Mangrove forest actinomycete secondary metabolite extraction device

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a mangrove forest actinomycete secondary metabolite extraction device which comprises a bottom plate, a supporting plate is fixed to the rear end of the upper side of the bottom plate, and a fixing box is fixed to the front side of the supporting plate. A water phase at the lower end can be discharged through a first discharge pipe, and after extract liquor collected for the first time in the extraction pipe is poured out, the water phase flows back to the extraction pipe again, so that an ethanol solution is added again for extraction, and the extraction effect of mangrove forest actinomycetes secondary metabolites is improved; and after the extraction process is finished, a cleaning water containing box at the upper end can be driven to move through a moving mechanism, so that cleaning water is injected into the extraction pipe through a second vertical pipe, and the cleaning water in the extraction pipe circularly flows through the work of a backflow mechanism and a first pump body, so that the cleaning effect is achieved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a device for extracting secondary metabolites of mangrove actinomycetes. Background Technology

[0002] The mangrove actinomycete secondary metabolite extraction device is an integrated equipment system whose main function is to separate and enrich bioactive secondary metabolites (such as antibiotics, antitumor substances, enzyme inhibitors, etc.) from mangrove actinomycete fermentation broth or mycelium, providing a material basis for subsequent structural identification, activity evaluation and drug development.

[0003] However, the existing extraction devices still have the following technical problems when in use: (1) In the existing extraction device, after the initial extraction, the lower aqueous phase is directly poured out, and after the lower aqueous phase is extracted again, a small amount of extract is still obtained, which easily leads to waste of extract. (2) The current extraction device is inconvenient to clean during use, so a small amount of extract or ethanol may adhere to the inside of the extraction device, which may cause further use of the extraction device.

[0004] To address these issues, a device for extracting secondary metabolites from mangrove actinomycetes is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a device for extracting secondary metabolites of mangrove actinomycetes, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a device for extracting secondary metabolites of mangrove actinomycetes, comprising a base plate, a support plate fixed to the upper rear end of the base plate, a fixed box fixed to the front side of the support plate, an extraction tube rotatably connected inside the fixed box, a first discharge pipe fixed to the lower side of the extraction tube, a moving mechanism fixed to the upper end of the front side of the support plate, an ethanol adding mechanism fixed to the upper left end of the moving mechanism, and a washing water adding mechanism fixed to the upper right end of the moving mechanism; A rotating mechanism is fixed to the right end inside the fixed box. An aqueous phase collection tank is fixed to the upper side of the base plate. A reflux mechanism is fixed to the left side of the aqueous phase collection tank. An extract collection tank is fixed to the right end of the upper side of the base plate. A first pump body is fixed to the left end of the extract collection tank on the upper side of the base plate. A first connecting pipe is fixed to the inlet end of the first pump body. A second connecting pipe is slidably connected to the other end of the first connecting pipe. The other end of the second connecting pipe extends into the interior of the extraction tube. A third connecting pipe is fixed to the outlet end of the first pump body. The other end of the third connecting pipe extends to the upper end of the extract collection tank.

[0007] Preferably, an observation slot is provided on the front side of the extraction tube, and a first switching valve is fixed to the wall of the first discharge tube.

[0008] Preferably, the moving mechanism includes a strip plate, which is fixedly connected to the rear end of the front side of the support plate. A groove is provided on the upper side of the strip plate, and a lead screw is rotatably connected to the inside of the groove via a bearing. A housing is fixed to the left side of the strip plate, and a first motor is fixed inside the housing. The left end of the lead screw is fixedly connected to the output end of the first motor. A moving block is threadedly connected to the wall of the lead screw. The lower side of the moving block is slidably connected to the inner wall of the groove, and a moving plate is fixed to the upper side of the moving block.

[0009] Preferably, the ethanol adding mechanism includes an ethanol container, the lower side of which is fixedly connected to the upper side of the movable plate, a first adding tube is fixedly fixed to the upper side of the ethanol container, and a first vertical pipe is fixedly fixed to the front side of the ethanol container.

[0010] Preferably, the cleaning water adding mechanism includes a cleaning water holding tank, which is fixedly connected to the upper side of the movable plate. A second adding pipe is fixed to the upper side of the cleaning water holding tank, and a second vertical pipe is fixed to the front side of the cleaning water holding tank.

[0011] Preferably, the rotating mechanism includes a second motor, which is fixedly connected to the upper side of the fixed box. A rotating rod is fixed to the output end of the second motor. A first gear is fixed to the wall of the rotating rod, and a second gear is fixed to the wall of the extraction tube. The first gear meshes with the second gear.

