Sterile pilot bioreactor based on plant cell culture and production of secondary metabolites

Abstract

The application relates to a sterile pilot culture device based on plant cell culture and secondary metabolite production, and relates to the field of plant cell culture device technology, which comprises a culture box body, the upper end of the culture box body is provided with an opening, a cover body is arranged at the opening and can be opened and closed, a plant cell culture bag is arranged in the culture box body, a cell liquid conveying mechanism is arranged on one side of the culture box body and is used for conveying plant cell protoplast suspension in the plant cell culture bag, a heating pad is arranged on the inner bottom wall of the culture box body and is used for heating the plant cell culture bag, a swing mechanism is arranged below the culture box body and is used for driving the culture box body to rotate in a circumferential direction, and a light source transformation mechanism is arranged on the cover body and is used for providing light to the plant cell culture bag and adjusting the brightness and color of the light source. The application has the effect of improving the problems of low applicability and few monitoring functions of the plant cell culture device.

Description

Technical Field

[0001] This application relates to the field of plant cell culture technology, and in particular to a sterile pilot-scale culture device based on plant cell culture and secondary metabolite production. Background Technology

[0002] Currently, plant cell culture is characterized by long cycles, weak cell shear resistance, and easy aggregation. Furthermore, the purpose of large-scale plant cell culture is to produce natural products, which are all cell growth metabolites. Therefore, the design of plant cell culture reactors must not only consider factors conducive to cell growth but also to product accumulation and separation. Generally speaking, a suitable reactor for plant cells should have appropriate oxygen transfer, good fluidity, and low shear force. Based on the characteristics of different plant cell growth and metabolite accumulation, various types of culture vessels are researched and designed for plant cell culture; the WAVE plant cell culture vessel is one such example. It mainly consists of a metal box containing plant cell culture bags. A pH meter and thermometer are connected to the plant cell culture bags to monitor the pH and temperature of the components within the bags at set intervals. A heating pad is located below the metal box to control the temperature of the nutrient solution in the plant cell culture bags, facilitating plant cell culture.

[0003] Regarding the aforementioned technologies, the inventors believe that current plant cell culture devices have limited capabilities in monitoring other components of plant cell culture bags, and different plant cells require different culture environments, thus making it impossible to more accurately understand the secondary metabolites produced during plant cell culture. Summary of the Invention

[0004] To address the issues of low applicability and limited monitoring capabilities of plant cell culture devices, this application provides a sterile pilot-scale culture device based on plant cell culture and secondary metabolite production.

[0005] The sterile pilot-scale culture device based on plant cell culture and secondary metabolite production provided in this application adopts the following technical solution: A sterile pilot-scale culture device based on plant cell culture and secondary metabolite production, comprising: A culture box, the culture box being hollow inside and open at the top, with a lid that opens and closes at the top opening of the culture box; A plant cell culture bag is placed inside a culture box to hold a suspension of plant cell protoplasts, and the plant cell culture bag is connected to a pH meter, a thermometer, and a UV detector. The cell sap delivery mechanism is installed on one side of the culture box and is used to deliver the plant cell protoplast suspension in the plant cell culture bag to the UV detector according to the set time. A heating pad is installed on the inner bottom wall of the culture box to heat the plant cell culture bag placed inside the culture box. The swing mechanism, installed below the culture box, is used to drive the culture box to rotate circumferentially on the horizontal plane; A light source conversion mechanism is installed on the cover. The light source conversion mechanism is used to provide light to the plant cell culture bag and can adjust the brightness and color of the light source.

[0006] Preferably, the cell fluid delivery mechanism includes: The support is installed on one side of the culture box. Liquid pump, mounted on a bracket; An infusion tube is connected between the inlet of the liquid pump and the plant cell culture bag; A branch pipe is connected to the output end of the liquid pump, and the two branches of the branch pipe are respectively connected to the UV detector. The timed start component, mounted on the bracket, is used to start the liquid pump after a set time to deliver the plant cell protoplast suspension in the plant cell culture bag to the UV detector.

