A tubular culture apparatus

Abstract

The application discloses a tubular culture device, which comprises a sample feeding container and a culture pipeline in communication with the sample feeding container and forming a circulation loop, wherein a circulation power assembly is arranged on the culture pipeline; the circulation power assembly is used for driving the fluid in the sample feeding container to flow in the culture pipeline at a controllable flow rate; and the culture pipeline comprises one or more parallel branch pipelines. The tubular culture device can be widely used for the culture of microorganisms, cells and the like, realizes the control and monitoring of the culture process, provides rich reference data for the scaled production of cells, reduces the cost and reduces the product development risk.

Description

Technical Field

[0001] This application relates to the field of cell culture device technology, specifically to a tubular culture device. Background Technology

[0002] In the fields of cell culture research and industrial production, traditional cell culture reactors mainly include multi-well plates (0.1-10 mL), shake flasks (10-1000 mL), fermenters (1-200 L), and wave bioreactors (1-200 L). These reactors share the common characteristic that their dissolved oxygen and mixing control relies on aeration, shaking, or stirring. However, the mass transfer parameters of reactors at different scales vary significantly. Therefore, during scale-up, extensive optimization of the culture process is required for each scale-up, consuming substantial human and material resources with low efficiency. Secondly, mammalian cells lack cell wall protection and are therefore more fragile. The enormous shear forces generated by stirring can damage the cells. Often, the process of accelerating stirring to improve mixing and dissolved oxygen further exacerbates this damage. Summary of the Invention

[0003] To address the mass transfer and detection problems inherent in traditional micro-reactors, this application proposes a novel tubular culture device based on permeable tubing. This device can be widely used for the culture of microorganisms, cells, etc., and enables the control and monitoring of the culture process. It provides abundant reference data for the scale-up production of cells, reduces costs, and minimizes product development risks.

[0004] This application provides the following technical solution.

[0005] 1. A tubular culture device, characterized in that it includes a sample inlet container and a culture pipeline connected to the sample inlet container and forming a circulation loop, wherein a circulation power component is provided on the culture pipeline; the circulation power component is used to drive the fluid in the sample inlet container to circulate in the culture pipeline at a controllable flow rate;

[0006] The culture tubing includes one or more parallel branch tubing.

[0007] 2. The tubular culture device according to item 1, characterized in that the inner diameter R of the branch pipe is 0.1-10mm, preferably 1-3mm, the wall thickness of the branch pipe is 0.1-1mm, preferably 0.1-0.5mm, and the length L of the branch pipe is 0.1-100m, preferably 0.5-5m.

[0008] 3. The tubular culture device according to item 2, characterized in that the flow rate of the fluid in the tubular culture device in the branch pipe is V (mL / min), and 0.01×R 2 ×n≤V≤500×R2 ×n, where n is the number of branch pipes.

[0009] 4. The tubular culture device according to item 1, characterized in that the branch pipeline is a permeable branch pipeline, and the gas mass transfer coefficient Kla of the branch pipeline is greater than 2 / hr.

[0010] 5. The tubular culture device according to item 1, characterized in that the material of the branch pipes is selected from one of polytetrafluoroethylene, fusible polytetrafluoroethylene, amino plastic or silicone, preferably polytetrafluoroethylene.

[0011] 6. The tubular culture device according to item 1, characterized in that the sample injection container is a sample injection bottle, and an air filter membrane is provided on the top of the sample injection bottle;

[0012] The pore size of the air filter membrane is 0.01-10 μm, preferably 0.02-0.22 μm.

[0013] 7. The tubular culture device according to any one of items 1-6, characterized in that the device further includes a gas partial pressure control mechanism, the gas partial pressure control mechanism includes a sealing component and a partial pressure control component, the partial pressure control component is in communication with the sealing component, and the sample injection container and the culture pipeline are located inside the sealing component.

[0014] 8. The tubular culture device according to item 7, characterized in that the sealed component is provided with a gas inlet and a gas outlet, and the pressure control component is connected to the sealed component through the gas inlet, and a switch is provided on the gas outlet.

[0015] 9. The tubular culture device according to item 7 or 8, characterized in that the device further includes a microfluidic detection chip and an optical detection mechanism, wherein the microfluidic detection chip is directly or indirectly connected to the culture tubing, and the optical detection mechanism is used to detect the fluid within the microfluidic detection chip.

