CN115521872A - Tubular cultivation equipment - Google Patents

Abstract

The present application discloses a tubular culture device, which includes a sample injection container and a culture pipeline that communicates with the sample injection container and forms a circulation loop. The culture pipeline is provided with a circulation power assembly; the circulation power assembly is used for Controllable flow rate drives the fluid in the sampling container to circulate in the culture pipeline; the culture pipeline includes one or more parallel branch pipelines. The tubular culture equipment proposed in this application can be widely used in the cultivation of microorganisms and cells, and can realize the control and monitoring of the cultivation process, provide abundant reference data for the enlarged production of cells, reduce costs, and reduce product development risks.

Description

A tube culture device

technical field

The present application relates to the technical field of cell culture devices, in particular to a tubular culture device.

Background technique

In the field of scientific research and industrial production of cell culture, traditional cell culture reactors are mainly porous plates (0.1-10mL), shake flasks (10-1000mL), fermenters (1-200L), bioreactor bags (wave bioreactor, 1 -200L), etc., the common feature of these reactors is that the control of dissolved oxygen and mixing depends on aeration, shaking or stirring. However, the mass transfer parameters of reactors of different scales are quite different. Therefore, in the process of step-by-step scale-up, a large number of cultivation process optimizations are required for each level of scale-up, which consumes a lot of manpower and material resources, and the efficiency is low. Secondly, for mammalian cells, the cells lack cell wall protection, so they are relatively fragile, and the huge shear force brought by stirring will cause damage to the cells. Usually, the stirring process is accelerated to improve mixing and dissolved oxygen, and further enhance this damage.

Contents of the invention

Aiming at the problems of mass transfer and detection in traditional micro-reactors, this application proposes a new tubular culture equipment based on gas-permeable pipelines, which can be widely used in the cultivation of microorganisms and cells, and realizes the control and monitoring of the cultivation process. The scale-up production provides rich reference data, reduces costs and reduces product development risks.

The application provides the following technical solutions.

1. A tubular culture device, characterized in that it includes a sample injection container and a culture pipeline that communicates with the sample injection container and forms a circulation loop, and the culture pipeline is provided with a circulation power assembly; the circulation power assembly It is used to control the flow rate to drive the fluid in the sampling container to circulate in the culture pipeline;

The culture pipeline includes one or more parallel branch pipelines.

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

3. The tubular culture device according to item 2, wherein the flow rate of the fluid in the tube culture device in the branch pipeline is V (mL/min), and 0.01×R ² ×n ≤V≤500×R ² ×n, where n is the number of branch pipelines.

4. The tubular culture equipment according to Item 1, wherein the branch pipeline is a gas-permeable branch pipeline, and the gas mass transfer coefficient Kla of the branch pipeline is greater than 2/hr.

5. The tubular culture equipment according to item 1, wherein the material of the branch pipeline is selected from one of polytetrafluoroethylene, fusible polytetrafluoroethylene, aminoplast or silica gel, preferably PTFE.

6. The tubular culture device according to item 1, wherein the sampling container is a sampling bottle, and the top of the sampling bottle is provided with an air filter;

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

7. The tubular culture device according to any one of Items 1-6, characterized in that the device also includes a gas partial pressure control mechanism, and the gas partial pressure control mechanism includes an airtight component and a partial pressure control component. The partial pressure control component communicates with the closed component, and the sampling container and the culture pipeline are located in the closed component.

8. The tubular culture device according to Item 7, wherein the sealing component is provided with a gas inlet and a gas outlet, and the partial pressure control component communicates with the sealing component through the gas inlet, so that The gas outlet is provided with a switch.

9. The tubular culture device according to item 7 or 8, characterized in that the device also includes a microfluidic detection chip and an optical detection mechanism, and the microfluidic detection chip is directly or indirectly connected to the culture pipeline In communication, the optical detection mechanism is used to detect the fluid in the microfluidic detection chip.

