Composite protection device and method for low-temperature preservation of haematococcus algae species

Abstract

The invention relates to the technical field of algae species preservation equipment, in particular to a composite protection device and method for low-temperature preservation of haematococcus algae species, and the composite protection device comprises a cold closet, a rotating table arranged in the cold closet, a conical flask placed above the rotating table and used for storing concentrated algae species, and a plurality of injection mechanisms arranged above the conical flask. According to the composite protection device and method for low-temperature preservation of haematococcus algae species, through the driving part, conical flasks can be automatically and sequentially conveyed to different protective agent injection sites, and the liquid passing part is matched with the injection mechanism to replace tedious manual adding operation, so that the problems of sequence errors, inaccurate dosage and cross contamination possibly occurring in manual operation are avoided; standardization and automation of the protective agent adding process are achieved, and a solid foundation is laid for the synergistic effect of the composite protective agent.

Description

Technical Field

[0001] This invention relates to the field of algae strain preservation equipment technology, and more specifically, to a composite protection device and method for low-temperature preservation of Haematococcus pluvialis strains. Background Technology

[0002] Cryopsus preservation of Haematococcus pluvialis strains is a key technology for maintaining their activity and genetic stability. It is usually carried out in an ultra-low temperature freezing environment of -196℃ liquid nitrogen or -80℃, combined with cryoprotectants such as glycerol and DMSO for pretreatment to avoid ice crystal damage to cell structure. This method can preserve algal strains for a long time, reduce the risk of contamination and mutation caused by frequent subculturing, and provide reliable germplasm resource guarantee for scientific research, breeding and industrial application.

[0003] Patent application number CN201610170366.2 discloses a method for cryopreservation of Haematococcus pluvialis after sterilization treatment. The cryopreservation method provided by this invention involves systematic sterilization before preservation. After sterilization, the Haematococcus pluvialis is subjected to specific low-temperature preservation conditions, resulting in a good recovery survival rate of 66.13% after thawing. Using the method provided by this invention, no bacterial colonies appear on LB agar plates after sterilization. After three subcultures, sterility testing on LB agar plates again shows no bacterial colonies. The sterilized algal strains are not prone to aging, thus maintaining the genetic stability of the germplasm and facilitating long-term preservation.

[0004] However, the existing process for adding cryoprotectants to Haematococcus pluvialis strains relies entirely on manual operation. This is not only cumbersome, but also makes it difficult to ensure the accuracy of the order and dosage of different cryoprotectants. Operational errors can easily prevent the compound cryoprotectants from achieving their optimal synergistic effect. Secondly, conventional containers need to be opened frequently when adding reagents, which cannot effectively isolate air and poses a risk of microbial contamination, endangering the purity and preservation safety of the algal strain. Finally, existing equipment lacks a complete temperature control system. The process of adding and mixing cryoprotectants is usually carried out at room temperature, and the cryoprotectants need to be pre-cooled before use.

[0005] In view of this, we propose a composite protection device and method for the low-temperature preservation of Haematococcus pluvialis species. Summary of the Invention

[0006] The purpose of this invention is to provide a composite protection device and method for cryopreservation of Haematococcus pluvialis strains. Through the drive unit, the conical flask can be automatically and sequentially transported to different protective agent injection sites. The liquid flow unit, in conjunction with the injection mechanism, replaces the cumbersome manual addition operation, thereby solving the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A composite protection device for low-temperature preservation of Haematococcus pluvialis algae includes a refrigerator, a rotating platform disposed inside the refrigerator, a conical flask placed above the rotating platform for storing concentrated algae, and several injection mechanisms disposed above the conical flask.

[0009] The conical flask includes a flask body, a stir bar placed inside it, a stopper located at the mouth of the flask body, and a sleeve located inside the stopper.

[0010] The rotating platform includes a drive unit, a turntable disposed above it, a magnetic disk and a liquid-passing unit disposed on the top surface of the turntable; the drive unit includes a motor, a rotating shaft driven by the motor, a disk rotating with the rotating shaft, a lever disposed on the top surface of the disk, and a grooved wheel whose central shaft is engaged with the bottom surface of the turntable.