[0007] Preferably, the timed start component includes: A long, narrow storage box is mounted on a support and has openings at both the top and bottom. The long, narrow storage box has multiple square storage cavities of the same volume along its length. Each square storage cavity contains an iron ball, and the diameter of the iron ball is the same as the width of the square storage cavity. A hinged plate is slidably disposed at the lower opening of the elongated storage box, and the hinged plate is used to cover the lower opening of the square storage cavity; The driving component, mounted on the bracket, is used to drive the opening and closing plate to move along the length of the long strip placement box; The funnel is mounted on the bracket directly below the long strip box, and the upper opening of the funnel completely covers the lower opening of the long strip box in the vertical direction. A vertical tube is installed vertically at the lower end of the funnel, and the inner diameter of the vertical tube is the same as the diameter of the iron ball; A pressure-conducting element is installed between the bracket and the vertical pipe. When the iron ball falls directly into the vertical pipe and touches a part of the pressure-conducting element, the liquid pump is activated.

[0008] Preferably, the driving element includes: The rack is mounted on the bracket and its length direction is consistent with the length direction of the opening and closing plate. The guide rail is mounted on the bracket and its length direction is consistent with the length direction of the rack. The slider is slidably mounted on the guide rail. A stepper motor is mounted on the slider; The gear is coaxially mounted on the output shaft of the stepper motor and meshes with the rack. The connecting rod is mounted on the stepper motor and connected to the opening and closing plate.

[0009] Preferably, the lower opening of the long strip box is provided with slide rails on both sides and along the length of the long strip box, and the opening and closing plate is slidably disposed between the two slide rails.

[0010] Preferably, the pressure-conducting element includes: The battery is mounted on a bracket; The first conductive plate is slidably installed inside the vertical tube and is electrically connected to the battery. The second conductive plate is slidably installed inside the vertical pipe below the first conductive plate and is electrically connected to the liquid pump. The first spring is vertically installed between the first conductive plate and the bottom wall of the vertical tube, and the second conductive plate has an opening for the first spring to pass through. The second spring is vertically installed between the second conductive plate and the bottom wall of the vertical tube, and the second spring is offset from the first spring.

[0011] When the first spring and the second spring are in their natural state, there is a gap between the first conductive plate and the second conductive plate.

[0012] Preferably, an inclined discharge pipe is connected to the side wall of the vertical pipe located below the second conductive plate, and an inclined guide plate is provided above the first conductive plate, with the inclined surface of the inclined guide plate facing the opening end of the inclined discharge pipe; when the iron ball presses against the inclined guide plate, forcing the first conductive plate to move vertically downward so that the iron ball is facing the opening end of the inclined guide plate, the iron ball rolls from the inclined guide plate into the inclined discharge pipe under the action of natural gravity.

[0013] Preferably, a storage tank is connected to the lowest end of the inclined discharge pipe.

[0014] Preferably, the swing mechanism includes: The base is installed at the bottom of the culture box. A rotating motor is installed inside the base, and the rotating shaft of the rotating motor extends vertically upward out of the base; A rotating disk is coaxially mounted at the end of the output shaft of a rotating motor, and a rotating shaft is vertically arranged below the culture box. The rotating shaft is connected to the rotating disk, and the rotating shaft and the output shaft of the rotating motor are on different rotation axes.

[0015] Preferably, the light source conversion mechanism includes: The frame is installed on the outer top of the cover; The lamp body is mounted on the frame; The louvers are installed on the cover and located below the lamp body; The color-changing cover is detachably mounted on the frame to cover the lamp body.

[0016] In summary, this application includes at least one of the following beneficial technical effects: 1. The plant cell culture bag is equipped with a pH meter and a thermometer to monitor the pH value and temperature of the components in the plant cell culture bag at set times; the heating pad located at the bottom of the culture box can be used to control the temperature of the nutrient solution in the plant cell culture bag, which is beneficial to the cultivation of plant cells. 2. The plant cell culture bag is connected to a device for detecting UV and viability. The specific operation is as follows: a timer is set, and a sample is taken from the suspension culture medium in the plant cell culture bag for testing at regular intervals. The testing must be carried out under sterile conditions to accurately obtain data related to secondary metabolites. 3. When different plant cell culture bags require different lighting and light intensities, color-changing covers of different colors can be installed according to the plant cells being cultured on-site, so that the lamp emits light colors suitable for the plant cells being cultured at that time; and by adjusting the opening angle of the louvers, the light intensity emitted by the lamp can be adjusted to enter the culture box, thereby making the environment for the culture of plant cell protoplast suspension more suitable. Attached Figure Description

[0017] Figure 1 This is a structural schematic diagram of an embodiment of this application; Figure 2 This is a partial cross-sectional view of an embodiment of the present application, illustrating the cell fluid delivery mechanism; Figure 3 yes Figure 2 Enlarged view of part A in the image; Figure 4 This is a front sectional view of an embodiment of this application; Figure 5 yes Figure 4 Enlarged view of part B in the image; Figure 6 This is a partial cross-sectional view of an embodiment of the present application, used to show the interior of the culture box.