[0016] 10. The tubular culture device according to item 9, wherein the microfluidic detection chip is located outside the sealed component.

[0017] 11. The tubular culture device according to item 10, characterized in that each of the branch pipes includes a first pipe and a second pipe directly or indirectly connected to the first pipe.

[0018] 12. The tubular culture device according to item 11, characterized in that: the first end of the first tubing is connected to the bottom of the sample bottle, and the second end of the first tubing is directly or indirectly connected to the first end of the microfluidic detection chip; the first end of the second tubing is connected to the side wall or top of the sample bottle, and the second end of the second tubing is directly or indirectly connected to the second end of the microfluidic detection chip.

[0019] 13. The tubular culture device according to item 11, characterized in that the microfluidic detection chip is provided with multiple through channels, and the first end and the second end of the microfluidic detection chip are both connected to the channels.

[0020] 14. The tubular culture device according to item 13, characterized in that the microfluidic chip is provided with an optical sensor, the optical sensor being a DO fluorescence sensor and / or a pH fluorescence sensor.

[0021] 15. The tubular culture device according to item 9, characterized in that the optical detection mechanism includes an optical detection platform, a visible light absorbance detection device and / or a fluorescence detection device, wherein the visible light absorbance detection device and / or the fluorescence detection device are disposed on the optical detection platform.

[0022] 16. The tubular culture device according to item 15, characterized in that the microfluidic detection chip is placed on the optical detection platform of the optical detection mechanism.

[0023] 17. The tubular culture device according to item 13, characterized in that the height of the flow channel is 0.2-5 mm, preferably 1-2 mm, and the width of the flow channel is 0.2-5 mm, preferably 1-2 mm.

[0024] The tubular culture device provided in this application allows for the placement of a cell suspension within the device during cell culture. The cell suspension circulates within the device, ensuring adequate growth. The circulation power component controls the flow rate of the cell suspension, preventing either excessively low flow rates that reduce mass transfer efficiency or excessively high flow rates that cause cell damage due to high fluid shear forces. Simultaneously, the sealing component provides a constant temperature and humidity environment for cell culture, and the partial pressure control component provides the necessary gases for cell culture.

[0025] The tubular culture device provided in this application can detect the pH and dissolved oxygen levels of the cell suspension through the microfluidic detection chip, and the light absorbance of the cell suspension through the optical detection mechanism. Therefore, this reaction device can achieve flexible control and real-time online monitoring of the cell culture process, providing abundant data and experience for subsequent reactor scale-up. Attached Figure Description

[0026] The accompanying drawings are provided to better understand this application and do not constitute an undue limitation thereof. Wherein:

[0027] Figure 1 This is a schematic diagram of the tubular culture device provided in this application.

[0028] Figure 2 This is a schematic diagram of the tubular culture device provided in this application.

[0029] Figure 3 This is a schematic diagram of the tubular culture device provided in this application.

[0030] Figure 4 This is the growth curve of Escherichia coli in a tubular culture device in Example 1 of this application.

[0031] Figure 5 This is the growth curve of Escherichia coli in a tubular culture device in Example 2 of this application.

[0032] Figure 6 This is the growth curve of lactic acid bacteria in Example 10 of this application and the change of pH / OD / DO during the culture process. Detailed Implementation

[0033] The following description provides exemplary embodiments of this application, including various details to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0034] like Figure 1 This application provides a tubular culture device, including a sample inlet container and a culture tubing (both ends of the culture tubing are connected to the sample inlet container) that forms a circulation loop with the sample inlet container. A circulation power component is installed on the culture tubing (the circulation power component is located at any position on the culture tubing). The circulation power component is used to controllably drive the fluid in the sample inlet container to circulate within the culture tubing. Driven by the circulation power component, the fluid (cell suspension) can circulate within the culture tubing, promoting mixing, mass transfer, and detection. The circulation power component can be a pressure pump, syringe pump, diaphragm pump, peristaltic pump, etc., preferably a peristaltic pump for ease of installation and sterilization. The circulation power component allows control of the flow rate of the cell suspension, thereby avoiding situations where the flow rate is too low, reducing mass transfer efficiency, or too high, causing cell damage due to high fluid shear force.