[0011] This setting allows the motor to drive the rotating shaft, causing the disc to rotate one revolution. The lever then moves the grooved wheel, driving the disc to rotate 90°.

[0012] The fluid passage includes an electric actuator and a bending rod driven by it.

[0013] The injection mechanism consists of a liquid storage bottle, a solenoid valve, and a movable frame from top to bottom. An insertion tube is fitted inside the movable frame. After the turntable rotates 90°, the top of the bent rod moves into the movable frame.

[0014] After the electric actuator drives the bending rod to move the movable frame downward, it drives the insertion tube to be inserted into the sleeve. At the same time, the magnetic disk and solenoid valve are opened to drip the liquid in the storage bottle into the conical flask, and the solution is mixed by the stir bar.

[0015] In the technical solution of the present invention, the sleeve is snapped and fixed inside the round hole of the bottle stopper, and a through groove is opened on the outer wall of the sleeve near the middle position, and a piston is slidably connected inside the sleeve.

[0016] In the technical solution of the present invention, a first spring is attached to the bottom of the piston, and the bottom end of the first spring is attached to the inner bottom surface of the sleeve. The elastic force provided by the first spring pushes the piston to move upward.

[0017] With the above setup, once the piston moves down to the bottom of the channel, the bottle can be connected to the outside. After the liquid is injected, the piston can be reset by the elastic force of the first spring, so that the algae liquid is always isolated from the outside.

[0018] In the technical solution of the present invention, the rotating platform further includes a square frame placed on the bottom surface of the refrigerator and a cover plate snapped and fixed to the top surface of the square frame. The turntable is rotatably connected to the top surface of the cover plate, and the magnetic disk is snapped and fixed to the turntable.

[0019] In the technical solution of the present invention, the motor is fixedly connected to the inner bottom surface of the square frame by screws, the rotating shaft is coaxially connected to the output shaft of the motor, a worm gear is snapped and fixed on the outer wall of the rotating shaft, a worm wheel that meshes with the worm gear is snapped and fixed on the outer wall of the central shaft of the disc, the lever is heat-fused to the top surface of the disc, and the disc and the grooved wheel are both rotatably connected to the inner bottom surface of the square frame through the central shaft.

[0020] The above configuration, through the drive unit, enables the conical flasks to stop sequentially directly below the injection mechanism containing different solutions, providing the prerequisite for the automated addition of different solvents.

[0021] In the technical solution of the present invention, the electric actuator is fixedly connected to the top surface of the turntable by screws, and the bending rod is snapped and fixed to the end of the electric actuator telescopic rod.

[0022] In the technical solution of the present invention, the liquid passage part further includes a slide rod fixedly connected to the center of the top surface of the turntable by screws, a limiting plate for limiting the position of the conical bottle by hot-melt connection to the outer wall of the slide rod, and two positioning frames integrally formed with the slide rod.

[0023] The above setup, with the use of two limit blocks, ensures the accurate positioning of the bending rod after each movement.

[0024] In the technical solution of the present invention, the liquid storage bottle is snapped and fixed to the top surface of the internal partition of the refrigerator, the solenoid valve is fixedly connected to the bottom surface of the internal partition of the refrigerator by screws, the solenoid valve has a round tube connected to the bottom of the liquid storage bottle, the outer walls of both sides of the movable frame are provided with limit grooves, the insertion tube is snapped and fixed in the movable frame and slidably connected to the outside of the round tube inside the solenoid valve, the outer wall of the insertion tube is provided with several drainage grooves that are connected inside and outside, along the rotation direction of the conical flask, the three liquid storage bottles sequentially store trehalose solution, glycerin and ascorbic acid solution.

[0025] In the technical solution of the present invention, the injection mechanism further includes a limiting frame that is snapped and fixed to the bottom surface of the internal partition of the refrigerator and sleeved on the outside of the movable frame, two limiting blocks that are slidably connected in the groove of its inner wall, and a second spring that is adhered to the inner wall of the limiting block. The other end of the second spring is adhered to the groove of the inner wall of the limiting frame, and the elastic force provided by the second spring pushes the limiting block to move towards the limiting groove.