[0018] In the diagram, 1. Culture box body; 11. Rotating shaft; 2. Cover; 3. Plant cell culture bag; 31. pH meter; 32. Thermometer; 33. UV detector; 4. Cell sap delivery mechanism; 41. Support; 42. Liquid pump; 43. Infusion tube; 44. Bifurcation tube; 45. Timer start assembly; 451. Long strip placement box; 4511. Square placement cavity; 452. Opening and closing plate; 453. Drive component; 4531. Rack; 4532. Guide rail; 4533. Slider; 4534. Stepper motor; 4535. Gear; 4536. 454. Connecting rod; 455. Funnel; 456. Vertical pipe; 457. Press-down power supply component; 4581. Storage battery; 4582. First conductive plate; 45821. Inclined guide plate; 4583. Second conductive plate; 4584. First spring; 4585. Second spring; 5. Heating pad; 6. Swinging mechanism; 61. Base; 62. Rotating motor; 63. Rotating disk; 7. Light source changing mechanism; 71. Frame; 72. Lamp body; 73. Louver; 74. Color-changing cover; 8. Iron ball; 9. Slide rail; 10. Inclined discharge pipe; 101. Storage box. Detailed Implementation

[0019] The following is in conjunction with the appendix Figure 1 - Appendix Figure 6 This application will be described in further detail below.

[0020] A sterile pilot-scale culture device based on plant cell culture and secondary metabolite production, referring to Figure 1 , 2The system includes a culture box 1, a plant cell culture bag 3, a heating pad 5, a cell sap delivery mechanism 4, a oscillating mechanism 6, and a light source conversion mechanism 7. The culture box 1 is hollow inside with an opening at the top. A cover 2 is installed at the top opening of the culture box 1. The plant cell culture bag 3 is placed inside the culture box 1 and is used to hold a suspension of plant cell protoplasts. When the cover 2 is closed, the culture box 1 forms a culture chamber for culturing the plant cell culture bag 3. The heating pad 5 is installed on the inner bottom wall of the culture box 1 and is used to heat the plant cell culture bag 3 placed inside the culture box 1. The main function of the heating pad 5 is to regulate the room temperature inside the culture box 1, ensuring that the temperature inside the culture box 1 is at a suitable culture temperature. A pH meter 31 and a thermometer 32 are connected to the plant cell culture bag 3. The device includes a 32-inch screen and a UV detector 33; a cell sap delivery mechanism 4 is installed on one side of the culture box 1, and the cell sap delivery mechanism 4 is used to deliver the plant cell protoplast suspension in the plant cell culture bag 3 to the UV detector 33 at a set time, so as to obtain various data results of the plant cell culture medium at a set time; a swing mechanism 6 is installed below the culture box 1, and is used to drive the culture box 1 to rotate circumferentially on the horizontal plane, so that the nutrient solution in the plant cell culture bag 3 can fully contact the heating pad 5, and the plant cells can absorb nutrients more fully; a light source changing mechanism 7 is installed on the cover 2, and the light source changing mechanism 7 is used to provide light to the plant cell culture bag 3 and can adjust the brightness and color of the light source, so as to provide different light intensity and color according to the different plant cell culture environment.

[0021] Reference Figure 1 , 2 The cell sap delivery mechanism 4 includes a support 41, a liquid pump 42, an infusion tube 43, a branch tube 44, and a timer start component 45. The support 41 is installed on one side of the culture box 1, the liquid pump 42 is installed on the support 41, and the infusion tube 43 is connected between the input end of the liquid pump 42 and the plant cell culture bag 3, that is, one end of the infusion tube 43 is connected to the input end of the liquid pump 42, and the other end is connected to the inside of the plant cell culture bag 3. The branch tube 44 is connected to the output end of the liquid pump 42. The branch tube 44 includes a main tube and two branch tubes connected to the main tube. That is, one end of the main tube is connected to the output end of the liquid pump 42, and the end of one branch tube away from the main tube is connected to the UV detector 33, and the end of the other branch tube away from the main tube is also connected to the UV detector 33. The timer start component 45 is also installed on the support 41. The timer start component 45 is used to start the liquid pump 42 after a set time to deliver the plant cell protoplast suspension in the plant cell culture bag 3 to the UV detector 33.