[0035] In this application, the culture tubing includes one or more parallel branch tubing. The number of branch tubing can be determined according to actual needs. The multiple branch tubing do not affect each other, and the first end of each of the multiple branch tubing is connected to the bottom of the sample injection container, and the second end of each of the multiple branch tubing is connected to the side wall or top of the sample injection container.

[0036] The inner diameter R of the branch pipe is 0.1-10mm, preferably 1-3mm, the wall thickness of the branch pipe is 0.1-1mm, preferably 0.1-0.5mm, and the length L of the branch pipe is 0.1-100m, preferably 0.5-5m.

[0037] The inner diameter R of the branch pipe can be 0.1mm, 0.5mm, 1mm, 1.5mm, 2.0mm, 2.5mm, 3.0mm, 3.5mm, 4mm, 4.5mm, 5mm, 5.5mm, 6mm, 6.5mm, 7mm, 7.5mm, 8mm, 8.5mm, 9mm, 9.5mm or 10mm.

[0038] The wall thickness of the branch pipe is 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm or 1mm.

[0039] The length L of the branch pipe is 0.1m, 1m, 2m, 3m, 4m, 5m, 6m, 7m, 8m, 9m, 10m, 11m, 12m, 13m, 14m, 15m, 16m, 17m, 18m, 19m, 20m, 21m, 22m, 23m, 24m, 25m, 26m, 27m, 28m, 29m, 30m, 31m, 32m, 33m, 34m, 35m, 36m, 37m, 38m, 39m, 40m, 41m, 42m, 43m, 44m, 45m, 46m, 47m, 48m, 49m, 5 0m, 51m, 52m, 53m, 54m, 55m, 56m, 57m, 58m, 59m, 60m, 61m, 62m, 63m, 64m, 65m, 66m, 67m, 68m, 69m, 70m, 71m, 72m, 73m, 74m, 75m, 76m, 77m, 78m, 79m, 80m, 81m, 82m, 83m, 84m, 85m, 86m, 87m, 88m, 89m, 90m, 91m, 92m, 93m, 94m, 95m, 96m, 97m, 98m, 99m or 100m.

[0040] The flow velocity V (mL / min) of the fluid in the branch pipe of the tubular culture device is 0.01×R. 2 ×n≤V≤500×R2 ×n, where n is the number of branch pipes.

[0041] The branch pipe is a permeable branch pipe, allowing gases such as oxygen, carbon dioxide, hydrogen, nitrogen, and ammonia to pass through. The gas mass transfer coefficient Kla of the branch pipe is greater than 2 / hr. When the device is culturing cells, the required gas can be determined according to the culture conditions of the cells, and then the branch pipe is placed in this atmosphere. The gas can enter the branch pipe through the pipe wall, thereby enabling the cells in the branch pipe to grow sufficiently.

[0042] The branch tubing is made of a material selected from polytetrafluoroethylene (PTFE), fusible PTFE, amino plastics, or silicone, preferably PTFE, and more preferably TEFLONAF-2400, which has the best gas permeability compared to tubing made of other materials. Furthermore, as gas passes through the tubing wall into the cell suspension, the thicker the tubing wall, the greater the mass transfer resistance.

[0043] In this application, the sample injection container is a sample injection bottle, and an air filter membrane is provided at the top of the sample injection bottle; the pore size of the air filter membrane is 0.2-10 μm, preferably 0.22 μm; it can effectively filter impurities and microorganisms in the gas, preventing contamination of the cell suspension in the sample injection bottle. The filter membrane is embedded in the bottle cap, and the bottle cap fits the bottle body to form a good seal, allowing only air to enter the sample injection bottle through the filter membrane. A miniature electrochemical detection sensor can be placed in the sample injection bottle to detect the liquid in the sample injection bottle.

[0044] The volume of the sample vial is 0-100 ml, preferably 0-10 ml, excluding 0; a detection electrode is placed in the sample vial, and the detection electrode is selected from one or more of ammonium ion detection electrodes, calcium ion detection electrodes, and hydrogen ion detection electrodes.

