The third pump body 63 can provide power for the flow of temperature control liquid in the temperature control component 6 and the temperature control cavity 14, and can adjust the flow rate.
[0072] The bacterial cell enrichment and purification device can also be equipped with a thermometer 223 for detecting the temperature of the fermentation broth. The location of the thermometer 223 is not limited here; in practice, those skilled in the art can adjust it according to actual needs, as long as the temperature detection requirements of the fermentation broth are met. In the embodiment shown in the attached drawings, as... Figure 1 As shown, thermometer 223 can be installed in the outflow pipe 22 to detect the temperature of the fermentation liquid flowing back into the storage chamber 11.
[0073] In fact, temperature component 6 can also be equipped with a thermometer to detect the temperature of the temperature-controlled liquid.
[0074] The present invention also provides a fermentation system, including a fermentation device and a cell enrichment and purification device. The cell enrichment and purification device is the cell enrichment and purification device involved in the foregoing embodiments. The fermentation device has a fermentation broth outlet, and the fermentation broth outlet is connected to the storage chamber 11.
[0075] In addition to the technical effects of the aforementioned cell enrichment and purification device, the fermentation system can also achieve the integration of fermentation and cell processing, which can improve efficiency. At the same time, it can also avoid the problem of contamination caused by the separation of the fermentation device and the cell enrichment and purification device. In other words, the fermentation system is conducive to improving the survival rate and purity of the cells.
[0076] In some embodiments, the fermentation system can also be connected to downstream devices, such as cell collection containers or freeze dryers, via the outlet pipe 41 of the cell enrichment and purification device, for collecting or further processing the cells obtained by the cell enrichment and purification device.
[0077] Please refer to Figure 2 , Figure 2 This is a schematic diagram of the usage method of the bacterial cell enrichment and purification device provided by the present invention.
[0078] like Figure 2 As shown, the present invention also provides a method of using the bacterial cell enrichment and purification device, which is applicable to the bacterial cell enrichment and purification device involved in the foregoing embodiments, and the method of use includes the following steps.
[0079] Step S1: Configure the bacterial cell enrichment and purification device. The specific structure of the bacterial cell enrichment and purification device can be found in the preceding description and will not be repeated here.
[0080] The configuration of the bacterial cell enrichment and purification device described here mainly refers to the acquisition, disinfection, cleaning, and temperature control of the device. The following embodiments of the invention will further explain the specific steps for disinfection, cleaning, and temperature control.
[0081] The disinfection step specifically refers to disinfecting the bacterial enrichment and purification device using a set disinfection method. The set disinfection methods include high-temperature dry heat sterilization, high-temperature sterilization (such as pressure cooker cooking), high-temperature steam sterilization, and disinfectant solution sterilization. In specific implementation, those skilled in the art can select the appropriate disinfection method according to actual needs.
[0082] Taking disinfectant sterilization as an example, the disinfectant can be an ethanol solution, and its purity can be adjusted as needed, for example, to 75% or higher; of course, the composition of the disinfectant is not limited to an ethanol solution. Combined with the aforementioned... Figure 1 The disinfectant solution with the set composition can enter the storage chamber 11 of the fermentation broth storage tank 1 through the inlet pipe 12. Then, the third switch valve 222 and the first pump body 211 can be opened, and the first switch valve 31, the second switch valve 411, the fourth switch valve 511, the fifth switch valve 32, and the sixth switch valve 33 can be opened slightly (to allow the disinfectant solution to flow into the filter element 2). The opening of the third switch valve 222 is gradually adjusted so that disinfectant solution can be discharged from the filtrate outlet 23. This condition is maintained for a first set time, for example, 30 minutes. Then, the first switch valve 31, the fourth switch valve 511, and the fifth switch valve 32 are fully opened, and the second switch valve 411 and the sixth switch valve 33 are closed, so that the disinfectant solution can be collected in the filtrate collection element 5. The composition of the disinfectant solution is not limited here.
[0083] The cleaning step is usually performed after the disinfection step. Specifically, it involves cleaning the bacterial cell enrichment and purification device with a pre-defined cleaning solution. The type of cleaning solution is not limited here; for example, sterile water can be used. Specifically, the cleaning solution can be introduced into the storage chamber 11 through the inlet pipe 12. The third switch valve 222 and the first pump body 211 can be opened, and the first switch valve 31, the second switch valve 411, the fourth switch valve 511, the fifth switch valve 32, and the sixth switch valve 33 can be slightly opened. The opening of the third switch valve 222 is gradually adjusted so that cleaning solution can be discharged from the filtrate outlet 23. After maintaining this condition for a second set time (e.g., 5 minutes), the first switch valve 31, the fourth switch valve 511, and the fifth switch valve 32 are fully opened, and the second switch valve 411 and the sixth switch valve 33 are closed, allowing the cleaning solution to be collected in the filtrate collection component 5. The cleaning step can be repeated a set number of times (e.g., 2-3 times) to remove residual disinfectant.
