Device and method for producing dextranase by using chaetomium gracile fermentation

Abstract

The invention discloses a device and method for producing dextranase through chaetomium gracile fermentation, and relates to the technical field of dextranase production.The device comprises a tank body and further comprises an oscillation stirring part, the oscillation stirring part comprises a rotating shaft which rotates and slides up and down on the tank body, and the rotating shaft is connected with the oscillation stirring part; a stirrer is fixedly mounted at one end, positioned in the tank body, of the rotating shaft; comprising the following steps: S1, taking out a chaetomium graciliforme strain from a glycerol tube at-80 DEG C, inoculating the chaetomium graciliforme strain to a potato dextrose agar slant culture medium in an ultra-clean workbench, and culturing at 28 DEG C for 5 days until a bacterial colony is in an off-white villus shape. The device has the advantages that the growth of thalli and the synthesis of dextranase can be effectively improved through up-and-down reciprocating oscillation, a fermentation sample can be automatically and quantitatively extracted at regular time in the up-and-down reciprocating oscillation process, manual sampling is not needed, improper influence on subsequent fermentation degree judgment caused by misoperation can be avoided, and the working efficiency is improved. And a worker can more accurately analyze and judge the fermentation progress.

Description

Technical Field

[0001] This invention relates to the field of dextranase production technology, and in particular to an apparatus and method for producing dextranase using *Chaetomium spp.* fermentation. Background Technology

[0002] Dextranase (α-1,6-glucanase) is a class of hydrolases that can specifically hydrolyze the α-1,6 glycosidic bonds in dextran. By degrading high molecular weight dextran into low molecular weight dextran, dextranase is widely used in the food, pharmaceutical, and petroleum industries.

[0003] As a safe strain, *Chaetoceros spp.* effectively overcomes the problems of complex extraction and purification processes, high costs, and safety hazards caused by the production of toxins in the metabolism of other microbial strains such as bacteria and fungi. It has become the preferred strain for dextranase production. However, existing equipment for the production of enzyme preparations by microbial fermentation has technical defects in adapting to the characteristics of *Chaetoceros spp.* and achieving efficient enzyme production, which seriously restricts the industrial application of *Chaetoceros spp.*

[0004] As a filamentous fungus, *Chaetoceros spp.* exhibits strong self-flocculation characteristics during fermentation. Insufficient stirring can lead to oxygen deficiency within the mycelial clusters, preventing nutrient penetration, while excess nutrients on the outside may produce metabolic byproducts (such as organic acids), ultimately inhibiting cell growth and dextranase synthesis. Existing devices have limited stirring capabilities and cannot adequately agitate the fermentation broth. Furthermore, existing devices cannot automatically sample the fermentation broth during fermentation, relying on manual operation, which makes it difficult to strictly perform timed and quantitative sampling and testing. Consequently, the enzyme production potential of *Chaetoceros spp.* cannot be fully released. Therefore, it is necessary to design an apparatus and method for producing dextranase using *Chaetoceros spp.* fermentation. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an apparatus and method for producing dextranase using *Chaetoceros spp.* fermentation, thus solving the problems mentioned in the background section.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: An apparatus for producing dextranase using *Chaetomium spp.* fermentation includes a tank and further includes: The oscillating and stirring section includes a rotating shaft that rotates and slides up and down on the tank body, and an agitator is fixedly installed at one end of the rotating shaft located inside the tank body. An automatic sampling unit includes an incomplete gear ring and multiple sampling and preservation components. Each sampling and preservation component includes a fixing block fixedly installed on the side wall of the tank, and a lead screw is rotatably installed on the fixing block. A fixed gear that cooperates with the incomplete gear ring is fixedly installed on the lead screw. The main control drive unit includes a fixed frame fixedly mounted on the tank body, and a motor fixedly mounted on the fixed frame. The output end of the motor is fixedly connected to a drive shaft, and an oscillation component is installed between the drive shaft and the rotating shaft. A differential transmission component that cooperates with the drive shaft and the incomplete gear ring is installed on the tank body.

[0007] Furthermore, a feed hopper and a discharge pipe are fixedly installed on the tank, and a temperature control device is installed on the tank.

[0008] Furthermore, the oscillation assembly consists of a telescopic rod, a telescopic groove, a spring, an arc-shaped block, and a top block. The telescopic rod is fixedly installed at the bottom of the drive shaft, the telescopic groove is opened on the rotating shaft, the spring is installed between the telescopic rod and the telescopic groove, the arc-shaped block is fixedly installed on the side wall of the rotating shaft, and the top block is fixedly installed at the bottom of the fixed frame, and the top block cooperates with the arc-shaped block.