[0026] The above setup controls the injection volume of the solution via a solenoid valve, avoiding potential issues such as incorrect sequence, inaccurate dosage, and cross-contamination that may occur during manual operation.

[0027] On the other hand, the present invention also provides a composite protection method for cryopreservation of Haematococcus pluvialis strains, which uses the above-mentioned composite protection device for cryopreservation of Haematococcus pluvialis strains and includes the following steps:

[0028] S1. First, the operator pours the concentrated Haematococcus pluvialis seed into the conical flask, inserts the stopper into the mouth of the flask, and places the conical flask in a refrigerator where the internal temperature is maintained at 0℃-4℃, ensuring that the bottom of the flask is placed above the magnetic disk of the rotating platform.

[0029] S2. Next, close the refrigerator door and start the control program inside the refrigerator. The motor in the drive unit drives the rotating shaft to rotate and the worm gear meshes with the worm wheel. After the drive disc rotates one revolution, it stops. At this time, the grooved wheel rotates 90° under the push of the lever, moving the conical bottle to below the injection mechanism containing the trehalose solution.

[0030] S3. Subsequently, the magnetic disk starts and drives the stir bar to rotate on the bottom of the bottle, causing the concentrated Haematococcus pluvialis seed inside to rotate slowly. The electric push rod of the liquid passage section starts simultaneously, driving the bent rod that extends from the top into the movable frame to move down.

[0031] S4. The movable frame then moves the insertion tube downwards, and its bottom end contacts the piston, causing it to retract downwards into the inside of the sleeve. When the drainage groove on the insertion tube is connected to the through groove of the sleeve, the electric actuator stops.

[0032] S5. After that, the solenoid valve is activated, controlling the trehalose solution in the storage bottle to drip into the conical flask. After the trehalose solution and the Haematococcus pluvialis are fully mixed, the solenoid valve is closed, the electric push rod is activated again and drives the bent rod to reset its position. At this time, the piston in the conical flask moves upward again under the elastic force of the first spring, restoring the conical flask to a sealed state.

[0033] S6. Subsequently, the control program repeats the above operation, injecting glycerol and ascorbic acid solution into the Haematococcus pluvialis seed in sequence and mixing them thoroughly. The operator then opens the refrigerator, removes the restored conical flask from it, and distributes it into several freezer tubes. After cooling the tubes to -80°C in a programmable cooling device, the tubes are transferred to a low-temperature freezer for long-term storage.

[0034] Compared with the prior art, the beneficial effects of the present invention are:

[0035] 1. The composite protection device and method for cryopreservation of Haematococcus pluvialis strains can automatically and sequentially transport conical flasks to different protective agent injection sites through the drive unit. The liquid flow unit, in conjunction with the injection mechanism, replaces the cumbersome manual addition operation, avoiding the problems of sequence errors, inaccurate dosage and cross-contamination that may occur in manual operation. It realizes the standardization and automation of the protective agent addition process, and lays a solid foundation for the composite protective agent to exert a synergistic effect.

[0036] 2. The composite protection device and method for cryopreservation of *Rhodochophora* strains employs a specially designed conical flask with a sleeve structure containing a piston and spring inside the stopper, which precisely matches the insertion tube of the injection mechanism. When the insertion tube is pressed down to open the injection, the sleeve channel is temporarily opened. After the injection is completed, it immediately resets and seals under the action of the spring, keeping the algal solution in a state of isolation from the outside world. The sealed environment eliminates microbial contamination from the air and ensures the purity of the algal strain.