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

11. The tube culture device according to item 10, wherein each of the branch pipelines includes a first pipeline and a second pipeline directly or indirectly connected to the first pipeline.

12. The tube culture device according to item 11, characterized in that, the first end of the first pipeline is connected to the bottom of the sampling bottle, and the second end of the first pipeline is connected to the bottom of the sampling bottle. The first end of the microfluidic detection chip is directly or indirectly connected; the first end of the second pipeline is connected to the side wall or top of the sampling bottle, and the second end of the second pipeline is connected to the The second end of the microfluidic detection chip is connected directly or indirectly.

13. The tubular culture device according to item 11, wherein the microfluidic detection chip is provided with a plurality of through flow channels, the first end of the microfluidic detection chip and the microfluidic The second ends of the control detection chips are all in communication with the flow channel.

14. The tubular culture device according to item 13, wherein the microfluidic chip is provided with an optical sensor, and the optical sensor is a DO fluorescence sensor and/or a pH fluorescence sensor.

15. The tubular culture equipment according to Item 9, wherein the optical detection mechanism includes an optical detection platform, a visible light absorbance detection device and/or a fluorescence detection device, and the visible light absorbance detection device and/or fluorescence detection device The detection device is arranged on the optical detection platform.

16. The tubular culture device according to item 15, wherein the microfluidic detection chip is placed on the optical detection platform of the optical detection mechanism.

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

In the tube culture equipment provided by the present application, when culturing cells, the cell suspension is placed in the tube culture equipment, and the cell suspension circulates in the tube culture equipment for sufficient growth. The circulation power component can control the flow rate of the cell suspension, so as to avoid the flow rate being too small, which reduces the mass transfer efficiency, or avoiding the situation where the flow rate is too high and the fluid shear force is too large to cause cell damage. At the same time, the airtight component can provide a constant temperature and humidity environment for cell culture, and the partial pressure control component can provide gas required for cell culture for cell culture.

The tubular culture equipment provided in the present application can detect the pH value and dissolved oxygen of the cell suspension through the microfluidic detection chip, and detect the light absorbance of the cell suspension through the optical detection mechanism. Therefore, the reaction equipment can realize the functions of flexible regulation and real-time online detection of the cell culture process, and provide rich data and experience for the subsequent expansion of the reactor.

Description of drawings

The accompanying drawings are used for better understanding of the present application, and do not constitute an improper limitation of the present application. in:

Fig. 1 is a schematic structural diagram of the tubular culture equipment provided by the present application.

Fig. 2 is a schematic structural diagram of the tubular culture equipment provided by the present application.

Fig. 3 is a schematic structural diagram of the tubular culture equipment provided by the present application.

Fig. 4 is the growth curve of Escherichia coli in tube culture equipment in Example 1 of the present application.

Fig. 5 is the growth curve of Escherichia coli in tube culture equipment in Example 2 of the present application.

Fig. 6 is the growth curve of lactic acid bacteria in Example 10 of the present application and the pH/OD/DO change graph during the cultivation process.

detailed description

The following describes the exemplary embodiments of the present application, including various details of the embodiments of the present application to facilitate understanding, and they should be considered as exemplary only. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the application. Also, descriptions of well-known functions and constructions are omitted in the following description for clarity and conciseness.

As shown in Figure 1, the present application provides a tube culture device, including a sample injection container and a culture pipeline that communicates with the sample injection container and forms a circulation loop (both ends of the culture pipeline are connected to the sample injection container connected), the culture pipeline is provided with a circulation power assembly (the circulation power assembly is located at any position on the culture pipeline); the circulation power assembly is used to control the flow rate to drive the fluid in the sampling container Circulating flow in the culture pipeline, the fluid (cell suspension) can circulate in the culture pipeline under the drive of the circulation power assembly to promote mixing, mass transfer, detection and the like. The cycle power component can be a pressure pump, a syringe pump, a diaphragm pump, a peristaltic pump, etc., preferably a peristaltic pump, which is convenient for installation and sterilization. The flow rate of the cell suspension can be controlled by the circulation power assembly, so as to avoid the flow rate being too small to reduce the mass transfer efficiency, or avoid the occurrence of cell damage caused by excessive flow rate and fluid shear force.

