Continuous bacteriophage culture device, bacteriophage culture method and bacteriophage cleaning method

Abstract

The invention relates to a bacteriophage continuous culture device, a bacteriophage culture method and a cleaning method, flowing pipes and connecting pipes are alternately arranged, the two ends of each connecting pipe are fixedly connected and communicated with the flowing pipes respectively, the connecting pipes are used for changing the flowing direction of fluid in the two flowing pipes communicated with the connecting pipes, the pipeline at the most upstream is communicated with a feeding pump, and the pipeline at the most upstream is communicated with a discharging pump. The feeding pump is communicated with the host bacteria liquid tank, and the temperature control device is located on the outer side of the flowing pipe and used for changing the temperature of fluid in the flowing pipe. Compared with stirring of stirring blades in a stirring tank, the effect of flowing and mixing fluid in a pipeline through the phage liquid and the host bacteria liquid, the host bacteria liquid and the fermentation culture medium is better, and the yield of the phage and the host bacteria is higher; and the shearing force of rotary stirring of the stirring blades is avoided, the damage of the pipeline mixing mode to the bacteriophage and the host bacteria is smaller, the bacteriophage and the host bacteria are not prone to being broken and damaged, and the integrity and the activity of the produced bacteriophage are better.

Description

Technical Field

[0001] The invention relates to the field of bacteriophage culture, in particular to a bacteriophage continuous culture device, a bacteriophage culture method and a cleaning method. Background Art

[0002] The existing phage culture requires the host bacterial liquid and phage liquid to be cultured separately first, and then the phage is cultured in batches using a fermenter. The existing fermenter uses a stirring device to stir the liquid, but due to the structural insufficiency of the stirring structure itself (such as insufficient stirring at the bottom), the phage and the culture liquid are not mixed evenly, affecting the proliferation efficiency.

[0003] At the same time, the volume of the fermentation tank is limited, and after each batch of phage is produced, the fermentation tank needs to be cleaned and sterilized, which results in low efficiency in producing phage using the fermentation tank and cannot meet the demand for large-scale industrial production of phage. Summary of the invention

[0004] The present invention aims to solve the above problems, and provides a phage continuous culture device, a phage culture method and a cleaning method to solve the above problems.

[0005] A phage continuous culture device comprises: a flow tube, a connecting tube, a feed pump, a host bacteria liquid tank and a temperature control device, wherein the flow tube and the connecting tube are alternately arranged, and both ends of the connecting tube are respectively fixedly connected to and communicated with the flow tube, and the connecting tube is used to change the flow direction of the fluid in the two flow tubes connected thereto, the most upstream pipeline is connected to the feed pump, and the feed pump is connected to the host bacteria liquid tank, and the temperature control device is located outside the flow tube, and the temperature control device is used to change the temperature of the fluid in the flow tube.

[0006] Furthermore, the flow tubes and connecting tubes are divided into multiple tube groups, the flow tubes and connecting tubes in each tube group are arranged alternately, the flow tubes of each tube group are fixedly connected to the same temperature control device, and multiple tube groups are connected in series in sequence; the temperature control device is a box body with an inner cavity, the temperature control device is formed with a liquid inlet and a liquid outlet, and both ends of the flow tube pass through the end of the temperature control device.

[0007] Furthermore, it also includes a multi-way valve, a reflux pump and a separation device. The outlet of the tube group is connected to the multi-way valve. The multi-way valve connected to the outlet of the last tube group has two outlets, one of which is connected in series with the reflux pump and the separation device. The outlet of the separation device is connected to the multi-way valve corresponding to the outlet of the tube group upstream of the last tube group or the inlet of the first tube group.

[0008] Furthermore, it also includes a first valve, a second pump and a second batching tank, the connecting pipe is formed with a batching inlet, the batching inlet is fixedly connected and communicated with the first valve, the inlet of the second pump is communicated with the second batching tank, and the outlet of the second pump is communicated with the first valve; It also includes a sensor, the connecting pipe is formed with a mounting hole, the sensor is inserted into the mounting hole and fixedly connected to the mounting hole, and the sensor includes a temperature sensor, a PH sensor, a flow rate sensor, a pressure sensor, a water oxygen content sensor, and an ion sensor.