[0037] 3. The composite protection device and method for cryopreservation of Haematococcus pluvialis strains are carried out in a refrigerator at 0-4℃. The magnetic disk drives the stir bar to achieve uniform mixing at low temperature. The low temperature operating environment greatly reduces the chemical toxicity stress of protective agents such as glycerol on algal cells at room temperature, and comprehensively protects the activity of the algal cell cryopreservation solution during the preparation process, providing a highly active sample for subsequent successful cryopreservation. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the overall structure of the present invention;

[0039] Figure 2 This is a cross-sectional view of the conical flask structure in this invention;

[0040] Figure 3 For the present invention Figure 2 An enlarged schematic diagram of part A in the middle;

[0041] Figure 4 This is a structural breakdown diagram of the rotating platform in this invention;

[0042] Figure 5 This is one of the structural schematic diagrams of the drive unit in this invention;

[0043] Figure 6 This is a second schematic diagram of the drive unit in this invention;

[0044] Figure 7 This is a schematic diagram of the liquid passage section in this invention;

[0045] Figure 8 This is a schematic diagram of the injection mechanism in this invention;

[0046] Figure 9 This is one of the partial structural schematic diagrams of the injection mechanism in this invention;

[0047] Figure 10 This is a second schematic diagram of a portion of the injection mechanism in this invention;

[0048] Explanation of reference numerals in the attached figures:

[0049] 100. Refrigerated box;

[0050] 200. Conical flask; 210. Bottle body; 220. Stirrer; 230. Stopper; 240. Sleeve; 241. Through groove; 250. Piston; 260. First spring;

[0051] 300. Rotating table; 310. Square frame; 320. Drive unit; 321. Motor; 322. Rotating shaft; 323. Disc; 324. Lever; 325. Grooved wheel; 326. Worm gear; 327. Worm wheel; 330. Cover plate; 340. Turntable; 350. Magnetic disk; 360. Liquid passage unit; 361. Electric actuator; 362. Bending rod; 363. Slide rod; 364. Limiting plate; 365. Positioning frame;

[0052] 400, Injection mechanism; 410, Storage bottle; 420, Solenoid valve; 430, Movable frame; 431, Limiting groove; 440, Insertion tube; 441, Drainage groove; 450, Limiting frame; 460, Limiting block; 470, Second spring. Detailed Implementation

[0053] The technical solutions of this invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0054] Please see Figures 1-3 As shown, this embodiment provides a technical solution:

[0055] A composite protection device for low-temperature preservation of Haematococcus pluvialis algae includes a refrigerator 100, a rotating platform 300 disposed inside the refrigerator, a conical flask 200 placed above the rotating platform 300 for storing concentrated algae, and several injection mechanisms 400 disposed above the conical flask 200.

[0056] Specifically, the conical flask 200 includes a flask body 210, a stir bar 220 placed inside it, a stopper 230 disposed at the mouth of the flask body 210, and a sleeve 240 disposed inside the stopper 230.

[0057] Furthermore, the sleeve 240 is snapped into the round hole inside the bottle stopper 230, and a through groove 241 is provided on the outer wall of the sleeve 240 near the middle position, and a piston 250 is slidably connected inside the sleeve 240.

[0058] Furthermore, a first spring 260 is attached to the bottom of the piston 250. The bottom end of the first spring 260 is attached to the inner bottom surface of the sleeve 240. The elastic force provided by the first spring 260 pushes the piston 250 to move upward.

[0059] Furthermore, the bottle body 210 is used to store the concentrated Haematococcus pluvialis seed, the stir bar 220 is used to stir the Haematococcus pluvialis seed when the bottle body 210 is sealed, the sleeve 240 is used to provide a sliding range for the piston 250, and the elastic force provided by the first spring 260 is used to allow the piston 250 to return to its original position after it moves down. With this setting, when the piston 250 moves down to below the through groove 241, the bottle body 210 can be connected to the outside. After the liquid is injected, the piston 250 can return to its original position under the elastic force of the first spring 260, so that the algal liquid is always isolated from the outside.

[0060] Please see Figures 4-6 As shown, in this embodiment, the rotating table 300 includes a drive unit 320, a turntable 340 disposed above it, and a magnetic disk 350 and a liquid passage unit 360 disposed on the top surface of the turntable 340. The drive unit 320 includes a motor 321, a rotating shaft 322 driven by the motor 321, a disk 323 rotating with the rotating shaft 322, a lever 324 disposed on the top surface of the disk 323, and a grooved wheel 325 whose central shaft is engaged with the bottom surface of the turntable 340. The motor 321 drives the rotating shaft 322 to rotate the disk 323 one revolution, and the lever 324 moves the grooved wheel 325 to drive the turntable 340 to rotate 90°.