In the present application, the culture pipeline includes one or more parallel branch pipelines. The number of branch pipelines can be determined according to actual needs, and the multiple branch pipelines do not affect each other, and the first ends of the multiple branch pipelines are all connected to the bottom of the sampling container, and the multiple branch pipelines The second end of the passage communicates with the side wall or the top of the sampling container.

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

The inner diameter R of the branch pipeline 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.

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

The length L of the branch pipeline 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, 50m, 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.

The flow velocity V (mL/min) of the fluid in the tube culture equipment in the branch pipeline, and 0.01×R ² ×n≤V≤500×R ² ×n, n is the branch pipeline quantity.

The branch pipeline is a gas-permeable branch pipeline through which gases such as oxygen, carbon dioxide, hydrogen, nitrogen, and ammonia can permeate. The gas mass transfer coefficient Kla of the branch pipeline is greater than 2/hr. When the device is cultivating cells, the required gas can be determined according to the culture conditions of the cultured cells, and then the branch pipeline is placed in the atmosphere, and the gas can enter through the wall of the branch pipeline In the branch pipeline, so that the cells in the branch pipeline can fully grow.

The material of the branch pipeline is selected from one of polytetrafluoroethylene, fusible polytetrafluoroethylene, aminoplast or silica gel, preferably polytetrafluoroethylene, more preferably TEFLONAF-2400, compared with pipes of other materials The road has the best gas permeability. In addition, the gas enters the cell suspension through the tube wall, and the thicker the tube wall, the greater the mass transfer resistance.

In the present application, the sampling container is a sampling bottle, and the top of the sampling bottle is provided with an air filter membrane; the pore size on the air filter membrane is 0.2-10 μm, preferably 0.22 μm; it can effectively filter gas Impurities and microorganisms in the sample to prevent contamination of the cell suspension in the sample bottle. The filter membrane is embedded in the bottle cap, and the bottle cap is adapted to the bottle body to form a good seal, allowing only air to enter the sampling bottle through the filter membrane. A miniature electrochemical detection sensor can be placed in the sampling bottle to detect the liquid in the sampling bottle.

The volume of the sampling bottle is 0-100ml, preferably 0-10ml, excluding 0; the detection electrode is placed in the sampling bottle, and the detection electrode is selected from ammonium ion detection electrode, calcium ion detection electrode, hydrogen ion detection electrode One or more of the electrodes.

As shown in Figure 2, the present application also provides a tubular culture device, including a sample injection container, a culture pipeline, and a gas partial pressure control mechanism, the gas partial pressure control mechanism includes an airtight component and a partial pressure control component, the The partial pressure control component communicates with the closed component, and the sampling container and the culture pipeline are located in the closed component. When the device is in use, the cell suspension is placed in the tube culture device, and the cell suspension circulates in the tube culture device for sufficient growth, and the airtight assembly can provide cell culture Constant temperature and humidity environment, partial pressure control components can provide the gas needed for cell culture for cell culture.

As shown in Figure 3, the present application also provides a tubular culture device, including a sample injection container, a culture pipeline, a gas partial pressure control mechanism, a microfluidic detection chip, and an optical detection mechanism. The gas partial pressure control mechanism includes An airtight assembly and a partial pressure control assembly, the partial pressure control assembly communicates with the airtight assembly, the sample injection container and the culture pipeline are located in the airtight assembly, the microfluidic detection chip is connected to the culture pipeline directly or indirectly, the microfluidic detection chip is connected with the optical detection mechanism. The microfluidic detection chip is located outside the airtight assembly.