[0012] Preferably, the reflux mechanism includes a second pump body, which is fixedly connected to the upper side of the base plate. A first reflux pipe is fixed to the liquid inlet end of the second pump body, and the other end of the first reflux pipe extends into the interior of the aqueous phase collection tank. A second reflux pipe is fixed to the liquid outlet end of the second pump body, and the other end of the second reflux pipe extends to the upper end of the extraction tube.

[0013] Preferably, the wall of the first connecting pipe is threaded with a fastening bolt, and one end of the fastening bolt extending into the inside of the first connecting pipe is engaged with the wall of the second connecting pipe; a second discharge pipe is fixed to the lower front side of the water phase collection tank, and a second switch valve is fixed to the wall of the second discharge pipe.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1) In the preliminary mixed extraction of secondary metabolites of mangrove actinomycetes by ethanol solution, the aqueous phase at the bottom can be discharged through the first discharge tube. After the extract collected in the extraction tube for the first time is poured out, the aqueous phase is refluxed back into the extraction tube, and then ethanol solution is added again for extraction, thereby improving the extraction effect of secondary metabolites of mangrove actinomycetes. 2) After the extraction process is completed, the upper cleaning water container can be moved by the moving mechanism, thereby injecting cleaning water into the extraction tube through the second vertical pipe. Through the operation of the reflux mechanism and the first pump, the cleaning water inside the extraction tube is circulated to achieve the cleaning effect. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the structure of the present invention; Figure 2 for Figure 1 A schematic diagram of the front structure; Figure 3 This is a top sectional view of the fixed box; Figure 4 This is a sectional view of the strip. Figure 5 for Figure 1 A magnified structural diagram of part A in the middle.

[0016] In the diagram: 1. Base plate, 2. Support plate, 3. Fixing box, 4. Extraction tube, 5. First discharge tube, 6. Aqueous phase collection tube, 7. Extraction liquid collection box, 8. First pump body, 9. First connecting tube, 10. Second connecting tube, 11. Third connecting tube, 12. Strip plate, 13. Lead screw, 14. Chassis, 15. First motor, 16. Moving block, 17. Moving plate, 18. Ethanol container, 19. First addition tube, 20. First vertical tube, 21. Cleaning water container, 22. Second addition tube, 23. Second vertical tube, 24. Second motor, 25. Rotating rod, 26. First gear, 27. Second gear, 28. Second pump body, 29. First reflux tube, 30. Second reflux tube, 31. Second discharge tube. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] In the description of this invention, it should be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0019] Example: Please see Figure 1-5 The present invention provides a technical solution: A device for extracting secondary metabolites of mangrove actinomycetes includes a base plate 1, a support plate 2 fixed to the upper rear end of the base plate 1, a fixed box 3 fixed to the front of the support plate 2, and an extraction tube 4 rotatably connected inside the fixed box 3. When the pretreated metabolites are poured into the extraction tube 4 and an ethanol solution is added accordingly, the extraction tube 4 is rotated by a rotating mechanism, thereby achieving the mixing of the ethanol solution and metabolites inside the extraction tube 4. A first discharge pipe 5 is fixed to the lower side of the extraction tube 4. A moving mechanism is fixed to the upper end of the front side of the support plate 2. An ethanol adding mechanism is fixed to the upper left end of the moving mechanism, and a cleaning water adding mechanism is fixed to the upper right end of the moving mechanism. The moving mechanism can drive the ethanol adding mechanism and the cleaning water adding mechanism to move. When it is necessary to inject ethanol solution into the device, the ethanol adding mechanism can be moved to the upper end of the extraction tube 4. When the extraction device is cleaned after use, the cleaning water adding mechanism can be moved to the upper end of the extraction tube 4. A rotating mechanism is fixed to the right end of the fixed box 3. An aqueous phase collection tank 6 is fixed to the upper side of the base plate 1. A reflux mechanism is fixed to the left side of the aqueous phase collection tank 6. An extract collection tank 7 is fixed to the right end of the upper side of the base plate 1. A first pump body 8 is fixed to the left end of the extract collection tank 7 on the upper side of the base plate 1. When the metabolites are mixed with the added ethanol for the first time, the lower aqueous phase will be discharged and collected through the aqueous phase collection tank 6. Finally, the collected aqueous phase is refluxed back to the interior of the extraction tube 4 through the reflux mechanism. Ethanol solution needs to be added a second time for extraction. A first connecting pipe 9 is fixed to the inlet end of the first pump body 8. A second connecting pipe 10 is slidably connected to the other end of the first connecting pipe 9. The other end of the second connecting pipe 10 extends into the interior of the extraction tube 4. A third connecting pipe 11 is fixed to the outlet end of the first pump body 8. The other end of the third connecting pipe 11 extends to the upper end of the extract collection tank 7.