[0022] It is worth noting that in this embodiment, a valve A and a liquid inlet pipe A are provided on one branch of the branch pipe 44; a valve B, a liquid inlet pipe B, and a crushing and filtering assembly are provided on the other branch of the branch pipe 44; the crushing and filtering assembly consists of a wall-breaking machine and a plant cell filter, that is, the other branch of the branch pipe 44 is connected to the UV detector via valve B, the wall-breaking machine, and the plant cell filter; both liquid inlet pipe A and liquid inlet pipe B are equipped with sealing caps, that is, when the detection reagent needs to be dripped in, the sealing caps can be opened to drip in. When testing the viability of a plant cell protoplast suspension, close valve B, open valve A, and then add the detection reagent into the addition tube A. The viability of the plant cell protoplast suspension is then detected using the TCC method. When testing the secondary metabolic content of a plant cell protoplast suspension, close valve A, open valve B, add the detection reagent into the addition tube B, and then break and filter the plant cell protoplast suspension before passing it into a UV detector. This allows for the detection of the secondary metabolic content of the plant cell protoplast suspension.

[0023] Since the plant cell culture bag 3 needs to check various data of the plant cell protoplast suspension after a period of culture, the culture time interval can be set. The timer start component 45 will automatically start the liquid pump 42 after the time is up. At this time, the liquid pump 42 can draw the plant cell protoplast suspension in the plant cell culture bag 3 into the UV detector 33, so that the UV detector 33 can analyze the various data of the plant cell protoplast suspension. That is, the timer start component 45 can automatically draw the plant cell protoplast suspension for detection at the time, without the need for manual timing.

[0024] Combination Figure 2 , 3 Specifically, the timed start component 45 includes a long strip box 451, an opening and closing plate 452, a drive component 453, a funnel 454, a vertical tube 455, and a downward pressure energizing component 456; combined with Figure 4 , 5A long, narrow storage box 451 is mounted on a bracket 41. The long, narrow storage box 451 is hollow inside and open at both the top and bottom. Multiple square storage cavities 4511 are provided along the length of the long, narrow storage box 451, each with the same volume. A hinged plate 452 is slidably positioned at the lower opening of the long, narrow storage box 451, on both sides of the lower opening and along the length of the long, narrow storage box 451. Slide rails 9 are provided between the two slide rails 9. An iron ball 8 is placed in each square storage cavity 4511, the diameter of which is the same as that of the square storage cavity 4511. The width of 11 is the same; the driving component 453 is installed on the bracket 41 and is used to drive the opening and closing plate 452 to move along the length direction of the long strip placement box 451, so that the lower opening of the long strip placement box 451 is gradually exposed; the funnel 454 is installed on the bracket 41 directly below the long strip placement box 451, and the upper opening of the funnel 454 completely covers the lower opening of the long strip placement box 451 in the vertical direction; the vertical tube 455 is vertically connected and installed at the lower end of the funnel 454, and the inner diameter of the vertical tube 455 is the same as the diameter of the iron ball 8; the downward energizing component 456 is installed between the bracket 41 and the vertical tube 455. When the iron ball 8 falls directly into the vertical tube 455 and touches part of the downward energizing component 456, the liquid pump 42 is started.

[0025] After the drive unit 453 is activated, it will drive the opening and closing plate 452 to move along the length of the long strip placement box 451. As the opening and closing plate 452 moves, the lower opening of each square placement cavity 4511 will gradually be exposed, allowing the iron ball 8 to fall out of the square placement cavity 4511. The drive unit 453 is adjusted and set so that when the drive unit 453 drives the opening and closing plate 452 to move, the opening and closing plate 452 moves a distance equal to the width of the square placement cavity 4511 before the time for detecting the plant cell protoplast suspension is required. This ensures that the opening and closing plate 452 moves a distance equal to the width of the square placement cavity 4511. After the same distance, the lower opening of a square placement cavity 4511 will be fully open, and the iron ball 8 inside the square placement cavity 4511 will fall completely. After the iron ball 8 falls from the square placement cavity 4511, it will fall directly into the funnel 454, and then from the funnel 454 into the vertical tube 455. Finally, the iron ball 8 will touch the downward energizing component 456, which will drive the liquid pump 42 to run. The liquid pump 42 will then start to draw the plant cell protoplast suspension in the plant cell culture bag 3 to the UV detector 33 for detection, achieving the effect of automatically and periodically extracting the plant cell protoplast suspension for detection.