[0061] Specifically, the rotating table 300 also includes a square frame 310 placed on the bottom surface of the refrigerator 100 and a cover plate 330 snapped and fixed to the top surface of the square frame 310. The turntable 340 is rotatably connected to the top surface of the cover plate 330, and the magnetic disk 350 is snapped and fixed to the turntable 340.

[0062] Furthermore, the motor 321 is fixedly connected to the inner bottom surface of the frame 310 by screws, the rotating shaft 322 is coaxially connected to the output shaft of the motor 321, the worm gear 326 is snapped and fixed on the outer wall of the rotating shaft 322, the worm wheel 327 that meshes with the worm gear 326 is snapped and fixed on the outer wall of the central shaft of the disc 323, and the lever 324 is heat-fused to the top surface of the disc 323. The disc 323 and the grooved wheel 325 are both rotatably connected to the inner bottom surface of the frame 310 through the central shaft.

[0063] Furthermore, the motor 321 in the drive unit 320 drives the rotating shaft 322 to rotate, and the worm gear 326 meshes with the worm wheel 327, driving the disk 323 to rotate one revolution and then stop. At this time, the grooved wheel 325 rotates 90° under the action of the lever 324, moving the conical flask 200 below the injection mechanism 400 containing the trehalose solution. At the same time, the magnetic disk 350 is activated, driving the stir bar 220 to rotate on the bottom surface inside the bottle body 210, allowing the concentrated Haematococcus pluvialis seed inside to rotate slowly. This setting, through the drive unit 320, enables the conical flask 200 to stop directly below the injection mechanism 400 containing different solutions in sequence, providing the prerequisite for the automatic addition of different solvents.

[0064] Please see Figures 4-7 As shown, in this embodiment, the liquid-passing part 360 includes an electric actuator 361 and a bending rod 362 driven therefrom.

[0065] Specifically, the electric actuator 361 is fixedly connected to the top surface of the turntable 340 by screws, and the bending rod 362 is snapped and fixed to the end of the telescopic rod of the electric actuator 361.

[0066] Furthermore, the liquid conveying unit 360 also includes a slide bar 363 fixedly connected to the center of the top surface of the turntable 340 by screws, a limiting plate 364 heat-fused to the outer wall of the slide bar 363 for limiting the position of the conical bottle 200, and two positioning frames 365 integrally formed with the slide bar 363.

[0067] Furthermore, the electric actuator 361 of the liquid-conducting section 360 is activated synchronously, driving the bent rod 362, which extends to the movable frame 430 at its top, to move downward. The slide bar 363 is used to ensure the stability of the bent rod 362 when it moves downward. This setting, through the setting of two limiting plates 364, ensures the accuracy of the position of the bent rod 362 after each movement.

[0068] Please see Figures 4-10 As shown, in this embodiment, the injection mechanism 400 is provided with a liquid storage bottle 410, a solenoid valve 420 and a movable frame 430 from top to bottom. The movable frame 430 is fitted with an insertion tube 440. After the turntable 340 rotates 90°, the top of the bending rod 362 moves into the movable frame 430. The electric push rod 361 drives the bending rod 362 to move the movable frame 430 down, thereby driving the insertion tube 440 to be inserted into the sleeve 240. At the same time, the magnetic disk 350 and the solenoid valve 420 are opened, and the liquid in the liquid storage bottle 410 is dripped into the conical flask 200. The solution is then mixed by the stir bar 220.