When the tubular culture equipment described in the present application is in use, the cell suspension is placed in the sample injection container, and the cell suspension circulates in the sample injection container, the culture pipeline and the microfluidic detection chip to perform Sufficient growth, and the airtight component can provide a constant temperature and humidity environment for cell culture, the partial pressure control component can provide the gas needed for cell culture for cell culture, and through the cooperation of the microfluidic detection chip and the optical detection mechanism can Detect the pH, dissolved oxygen, absorbance, etc. of the cell suspension or carry out the relevant ion detection of the cell suspension through the built-in ion electrode in the sample injection bottle (the detection electrode is placed in the sample injection bottle, and the detection electrode is selected from ammonium ion one or more of detection electrodes, calcium ion detection electrodes, and hydrogen ion detection electrodes). Therefore, the reaction equipment can realize the functions of flexible regulation and real-time online detection of the cell culture process, and provide rich data and experience for the subsequent expansion of the reactor.

In the present application, the sealing component is provided with a gas inlet and a gas outlet, and the partial pressure control component communicates with the sealing component through the gas inlet, and a switch is provided on the gas outlet. The airtight assembly is closed and can be in any shape, and its volume range is 1-100L.

The partial pressure control component can be an oxygen partial pressure control box, and 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, so that Provide different degrees of gas supply for the cell suspension in the culture pipeline to achieve precise control of gas mass transfer during the culture process.

In the present application, each of the branch pipelines includes a first pipeline and a second pipeline directly or indirectly connected with the first pipeline, as shown in FIG. 2 , the first pipeline and the The second pipeline is directly connected. As shown in FIG. 3 , the first pipeline is indirectly connected with the second pipeline.

The first end of the first pipeline is connected to the bottom of the sampling bottle, and the second end of the first pipeline is directly or indirectly connected to the first end of the microfluidic detection chip; The first end of the second pipeline is connected to the side wall or the top of the sampling bottle, and the second end of the second pipeline communicates directly or indirectly with the second end of the microfluidic detection chip. During the circulating flow process, the cell suspension flows into the first pipeline from the bottom of the sampling bottle, then enters the microfluidic detection chip through the first end of the microfluidic detection chip, and then Enter the second pipeline through the second end of the microfluidic detection chip, and finally enter the sampling bottle from the side wall or top of the sampling bottle, and the cycle repeats.

Both the first pipeline and the second pipeline are made of a material with good air permeability, and various gases such as oxygen, carbon dioxide, and nitrogen in the airtight assembly can pass through the first pipeline and the second pipeline. The tube wall of the pipeline enters the lumen and then dissolves in the cell suspension to provide sufficient gas supply for cell growth. The lengths of the first pipeline and the second pipeline may be equal or unequal.

In the present application, the microfluidic detection chip is provided with a plurality of through flow channels, and the first end of the microfluidic detection chip and the second end of the microfluidic detection chip are connected to the flow channel. The flow channel is connected, and an optical sensor is arranged in the flow channel, and the optical sensor is a DO fluorescence sensor and/or a pH fluorescence sensor. The microfluidic detection chip is placed on an optical detection platform. The optical detection mechanism includes an optical detection platform, a visible light absorption detection device and/or a fluorescence detection device, and the visible light absorption detection device and/or fluorescence detection device are arranged on the optical detection platform for detecting the microscopic Fluidics detects the liquid inside the chip.

The fluorescence detection device is used to detect the fluorescence signal of the liquid detected by the DO fluorescence sensor and the pH fluorescence sensor in the microfluidic detection chip, so as to analyze the values of the two.

The cross section of the flow channel is rectangular, with a width of 0.2-5mm, preferably 1-2mm, and a height of 0.2-5mm, preferably 1-2mm. When the cell suspension passes through the microfluidic detection chip, the DO fluorescence sensor and/or the pH fluorescence sensor will respond to the dissolved oxygen and pH in the suspension, and generate a corresponding fluorescence signal under the excitation light of the fluorescence detection device. By analyzing the signal, the DO value and pH value can be obtained. The detection of OD depends on the optical 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.