[0026] Combination Figure 3 , 5The driving component 453 includes a stepper motor 4534, a gear 4535, a rack 4531, a guide rail 4532, a slider 4533, and a connecting rod 4536. The guide rail 4532 is mounted on the bracket 41 on one side of the long strip placement box 451, and the length direction of the guide rail 4532 is parallel to the length direction of the long strip placement box 451. The slider 4533 is mounted on the lower end of the stepper motor 4534 and slides with the guide rail 4532. In this embodiment, the slider 4533 is T-shaped, and the guide rail 4532 has a T-shaped groove along its length direction that matches the shape of the slider 4533. The gear 4535 is coaxially mounted on the stepper motor 4534. On the output shaft of stepper motor 4534, rack 4531 is mounted on bracket 41 on one side of guide rail 4532, and the length direction of rack 4531 is consistent with the length direction of guide rail 4532. Gear 4535 meshes with rack 4531. To ensure stable meshing between gear 4535 and rack 4531, a positioning frame is provided around rack 4531. The output shaft of stepper motor 4534 passes through the positioning frame, gear 4535 is located inside the positioning frame, and the positioning frame is also slidably set around rack 4531. Connecting rod 4536 is mounted on stepper motor 4534, and the end of connecting rod 4536 away from stepper motor 4534 is connected to opening and closing plate 452.

[0027] Since it is a stepper motor 4534, the speed of the stepper motor 4534 can be set by itself. The speed of the stepper motor 4534 can be set to the appropriate parameters to meet the detection time of the plant cell protoplast suspension. When the output shaft of the stepper motor 4534 drives the gear 4535 to rotate, the gear 4535 will move along the length of the rack 4531, thereby driving the opening plate 452 to move along the length of the long strip placement box 451. That is, the speed of the stepper motor 4534 is adjusted so that the time for the opening plate 452 to move so that the lower opening of a square placement cavity 4511 is fully exposed is equal to the time required to detect the plant cell protoplast suspension at intervals. This can achieve the effect of automatically drawing the plant cell protoplast suspension in the plant cell culture bag 3 to the UV detector 33 for detection after a certain interval. It is worth noting that how to adjust the speed of the stepper motor 4534 is a conventional adjustment method. This application does not need to disclose the specific adjustment method. Those skilled in the art can understand the specific implementation principle.

[0028] Reference Figure 3 , 5The downward-pressurized energizing component 456 includes a battery 4561, a first conductive plate 4562, a second conductive plate 4563, a first spring 4564, and a second spring 4565. The battery 4561 is mounted on the bracket 41 on one side outside the vertical tube 455. The first conductive plate 4562 is vertically slidably mounted inside the vertical tube 455 and is electrically connected to the battery 4561 via a spring wire. The first spring 4564 is vertically mounted between the first conductive plate 4562 and the inner bottom wall of the vertical tube 455, and is used to support the first conductive plate 4562. The second conductive plate 4563 is vertically slidably mounted inside the vertical tube 455, directly below the first conductive plate 4562, and is also electrically connected to the liquid pump 42 via a spring wire. The second conductive plate 4563 is mounted on the side wall of the vertical tube 455 along its length. A vertical strip-shaped hole is provided in the direction for the spring wire to pass through; an opening is provided on the second conductive plate 4563 for the first spring 4564 to pass through, and the size of the opening is sufficient for the first spring 4564 to pass through normally in both stretched and compressed states; the second spring 4565 is vertically installed between the second conductive plate 4563 and the bottom wall of the vertical tube 455, and the second spring 4565 is used to support the second conductive plate 4563, and the second spring 4565 is offset from the first spring 4564. When the first spring 4564 and the second spring 4565 are in their natural state, there is a gap between the supported first conductive plate 4562 and the second conductive plate 4563; in this embodiment, one second spring 4565 is provided on each side of the first spring 4564, and the first spring 4564 and the second spring 4565 do not interfere with each other. The weight of the iron ball 8 is greater than the sum of the weight and elastic force of the first conductive plate 4562, the second conductive plate 4563, the first spring 4564, and the two second springs 4565.