[0009] Furthermore, it also includes a first pump and a first batching tank, the inlet of the first pump is connected to the first batching tank, the outlet of the first pump is connected to the first valve, and the first valve connected to the first pump is upstream of the first valve connected to the second pump.

[0010] Furthermore, it also includes a phage liquid tank, which is connected to a feed pump or a flow pipe.

[0011] Furthermore, it also includes a second valve, a high-pressure water pump and a water tank, the connecting pipe is formed with a liquid inlet and outlet, the liquid inlet and outlet are fixedly connected and communicated with the second valve, the liquid outlet pipe of the second valve is respectively communicated with the outlet of the high-pressure water pump, the inlet of the high-pressure water pump is connected to the water tank, and an ultrasonic bubble generator is installed in the water tank.

[0012] Furthermore, it also includes a third valve and a breathable membrane, a breathable opening is formed on the top of the connecting pipe, and the breathable membrane covers the breathable opening and is fixedly connected to the connecting pipe and the third valve.

[0013] A phage cultivation method using the phage continuous cultivation device comprises the following steps: Step S1: culturing host bacteria; Step S2: adding a phage solution containing phages into the flow tube; Step S3: culturing bacteriophage; Step S4: a portion of the prepared fermentation liquid leaves the culture device, and another portion passes through a separation device, and the separation device removes impurities and host bacteria; Step S5: The purified fermentation liquid is driven by a reflux pump back into the flow tube.

[0014] A method for cleaning the phage continuous culture device comprises the following steps: Step A1: adding cleaning liquid and particulate matter into the water tank, opening the second valve, and spraying high-pressure liquid containing cleaning liquid and particulate matter into the second valve by the high-pressure water pump; Step A2: The liquid containing the cleaning liquid and the particles is discharged from the outlet of the last pipe; Step A3: The water tank prepares micro-nano bubble water, and the high-pressure water pump sprays the micro-nano bubble water out from the second valve at high pressure; Step A4: The micro-nano bubble water is discharged from the outlet of the last pipe; Step A5: The temperature control device heats the flow pipe, the hot air blower blows hot air toward the second valve, and the gas is discharged from the outlet of the last pipe; Step A6: Close the second valve.

[0015] The present invention has the following advantages: 1. Compared with stirring in a stirring tank using stirring blades, the effect of mixing the fluid by flowing the phage liquid and the host bacterial liquid, or the host bacterial liquid and the fermentation medium in the pipeline is better, and the yield of phage and host bacteria is higher; 2. Without the shear force of the rotating stirring blades, the pipeline mixing method is less harmful to the phage and host bacteria, and is less likely to cause phage and host bacteria to be broken and damaged, making the integrity and activity of the produced phage better; 3. The host bacteria and bacteriophages are continuously produced in the pipeline. In normal production, there is no need to stop production to take out the fermentation liquid or clean the fermentation tank. The production is uninterrupted, and the output is larger and more stable; 4. Fill oxygen into the solution used to adjust the pH value and ion concentration of the liquid in the pipeline, so that the oxygen is mixed more evenly in the pipeline, which is conducive to increasing the production of host bacteria and bacteriophages; 5. The temperature control device directly exchanges heat with each flow tube. The inner diameter of the flow tube is much smaller than the inner diameter of the fermenter, so that the liquid temperature in each flow tube is more uniform, and there will be no uneven temperature of the liquid in the fermenter, which is conducive to the proliferation of host bacteria and bacteriophages; 6. Use a high-pressure pump with micro-nano bubble water for cleaning. There is no need to disassemble the pipes. The cleaning steps are simple and the cleaning efficiency is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0017] Figure 1 : A top view of the first embodiment of the present invention; Figure 2 : A front cross-sectional view of the connecting pipe of the present invention; Figure 3 : A top view of the second embodiment of the present invention. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with the accompanying drawings and examples: Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0019] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be directly connected or indirectly connected through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0020] In the description of the present invention, it should be understood that the terms "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside" and "outside" etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention. Embodiment 1: like Figure 1 and Figure 2 As shown, a phage continuous culture device comprises: a flow tube 1, a connecting tube 2, a feed pump 3, a host bacteria liquid tank 4 and a temperature control device 9, wherein the flow tube 1 and the connecting tube 2 are alternately arranged, and both ends of the connecting tube 2 are respectively fixedly connected to and communicated with the flow tube 1, and the connecting tube 2 is used to change the flow direction of the fluid in the two flow tubes 1 connected thereto, and the most upstream pipeline (the pipeline includes the flow tube 1 and the connecting tube 2, which are collectively referred to as the two) is connected to the feed pump 3, and the feed pump 3 is connected to the host bacteria liquid tank 4, and the temperature control device 9 is located outside the flow tube 1, and the temperature control device 9 is used to change the temperature of the fluid in the flow tube 1.