[0069] Specifically, the storage bottle 410 is snapped and fixed to the top surface of the internal partition of the refrigerator 100, and the solenoid valve 420 is fixedly connected to the bottom surface of the internal partition of the refrigerator 100 by screws. The solenoid valve 420 has a round tube that is connected to the bottom of the storage bottle 410. Limiting grooves 431 are opened on the outer walls of both sides of the movable frame 430. The insertion tube 440 is snapped and fixed inside the movable frame 430 and slidably connected to the outside of the round tube inside the solenoid valve 420. Several drainage grooves 441 that are connected inside and outside are opened on the outer wall of the insertion tube 440. Along the rotation direction of the conical flask 200, the three storage bottles 410 contain trehalose solution, glycerol and ascorbic acid solution in sequence. Trehalose solution can form a water-like protective layer around the protein and phospholipid molecules on the cell membrane surface, replacing water molecules to form hydrogen bonds, thereby preventing the cell membrane from rupturing, collapsing and fusing when it loses water. It physically stabilizes the cell membrane structure from the outside of the cell and helps the solution form a protective glassy state.

[0070] Glycerin, as a small-molecule penetrating protective agent, can freely pass through the cell membrane and enter the cell. By lowering the freezing point and balancing osmotic pressure, it reduces the formation of intracellular ice crystals and excessive dehydration. Ascorbic acid solution, through its own antioxidant properties, effectively neutralizes and removes harmful chemicals generated during the freezing process, protecting important cell components from oxidative damage and thus maintaining cell vitality.

[0071] Furthermore, the injection mechanism 400 also includes a limiting frame 450 that is snapped and fixed to the bottom surface of the internal partition of the refrigerator 100 and sleeved on the outside of the movable frame 430, two limiting blocks 460 that are slidably connected in the grooves of its inner wall, and a second spring 470 that is adhered to the inner wall of the limiting block 460. The other end of the second spring 470 is adhered to the groove of the inner wall of the limiting frame 450, and the elastic force provided by the second spring 470 pushes the limiting block 460 to move in the direction of the limiting groove 431.

[0072] Furthermore, the movable frame 430 drives the insertion tube 440 to move downwards, and its bottom end abuts against the piston 250, causing it to retract downwards into the interior of the sleeve 240. When the drainage groove 441 on the insertion tube 440 is connected to the through groove 241 of the sleeve 240, the electric push rod 361 stops. Then, the solenoid valve 420 is activated, controlling the solution in the storage bottle 410 to drip into the interior of the conical flask 200. After the solution is fully mixed with the Haematococcus pluvialis algae, the solenoid valve 420 is closed. This setting controls the injection volume of the solution through the solenoid valve 420, avoiding possible sequence errors, inaccurate dosages, and cross-contamination problems that may occur during manual operation.

[0073] This invention also provides a composite protection method for cryopreservation of *Rhodotorula pulmonarius* strains, using the aforementioned composite protection device for cryopreservation of *Rhodotorula pulmonarius* strains, comprising the following steps:

[0074] S1. First, the operator pours the concentrated Haematococcus pluvialis seed into the conical flask 200, inserts the stopper 230 into the mouth of the flask body 210, and places the conical flask 200 into the refrigerator 100 where the internal temperature is maintained at 0℃-4℃, and ensures that the bottom of the flask is placed above the magnetic disk 350 of the rotating table 300.

[0075] S2. Next, close the door of the refrigerator 100 and start the control program inside the refrigerator 100. The motor 321 in the drive unit 320 drives the rotating shaft 322 to rotate, and the worm gear 326 meshes with the worm wheel 327, driving the disc 323 to rotate one revolution and then stop. At this time, the grooved wheel 325 rotates 90° under the action of the lever 324, moving the conical bottle 200 below the injection mechanism 400 containing the trehalose solution.

[0076] S3. Subsequently, the magnetic disk 350 is activated, which drives the stir bar 220 to rotate on the bottom surface inside the bottle 210, allowing the concentrated Haematococcus pluvialis seed inside to rotate slowly. The electric push rod 361 of the liquid passage section 360 is activated simultaneously, driving the bent rod 362, which extends from the top to the movable frame 430, to move down.

[0077] S4. The movable frame 430 then drives the insertion tube 440 to move downward, and its bottom end touches the piston 250 to retract downward into the interior of the sleeve 240. When the drainage groove 441 on the insertion tube 440 is connected to the through groove 241 of the sleeve 240, the electric push rod 361 stops.