[0029] like Figure 3 , 5 As shown, an inclined discharge pipe 10 is connected to the side wall of the vertical pipe 455 below the second conductive plate 4563. An inclined guide plate 45621 is provided above the first conductive plate 4562, and the inclined surface of the inclined guide plate 45621 faces the opening end of the inclined discharge pipe 10. When the iron ball 8 presses against the inclined guide plate 45621 and forces the first conductive plate 4562 to move vertically downward so that the iron ball 8 is facing the opening end of the inclined guide plate 45621, the iron ball 8 rolls from the inclined guide plate 45621 into the inclined discharge pipe 10 under the action of natural gravity.

[0030] After the opening plate 452 moves and fully exposes the lower opening of a square placement cavity 4511, the iron ball 8 falls vertically from the square placement cavity 4511 into the funnel 454, and then enters the vertical tube 455 from the funnel 454. Inside the vertical tube 455, it falls directly onto the inclined guide plate 45621 and, under the influence of gravity, presses down on the first conductive plate 4562, causing the first conductive plate 4562 to move vertically downwards within the vertical tube 455. Because there is a gap between the first conductive plate 4562 and the second conductive plate 4563, the ball 8 briefly... After the first conductive plate 4562 and the second conductive plate 4563 move downwards, they will adhere together, thereby energizing the battery 4561 and the liquid pump 42. The liquid pump 42 will then start to pump the plant cell protoplast suspension in the plant cell culture bag 3 into the UV detector 33. As the first conductive plate 4562 and the second conductive plate 4563 continue to move downwards, the iron ball 8 will move to a position where it is directly opposite the opening of the inclined discharge pipe 10. At this point, the iron ball 8 is no longer restricted by the wall of the vertical pipe 455 and tilts upwards onto the inclined guide plate 45621. The ball slid obliquely into the inclined discharge pipe 10. It is worth noting that the inner diameter of the inclined discharge pipe 10 is larger than the diameter of the iron ball 8. This ensures that the iron ball 8 remains within the diameter range of the inclined discharge pipe 10's opening during its oblique movement on the inclined guide plate 45621. Once the iron ball 8 enters the inclined discharge pipe 10, the first conductive plate 4562 and the second conductive plate 4563 are no longer compressed. Under the elastic force of the first spring 4564 and the second spring 4565, the first conductive plate 4562 and the second conductive plate 4563... 563 will immediately return to its original state, that is, to a state where there is a gap between the first conductive plate 4562 and the second conductive plate 4563. At this time, the liquid pump 42 will be de-energized and will not start again, so that the liquid pump 42 only briefly extracts a small portion of the plant cell protoplast suspension. Similarly, as the opening and closing plate 452 moves continuously, the iron balls 8 in each square placement cavity 4511 will fall at the same time interval, thereby achieving the effect of the liquid pump 42 starting to extract the plant cell protoplast suspension at the same time interval. In this embodiment, the same time interval means approximately the same. Since the falling time of the iron balls 8 cannot be exactly the same, and generally under experimental conditions, the time interval is more than 24 hours, the difference of a few seconds in the time interval can be ignored.

[0031] like Figure 4 , 6As shown, the swing mechanism 6 includes a base 61, a rotating motor 62, and a rotating disk 63; the base 61 is installed below the culture box 1; the rotating motor 62 is installed inside the base 61, and the rotating shaft of the rotating motor 62 extends vertically upward out of the base 61; the rotating disk 63 is coaxially installed at the end of the output shaft of the rotating motor 62, and a rotating shaft 11 is vertically arranged below the culture box 1, the rotating shaft 11 is connected to the rotating disk 63, and the rotating shaft 11 and the output shaft of the rotating motor 62 are on different rotation axes.

[0032] After the rotating motor 62 is started, the output shaft of the rotating motor 62 will drive the rotating disk 63 to rotate, thereby causing the rotating shaft 11 to drive the culture box 1 to rotate. Since the rotating shaft 11 and the output shaft of the rotating motor 62 are on different rotation axes, the rotation of the culture box 1 will cause the plant cell protoplast suspension in the plant cell culture bag 3 to shake, allowing the plant cell protoplast suspension to better contact the heating pad 5.