[0021] Preferably, the height of the most downstream pipeline is higher than that of the most upstream pipeline, and the height of the liquid gradually rises in the pipeline, thereby preventing the liquid from flowing too fast in the pipeline.

[0022] Furthermore, the temperature control device 9 is an electric heating wire, which maintains the liquid in the flow tube 1 at the most suitable fermentation temperature by heating. The heating amount of the temperature control device 9 is adjusted by the control device according to the feedback of the temperature sensor.

[0023] Furthermore, it also includes a first valve 10, a second pump 17 and a second batching tank 18, the connecting pipe 2 is formed with a batching inlet 21, the batching inlet 21 is fixedly connected and communicated with the first valve 10, the inlet of the second pump 17 is communicated with the second batching tank 18, and the outlet of the second pump 17 is communicated with the first valve 10. The second pump 17 can be connected to a plurality of second batching tanks 18 filled with different solutions, and different solutions can be added according to the conditions of the liquid in the pipeline to increase the yield of bacteriophages.

[0024] Furthermore, the sensor 12 is also included. The connecting pipe 2 is formed with a mounting hole 23. The sensor 12 is inserted into the mounting hole 23 and fixedly connected to the mounting hole 23. The sensor 12 includes a temperature sensor, a pH sensor, a flow rate sensor, a pressure sensor, a water oxygen content sensor, and an ion sensor. According to the feedback of the pH sensor, the controller causes the second pump 17 to pump an acidic or alkaline solution into the first valve 10 to achieve the optimal pH value required for proliferation; the controller adjusts the speed of the feed pump 3 according to the feedback of the flow rate sensor, thereby changing the flow rate of the pumped liquid to achieve the best mixing effect between the liquids; according to the feedback of the water oxygen content sensor, the controller causes the second pump 17 to pump oxygen or a solution containing dissolved oxygen into the first valve 10. The solution can be made by oxygenating on the basis of other required solutions (such as pH value solutions or ion solutions); the ion sensor detects the concentration of specified ions (such as calcium ions and magnesium ions, etc.) so that the concentration of specified ions in the pipeline reaches the optimal value required for proliferation.

[0025] Furthermore, it also includes a phage liquid tank 8, which is connected to the feed pump 3 or the flow tube 1. The phage liquid tank 8 stores phage liquid, and the phage is used to infect the host bacteria, thereby multiplying the phage.

[0026] Furthermore, it also includes a third valve 19 and a breathable membrane 25. A breathable port 24 is formed on the top of the connecting pipe 2. The breathable membrane 25 covers the breathable port 24 and is fixedly connected to the connecting pipe 2 and the third valve 19. The breathable membrane 25 allows gas to pass through but does not allow liquid to pass through. A cavity is formed between the connecting pipe 2 and the third valve 19. An air pressure sensor (not shown in the figure) is installed in the cavity. When the air pressure sensor detects that the air pressure is too high, the third valve 19 opens to connect the cavity with the outside world and release excess gas.

[0027] When working, the feed pump 3 continuously pumps the host bacterial liquid containing host bacteria and the fermentation medium in the host bacterial liquid tank 4 into the flow tube 1. Then, the phage liquid containing bacteriophage is added to the flow tube 1. The phage liquid and the host bacterial liquid flow and mix in the flow tube 1 and the connecting tube 2, and the bacteriophage infects the host bacteria. Finally, the prepared fermentation liquid leaves the culture device and enters the next step.