[0020] An observation slot is provided on the front side of the extraction tube 4, and a first switch valve is fixed on the wall of the first discharge tube 5, so that the separation interface between the lower aqueous phase and the upper extract can be observed through the observation slot.

[0021] The moving mechanism includes a strip plate 12, which is fixedly connected to the rear end of the front side of the support plate 2. A groove is provided on the upper side of the strip plate 12, and a lead screw 13 is rotatably connected to the inside of the groove through a bearing. A housing 14 is fixed on the left side of the strip plate 12, and a first motor 15 is fixed inside the housing 14. The left end of the lead screw 13 is fixedly connected to the output end of the first motor 15. A moving block 16 is threadedly connected to the rod wall of the lead screw 13. The lower side of the moving block 16 is slidably connected to the inner wall of the groove. A moving plate 17 is fixed on the upper side of the moving block 16. The first motor 15 is a forward and reverse motor. The first motor 15 drives the lead screw 13 at the output end to rotate, and the lead screw 13 drives the moving block 16 threadedly connected to the rod wall to move. The moving block 16 drives the moving plate 17 on the upper side, as well as the ethanol adding mechanism and the cleaning water adding mechanism, to move left and right.

[0022] The ethanol addition mechanism includes an ethanol container 18, the lower side of which is fixedly connected to the upper side of a movable plate 17. A first addition tube 19 is fixedly attached to the upper side of the ethanol container 18, and a first vertical tube 20 is fixedly attached to the front side of the ethanol container 18.

[0023] The cleaning water adding mechanism includes a cleaning water container 21, which is fixedly connected to the upper side of the movable plate 17. A second adding pipe 22 is fixedly attached to the upper side of the cleaning water container 21, and a second vertical pipe 23 is fixedly attached to the front side of the cleaning water container 21.

[0024] The rotating mechanism includes a second motor 24, which is fixedly connected to the upper side of the fixed box 3. A rotating rod 25 is fixed to the output end of the second motor 24. A first gear 26 is fixed to the rod wall of the rotating rod 25. A second gear 27 is fixed to the tube wall of the extraction tube 4. The first gear 26 and the second gear 27 mesh.

[0025] The reflux mechanism includes a second pump body 28, which is fixedly connected to the upper side of the base plate 1. A first reflux pipe 29 is fixed to the liquid inlet end of the second pump body 28, and the other end of the first reflux pipe 29 extends into the interior of the aqueous phase collection tank 6. A second reflux pipe 30 is fixed to the liquid outlet end of the second pump body 28, and the other end of the second reflux pipe 30 extends into the upper end of the extraction tube 4.

[0026] The first connecting pipe 9 has a threaded fastening bolt on its wall. One end of the fastening bolt extends into the inside of the first connecting pipe 9 and engages with the wall of the second connecting pipe 10. When it is necessary to draw the extract from the upper end, the second connecting pipe 10 inside the first connecting pipe 9 is moved downward so that the lower end of the second connecting pipe 10 is inserted into the extract, and the extract is drawn and discharged into the extract collection tank 7. The lower front end of the aqueous phase collection tank 6 is fixed with a second discharge pipe 31, and a second switch valve is fixed to the wall of the second discharge pipe 31.

[0027] Working principle: Material pretreatment: Separation of mangrove actinomycete fermentation broth from mycelium: Take the mangrove actinomycete fermentation broth (fermentation cycle is usually 7-14 days, with the highest product yield after optimization), pour it into centrifuge tubes, and centrifuge at 4℃ and 8000r / min for 20min using a high-speed refrigerated centrifuge to separate the supernatant (containing extracellular metabolites) and precipitated mycelium (containing intracellular metabolites), and collect them separately for later use. If the salinity of the fermentation broth is high (mangrove actinomycete fermentation requires simulating a high-salt environment, with a salinity of 5-35‰), the mycelium precipitate needs to be washed 2-3 times with sterile deionized water (each time adding 5 times the volume of deionized water, vortexing for 1min, and then centrifuging) to remove surface salts (to avoid salt ions affecting extraction efficiency or causing product precipitation during subsequent concentration). Intracellular product disruption: The cell walls of *Actinomyces lindica* are relatively thick (containing peptidoglycan and lipopolysaccharide), necessitating cell wall disruption to release intracellular products. Efficient cell wall disruption methods are as follows: Ultrasonic disruption method: Add the mycelial precipitate to sterile deionized water to prepare a bacterial suspension of 100 g / L, and place it in an ultrasonic disruptor (power 300W, ultrasonic time 3s, interval time 5s, total duration 20min). Cool the product in an ice bath during the disruption process (keep the temperature ≤20℃) to avoid thermal degradation of the product. After cell wall disruption, the bacterial suspension was centrifuged again (4℃, 8000r / min, 15min), and the supernatant (intracellular product extract) was collected and combined with the extracellular product supernatant from step (1) to obtain the total crude extract. The mycelial residue can be retained for later use (to avoid product residue).