Example

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1: Culture of microorganism + single pipeline + spectroscopic (OD) detection (10mL)

In this implementation case, the volume of Escherichia coli culture solution is 10ml, and the culture pipeline is made of TEFLONAF-2400 material, the inner diameter R is 2mm, the outer diameter is 2.3mm, the length L is 3.5m, n=1, The culture pipeline is in communication with the sampling bottle, the circulation power component is located on the culture pipeline, the circulation power component is a peristaltic pump, one side of the silicone tube in the peristaltic pump is connected to the culture pipeline, and the other side is connected to the culture pipeline. It is connected with the injection bottle, and a visible light absorbance (OD) detection device is installed on the silicone tube on this side, which is used to detect the OD growth change of Escherichia coli.

The experimental process is as follows: the monoclonal Escherichia coli on the plate was picked and inserted into LB medium, cultured with shaking for 4 hours, and the growth of Escherichia coli entered the logarithmic phase. Take 0.2 mL of the logarithmic phase bacterial solution, add it to 9.8 mL of fresh LB medium, shake it well, and then add it to the sample injection bottle of this application. The sample bottle and branch pipeline were placed in an incubator, and the peristaltic pump was started, and the Escherichia coli suspension was driven by the peristaltic pump to circulate in the pipeline to grow. The growth data of E. coli can be obtained by the OD detection system. The results are shown in Table 1 and Figure 4. With the growth of culture time, the OD value (absorbance) of Escherichia coli gradually increases, and enters the logarithmic phase of rapid growth through a short adaptation period. Accumulation of pollutants or harmful substances, the growth of bacteria enters a plateau period and lasts for a long time.

Embodiment 2: Microbiological culture multi-pipeline parallel reactor+spectral (OD) detection (1L)

In this implementation case, the volume of Escherichia coli culture solution is 10mL, n=100, that is, the culture pipeline includes 100 branch pipelines, and each branch pipeline is made of TEFLONAF-2400 material culture pipeline, and its inner diameter R The diameter is 2mm, the outer diameter is 2.3mm, the length L is 3.5m, and 100 branch pipes are connected in parallel to form a tubular reactor with a total volume of 1L. After parallel connection, both ends of the branch pipes have common areas, and the first end The public area of the sample bottle is connected with the sample bottle, and the public area at the second end is connected with the circulation power assembly. The side is connected with the sample bottle, and a visible light absorbance (OD) detection device is installed on the silicone tube on this side, which is used to detect the OD growth change of Escherichia coli.

The experimental process is as follows: the monoclonal Escherichia coli on the plate was picked and inserted into LB medium, cultured with shaking for 4 hours, and the growth of Escherichia coli entered the logarithmic phase. Take 0.02L of the logarithmic phase bacterial solution, add it to 0.98L of fresh LB medium, shake it well, and then add it to the sample injection bottle of this application. The sample bottle and branch pipeline were placed in an incubator, and the peristaltic pump was started, and the Escherichia coli suspension was driven by the peristaltic pump to circulate in the pipeline to grow. The growth data of E. coli can be obtained by the OD detection system.

The results are shown in Table 1 and Figure 5. With the growth of culture time, the OD value (absorbance) of Escherichia coli gradually increases, and enters the logarithmic phase of rapid growth through a short adaptation period. With the consumption of nutrients and metabolites Or the accumulation of harmful substances, the growth of bacteria enters the plateau period and lasts for a long time.

The difference between Embodiment 3 and Embodiment 1 is that the number of branch pipelines is different, see Table 1 for details.

Embodiment 4-Embodiment 9 differs from Embodiment 2 in that the flow velocity of the fluid in the branch pipeline is different, see Table 1 for details.

Embodiment 10-Embodiment 14 differs from Embodiment 2 in that the inner diameters of the branch pipelines are different, see Table 1 for details.