[0028] During cleaning, the following steps are included: Step A1: Add cleaning liquid and particulate matter to the water tank 14, open the second valve 11, and the high-pressure water pump 13 sprays high-pressure liquid containing cleaning liquid and particulate matter towards the second valve 11; Step A2: The liquid containing cleaning liquid and particulate matter is discharged from the outlet of the last pipe; Step A3: The water tank 14 prepares micro-nano bubble water, and the high-pressure water pump 13 sprays the micro-nano bubble water from the second valve 11 under high pressure; Step A4: The micro-nano bubble water is discharged from the outlet of the last pipe; Step A5: The temperature control device 9 heats the flow pipe 1, the hot air blower blows hot air towards the second valve 11, and the gas is discharged from the outlet of the last pipe; Step A6: After the inner wall of the pipe is dried, close the second valve 11.

[0029] Example Two: As Figure 2 and Figure 3 shown, a phage continuous culture device includes: a flow pipe 1, a connecting pipe 2, a feed pump 3, a host bacterial liquid tank 4, and a temperature control device 9. The flow pipe 1 and the connecting pipe 2 are arranged alternately. The two ends of the connecting pipe 2 are respectively fixedly connected to and communicate with the flow pipe 1. The connecting pipe 2 is used to change the flow direction of the fluid in the two flow pipes 1 connected to it. The uppermost upstream pipe (the pipe includes the flow pipe 1 and the connecting pipe 2, which is a general term for the two) communicates with the feed pump 3, the feed pump 3 communicates with the host bacterial liquid tank 4, the temperature control device 9 is located outside the flow pipe 1, and the temperature control device 9 is used to change the temperature of the fluid in the flow pipe 1.

[0030] Compared with using a stirring blade to stir in a stirring tank, the effect of mixing the fluid through the pipeline flow is better; at the same time, there is no shear force of rotational stirring, and the pipeline mixing method causes less damage to phages and host bacteria, and it is less likely to cause fragmentation and damage to phages and host bacteria.

[0031] Preferably, the cross-sectional areas of the flow pipe 1 and the connecting pipe 2 are different, and the cross-sectional area of the connecting pipe 2 is larger than that of the flow pipe 1, which is convenient for leaving enough space in the connecting pipe 2 to install equipment. At the same time, the sudden change in the cross-sectional area between the flow pipe 1 and the connecting pipe 2 will enhance the turbulence of the liquid in the pipeline and enhance the liquid mixing effect. Preferably, the inner diameter of the flow pipe 1 is greater than or equal to 5 mm and less than or equal to 20 mm.

[0032] Preferably, the height of the lowermost downstream pipe is higher than that of the uppermost upstream pipe, and the height of the liquid gradually rises in the pipeline, so as to avoid too fast flow velocity of the liquid in the pipeline.

[0033] Furthermore, the flow tube 1 and the connecting tube 2 are divided into a plurality of tube groups, the flow tubes 1 and the connecting tubes 2 in each tube group are arranged alternately, the flow tube 1 of each tube group is fixedly connected to the same temperature control device 9, and the plurality of tube groups are sequentially connected in series; the temperature control device 9 is a box body with an inner cavity, the temperature control device 9 is formed with a liquid inlet 91 and a liquid outlet 92, and the two ends of the flow tube 1 pass through the ends of the temperature control device 9. The heat exchange liquid for temperature regulation enters from the liquid inlet 91 and is discharged from the liquid outlet 92, and the temperature of the flow tube 1 and the liquid inside thereof is changed through heat exchange to reach the optimal temperature.

[0034] Furthermore, it also includes a multi-way valve 5, a reflux pump 6 and a separation device 7. The outlet of the pipe group is connected to the multi-way valve 5. The multi-way valve 5 connected to the outlet of the last pipe group has two outlets, one of which is connected in series with the reflux pump 6 and the separation device 7. The outlet of the separation device 7 is connected to the multi-way valve 5 corresponding to the outlet of the pipe group upstream of the last pipe group or the inlet of the first pipe group. The separation device 7 is a filter or a centrifuge. The multi-way valve 5 can have a channel to discharge the liquid in the pipeline to the outside, which is convenient for sampling and testing.

[0035] It also includes a sensor 12. The connecting pipe 2 is formed with a mounting hole 23. The sensor 12 is inserted into the mounting hole 23 and fixedly connected to the mounting hole 23. The sensor 12 includes a temperature sensor, a PH sensor, a flow rate sensor, a pressure sensor, a water oxygen content sensor, and an ion sensor.