[0028] Reagent and equipment preparation: Ethanol solution: The concentration is selected according to the polarity of the target product. Commonly used gradient concentrations are 30%, 50%, 70%, and 95% (anhydrous ethanol). All are prepared with anhydrous ethanol and sterile deionized water and should be used immediately after preparation. Equipment: Separating funnel (500mL / 1000mL), high-speed refrigerated centrifuge, rotary evaporator, constant temperature water bath, ultrasonic cleaner, electronic balance, vacuum drying oven; Auxiliary reagents: anhydrous sodium sulfate (desiccant), filter membrane (0.45μm, organic phase).

[0029] Gradient extraction with ethanol solution: Single extraction: Take 100 mL of the total crude extract and pour it into a 500 mL extraction tube 4, according to a material-to-liquid ratio of 1:1 (V / V). Add the corresponding concentration of ethanol solution, start the second motor 24, the second motor 24 drives the output rod 25 to rotate, the rod 25 drives the lower first gear 26 to rotate, the first gear 26 drives the second gear 27 to rotate, the second gear 27 drives the extraction tube 4 to rotate, so that the extract and ethanol solution are shaken and mixed for 5 minutes; let the extraction tube 4 stand to separate the layers (stand at room temperature for 30 minutes, or refrigerate at 4℃ for 60 minutes to avoid product oxidation). At this time, the solution is divided into two layers, the upper layer is the ethanol extract phase (containing the target product), and the lower layer is the aqueous phase (containing salt and water-soluble impurities); slowly release the lower aqueous phase through the first discharge pipe 5, and collect the upper ethanol extract through the cooperation of the first pump body 8, the first connecting pipe 9, the second connecting pipe 10 and the third connecting pipe 11, and discharge it into the extract collection tank 7; the lower aqueous phase is discharged back into the interior of the extraction tube through the reflux mechanism, and 50 mL of the same concentration of ethanol solution is added again to repeat the extraction once, and the same steps are repeated, and the two extracts are combined.

[0030] Subsequent processing, dehydration and impurity removal: Add anhydrous sodium sulfate (5-10% of the extract volume, e.g., 5-10g per 100mL extract) to the combined ethanol extract, shake well and let stand for 30min. The anhydrous sodium sulfate will adsorb trace amounts of water in the extract. Filter with a 0.45μm organic phase filter membrane to remove anhydrous sodium sulfate and a small amount of insoluble impurities, and obtain a clear ethanol extract. Concentration under reduced pressure: Pour the clarified extract into the distillation flask of a rotary evaporator, set the parameters to water bath temperature 35-40℃ and vacuum degree 0.08-0.1MPa, start the rotary evaporator, and slowly concentrate until the volume of the extract is 1 / 10 of the original volume; if a solid crude extract is required, continue to concentrate until no ethanol is distilled off (ethanol recovery rate ≥90%), and a viscous paste is obtained. Then place it in a vacuum drying oven (40℃, vacuum degree 0.09MPa) to dry for 24 hours, and grind it to obtain a solid crude extract.

[0031] It should be noted that the electrical components mentioned in this invention have had their wiring harnesses combed according to actual conditions during manufacturing, so that the wiring harnesses will not be tangled or affect the operation. All standard parts used in this invention can be purchased from the market, and irregularly shaped parts can be customized according to the description and drawings. The specific connection methods of each part adopt conventional methods such as bolts, rivets, and welding that are mature in the prior art. The machinery, parts, and equipment all adopt conventional models in the prior art, and the circuit connection adopts conventional connection methods in the prior art, which will not be described in detail here. The control method of this invention is through a controller. The control circuit of the controller can be implemented by those skilled in the art through simple programming. It should be noted that the electrical components mentioned in this invention have been sorted according to the actual situation during manufacturing, so as not to cause the wire harness to become tangled or affect the operation. The contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0032] In the description of this invention, it should be understood that the indicated orientation or positional relationship is based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing this invention and simplifying the description, and is not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0033] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