[0045] like Figure 2 As shown, this application also provides a tubular culture device, including a sample inlet container, a culture tubing, and a gas partial pressure control mechanism. The gas partial pressure control mechanism includes a sealing component and a partial pressure control component, which communicates with the sealing component. The sample inlet container and the culture tubing are located within the sealing component. In use, a cell suspension is placed within the tubular culture device, where it circulates and grows sufficiently. The sealing component provides a constant temperature and humidity environment for cell culture, and the partial pressure control component provides the necessary gases for cell culture.

[0046] like Figure 3As shown, this application also provides a tubular culture device, including a sample inlet container, a culture tubing, a gas partial pressure control mechanism, a microfluidic detection chip, and an optical detection mechanism. The gas partial pressure control mechanism includes a sealing component and a pressure control component, which communicate with the sealing component. The sample inlet container and the culture tubing are located inside the sealing component. The microfluidic detection chip is directly or indirectly connected to the culture tubing and is connected to the optical detection mechanism. The microfluidic detection chip is located outside the sealing component.

[0047] The tubular culture device described in this application, during use, places the cell suspension in the sample inlet container. The cell suspension circulates within the sample inlet container, culture tubing, and microfluidic detection chip, allowing for sufficient growth. Furthermore, the sealed assembly provides a constant temperature and humidity environment for cell culture, and the partial pressure control assembly provides the necessary gases for cell culture. The microfluidic detection chip and optical detection mechanism work together to detect the pH, dissolved oxygen, absorbance, etc., of the cell suspension, or to perform relevant ion detection using an ion electrode built into the sample inlet (the sample inlet contains a detection electrode selected from one or more of ammonium ion, calcium ion, and hydrogen ion detection electrodes). Therefore, this reaction device enables flexible control and real-time online monitoring of the cell culture process, providing abundant data and experience for subsequent reactor scale-up.

[0048] In this application, the hermetic assembly is provided with a gas inlet and a gas outlet, and the pressure control assembly is connected to the hermetic assembly through the gas inlet, and a switch is provided on the gas outlet. The hermetic assembly is closed and can be of any shape, with a volume ranging from 1 to 100 L.

[0049] The partial pressure control component can be an oxygen partial pressure control box. The oxygen partial pressure control box controls the concentration and pressure of oxygen, carbon dioxide, nitrogen, methane, hydrogen, etc. inside the sealed component through the gas inlet, thereby providing different levels of gas supply to the cell suspension in the culture pipeline and realizing precise control of gas mass transfer during the culture process.

[0050] In this application, each of the branch pipes includes a first pipe and a second pipe that is directly or indirectly connected to the first pipe, such as... Figure 2 As shown, the first pipeline is directly connected to the second pipeline, as... Figure 3 As shown, the first pipeline is indirectly connected to the second pipeline.

[0051] The first end of the first conduit is connected to the bottom of the sample vial, and the second end of the first conduit is directly or indirectly connected to the first end of the microfluidic detection chip. The first end of the second conduit is connected to the side wall or top of the sample vial, and the second end of the second conduit is directly or indirectly connected to the second end of the microfluidic detection chip. During the circulation process, the cell suspension flows from the bottom of the sample vial into the first conduit, then through the first end of the microfluidic detection chip into the microfluidic detection chip, then through the second end of the microfluidic detection chip into the second conduit, and finally through the side wall or top of the sample vial into the sample vial, repeating the cycle continuously.

[0052] Both the first and second conduits are made of a highly breathable material. Various gases, such as oxygen, carbon dioxide, and nitrogen, within the sealed assembly can permeate through the walls of the first and second conduits into the lumen, dissolving in the cell suspension and providing a sufficient gas supply for cell growth. The lengths of the first and second conduits can be equal or unequal.

[0053] In this application, the microfluidic detection chip has multiple through channels. Both the first and second ends of the microfluidic detection chip are connected to these channels. Optical sensors, specifically DO fluorescence sensors and / or pH fluorescence sensors, are disposed within the channels. The microfluidic detection chip is placed on an optical detection platform. The optical detection mechanism includes the optical detection platform, a visible light absorbance detection device, and / or a fluorescence detection device. The visible light absorbance detection device and / or the fluorescence detection device are disposed on the optical detection platform and are used to detect the liquid within the microfluidic detection chip.

[0054] The fluorescence detection device is used to detect the fluorescence signals of the liquid detected by the DO fluorescence sensor and pH fluorescence sensor in the microfluidic detection chip, thereby analyzing the magnitude of their values.