[0084] The temperature control step mainly involves introducing temperature-controlled liquid into the temperature control chamber 14 to provide a specific temperature environment for the liquid storage chamber 11 and the fermentation liquid inside the liquid storage chamber 11, thereby maintaining the temperature of the fermentation liquid and improving the survival rate of the cells.
[0085] It should be understood that for continuous production processes, disinfection and cleaning steps are not mandatory, and temperature control steps may be implemented or not, depending on the specific circumstances.
[0086] Step S2: A set volume of fermentation liquid is introduced into the storage chamber 11, and the fermentation liquid is controlled to circulate between the storage chamber 11 and the filter component 2.
[0087] Step S3: Obtain the discharge volume of the filtrate discharged from the filtrate discharge port 23, and calculate the cell concentration by using the discharge volume and the set volume.
[0088] The discharge volume can be obtained in two ways: firstly, by using the filtrate collection component 5 to collect the filtrate and then calculating the discharge volume; secondly, by configuring a flow meter to directly obtain the discharge volume.
[0089] Step S4: Determine whether the cell concentration has reached the set concentration. If so, a fermentation broth with a cell concentration reaching the set concentration can be obtained.
[0090] Furthermore, after step S4, the method provided by the present invention may further include step S5: introducing buffer solution into the storage chamber 11, and controlling the inflow rate of the buffer solution to be consistent with the outflow rate of the filtrate. Step S5 can wash and resuspend the bacterial cells, thereby improving the purity of the bacterial cells. It should be understood that "consistent" here means substantially consistent, allowing for a certain range of error, while the specific size of the error range is not limited here.
[0091] The aforementioned buffer solution has set parameters that can be used to characterize the properties of the buffer solution. These set parameters can specifically be parameters such as osmotic pressure and conductivity.
[0092] After step S5 is executed, steps S6-S8 can also be executed.
[0093] Step S6: Obtain the real-time parameters of the filtrate. These real-time parameters characterize the properties of the filtrate and are used to compare with the aforementioned set parameters. The two should be the same parameters; that is, when the set parameters include osmotic pressure and conductivity, the real-time parameters should also include osmotic pressure and conductivity.
[0094] Step S7: Determine whether the real-time parameters and the set parameters are the same. If they are the same, it means that washing and resuspension can be completed, and the fermentation broth basically does not contain residual culture medium components, cell debris, etc., and the purity of the cells is guaranteed.
[0095] Then, step S8 can be performed to stop the flow of buffer solution into the reservoir.
[0096] Experimental verification: At the end of the fermentation of batch RF20220725, the viable cell concentration of the fermentation broth was obtained by viable cell counting according to the national standard plate count method, which was 2.92E9 / mL. The fermentation broth was divided into two portions, each 1210mL. One portion was processed using the cell enrichment and purification device provided in this invention, and the other portion was collected by floor centrifugation. Then, the cells were washed and resuspended in an ultra-clean workbench by pipetting. The survival rate of the cells collected by centrifugation was 89.9%, while the survival rate of the cells collected by the cell enrichment and purification device provided in this invention was 99.2%. The comparison shows that the cell enrichment and purification device provided in this invention can significantly improve the cell survival rate.
[0097] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A bacterial cell enrichment and purification device, characterized in that, include: Fermentation broth storage tank (1) includes a storage chamber (11) for containing fermentation broth; The filter component (2) is equipped with an inlet pipe (21) and an outlet pipe (22). Both the inlet pipe (21) and the outlet pipe (22) are connected to the storage chamber (11) for circulating filtration of the fermentation liquid between the storage chamber (11) and the filter component (2). The filter component (2) is also equipped with a filtrate outlet (23). The bypass pipeline (3) is connected to the liquid storage chamber (11) at both ends, and the bypass pipeline (3) is equipped with a first switching valve (31). Both the inlet pipe (21) and the outlet pipe (22) are equipped with pressure detection components; the pressure detection component of the inlet pipe (21) is a first pressure gauge (212) used to detect the inlet pressure; the pressure detection component of the outlet pipe (22) is a second pressure gauge (221) used to detect the outlet pressure; the difference between the inlet pressure and the outlet pressure is the pressure difference on both sides of the filter component (2), and the opening degree of the first switching valve (31) is adjusted by the pressure difference.
2. The bacterial cell enrichment and purification device according to claim 1, characterized in that, The bypass pipe (3) and the inlet pipe (21) are connected to form a first connection point (A). The inlet pipe (21) is equipped with a first pump body (211), which is located upstream of the first connection point (A).
3. The bacterial cell enrichment and purification device according to claim 2, characterized in that, The outflow pipe (22) is equipped with a third switching valve (222).