[0036] Furthermore, it also includes a first valve 10, a second pump 17 and a second batching tank 18, the connecting pipe 2 is formed with a batching inlet 21, the batching inlet 21 is fixedly connected and communicated with the first valve 10, the inlet of the second pump 17 is communicated with the second batching tank 18, and the outlet of the second pump 17 is communicated with the first valve 10. The second pump 17 and the second batching tank 18 transport a solution for adjusting the environment when culturing bacteriophages.

[0037] Furthermore, it also includes a first pump 15 and a first batching tank 16, wherein the inlet of the first pump 15 is connected to the first batching tank 16, and the outlet of the first pump 15 is connected to the first valve 10, and the first valve 10 connected to the first pump 15 is upstream of the first valve 10 connected to the second pump 17. The first pump 15 and the first batching tank 16 transport a solution for adjusting the environment when culturing host bacteria.

[0038] Furthermore, a phage liquid tank 8 is included, and the phage liquid tank 8 is connected with the feed pump 3 or the flow pipe 1 .

[0039] Furthermore, it also includes a third valve 19 and a breathable membrane 25. A breathable port 24 is formed on the top of the connecting pipe 2. The breathable membrane 25 covers the breathable port 24 and is fixedly connected to the connecting pipe 2 and the third valve 19. The breathable membrane 25 allows gas to pass through but does not allow liquid to pass through. A cavity is formed between the connecting pipe 2 and the third valve 19. An air pressure sensor (not shown in the figure) is installed in the cavity. When the air pressure sensor detects that the air pressure is too high, the third valve 19 opens to connect the cavity with the outside world and release excess gas.

[0040] Furthermore, the connecting pipe 2 is a U-shaped pipe, which changes the flow direction to reduce the space occupied by the entire pipeline and increase the turbulence between the liquids to achieve a better mixing degree.

[0041] A phage cultivation method using the phage continuous cultivation device comprises the following steps: Step S1: culturing host bacteria; Step S11: the feed pump 3 continuously pumps the host bacterial liquid containing host bacteria and the fermentation medium in the host bacterial liquid tank 4 into the flow tube 1, and the host bacterial liquid and the fermentation medium are evenly mixed during the flow process; wherein the fermentation medium can be mixed with the host bacterial liquid together in the host bacterial liquid tank 4, or can be placed in different tanks and mixed in the flow tube 1; Step S12: According to the feedback from the sensors 12 corresponding to the N upstream tube groups, the controller drives the first pump 15 to pump the solution used for adjusting the environment when culturing the host bacteria in the first batching tank 16 into the pipes of the N upstream tube groups corresponding to the host bacteria, and controls the temperature of the heat exchange liquid in the temperature control device 9 of the N upstream tube groups so that the pipes reach the optimal temperature for the proliferation of the host bacteria; wherein the solution is used to adjust the pH value of the liquid in the pipe, the concentration of specific ions (such as calcium ions, magnesium ions, etc.), the water oxygen content and other parameters, so that the environment is adapted to the growth of the host bacteria and the proliferation rate of the host bacteria is accelerated; Step S13: After the host bacteria proliferate to a certain extent, they enter the pipes of the M pipe groups downstream through the multi-way valve 5; Step S2: adding a phage liquid containing phages into the flow tube 1; the third pump 31 pumps the phages in the phage liquid tank 8 into the pipe of the most upstream pipe group among the M downstream pipe groups as an initial sample; Step S3: culturing bacteriophage; Step S31: The phage solution and the host bacterial solution flow and mix in the flow tube 1 and the connecting tube 2. Step S32: According to the feedback from the sensors 12 corresponding to the downstream M pipe groups, the controller drives the second pump 1 to pump the solution used for adjusting the environment for culturing bacteriophages in the second batching tank 18 into the pipes of the downstream M pipe groups corresponding to the culturing bacteriophages, and controls the temperature of the heat exchange liquid in the temperature control device 9 of the downstream M pipe groups so that the pipes reach the optimal temperature for the proliferation of bacteriophages; wherein the solution is used to adjust the pH value of the liquid in the pipe, the concentration of specific ions (such as calcium ions, magnesium ions, etc.), the water oxygen content and other parameters, so that the environment is adapted to the growth of bacteriophages and accelerates the proliferation of bacteriophages; Step S4: After the phage proliferation is completed, a portion of the prepared fermentation liquid leaves the culture device through the multi-way valve 5 of the last pipe group, and another portion passes through the separation device 7, and the separation device 7 removes impurities and host bacteria; Step S5: The purified fermentation liquid (without impurities and host bacteria) is driven by the reflux pump 6 to be refluxed into the flow tube 1 of the most upstream tube group among the downstream M tube groups. The phage in the fermentation liquid can partially or completely replace the phage liquid prepared in the phage liquid tank 8. The phage in the refluxed fermentation liquid has evolved with the host bacteria for a long time during multiple cycles of proliferation, and can adapt to more mutated host bacteria. The phage produced thereafter has stronger drug resistance when actually used in the medical and agricultural fields.