[0033] Combination Figure 1 , 6 The light source conversion mechanism 7 includes a frame 71, a lamp body 72, a louver 73, and a color-changing cover 74. The frame 71 is installed on the outer top of the cover 2, the lamp body 72 is installed on the frame 71, the louver 73 is installed on the cover 2 below the lamp body 72, and the color-changing cover 74 is detachably installed on the frame 71 and is used to cover the lamp body 72. In this embodiment, the color-changing cover 74 is actually a cover of different colors, used to cover the lamp body 72, so that the lamp body 72 can emit light of different colors. Moreover, the opening of the color-changing cover 74 is a constricted structure. After covering the lamp body 72, the opening can be locked to achieve installation, and it can be released to detach.

[0034] When different plant cell culture bags 3 require different lights and light intensities, color-changing covers 74 of different colors can be installed according to the plant cells being cultured on site, so that the light body 72 emits a light color suitable for the plant cells being cultured at the moment; and by adjusting the opening angle of the louvers 73, the light intensity of the light emitted by the light body 72 entering the culture box 1 can be adjusted, thereby making the environment for the culture of plant cell protoplast suspension more suitable.

[0035] The implementation principle of this application embodiment is as follows: First, plant cell protoplast suspension is filled into plant cell culture bag 3. Then, heating pad 5 is energized and heated. Color-changing cover 74 of the required color is put on lamp body 72. Lamp body 72 is turned on, and the opening angle of louver 73 is adjusted to the angle suitable for the current environment. Finally, the speed of stepper motor 4534 is adjusted so that the time for moving opening plate 452 to fully expose the lower opening of square placement cavity 4511 is equal to the time required to detect plant cell protoplast suspension.

[0036] The embodiments described in this specific implementation are preferred embodiments of this application and are not intended to limit the scope of protection of this application. Identical components are represented by the same reference numerals. Therefore, all equivalent changes made to the structure, shape, and principle of this application should be included within the scope of protection of this application.

Claims

1. A sterile pilot-scale culture device based on plant cell culture and secondary metabolite production, characterized in that, include: A culture box (1) is hollow inside and open at the top. A cover (2) is provided at the top opening of the culture box (1). A plant cell culture bag (3) is placed inside the culture box (1) and is used to load a suspension of plant cell protoplasts. A pH meter (31), a thermometer (32), and a UV detector (33) are connected to the plant cell culture bag (3). A cell fluid delivery mechanism (4) is installed on one side of the culture box (1) and is used to deliver the suspension of plant cell protoplasts in the plant cell culture bag (3) to the UV detector (33) at a set time. A heating pad (5) is installed on the inner bottom wall of the culture box (1) and is used to heat the plant cell culture bag (3) placed in the culture box (1). A swing mechanism (6) is installed below the culture box (1) and is used to drive the culture box (1) to rotate circumferentially on the horizontal plane. A light source conversion mechanism (7) is installed on the cover (2). The light source conversion mechanism (7) is used to provide light to the plant cell culture bag (3) and can adjust the brightness and color of the light source. The cell sap delivery mechanism (4) includes: a support (41) installed on one side of the culture box (1); a liquid pump (42) installed on the support (41); an infusion tube (43) connected between the input end of the liquid pump (42) and the plant cell culture bag (3); a branch tube (44) connected to the output end of the liquid pump (42), and the two branches of the branch tube (44) are respectively connected to the UV detector (33); and a timed start component (45) installed on the support (41) for starting the liquid pump (42) after a set time to deliver the plant cell protoplast suspension in the plant cell culture bag (3) to the UV detector (33). The timed start component (45) includes: a long strip placement box (451), which is mounted on a bracket (41) and has openings at both the top and bottom. The long strip placement box (451) has multiple square placement cavities (4511) of the same volume along its length. Each square placement cavity (4511) contains an iron ball (8), and the diameter of the iron ball (8) is the same as the width of the square placement cavity (4511); a hinged plate (452), which is slidably disposed at the lower opening of the long strip placement box (451) and is used to cover the lower opening of the square placement cavity (4511); and a drive unit (453), which is mounted on the bracket (41) and is used to drive the hinged plate (452) to move along the length of the long strip placement box (451). A funnel (454) is mounted on a bracket (41) directly below a long strip box (451), with the upper opening of the funnel (454) completely covering the lower opening of the long strip box (451) in the vertical direction; a vertical tube (455) is vertically connected and mounted at the lower end of the funnel (454), with the inner diameter of the vertical tube (455) being the same as the diameter of the iron ball (8); a pressure-conducting element (456) is mounted between the bracket (41) and the vertical tube (455), and the liquid pump (42) is activated when the iron ball (8) falls directly into the vertical tube (455) and touches a part of the pressure-conducting element (456).