[0009] Furthermore, a refrigerator and a extraction seat are fixedly installed on the side wall of the tank. A measuring cup is placed inside the refrigerator. A one-way discharge pipe is fixedly connected between the bottom of the extraction seat and the refrigerator. A one-way extraction pipe is fixedly connected between the extraction seat and the tank.

[0010] Furthermore, the tank is equipped with an opening and closing mechanism that cooperates with the one-way extraction tube. A moving block is threaded onto the lead screw, and an extraction mechanism that cooperates with the extraction seat is installed at the bottom of the moving block. The extraction mechanism cooperates with the opening and closing mechanism. A torsion spring is installed between the fixed gear and the fixed block. A touch switch that is electrically connected to the refrigerator is fixedly installed on the upper surface of the tank. A touch rod that cooperates with the touch switch is fixedly installed on the top of the incomplete gear ring.

[0011] Furthermore, the opening and closing mechanism consists of an arc-shaped groove, a round hole, an arc-shaped plate, and an alignment hole. The arc-shaped groove is formed inside the tank, the round hole is formed on the inner wall of the tank, and the round hole communicates with the arc-shaped groove. The round hole corresponds to the position of the one-way extraction tube away from the extraction seat. The arc-shaped plate is slidably installed in the arc-shaped groove, and the alignment hole is formed on the arc-shaped plate.

[0012] Furthermore, the extraction mechanism comprises a pull rod, a piston plate, a fixed rod, a lifting rod, and a limiting block. The pull rod is fixedly installed at the bottom of the moving block, the piston plate is slidably and sealed inside the extraction seat, the fixed rod is fixedly installed at the top of the piston plate, the lifting rod is slidably installed at the bottom of the pull rod and is fixedly connected to the fixed rod, the limiting block is fixedly installed on the top wall of the lifting rod, and a limiting groove that mates with the limiting block is provided inside the pull rod. A connecting block is fixedly installed on the side wall of the pull rod, an L-shaped rod is fixedly installed on the top of the arc plate, and a sliding groove that mates with the end of the L-shaped rod away from the arc plate is provided on the connecting block, and a spring is installed between the sliding groove and the L-shaped rod.

[0013] Furthermore, the differential transmission assembly comprises a transmission rod, a transmission gear, an internal gear ring, a connecting shaft, an incomplete gear, a connecting gear, and a belt drive structure. The transmission rod and the connecting shaft are rotatably mounted on the tank body. The transmission gear and the connecting gear are fixedly mounted on the transmission rod. The incomplete gear is fixedly mounted on the connecting shaft. The internal gear ring is fixedly mounted on the incomplete gear ring and meshes with the transmission gear. The connecting gear meshes with the incomplete gear. The belt drive structure is installed between the connecting shaft and the drive shaft.

[0014] Furthermore, the arc length of the toothed portion of the incomplete gear is equal to the circumference of the connecting gear.

[0015] A method for producing dextranase using *Chaetomium spp.* fermentation, employing the aforementioned apparatus for producing dextranase using *Chaetomium spp.* fermentation, includes the following steps: S1: Take out the *Chaetoceros spp.* strain from the -80℃ glycerol tube, inoculate it onto potato dextrose agar slant medium in a clean bench, and incubate at 28℃ for 5 days until the colonies are grayish-white and fluffy. Pick a single colony and transfer it to a new potato dextrose agar slant medium. Repeat the culture for 2-3 generations to ensure the viability of the strain. S2: Inoculate the activated strain into liquid seed culture medium (50 mL in a 250 mL Erlenmeyer flask, inoculation volume 3%), and incubate at 28℃ and 150 rpm for 48 hours until OD... 600 Reaching 0.8-1.0; S3: Take 25% by weight of crude dextran, 10% by weight of yeast extract, 2.5% by weight of KH2PO4, and 2.5% by weight of MgSO4·7H2O, dissolve them, add water to 100% to prepare crude dextran culture medium, adjust the pH to 5.5, then autoclave at 121℃ for 20 minutes, cool and aseptically dispense into tanks for fermentation. S4: During fermentation, inoculate 3% seed liquid into the tank, control the dissolved oxygen content in the tank to 20%-30%, the stirring speed of the stirrer to 100-200 r / min, the pH to be automatically maintained at 5.5, and the temperature to be controlled at 28℃. S5: The stirrer shakes and stirs the fermentation liquid in the tank to ensure the fermentation effect. At the same time, the extraction mechanism extracts the fermentation liquid in the tank into a measuring cup for storage in a timed and quantitative manner during the stirring process. S6: Measure the pH and OD of the fermentation broth in the measuring cup. 600 Adjust the pH value in the tank with 1M NaOH or HCl according to the pH change to maintain it at 5.5. At the same time, centrifuge the fermentation broth in the measuring cup and take the supernatant. Use the DNS method to detect enzyme activity. When the enzyme activity no longer increases significantly, stop the fermentation and discharge the fermentation broth in the tank from the discharge pipe and place it in a 4℃ refrigerator to terminate the reaction and avoid excessive degradation. S7: Centrifuge the cooled fermentation broth from S6 at 4℃ and 4000r / min for 15 minutes, collect the supernatant as crude enzyme solution, slowly add solid ammonium sulfate to the supernatant until 30% saturation, stir at 4℃ for 1 hour, centrifuge at 10000r / min for 20 minutes, and discard the precipitate. S8: Add ammonium sulfate to the supernatant until 60% saturation, centrifuge to collect the precipitate, dissolve the precipitate in 50mM sodium acetate buffer at pH 5.5, dialyze to remove salt for 24 hours, load the dialyzed enzyme solution onto a Sepharose 6B column, elute with 50mM sodium acetate buffer at pH 5.5 at a flow rate of 0.5 mL / min, and collect the activity peak.