Table 1

Summary: The tubular culture equipment described in this application can be enlarged by increasing the number of parallel branch pipelines, and the growth state of the cells cultured inside it is not affected by the number of branch pipelines (Example 1-3) . The applicant also found that when the flow rate was less than 0.01, the mass transfer mixing inside the branch pipeline was affected and reduced, resulting in a decline in the cell growth state (Example 4), and in a suitable flow rate range, the mass transfer was best guaranteed, and the cells The state remained stable and did not change with changes in flow rate (Examples 5-8). And when the flow rate exceeds the limit range, the internal shear force of the fluid is too large, which inhibits the cell growth (Example 9). In addition, for the internal diameter of the branch pipeline, within a certain range, the smaller the internal diameter, the larger the specific surface area, the more sufficient the mass transfer, and the better the growth state of bacteria (Example 10-12).

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

Growth curve of lactic acid bacteria under anaerobic culture conditions and determination of pH and dissolved oxygen (DO) changes during the culture process

In this implementation case, lactic acid bacteria, as facultative microorganisms, can grow in an anaerobic environment. Through the regulation of the oxygen partial pressure control box, the oxygen concentration in the confined space can be kept close to 0, and constant temperature and humidity conditions are provided for the growth of lactic acid bacteria. to cultivate. The volume of the culture solution is 5mL, the volume of the sample bottle is 10mL, and a miniature dissolved oxygen detection electrode with a diameter of 2mm and a length of 5mm is installed in the sample bottle to detect dissolved oxygen (DO) in the culture solution. The cycle power pack uses a peristaltic pump. The width and height of the channel in the microfluidic detection chip are both 2mm, and a pH fluorescent sensor is arranged in the channel. The culture pipeline includes a branch pipeline, and the branch pipeline includes the first pipeline and the second pipeline. The first pipeline and the second pipeline are the same and are made of AF-2400 material. The length is 0.8m, the inner diameter is 2mm, and the outer diameter is 2.6mm. The optical detection mechanism includes a visible light absorbance (OD) detection device and a fluorescence excitation detection device.

The experimental process is as follows: Pick the monoclonal lactic acid bacteria on the plate and insert them into the MRS medium, shake and culture for 4 hours, and the growth of the lactic acid bacteria enters the logarithmic phase. Take 50 μl of the logarithmic phase bacterial solution, add it to 5 mL of fresh MRS medium, shake it well, and then add it to the device of this application. Start the peristaltic pump and the optical detection mechanism, the lactic acid bacteria suspension circulates in the sampling bottle, the first pipeline and the second pipeline at a flow rate of 0.5mL/min, and passes through the tube wall and the closed component (the gas in the closed component is air ) for mass-energy exchange. When the bacterial liquid flows through the microfluidic detection chip, its OD, pH, and DO are continuously detected and recorded. After culturing for 24 hours and processing the data, the obtained OD value, pH and DO changes are shown in Figure 6. It can be seen from Figure 6 that with the growth of culture time, the OD value (absorbance) of lactic acid bacteria gradually increases, and enters the logarithmic phase of rapid growth after a short adaptation period. With the consumption of nutrients and the accumulation of metabolites or harmful substances, bacteria Seed growth enters a plateau and lasts for a long time. In the initial stage, due to less lactic acid, the pH value is maintained at a good level. As the culture time increases, the lactic acid produced by lactic acid bacteria gradually accumulates, resulting in a decrease in pH value. The culture device is placed in an anaerobic environment as a whole (the oxygen concentration is extremely low), and the dissolved oxygen is maintained at a low level.

Although the embodiments of the present application have been described above, the present application is not limited to the above-mentioned specific embodiments and application fields, and the above-mentioned specific embodiments are only illustrative, instructive, and not restrictive. Those skilled in the art can also make many forms under the enlightenment of this description and without departing from the protection scope of the claims of the application, and these all belong to the protection list of the application.