[0055] The cross-section of the flow channel is rectangular, with a width of 0.2-5 mm, preferably 1-2 mm, and a height of 0.2-5 mm, preferably 1-2 mm. When the cell suspension passes through the microfluidic detection chip, the DO fluorescence sensor and / or pH fluorescence sensor respond to the dissolved oxygen and pH in the suspension, generating corresponding fluorescence signals under the excitation light of the fluorescence detection device. By analyzing the signals, the DO and pH values ​​can be obtained. OD detection relies on the light absorbance detection device; when the suspension flows through the microfluidic detection chip, its spectral signal is collected, and its OD value is obtained through analysis.

[0056] Example

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0058] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0059] Example 1: Microbial culture in a single tube followed by spectral (OD) detection (10 mL)

[0060] In this embodiment, the culture medium volume of Escherichia coli is 10 ml, and the culture tubing is made of TEFLONAF-2400 material with an inner diameter R of 2 mm, an outer diameter of 2.3 mm, a length L of 3.5 m, and n = 1. The culture tubing is connected to the sample bottle, and the circulation power component is located on the culture tubing. The circulation power component is a peristaltic pump. One side of the silicone tube in the peristaltic pump is connected to the culture tubing, and the other side is connected to the sample bottle. A visible light absorbance (OD) detection device is installed on the silicone tube on the other side to detect the OD growth changes of Escherichia coli.

[0061] The experimental procedure is as follows: Single-clone *E. coli* bacteria were picked from the plate and inoculated into LB medium. After shaking and incubation for 4 hours, the *E. coli* entered the logarithmic growth phase. 0.2 mL of this logarithmic growth phase bacterial suspension was added to 9.8 mL of fresh LB medium, shaken well, and then added to the sample vial provided in this application. The sample vial and branch tubing were placed in an incubator, and the peristaltic pump was started. The *E. coli* suspension circulated in the tubing under the drive of the peristaltic pump, allowing for growth. Growth data of *E. coli* could be obtained using an OD detection system. The results are shown in Table 1 and [other tables not provided]. Figure 4 As shown, with the increase of culture time, the OD value (absorbance) of Escherichia coli gradually increases, and after a short adaptation period, it enters the logarithmic growth phase of rapid growth. With the consumption of nutrients and the accumulation of metabolites or harmful substances, the growth of the strain enters the plateau phase and lasts for a long time.

[0062] Example 2: Microbial culture multi-tube parallel reactor + spectral (OD) detection (1L)

[0063] In this embodiment, the culture medium volume of E. coli is 10 mL, and n = 100, meaning the culture tubing includes 100 branch tubings. Each branch tubing is made of TEFLONAF-2400 material, with an inner diameter R of 2 mm, an outer diameter of 2.3 mm, and a length L of 3.5 m. The 100 branch tubings are connected in parallel to form a tubular reactor with a total volume of 1 L. Both ends of the parallel branch tubing have a common area. The common area at the first end is connected to the sample bottle, and the common area at the second end is connected to the circulation power unit. The circulation power unit uses a peristaltic pump. One side of the silicone tube in the peristaltic pump is connected to the common area at the second end of the branch tubing, and the other side is connected to the sample bottle. A visible light absorbance (OD) detection device is installed on the silicone tube on this side to detect changes in the OD growth of E. coli.

[0064] The experimental procedure is as follows: A single clone of *E. coli* was picked from the plate and inoculated into LB medium. After shaking and incubation for 4 hours, the *E. coli* entered the logarithmic growth phase. 0.02 L of this logarithmic growth phase bacterial suspension was added to 0.98 L of fresh LB medium, shaken well, and then added to the sample vial provided in this application. The sample vial and branch tubing were placed in an incubator, and the peristaltic pump was started. The *E. coli* suspension circulated within the tubing under the drive of the peristaltic pump, allowing for growth. Growth data of the *E. coli* could be obtained using an OD detection system.

[0065] The results are shown in Table 1 and Figure 5 As shown, with the increase of culture time, the OD value (absorbance) of Escherichia coli gradually increases, and after a short adaptation period, it enters the logarithmic growth phase of rapid growth. With the consumption of nutrients and the accumulation of metabolites or harmful substances, the growth of the strain enters the plateau phase and lasts for a long time.