[0016] Compared with existing technologies, the advantages of this invention are: 1. By cooperating with the drive shaft and the oscillation assembly, the stirrer can rotate and oscillate up and down in the tank while the motor is working, thereby improving the stirring effect of the fermentation liquid in the tank, making the seed liquid and the culture liquid fully mixed, promoting oxygen transfer in the tank, facilitating full contact between the *Trichoderma viride* and nutrients, promoting the growth of the cells and the synthesis of dextranase, and effectively improving the fermentation efficiency and fermentation effect.

[0017] 2: By combining the sampling and preservation components, differential transmission components, and incomplete gear ring, the motor can drive the incomplete gear ring to rotate intermittently, enabling timed and quantitative sampling of fermentation samples. This eliminates the need for manual sampling, avoiding operational errors that could negatively impact subsequent fermentation assessments. It also allows staff to more accurately analyze and judge the fermentation progress, thus enabling the development of large-scale production parameters.

[0018] In summary, reciprocating oscillation can effectively improve cell growth and dextranase synthesis. Furthermore, the reciprocating oscillation process can automatically extract fermentation samples at regular intervals and in quantitative quantities, eliminating the need for manual sampling. This avoids operational errors that could negatively impact subsequent fermentation assessments, allowing staff to more accurately analyze and judge the fermentation progress. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the apparatus for producing dextran anhydrase using *Chaetoceros spp.* fermentation, as proposed in this invention. Figure 2 for Figure 1 Schematic diagram of the structure of the middle tank; Figure 3 for Figure 1 A schematic diagram of the structure after removing the tank body; Figure 4 for Figure 1 Top view; Figure 5 for Figure 4 Schematic diagram of the structure of surface AA; Figure 6 for Figure 5 A schematic diagram of the three-dimensional structure; Figure 7 for Figure 6 Enlarged structural diagram of part a; Figure 8 for Figure 3 Exploded view of the structure at the stirrer; Figure 9 for Figure 8 Enlarged structural diagram of part b in the middle; Figure 10 for Figure 3 A schematic diagram showing the structure after removing the agitator; Figure 11 for Figure 10 An enlarged schematic diagram of the structure at one of the tie rods; Figure 12 for Figure 11 A structural decomposition diagram; Figure 13 for Figure 12 Enlarged schematic diagram of the structure at the piston plate.

[0020] In the diagram: 1. Tank body; 2. Feed hopper; 3. Discharge pipe; 4. Fixing frame; 5. Motor; 6. Drive shaft; 7. Rotating shaft; 8. Agitator; 9. Telescopic rod; 10. Telescopic groove; 11. Spring 1; 12. Arc-shaped block; 13. Top block; 14. Incomplete gear ring; 15. Fixing block; 16. Lead screw; 17. Fixed gear; 18. Torsion spring; 19. Moving block; 20. Extraction seat; 21. One-way extraction pipe; 22. Piston plate; 23. Pull rod; 24. Refrigerated display case; 25. One-way discharge pipe; 26. Measuring cup; 27. Arc groove; 28. Round hole; 29. ​​Arc plate; 30. Alignment hole; 31. L-shaped rod; 32. Connecting block; 33. Slide groove; 34. Spring II; 35. Fixing rod; 36. Lifting rod; 37. Limiting block; 38. Transmission rod; 39. Transmission gear; 40. Internal gear ring; 41. Connecting shaft; 42. Incomplete gear; 43. Connecting gear; 44. Belt drive structure; 45. Touch switch. Detailed Implementation

[0021] Reference Figure 1 An apparatus for producing dextranase using *Chaetoceros spp.* fermentation includes a tank 1 for fermentation. A feed hopper 2 and a discharge pipe 3 are fixedly installed on the tank 1. The feed hopper 2 is used to feed sterilized crude dextran culture medium and seed culture into the tank 1. The discharge pipe 3 is used to discharge the fermentation broth from the tank 1 for further processing. A valve is installed on the discharge pipe 3 to control its opening and closing. A temperature control device is installed on the tank 1 to monitor and control the temperature inside the tank 1. The temperature control device is an existing product, and its working principle and specific structure are not described here.