[0078] S5. Then, the solenoid valve 420 is activated, controlling the trehalose solution in the storage bottle 410 to drip into the conical flask 200. After the trehalose solution and the Haematococcus pluvialis are fully mixed, the solenoid valve 420 is closed, the electric push rod 361 is activated again and drives the bent rod 362 to reset its position. At this time, the piston 250 in the conical flask 200 moves upward again under the elastic force of the first spring 260, restoring the conical flask 200 to a sealed state.

[0079] S6. Subsequently, the control program repeats the above operation, injecting glycerol and ascorbic acid solution into the Haematococcus pluvialis seed in sequence and mixing them thoroughly. Then, the operator opens the refrigerator 100, takes out the restored conical flask 200, and distributes it into several freezing tubes. After cooling to -80°C in a programmable cooling device, the tubes are transferred to a low-temperature freezer for long-term storage.

[0080] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the specification and its equivalents.

Claims

1. A composite protection device for the cryopreservation of Haematococcus pluvialis strains, characterized in that: It includes a refrigerator, a rotating platform inside it, conical flasks placed on top of the rotating platform for storing concentrated algal cultures, and several injection mechanisms placed on top of the conical flasks. The conical flask includes a flask body, a stir bar placed inside it, a stopper located at the mouth of the flask body, and a sleeve located inside the stopper. The rotating platform includes a drive unit, a turntable disposed above it, and a magnetic disk and a liquid-passing unit disposed on the top surface of the turntable; the drive unit includes a motor, a rotating shaft driven by the motor, a disc rotating with the rotating shaft, a lever disposed on the top surface of the disc, and a grooved wheel whose central shaft is engaged with the bottom surface of the turntable. The motor drives the rotating shaft to rotate the disc one revolution, and the lever moves the grooved wheel to drive the turntable to rotate 90°. The fluid passage includes an electric actuator and a bending rod driven by it; The injection mechanism consists of a storage bottle, a solenoid valve, and a movable frame, arranged from top to bottom. An insertion tube is fitted inside the movable frame. After the turntable rotates 90°, the top of the bending rod moves into the movable frame. The electric actuator drives the bending rod to move the movable frame downward, thereby driving the insertion tube to be inserted into the sleeve. At the same time, the magnetic disk and the solenoid valve are opened, dripping the liquid in the storage bottle into the conical flask. The solution is then mixed by a stir bar.

2. The composite protection device for cryopreservation of *Rhodotorula pulmonata* strains according to claim 1, characterized in that: The sleeve is snapped and fixed inside the round hole of the bottle stopper. A through groove is opened on the outer wall of the sleeve near the middle position, and a piston is slidably connected inside the sleeve.

3. The composite protection device for low-temperature preservation of *Rhodotorula pulmonata* strains according to claim 2, characterized in that: A first spring is attached to the bottom of the piston, and the bottom end of the first spring is attached to the inner bottom surface of the sleeve. The elastic force provided by the first spring pushes the piston to move upward.

4. The composite protection device for low-temperature preservation of *Rhodotorula pulmonarius* strains according to claim 3, characterized in that: The rotating platform also includes a square frame placed on the bottom of the refrigerator and a cover plate snapped onto the top surface of the square frame. The turntable is rotatably connected to the top surface of the cover plate, and the magnetic disk is snapped onto the turntable.

5. The composite protection device for low-temperature preservation of *Rhodotorula pulmonata* strains according to claim 4, characterized in that: The motor is fixedly connected to the inner bottom surface of the frame by screws. The rotating shaft is coaxially connected to the output shaft of the motor. A worm gear is snapped and fixed on the outer wall of the rotating shaft. A worm wheel that meshes with the worm gear is snapped and fixed on the outer wall of the central shaft of the disc. The lever is heat-fused to the top surface of the disc. The disc and the grooved wheel are both rotatably connected to the inner bottom surface of the frame through the central shaft.