[0066] The difference between Example 3 and Example 1 is that the number of branch pipes is different, as detailed in Table 1.

[0067] The difference between Examples 4-9 and Example 2 is that the flow rate of the fluid in the branch pipes is different, as detailed in Table 1.

[0068] The difference between Examples 10-14 and Example 2 is that the inner diameter of the branch pipes is different, as detailed in Table 1.

[0069] Table 1

[0070]

[0071] Summary: The tubular culture device described in this application can be scaled up by increasing the number of parallel branch pipes, and the growth state of the cultured cells inside is not affected by the number of branch pipes (Examples 1-3). The applicant also found that when the flow rate is less than 0.01, the mass transfer mixing inside the branch pipes is affected and reduced, leading to a decline in cell growth (Example 4). However, within a suitable flow rate range, mass transfer is optimally guaranteed, and the cell state remains stable and does not change with the flow rate (Examples 5-8). When the flow rate exceeds the limit, the internal shear force of the fluid is too large, inhibiting cell growth (Example 9). In addition, regarding the inner diameter of the branch pipes, within a certain range, the smaller the inner diameter, the larger its specific surface area, the more sufficient the mass transfer, and the better the bacterial growth state (Examples 10-12).

[0072] Example 10: Oxygen partial pressure control + tube culture + OD + DO

[0073] Growth curves of lactic acid bacteria under anaerobic culture conditions and measurement of pH and dissolved oxygen (DO) changes during culture.

[0074] In this embodiment, lactic acid bacteria, as facultative microorganisms, can grow in an anaerobic environment. By regulating the oxygen partial pressure control box, the oxygen concentration in the sealed space can be maintained close to 0, providing constant temperature and humidity conditions for the cultivation of lactic acid bacteria. The culture medium volume is 5 mL, the sample bottle volume is 10 mL, and a miniature dissolved oxygen detection electrode with a diameter of 2 mm and a length of 5 mm is placed in the sample bottle to detect the dissolved oxygen (DO) in the culture medium. A peristaltic pump is used as the circulation power component. The width and height of the flow channel in the microfluidic detection chip are both 2 mm, and a pH fluorescence sensor is installed in the flow channel. The culture pipeline includes a branch pipeline, which includes the first pipeline and the second pipeline. The first pipeline and the second pipeline are identical, both made of AF-2400 material, with a length of 0.8 m, an inner diameter of 2 mm, and an outer diameter of 2.6 mm. The optical detection mechanism includes a visible light absorbance (OD) detection device and a fluorescence excitation detection device.

[0075] The experimental procedure is as follows: Monoclonal lactic acid bacteria were picked from a plate and inoculated into MRS medium. After shaking and incubation for 4 hours, the lactic acid bacteria entered the logarithmic growth phase. 50 μl of this logarithmic growth phase bacterial suspension was added to 5 mL of fresh MRS medium, shaken well, and then added to the device described in this application. The peristaltic pump and optical detection mechanism were started, and the lactic acid bacteria suspension circulated in the sample bottle, the first tubing, and the second tubing at a flow rate of 0.5 mL / min, exchanging mass and energy with the gas in the sealed assembly (the gas inside the sealed assembly was air) through the tube walls. As the bacterial suspension flowed through the microfluidic detection chip, its OD, pH, and DO were continuously detected and recorded. After 24 hours of incubation, the data were processed, and the obtained OD values ​​and changes in pH and DO are shown below. Figure 6 As shown. By Figure 6 It can be seen that as the cultivation time increases, the OD value (absorbance) of lactic acid bacteria gradually increases, undergoing a brief adaptation period before entering a rapid logarithmic growth phase. With the consumption of nutrients and the accumulation of metabolites or harmful substances, the growth of the strain enters a plateau phase, which lasts for a relatively long time. In the initial stage, due to the low amount of lactic acid, the pH value is maintained at a good level. As the cultivation time increases, the lactic acid produced by the lactic acid bacteria gradually accumulates, leading to a decrease in pH. The entire cultivation device is placed in an anaerobic environment (extremely low oxygen concentration), and dissolved oxygen is maintained at a low level.

[0076] Although the embodiments of this application have been described above in conjunction with the specific embodiments described, this application is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely illustrative and instructive, and not restrictive. Those skilled in the art can make many other forms based on the teachings of this specification and without departing from the scope of protection of the claims of this application, and these are all within the scope of protection of this application.