[0034] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A device for extracting secondary metabolites of mangrove actinomycetes, comprising a base plate (1), characterized in that, A support plate (2) is fixed to the upper rear end of the base plate (1), a fixed box (3) is fixed to the front side of the support plate (2), an extraction tube (4) is rotatably connected inside the fixed box (3), a first discharge pipe (5) is fixed to the lower side of the extraction tube (4), a moving mechanism is fixed to the upper end of the front side of the support plate (2), an ethanol adding mechanism is fixed to the upper left end of the moving mechanism, and a cleaning water adding mechanism is fixed to the upper right end of the moving mechanism. A rotating mechanism is fixed to the right end inside the fixed box (3). An aqueous phase collection box (6) is fixed to the upper side of the base plate (1). A reflux mechanism is fixed to the left side of the aqueous phase collection box (6). An extract collection box (7) is fixed to the right end of the upper side of the base plate (1). A first pump body (8) is fixed to the left end of the extract collection box (7) on the upper side of the base plate (1). A first connecting pipe (9) is fixed to the inlet end of the first pump body (8). A second connecting pipe (10) is slidably connected to the other end of the first connecting pipe (9). The other end of the second connecting pipe (10) extends into the interior of the extraction tube (4). A third connecting pipe (11) is fixed to the outlet end of the first pump body (8). The other end of the third connecting pipe (11) extends into the upper end of the extract collection box (7).

2. The apparatus for extracting secondary metabolites of mangrove actinomycetes according to claim 1, characterized in that: The extraction tube (4) has an observation slot on its front side, and the first discharge tube (5) has a first switch valve fixed to its wall.

3. The apparatus for extracting secondary metabolites of mangrove actinomycetes according to claim 1, characterized in that: The moving mechanism includes a strip plate (12), which is fixedly connected to the rear end of the front side of the support plate (2). A sliding groove is provided on the upper side of the strip plate (12), and a lead screw (13) is rotatably connected to the inside of the sliding groove through a bearing. A housing (14) is fixed on the left side of the strip plate (12), and a first motor (15) is fixed inside the housing (14). The left end of the lead screw (13) is fixedly connected to the output end of the first motor (15). A moving block (16) is threadedly connected to the rod wall of the lead screw (13). The lower side of the moving block (16) is slidably connected to the inner wall of the sliding groove, and a moving plate (17) is fixed on the upper side of the moving block (16).

4. The apparatus for extracting secondary metabolites of mangrove actinomycetes according to claim 3, characterized in that: The ethanol addition mechanism includes an ethanol container (18), the lower side of which is fixedly connected to the upper side of a movable plate (17), a first addition tube (19) is fixedly attached to the upper side of the ethanol container (18), and a first vertical tube (20) is fixedly attached to the front side of the ethanol container (18).

5. The apparatus for extracting secondary metabolites of mangrove actinomycetes according to claim 1, characterized in that: The cleaning water adding mechanism includes a cleaning water container (21), which is fixedly connected to the upper side of the movable plate (17). A second adding pipe (22) is fixed to the upper side of the cleaning water container (21), and a second vertical pipe (23) is fixed to the front side of the cleaning water container (21).

6. The apparatus for extracting secondary metabolites of mangrove actinomycetes according to claim 1, characterized in that: The rotating mechanism includes a second motor (24), which is fixedly connected to the upper side of the fixed box (3). A rotating rod (25) is fixed to the output end of the second motor (24). A first gear (26) is fixed to the rod wall of the rotating rod (25), and a second gear (27) is fixed to the tube wall of the extraction tube (4). The first gear (26) meshes with the second gear (27).

7. The apparatus for extracting secondary metabolites of mangrove actinomycetes according to claim 1, characterized in that: The reflux mechanism includes a second pump body (28), which is fixedly connected to the upper side of the base plate (1). The inlet end of the second pump body (28) is fixed with a first reflux pipe (29), and the other end of the first reflux pipe (29) extends into the interior of the aqueous phase collection tank (6). The outlet end of the second pump body (28) is fixed with a second reflux pipe (30), and the other end of the second reflux pipe (30) extends into the upper end of the extraction tube (4).

8. The apparatus for extracting secondary metabolites of mangrove actinomycetes according to claim 1, characterized in that: The first connecting pipe (9) has a threaded fastening bolt on its wall. One end of the fastening bolt extends into the inside of the first connecting pipe (9) and is engaged with the wall of the second connecting pipe (10). The lower front end of the water phase collection tank (6) is fixed with a second discharge pipe (31), and the wall of the second discharge pipe (31) is fixed with a second switch valve.

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