4. The bacterial cell enrichment and purification device according to claim 1, characterized in that, It also includes a filtrate collection component (5), which is equipped with a filtrate collection pipeline (51) and is connected to the filtrate outlet (23).
5. The bacterial cell enrichment and purification device according to claim 4, characterized in that, The filtrate collection line (51) and the bypass line (3) are connected to form a second connection point (B). The filtrate collection line (51) is also equipped with a fourth switch valve (511). The bypass line (3) is also equipped with a fifth switch valve (32) and a sixth switch valve (33). The fourth switch valve (511) is located downstream of the second connection point (B). The fifth switch valve (32) and the sixth switch valve (33) are located upstream and downstream of the second connection point (B), respectively.
6. The bacterial cell enrichment and purification device according to claim 4, characterized in that, The filtrate collection line (51) and the bypass line (3) are independent of each other.
7. The bacterial cell enrichment and purification device according to claim 1, characterized in that, It also includes a cell outlet pipe (41), which is connected to the liquid storage chamber (11), and the cell outlet pipe (41) is equipped with a second switching valve (411).
8. The bacterial cell enrichment and purification device according to claim 7, characterized in that, The bacterial cell outlet pipe (41) and the bypass pipe (3) are connected.
9. The bacterial cell enrichment and purification apparatus according to any one of claims 1-8, characterized in that, The fermentation broth storage tank (1) is also equipped with an inlet pipe (12), which is connected to the storage chamber (11).
10. The bacterial cell enrichment and purification apparatus according to claim 9, characterized in that, The inlet pipeline (12) is also equipped with a second pump body (121).
11. The bacterial cell enrichment and purification apparatus according to any one of claims 1-8, characterized in that, The fermentation broth storage tank (1) is also equipped with a pressure balancing component (13) to balance the pressure inside and outside the storage chamber (11).
12. The bacterial cell enrichment and purification apparatus according to any one of claims 1-8, characterized in that, The fermentation broth storage tank (1) also includes a temperature control chamber (14) isolated from the storage chamber (11), and the cell enrichment and purification device also includes a temperature control component (6), which is connected to the temperature control chamber (14); It also includes a thermometer (223) for detecting the temperature of the fermentation broth.
13. A fermentation system, characterized in that, It includes a fermentation device and a cell enrichment and purification device, wherein the cell enrichment and purification device is the cell enrichment and purification device according to any one of claims 1-12, the fermentation device has a fermentation broth outlet, and the fermentation broth outlet is connected to the storage chamber (11).
14. A method of using a bacterial cell enrichment and purification device, characterized in that, The method of using the bacterial cell enrichment and purification apparatus according to any one of claims 1-12 includes the following steps: Configure the bacterial cell enrichment and purification device; A set volume of fermentation liquid is introduced into the storage chamber (11), and the fermentation liquid is controlled to circulate between the storage chamber (11) and the filter component (2); Obtain the discharge volume of the filtrate discharged from the filtrate discharge port (23), and calculate the cell concentration based on the discharge volume and the set volume; Determine whether the bacterial cell concentration has reached the set concentration. If so, a fermentation broth with a bacterial cell concentration reaching the set concentration can be obtained.
15. The method of using the bacterial cell enrichment and purification device according to claim 14, characterized in that, After the bacterial cell concentration reaches the set concentration, the method of use further includes: A buffer solution is introduced into the storage chamber (11), and the flow rate of the buffer solution is controlled to be consistent with the flow rate of the filtrate. The buffer solution has set parameters that characterize the properties of the buffer solution. The real-time parameters of the filtrate are obtained, and the real-time parameters can characterize the properties of the filtrate. Determine whether the real-time parameters and the set parameters are consistent. If they are, stop the flow of the buffer solution.
16. The method of using the bacterial cell enrichment and purification device according to claim 14 or 15, characterized in that, The configuration of the bacterial cell enrichment and purification device includes a disinfection step and a cleaning step. The disinfection step specifically involves disinfecting the bacterial cell enrichment and purification device by setting a disinfection method, and the cleaning step specifically involves cleaning the bacterial cell enrichment and purification device with a cleaning solution.
17. The method of using the bacterial cell enrichment and purification device according to claim 16, characterized in that, The specified disinfection methods include high-temperature dry heat sterilization, high-temperature steam sterilization, and disinfectant solution sterilization, wherein the disinfectant solution is ethanol with a purity of 75% or higher.
18. The method of using the bacterial cell enrichment and purification device according to claim 16, characterized in that, The cleaning solution is sterile water, sterile saline, or sterile buffer solution.
19. The method of using the bacterial cell enrichment and purification device according to claim 14 or 15, characterized in that, The configuration of the bacterial cell enrichment and purification device also includes a temperature control step for controlling the temperature of the fermentation broth.