6. The composite protection device for cryopreservation of *Rhodotorula pulmonata* strains according to claim 5, characterized in that: The electric actuator is fixedly connected to the top surface of the turntable by screws, and the bending rod is snapped and fixed to the end of the electric actuator telescopic rod.

7. The composite protection device for cryopreservation of *Rhodotorula pulmonata* strains according to claim 6, characterized in that: The liquid-passing section also includes a slide rod fixed to the center of the top surface of the turntable by screws, a limiting plate heat-fused to the outer wall of the slide rod to limit the position of the conical bottle, and two positioning frames integrally formed with the slide rod.

8. The composite protection device for low-temperature preservation of *Rhodotorula pulmonata* strains according to claim 7, characterized in that: The storage bottle is snapped and fixed to the top surface of the internal partition of the refrigerator. The solenoid valve is fixed to the bottom surface of the internal partition of the refrigerator by screws. A round tube connected to the bottom of the storage bottle is snapped inside the solenoid valve. Limiting grooves are opened on the outer walls of both sides of the movable frame. The insertion tube is snapped and fixed inside the movable frame and slidably connected to the outside of the round tube inside the solenoid valve. Several drainage grooves with internal and external connections are opened on the outer wall of the insertion tube. Along the rotation direction of the conical flask, the three storage bottles contain trehalose solution, glycerin and ascorbic acid solution in sequence.

9. The composite protection device for cryopreservation of *Rhodotorula pulmonata* strains according to claim 8, characterized in that: The injection mechanism also includes a limiting frame that is snapped and fixed to the bottom surface of the internal partition of the refrigerator and sleeved on the outside of the movable frame, two limiting blocks that are slidably connected in the grooves of its inner wall, and a second spring that is adhered to the inner wall of the limiting block. The other end of the second spring is adhered to the groove of the inner wall of the limiting frame, and the elastic force provided by the second spring pushes the limiting block to move towards the limiting groove.

10. A composite protection method for cryopreservation of *Rhodotorula pulmonarius* strains, using the composite protection device for cryopreservation of *Rhodotorula pulmonarius* strains as described in claim 9, characterized in that... Includes the following steps: S1. First, the operator pours the concentrated Haematococcus pluvialis seed into the conical flask, inserts the stopper into the mouth of the flask, and places the conical flask in a refrigerator where the internal temperature is maintained at 0℃-4℃, ensuring that the bottom of the flask is placed above the magnetic disk of the rotating platform. S2. Next, close the refrigerator door and start the control program inside the refrigerator. The motor in the drive unit drives the rotating shaft to rotate and the worm gear meshes with the worm wheel. After the drive disc rotates one revolution, it stops. At this time, the grooved wheel rotates 90° under the push of the lever, moving the conical bottle to below the injection mechanism containing the trehalose solution. S3. Subsequently, the magnetic disk starts and drives the stir bar to rotate on the bottom of the bottle, causing the concentrated Haematococcus pluvialis seed inside to rotate slowly. The electric push rod of the liquid passage section starts simultaneously, driving the bent rod that extends from the top into the movable frame to move down. S4. The movable frame then moves the insertion tube downwards, and its bottom end contacts the piston, causing it to retract downwards into the inside of the sleeve. When the drainage groove on the insertion tube is connected to the through groove of the sleeve, the electric actuator stops. S5. After that, the solenoid valve is activated, controlling the trehalose solution in the storage bottle to drip into the conical flask. After the trehalose solution and the Haematococcus pluvialis are fully mixed, the solenoid valve is closed, the electric push rod is activated again and drives the bent rod to reset its position. At this time, the piston in the conical flask moves upward again under the elastic force of the first spring, restoring the conical flask to a sealed state. S6. Subsequently, the control program repeats the above operation, injecting glycerol and ascorbic acid solution into the Haematococcus pluvialis seed in sequence and mixing them thoroughly. The operator then opens the refrigerator, removes the restored conical flask from it, and distributes it into several freezer tubes. After cooling the tubes to -80°C in a programmable cooling device, the tubes are transferred to a low-temperature freezer for long-term storage.

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