[0022] Reference Figures 1-13 An apparatus for producing dextranase using *Chaetoceros spp.* fermentation further includes a shaking and stirring unit. The shaking and stirring unit is used to shake and stir the fermentation broth located in the tank 1. The shaking and stirring unit includes a rotating shaft 7 that rotates and slides up and down on the tank 1. A stirrer 8 is fixedly installed at one end of the rotating shaft 7 located inside the tank 1. When the rotating shaft 7 rotates, it can drive the stirrer 8 to rotate inside the tank 1, thereby stirring the fermentation broth located in the tank 1, making it evenly mixed, and improving the fermentation effect and fermentation efficiency.

[0023] An automatic sampling unit is used to automatically and quantitatively sample during fermentation. The automatic sampling unit includes an incomplete gear ring 14 and multiple sampling and preservation components. The sampling and preservation components are used to extract and preserve samples at low temperatures. The sampling and preservation components include a fixing block 15 fixedly installed on the side wall of the tank 1, and a lead screw 16 is rotatably installed on the fixing block 15. A fixed gear 17 that cooperates with the incomplete gear ring 14 is fixedly installed on the lead screw 16. When the incomplete gear ring 14 rotates, the lead screw 16 can be driven to rotate through the meshing of its toothed part with the fixed gear 17. A torsion spring 18 is installed between the fixed gear 17 and the fixing block 15. Under the action of the torsion spring 18, when the fixed gear 17 is not driven by the incomplete gear ring 14, the stability of the lead screw 16 installed on the fixing block 15 can be maintained.

[0024] A refrigerator 24 and a sampling seat 20 are fixedly installed on the side wall of the tank body 1. A measuring cup 26 is placed inside the refrigerator 24. A touch switch 45 electrically connected to the refrigerator 24 is fixedly installed on the upper surface of the tank body 1. A touch rod that cooperates with the touch switch 45 is fixedly installed on the top of the incomplete gear ring 14. The refrigerator 24 is used for refrigerating the sample, and the measuring cup 26 is used to hold the sample, making it convenient for staff to take the sample out of the refrigerator 24 for testing and analysis. The touch switch 45 is used to control the opening and closing of the refrigerator 24. When the incomplete gear ring 14 rotates, the touch rod engages with the touch switch 45. When activated, the refrigerator 24 will begin cooling. By placing the touch lever at the front end of the toothed portion of the incomplete gear ring 14 along the rotation direction and adjusting the installation positions of the touch switch 45 and the corresponding fixed gear 17, the refrigerator 24 can be cooled before the incomplete gear ring 14 drives the fixed gear 17 to rotate. This ensures the effective preservation of subsequently extracted samples and avoids increased operating costs caused by prolonged operation of the refrigerator 24. Both the touch switch 45 and the refrigerator 24 are existing products, and their working principles and specific structures will not be described here.

[0025] A one-way discharge pipe 25 is fixedly connected between the bottom of the extraction seat 20 and the refrigerator 24, and a one-way extraction pipe 21 is fixedly connected between the extraction seat 20 and the tank 1. Through the design of the one-way extraction pipe 21, the fermentation liquid in the tank 1 can be drawn into the extraction seat 20 in one direction. Then, under the design of the one-way discharge pipe 25, the fermentation liquid drawn into the extraction seat 20 is discharged into the measuring cup 26 in the refrigerator 24 for storage. A moving block 19 is threaded on the screw 16, and an extraction mechanism that cooperates with the extraction seat 20 is installed at the bottom of the moving block 19. The extraction mechanism consists of a pull rod 23, a piston plate 22, a fixed rod 35, a lifting rod 36, and a limiting block 37. The pull rod 23 is fixedly installed at the bottom of the moving block 19. The piston plate 22 is slidably and sealed inside the extraction seat 20. The fixed rod 35 is fixedly installed at the top of the piston plate 22. The lifting rod 36 is slidably installed at the bottom of the pull rod 23 and is fixedly connected to the fixed rod 35. The limiting block 37 is fixedly installed on the top wall of the lifting rod 36, and a limiting groove that mates with the limiting block 37 is provided inside the pull rod 23. When the lead screw 16 rotates due to the rotation of the incomplete gear ring 14, it drives the pull rod 23 to move upward through the moving block 19. At this time, the pull rod 23 moves upward relative to the fixed rod 35. When the fixed rod 35 rises... When the lowering rod 36 moves to its maximum distance relative to the pull rod 23, the upward movement of the pull rod 23 causes the fixed rod 35 to move upward simultaneously. At this time, the piston plate 22 moves upward within the extraction seat 20, allowing the fermentation liquid in the tank 1 to be extracted. When the incomplete gear ring 14 separates from the fixed gear 17, the elastic force of the torsion spring 18 causes the fixed gear 17 to reset and rotate. At this time, the rotation of the lead screw 16 causes the moving block 19 to drive the pull rod 23 downward. Similarly, when the pull rod 23 moves downward, it does not drive the piston plate 22 downward at first. After the pull rod 23 moves downward a certain distance, causing the lifting rod 36 to move to its maximum distance within the pull rod 23, the piston plate 22 moves downward as well, allowing the fermentation liquid in the extraction seat 20 to be input into the measuring cup 26 through the one-way discharge pipe 25.