2. The sterile pilot-scale culture apparatus based on plant cell culture and secondary metabolite production according to claim 1, characterized in that, The driving component (453) includes: a rack (4531) mounted on the bracket (41) with its length direction aligned with the length direction of the opening and closing plate (452); a guide rail (4532) mounted on the bracket (41) with its length direction aligned with the length direction of the rack (4531); a slider (4533) slidably mounted on the guide rail (4532); a stepper motor (4534) mounted on the slider (4533); a gear (4535) coaxially mounted on the output shaft of the stepper motor (4534) and meshing with the rack (4531); and a connecting rod (4536) mounted on the stepper motor (4534) and connected to the opening and closing plate (452).

3. The sterile pilot-scale culture apparatus based on plant cell culture and secondary metabolite production according to claim 1, characterized in that, The lower opening of the long strip box (451) is provided with slide rails (9) on both sides and along the length of the long strip box (451), and the opening and closing plate (452) is slidably disposed between the two slide rails (9).

4. The sterile pilot-scale culture apparatus based on plant cell culture and secondary metabolite production according to claim 1, characterized in that, The pressure-conducting component (456) includes: a battery (4561) mounted on a bracket (41); a first conductive plate (4562) slidably mounted inside a vertical tube (455) and electrically connected to the battery (4561); a second conductive plate (4563) slidably mounted inside the vertical tube (455) below the first conductive plate (4562) and electrically connected to a liquid pump (42); a first spring (4564) vertically mounted between the first conductive plate (4562) and the bottom wall of the vertical tube (455), and the second conductive plate (4563) has an opening for the first spring (4564) to pass through; and a second spring (4565) vertically mounted between the second conductive plate (4563) and the bottom wall of the vertical tube (455), and the second spring (4565) is offset from the first spring (4564). When the first spring (4564) and the second spring (4565) are in their natural state, there is a gap between the first conductive plate (4562) and the second conductive plate (4563).

5. The sterile pilot-scale culture apparatus based on plant cell culture and secondary metabolite production according to claim 4, characterized in that, An inclined discharge pipe (10) is connected to the side wall of the vertical pipe (455) below the second conductive plate (4563). An inclined guide plate (45621) is provided above the first conductive plate (4562). The inclined surface of the inclined guide plate (45621) faces the opening end of the inclined discharge pipe (10). When the iron ball (8) presses against the inclined guide plate (45621) and forces the first conductive plate (4562) to move vertically downward so that the iron ball (8) is facing the opening end of the inclined guide plate (45621), the iron ball (8) rolls from the inclined guide plate (45621) into the inclined discharge pipe (10) under the action of natural gravity.

6. The sterile pilot-scale culture apparatus based on plant cell culture and secondary metabolite production according to claim 5, characterized in that, The lowest end of the inclined discharge pipe (10) is connected to a storage tank (101).

7. The sterile pilot-scale culture apparatus based on plant cell culture and secondary metabolite production according to claim 1, characterized in that, The swing mechanism (6) includes: a base (61) installed below the culture box (1); a rotating motor (62) installed inside the base (61), with the rotating shaft of the rotating motor (62) extending vertically upward out of the base (61); and a rotating disk (63) coaxially installed at the end of the output shaft of the rotating motor (62), with a rotating shaft (11) vertically arranged below the culture box (1), the rotating shaft (11) being connected to the rotating disk (63), and the rotating shaft (11) and the output shaft of the rotating motor (62) being on different rotation axes.

8. The sterile pilot-scale culture apparatus based on plant cell culture and secondary metabolite production according to claim 1, characterized in that, The light source conversion mechanism (7) includes: a frame (71) installed on the top of the cover (2); a lamp body (72) installed on the frame (71); a louver (73) installed on the cover (2) below the lamp body (72); and a color-changing cover (74) detachably installed on the frame (71) for covering the lamp body (72).

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