[0042] The present application continuously mixes and cultures host bacteria and bacteriophages in the pipeline. Since the liquid in the pipeline continuously flows in a specified direction from upstream to downstream and then is discharged, there is little waste accumulated inside, and it can be operated continuously for a long time without cleaning. Only when the liquid is detected to be unqualified in the sampling of the second valve 11 or the multi-way valve 5 (such as the bacteriophage content in the pipelines of the downstream M pipe groups is detected to be significantly reduced compared with the past), the inside of the pipeline needs to be cleaned.

[0043] Furthermore, it also includes a second valve 11, a high-pressure water pump 13 and a water tank 14. The connecting pipe 2 is formed with a liquid inlet and outlet 22, and the liquid inlet and outlet 22 are fixedly connected and communicated with the second valve 11. The liquid outlet pipe of the second valve 11 is respectively communicated with the outlet of the high-pressure water pump 13, and the inlet of the high-pressure water pump 13 is communicated with the water tank 14. An ultrasonic bubble generator is installed in the water tank 14.

[0044] When cleaning, the following steps are included: Step A1: Add cleaning liquid and particulate matter into the water tank 14, open the first valve 10, and the high-pressure water pump 13 sprays high-pressure liquid containing cleaning liquid and particulate matter into the first valve 10; wherein the cleaning liquid may include an acidic solution, an alkaline solution, a surfactant, an enzyme, etc., which is used to kill host bacteria in the pipeline and remove organic and inorganic waste in the pipeline.

[0045] Step A2: The liquid containing the cleaning liquid and the particles is discharged from the outlet of the last pipe; Step A3: the water tank 14 prepares micro-nano bubble water, and the high-pressure water pump 13 sprays the micro-nano bubble water out of the first valve 10 at high pressure; preferably, the liquid inlet and outlet 22 are oriented toward the flow pipe 1 downstream of the connecting pipe 2, thereby driving the liquid to flow; Step A4: The micro-nano bubble water is discharged from the outlet of the last pipe, and the micro-nano bubble water is used to clean the pipe wall to remove residual waste and cleaning liquid; Step A5: The temperature control device 9 heats the flow tube 1, and the hot air blower blows hot air into the first valve 10, and the gas is discharged from the outlet of the last pipe; wherein, the heating of the flow tube 1 and the hot air cooperate to achieve the effect of disinfection and sterilization; Step A6: After the inner wall of the pipeline is dried, close the first valve 10.

[0046] It should be noted that M and N are respectively greater than or equal to 1.

[0047] The present invention is described above by way of examples, but the present invention is not limited to the above specific embodiments, and any changes or modifications made based on the present invention belong to the scope of protection claimed by the present invention.

Claims

1. A phage continuous culture device, characterized in that: include: A flow tube (1), a connecting tube (2), a feed pump (3), a host bacterial liquid tank (4) and a temperature control device (9), wherein the flow tube (1) and the connecting tube (2) are arranged alternately, and both ends of the connecting tube (2) are respectively fixedly connected to and communicate with the flow tube (1), and the connecting tube (2) is used to change the flow direction of the fluid in the two flow tubes (1) connected thereto, and the most upstream pipeline is connected to the feed pump (3), and the feed pump (3) is connected to the host bacterial liquid tank (4), and the temperature control device (9) is located outside the flow tube (1), and the temperature control device (9) is used to change the temperature of the fluid in the flow tube (1).

2. A phage continuous culture device according to claim 1, characterized in that: The flow tubes (1) and connecting tubes (2) are divided into a plurality of tube groups, the flow tubes (1) and connecting tubes (2) in each tube group are arranged alternately, the flow tubes (1) in each tube group are fixedly connected to the same temperature control device (9), and the plurality of tube groups are sequentially connected in series; the temperature control device (9) is a box body having an inner cavity, the temperature control device (9) having a liquid inlet (91) and a liquid outlet (92), and both ends of the flow tube (1) pass through the ends of the temperature control device (9).