[0026] The tank body 1 is equipped with an opening and closing mechanism that cooperates with the one-way extraction tube 21. The opening and closing mechanism consists of an arc groove 27, a round hole 28, an arc plate 29, and an alignment hole 30. The arc groove 27 is opened inside the tank body 1, and the round hole 28 is opened on the inner wall of the tank body 1. The round hole 28 and the arc groove 27 are interconnected. The round hole 28 corresponds to the end of the one-way extraction tube 21 away from the extraction seat 20. The arc plate 29 is slidably installed in the arc groove 27. The alignment hole 30 is opened on the arc plate 29. When the position of the arc plate 29 in the arc groove 27 remains unchanged, the positions of the alignment hole 30 and the round hole 28 are staggered. At this time, the round hole 28 is in a blocked state, and the one-way extraction tube 21 and the tank body 1 are in a closed state. This can avoid the problem that the rotation of the stirrer 8 during the fermentation process causes the fermentation liquid to shake, resulting in some sediment adhering to the opening of the one-way extraction tube 21, which leads to poor subsequent sampling or inconsistent sampling volume each time.

[0027] A connecting block 32 is fixedly installed on the side wall of the pull rod 23, and an L-shaped rod 31 is fixedly installed on the top of the arc plate 29. A groove 33 is provided on the connecting block 32 to mate with the end of the L-shaped rod 31 away from the arc plate 29. A spring 34 is installed between the groove 33 and the L-shaped rod 31. Through the cooperation of the connecting block 32 and the spring 34, the L-shaped rod 31 can move a certain distance on the connecting block 32. Furthermore, through the sliding cooperation between the lifting rod 36 and the pull rod 23, the L-shaped rod 31 can move upwards by first passing through the groove 32. The rod 31 drives the arc plate 29 to move upward, so that the alignment hole 30 and the round hole 28 on it correspond to maintain the connection between the one-way extraction tube 21 and the tank 1. Then the sample extraction process is carried out. During the process of transferring the sample to the measuring cup 26 after the extraction is completed, the pull rod 23 moves downward to drive the arc plate 29 to move downward and reset to maintain the closure between the one-way extraction tube 21 and the tank 1 before the sample is transferred. This can avoid the pressure change in the extraction seat 20 during the sample extraction process from having an adverse effect on the fermentation of the fermentation liquid in the tank 1.

[0028] The main control drive unit provides independent drive for the rotation of the rotating shaft 7 and the incomplete gear ring 14. The main control drive unit includes a fixed frame 4 fixedly mounted on the tank body 1, and a motor 5 fixedly mounted on the fixed frame 4. The output end of the motor 5 is fixedly connected to a drive shaft 6, and an oscillation assembly is installed between the drive shaft 6 and the rotating shaft 7. The oscillation assembly consists of a telescopic rod 9, a telescopic groove 10, a spring 11, an arc-shaped block 12, and a top block 13. The telescopic rod 9 is fixedly mounted on the bottom of the drive shaft 6, the telescopic groove 10 is formed on the rotating shaft 7, the spring 11 is installed between the telescopic rod 9 and the telescopic groove 10, the arc-shaped block 12 is fixedly mounted on the side wall of the rotating shaft 7, and the top block 13 is fixedly mounted on the bottom of the fixed frame 4, with the top block 13 cooperating with the arc-shaped block 12. The oscillation assembly extends... The cooperation between the retractable rod 9 and the telescopic groove 10 allows the rotating shaft 7 to move up and down relative to the drive shaft 6. With the cooperation of the top block 13 and the arc-shaped block 12, when the drive shaft 6 rotates, causing the rotating shaft 7 to rotate, the arc-shaped block 12 also rotates relative to the top block 13. At this time, the pushing effect of the top block 13 on the arc-shaped block 12 and the elastic force of the spring 11 cause the rotating shaft 7 to reciprocate up and down relative to the drive shaft 6. Combined with the rotation of the drive shaft 6, this allows the stirrer 8 to rotate and oscillate up and down within the tank 1, thereby improving the stirring effect on the fermentation liquid within the tank 1 and facilitating better fermentation. Specifically, the motor 5 can be a common unidirectional motor used in daily work, with its operating direction set to counterclockwise (towards...). Figure 4 (Taking the direction shown as an example).