Claims (15)

1. The tubular culture equipment is characterized by comprising a sample introduction container and a culture pipeline which is communicated with the sample introduction container and forms a circulation loop, wherein a circulation power assembly is arranged on the culture pipeline; the circulating power assembly is used for driving the fluid in the sample introduction container to circularly flow in the culture pipeline at a controllable flow rate;

the culture line comprises one or more branch lines connected in parallel.

2. The tubular cultivation apparatus according to claim 1, wherein the branch line has an inner diameter R of 0.1 to 10mm, preferably 1 to 3mm, a wall thickness of 0.1 to 1mm, preferably 0.1 to 0.5mm, and a length L of 0.1 to 100m, preferably 0.5 to 5m.

3. The tube cultivation apparatus according to claim 2, wherein the flow rate of the fluid in the tube cultivation apparatus in the branch line is V (mL/min) and 0.01 XR ² ×n≤V≤500×R ² And x n is the number of the branch pipelines.

4. The tubular cultivation apparatus according to claim 1, wherein the branch line is a gas-permeable branch line, and the gas mass transfer coefficient Kla of the branch line is more than 2/hr.

5. The tubular culture apparatus of claim 1, wherein the branch conduit is made of one material selected from the group consisting of polytetrafluoroethylene, fusible polytetrafluoroethylene, aminoplast, and silica gel, preferably polytetrafluoroethylene.

6. The tubular cultivation apparatus according to claim 1, wherein the sample introduction container is a sample introduction bottle, and an air filter membrane is disposed on the top of the sample introduction bottle;

the pore diameter on the air filter membrane is 0.01-10 μm, preferably 0.02-0.22 μm.

7. The tubular cultivation apparatus according to any one of claims 1 to 6, further comprising a gas partial pressure control mechanism, wherein the gas partial pressure control mechanism comprises a sealing component and a partial pressure control component, the partial pressure control component is communicated with the sealing component, and the sample introduction container and the cultivation pipeline are located in the sealing component.

8. The tubular cultivation apparatus according to claim 7, wherein the sealing member is provided with a gas inlet and a gas outlet, the partial pressure control member is communicated with the sealing member through the gas inlet, and the gas outlet is provided with a switch.

9. The tubular cultivation apparatus according to claim 7 or 8, further comprising a microfluidic detection chip directly or indirectly communicating with the cultivation pipeline, and an optical detection mechanism for detecting a fluid in the microfluidic detection chip.

10. The tubular culture apparatus of claim 9, wherein the microfluidic detection chip is located outside the containment assembly.

11. The tube culturing apparatus according to claim 10, wherein each of the branch lines comprises a first line and a second line directly or indirectly communicating with the first line.

12. The tube culturing device of claim 11, wherein a first end of the first tube is connected to the bottom of the sample bottle, and a second end of the first tube is in direct or indirect communication with a first end of the microfluidic detection chip; the first end of the second pipeline is connected to the side wall or the top of the sample feeding bottle, and the second end of the second pipeline is directly or indirectly communicated with the second end of the microfluidic detection chip.

13. The tubular culture apparatus of claim 11, wherein a plurality of flow channels are disposed through the microfluidic detection chip, and a first end of the microfluidic detection chip and a second end of the microfluidic detection chip are both in communication with the flow channels;

preferably, the height in the flow channel is 0.2-5mm, preferably 1-2mm, and the width in the flow channel is 0.2-5mm, preferably 1-2mm.

14. The tubular cultivation apparatus according to claim 13, wherein an optical sensor is disposed in the microfluidic chip, and the optical sensor is a DO fluorescence sensor and/or a pH fluorescence sensor.

15. The tubular cultivation apparatus according to claim 9, wherein the optical detection mechanism comprises an 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 being disposed on the optical detection platform;

preferably, the microfluidic detection chip is placed on an optical detection platform of the optical detection mechanism.

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