Claims

1. A tubular culture apparatus, characterized by, It includes a sample inlet container and a culture tubing connected to the sample inlet container and forming a circulation loop. The culture tubing is equipped with a circulation power component. The circulation power component is used to drive the fluid in the sample inlet container to circulate in the culture tubing at a controllable flow rate. The culture tubing includes multiple parallel branch tubing; The inner diameter R of the branch pipe is 0.8-3mm, the wall thickness of the branch pipe is 0.1-1mm, and the length L of the branch pipe is 0.1-100m. The flow rate of the fluid in the pipe culture apparatus in the branch pipes is V (mL / min), and 0.01 x R 2 x n ≤ V ≤ 500 x R 2 x n, n is the number of the branch pipes; The sample inlet container is a sample inlet bottle, and an air filter membrane is provided on the top of the sample inlet bottle; The pore size of the air filter membrane is 0.01-10 μm; The device also includes a gas partial pressure control mechanism, which includes a sealing component and a partial pressure control component. The partial pressure control component is in communication with the sealing component, and the sample injection container and culture tubing are located inside the sealing component. The device also includes a microfluidic detection chip and an optical detection mechanism. The microfluidic detection chip is directly or indirectly connected to the culture tubing, and the optical detection mechanism is used to detect the fluid inside the microfluidic detection chip. Each of the branch pipes includes a first pipe and a second pipe that is directly or indirectly connected to the first pipe; The first end of the first conduit is connected to the bottom of the sample vial, and the second end of the first conduit is directly or indirectly connected to the first end of the microfluidic detection chip; the first end of the second conduit is connected to the side wall or top of the sample vial, and the second end of the second conduit is directly or indirectly connected to the second end of the microfluidic detection chip.

2. The tubular culture device according to claim 1, characterized in that, The inner diameter R of the branch pipe is 1-3mm.

3. The tubular culture device according to claim 1, characterized in that, The wall thickness of the branch pipeline is 0.1-0.5 mm.

4. The tubular culture device according to claim 1, characterized in that, The length L of the branch pipeline is 0.5-5m.

5. The tubular culture device according to claim 1, characterized in that, The branch pipeline is a permeable branch pipeline, and the gas mass transfer coefficient Kla of the branch pipeline is greater than 2 / hr.

6. The tubular culture device according to claim 1, characterized in that, The branch pipe is made of one of the following materials: polytetrafluoroethylene, fusible polytetrafluoroethylene, amino plastic, or silicone.

7. The tubular culture device according to claim 1, characterized in that, The branch pipe is made of polytetrafluoroethylene.

8. The tubular culture device according to claim 1, characterized in that, The pore size of the air filter membrane is 0.02-0.22 μm.

9. The tubular culture device according to claim 1, characterized in that, The sealed assembly is provided with a gas inlet and a gas outlet, and the pressure control assembly is connected to the sealed assembly through the gas inlet, and a switch is provided on the gas outlet.

10. The tubular culture device according to claim 1, characterized in that, The microfluidic detection chip is located outside the sealed component.

11. The tubular culture device according to claim 1, characterized in that, The microfluidic detection chip has multiple through channels, and both the first end and the second end of the microfluidic detection chip are connected to the channels.

12. The tubular culture device according to claim 11, characterized in that, The height within the flow channel is 0.2-5 mm.

13. The tubular culture device according to claim 11, characterized in that, The height inside the flow channel is 1-2 mm.

14. The tubular culture device according to claim 11, characterized in that, The width of the flow channel is 0.2-5mm.

15. The tubular culture device according to claim 11, characterized in that, The width of the flow channel is 1-2 mm.

16. The tubular culture apparatus according to any one of claims 11-15, characterized in that, The microfluidic detection chip is equipped with an optical sensor, which is a DO fluorescence sensor and / or a pH fluorescence sensor.

17. The tubular culture device according to claim 1, characterized in that, The optical detection mechanism includes an optical detection platform, a visible light absorption detection device and / or a fluorescence detection device, wherein the visible light absorption detection device and / or the fluorescence detection device are disposed on the optical detection platform.

18. The tubular culture device according to claim 17, characterized in that, The microfluidic detection chip is placed on the optical detection platform of the optical detection mechanism.

Images