[0029] A differential transmission assembly is installed on the tank body 1, which meshes with the drive shaft 6 and the incomplete gear ring 14. The differential transmission assembly is used to allow the incomplete gear ring 14 to rotate intermittently in fixed angular increments. The differential transmission assembly consists of a transmission rod 38, a transmission gear 39, an internal gear ring 40, a connecting shaft 41, an incomplete gear 42, a connecting gear 43, and a belt drive structure 44. The transmission rod 38 and the connecting shaft 41 are rotatably mounted on the tank body 1. The transmission gear 39 and the connecting gear 43 are fixedly mounted on the transmission rod 38. The incomplete gear 42 is fixedly mounted on the connecting shaft 41. The internal gear ring 40 is fixedly mounted on the incomplete gear ring 14, and the internal gear ring 40 is meshed with the transmission gear 39. The connecting gear 43 meshes with the incomplete gear 42. The belt drive structure 44 is installed between the connecting shaft 41 and the drive shaft 6. The arc length of the toothed part on the incomplete gear 42 is equal to the circumference of the connecting gear 43. During the rotation of the drive shaft 6, the belt drive structure 44 drives the connecting shaft 41 to rotate simultaneously. When the connecting shaft 41 rotates at a certain angle so that the incomplete gear 42 meshes with the connecting gear 43, the connecting shaft 41 drives the transmission rod 38 to rotate simultaneously. Then, the meshing effect of the transmission gear 39 and the internal gear ring 40 causes the incomplete gear ring 14 to rotate. The meshing effect of the toothed part on the incomplete gear ring 14 and the fixed gear 17 drives the lead screw 16 to rotate. The belt drive structure 44 consists of pulley one, pulley two, and a belt. Pulley one is fixed on the drive shaft 6, pulley two is fixed on the connecting shaft 41, and the belt is installed between pulley one and pulley two. Figure 9 As can be seen, the diameter of pulley one is smaller than that of pulley two, the diameter of the incomplete gear 42 is larger than that of the connecting gear 43, and the diameter of the transmission gear 39 is smaller than that of the connecting gear 43. Therefore, through the above-mentioned size design, the drive shaft 6 can slowly drive the connecting shaft 41 to rotate during rotation. The design that the size of the incomplete gear 42 is larger than that of the connecting gear 43 allows the incomplete gear 42 to drive the transmission rod 38 to rotate faster when meshing with the connecting gear 43. The larger size of the transmission gear 39 allows the incomplete gear ring 14 to rotate at a relatively fast speed under the driving action of the transmission rod 38. Through the above-mentioned different transmission effect design, the incomplete gear ring 14 can have sufficient interval rotation time each time, and can drive the lead screw 16 to rotate quickly to complete the sampling process. Thus, it can not only extract samples in a timely and quantitative manner according to the fermentation process, but also avoid the problem that the large time span corresponding to the sample due to the excessively long sampling time each time affects the accuracy of the test results.