3. A phage continuous culture device according to claim 2, characterized in that: It also comprises a multi-way valve (5), a reflux pump (6) and a separation device (7); the outlet of the tube group is connected to the multi-way valve (5); the multi-way valve (5) connected to the outlet of the last tube group has two outlets, one of which is connected in series with the reflux pump (6) and the separation device (7); the outlet of the separation device (7) is connected to the multi-way valve (5) corresponding to the outlet of the tube group upstream of the last tube group or the inlet of the first tube group.

4. The phage continuous culture device according to claim 1, characterized in that: It also comprises a first valve (10), a second pump (17) and a second ingredient tank (18), the connecting pipe (2) is formed with an ingredient inlet (21), the ingredient inlet (21) is fixedly connected to and communicates with the first valve (10), the inlet of the second pump (17) is communicated with the second ingredient tank (18), and the outlet of the second pump (17) is communicated with the first valve (10); It also comprises a sensor (12), the connecting pipe (2) being formed with a mounting hole (23), the sensor (12) being inserted into the mounting hole (23) and fixedly connected to the mounting hole (23), the sensor (12) comprising a temperature sensor, a pH sensor, a flow rate sensor, a pressure sensor, a water oxygen content sensor, and an ion sensor.

5. The phage continuous culture device according to claim 4, characterized in that: The invention also comprises a first pump (15) and a first material tank (16), wherein the inlet of the first pump (15) is connected to the first material tank (16), the outlet of the first pump (15) is connected to the first valve (10), and the first valve (10) connected to the first pump (15) is upstream of the first valve (10) connected to the second pump (17).

6. The phage continuous culture device according to claim 1, characterized in that: It also includes a bacteriophage liquid tank (8), wherein the bacteriophage liquid tank (8) is connected to the feed pump (3) or the flow pipe (1).

7. The phage continuous culture device according to claim 4, characterized in that: It also comprises a second valve (11), a high-pressure water pump (13) and a water tank (14); the connecting pipe (2) is formed with a liquid inlet and outlet (22); the liquid inlet and outlet (22) are fixedly connected to and communicate with the second valve (11); the liquid outlet pipe of the second valve (11) is communicated with the outlet of the high-pressure water pump (13); the inlet of the high-pressure water pump (13) is communicated with the water tank (14); an ultrasonic bubble generator is installed in the water tank (14).

8. The phage continuous culture device according to claim 1, characterized in that: It also comprises a third valve (19) and a breathable membrane (25); a breathable opening (24) is formed at the top of the connecting pipe (2); the breathable membrane (25) covers the breathable opening (24) and is fixedly connected to the connecting pipe (2) and the third valve (19).

9. A method for culturing bacteriophage using the bacteriophage continuous culturing device according to claim 1, characterized in that: The following steps are involved: Step S1: culturing host bacteria; Step S2: adding a phage solution containing phages into the flow tube (1); Step S3: Cultivating phage Step S4: a portion of the prepared fermentation liquid leaves the culture device, and another portion passes through the separation device (7), and the separation device (7) removes impurities and host bacteria; Step S5: The purified fermentation liquid is driven by the reflux pump (6) to return to the flow pipe (1).

10. A method for cleaning the phage continuous culture device according to claim 7, characterized in that: The following steps are involved: Step A1: adding cleaning liquid and particulate matter into the water tank (14), opening the second valve (11), and causing the high-pressure water pump (13) to spray high-pressure liquid containing the cleaning liquid and particulate matter toward the second valve (11); Step A2: The liquid containing the cleaning liquid and the particles is discharged from the outlet of the last pipe; Step A3: the water tank (14) prepares micro-nano bubble water, and the high-pressure water pump (13) sprays the micro-nano bubble water out from the second valve (11) at high pressure; Step A4: The micro-nano bubble water is discharged from the outlet of the last pipe; Step A5: the temperature control device (9) heats the flow pipe (1), the hot air blower blows hot air toward the second valve (11), and the gas is discharged from the outlet of the last pipe; Step A6: Close the second valve (11).

Images