[0030] This invention discloses a method for producing dextranase using *Chaetomium spp.* fermentation, comprising the following steps: S1: Take out the *Chaetoceros spp.* strain from the -80℃ glycerol tube, inoculate it onto potato dextrose agar slant medium in a clean bench, and incubate at 28℃ for 5 days until the colonies are grayish-white and fluffy. Pick a single colony and transfer it to a new potato dextrose agar slant medium. Repeat the culture for 2-3 generations to ensure the viability of the strain. S2: Inoculate the activated strain into liquid seed culture medium (50 mL in a 250 mL Erlenmeyer flask, inoculation volume 3%), and incubate at 28℃ and 150 rpm for 48 hours until OD... 600 Reaching 0.8-1.0; S3: Take 25% by weight of crude dextran, 10% by weight of yeast extract, 2.5% by weight of KH2PO4, and 2.5% by weight of MgSO4·7H2O, dissolve them, add water to 100% to prepare crude dextran culture medium, adjust the pH to 5.5, then autoclave at 121℃ for 20 minutes, cool and aseptically dispense into tanks for fermentation. S4: During fermentation, inoculate 3% seed liquid into the tank, control the dissolved oxygen content in the tank to 20%-30%, the stirring speed of the stirrer to 100-200 r / min, the pH to be automatically maintained at 5.5, and the temperature to be controlled at 28℃. S5: The stirrer shakes and stirs the fermentation liquid in the tank to ensure the fermentation effect. At the same time, the extraction mechanism extracts the fermentation liquid in the tank into a measuring cup for storage in a timed and quantitative manner during the stirring process. S6: Measure the pH and OD of the fermentation broth in the measuring cup. 600 Adjust the pH value in the tank with 1M NaOH or HCl according to the pH change to maintain it at 5.5. At the same time, centrifuge the fermentation broth in the measuring cup and take the supernatant. Use the DNS method to detect enzyme activity. When the enzyme activity no longer increases significantly, stop the fermentation and discharge the fermentation broth in the tank from the discharge pipe and place it in a 4℃ refrigerator to terminate the reaction and avoid excessive degradation. S7: Centrifuge the cooled fermentation broth from S6 at 4℃ and 4000r / min for 15 minutes, collect the supernatant as crude enzyme solution, slowly add solid ammonium sulfate to the supernatant until 30% saturation, stir at 4℃ for 1 hour, centrifuge at 10000r / min for 20 minutes, and discard the precipitate. S8: Add ammonium sulfate to the supernatant until 60% saturation, centrifuge to collect the precipitate, dissolve the precipitate in 50mM sodium acetate buffer at pH 5.5, dialyze to remove salt for 24 hours, load the dialyzed enzyme solution onto a Sepharose 6B column, elute with 50mM sodium acetate buffer at pH 5.5 at a flow rate of 0.5 mL / min, and collect the activity peak.

[0031] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. An apparatus for producing dextranase using *Chaetoceros spp.* fermentation, comprising a tank, characterized in that, Also includes: The oscillating and stirring section includes a rotating shaft that rotates and slides up and down on the tank body, and an agitator is fixedly installed at one end of the rotating shaft located inside the tank body. An automatic sampling unit includes an incomplete gear ring and multiple sampling and preservation components. Each sampling and preservation component includes a fixing block fixedly installed on the side wall of the tank, and a lead screw is rotatably installed on the fixing block. A fixed gear that cooperates with the incomplete gear ring is fixedly installed on the lead screw. The main control drive unit includes a fixed frame fixedly mounted on the tank body, and a motor fixedly mounted on the fixed frame. The output end of the motor is fixedly connected to a drive shaft, and an oscillation component is installed between the drive shaft and the rotating shaft. A differential transmission component that cooperates with the drive shaft and the incomplete gear ring is installed on the tank body.

2. The apparatus for producing dextranase using *Chaetoceros spp.* fermentation according to claim 1, characterized in that, The tank is fixedly equipped with a feed hopper and a discharge pipe, and a temperature control device is installed on the tank.

3. The apparatus for producing dextranase using *Chaetomium spp.* fermentation according to claim 1, characterized in that, The oscillation assembly consists of a telescopic rod, a telescopic groove, a spring, an arc-shaped block, and a top block. The telescopic rod is fixedly installed at the bottom of the drive shaft, the telescopic groove is opened on the rotating shaft, the spring is installed between the telescopic rod and the telescopic groove, the arc-shaped block is fixedly installed on the side wall of the rotating shaft, and the top block is fixedly installed at the bottom of the fixed frame, and the top block cooperates with the arc-shaped block.

4. The apparatus for producing dextranase using *Chaetomium spp.* fermentation according to claim 1, characterized in that, A refrigerator and a drawer are fixedly installed on the side wall of the tank. A measuring cup is placed inside the refrigerator. A one-way discharge pipe is fixedly connected between the bottom of the drawer and the refrigerator. A one-way draw pipe is fixedly connected between the drawer and the tank.

5. The apparatus for producing dextranase using *Chaetoceros spp.* fermentation according to claim 4, characterized in that, The tank is equipped with an opening and closing mechanism that cooperates with the one-way extraction tube. A moving block is threaded onto the lead screw, and an extraction mechanism that cooperates with the extraction seat is installed at the bottom of the moving block. The extraction mechanism cooperates with the opening and closing mechanism. A torsion spring is installed between the fixed gear and the fixed block. A touch switch that is electrically connected to the refrigerator is fixedly installed on the upper surface of the tank. A touch rod that cooperates with the touch switch is fixedly installed on the top of the incomplete gear ring.

6. The apparatus for producing dextranase using *Chaetoceros spp.* fermentation according to claim 5, characterized in that, The opening and closing mechanism consists of an arc-shaped groove, a round hole, an arc-shaped plate, and an alignment hole. The arc-shaped groove is formed inside the tank, and the round hole is formed on the inner wall of the tank. The round hole communicates with the arc-shaped groove. The round hole corresponds to the end of the one-way extraction tube away from the extraction seat. The arc-shaped plate is slidably installed in the arc-shaped groove, and the alignment hole is formed on the arc-shaped plate.

7. The apparatus for producing dextranase using *Chaetoceros spp.* fermentation according to claim 6, characterized in that, The extraction mechanism consists of a pull rod, a piston plate, a fixed rod, a lifting rod, and a limiting block. The pull rod is fixedly installed at the bottom of the moving block. The piston plate is slidably and sealingly installed inside the extraction seat. The fixed rod is fixedly installed at the top of the piston plate. The lifting rod is slidably installed at the bottom of the pull rod and is fixedly connected to the fixed rod. The limiting block is fixedly installed on the top wall of the lifting rod, and a limiting groove that mates with the limiting block is provided inside the pull rod. A connecting block is fixedly installed on the side wall of the pull rod. An L-shaped rod is fixedly installed on the top of the arc-shaped plate. A sliding groove that mates with the end of the L-shaped rod away from the arc-shaped plate is provided on the connecting block, and a spring is installed between the sliding groove and the L-shaped rod.

8. The apparatus for producing dextranase using *Chaetoceros spp.* fermentation according to claim 1, characterized in that, The differential transmission assembly consists of a transmission rod, a transmission gear, an internal gear ring, a connecting shaft, an incomplete gear, a connecting gear, and a belt drive structure. The transmission rod and the connecting shaft are rotatably mounted on the tank body. The transmission gear and the connecting gear are fixedly mounted on the transmission rod. The incomplete gear is fixedly mounted on the connecting shaft. The internal gear ring is fixedly mounted on the incomplete gear ring and meshes with the transmission gear. The connecting gear meshes with the incomplete gear. The belt drive structure is installed between the connecting shaft and the drive shaft.

9. The apparatus for producing dextranase using *Chaetomium spp.* fermentation according to claim 8, characterized in that, The arc length of the toothed portion of the incomplete gear is equal to the circumference of the connecting gear.

10. A method for producing dextranase by fermentation using *Chaetoceros spp.*, comprising the apparatus for producing dextranase by fermentation using *Chaetoceros spp.* as described in claim 9, characterized in that... Includes the following steps: S1: Take out the *Chaetoceros spp.* strain from the -80℃ glycerol tube, inoculate it onto potato dextrose agar slant medium in a clean bench, and incubate at 28℃ for 5 days until the colonies are grayish-white and fluffy. Pick a single colony and transfer it to a new potato dextrose agar slant medium. Repeat the culture for 2-3 generations to ensure the viability of the strain. S2: Inoculate the activated strain into liquid seed culture medium (50 mL in a 250 mL Erlenmeyer flask, inoculation volume 3%), and incubate at 28℃ and 150 rpm for 48 hours until OD... 600 Reaching 0.8-1.0; S3: Take 25% by weight of crude dextran, 10% by weight of yeast extract, 2.5% by weight of KH2PO4, and 2.5% by weight of MgSO4·7H2O, dissolve them, add water to 100% to prepare crude dextran culture medium, adjust the pH to 5.5, then autoclave at 121℃ for 20 minutes, cool and aseptically dispense into tanks for fermentation. S4: During fermentation, inoculate 3% seed liquid into the tank, control the dissolved oxygen content in the tank to 20%-30%, the stirring speed of the stirrer to 100-200 r / min, the pH to be automatically maintained at 5.5, and the temperature to be controlled at 28℃. S5: The stirrer shakes and stirs the fermentation liquid in the tank to ensure the fermentation effect. At the same time, the extraction mechanism extracts the fermentation liquid in the tank into a measuring cup for storage in a timed and quantitative manner during the stirring process. S6: Measure the pH and OD of the fermentation broth in the measuring cup. 600 Adjust the pH value in the tank with 1M NaOH or HCl according to the pH change to maintain it at 5.

5. At the same time, centrifuge the fermentation broth in the measuring cup and take the supernatant. Use the DNS method to detect enzyme activity. When the enzyme activity no longer increases significantly, stop the fermentation and discharge the fermentation broth in the tank from the discharge pipe and place it in a 4℃ refrigerator to terminate the reaction and avoid excessive degradation. S7: Centrifuge the cooled fermentation broth from S6 at 4℃ and 4000r / min for 15 minutes, collect the supernatant as crude enzyme solution, slowly add solid ammonium sulfate to the supernatant until 30% saturation, stir at 4℃ for 1 hour, centrifuge at 10000r / min for 20 minutes, and discard the precipitate. S8: Add ammonium sulfate to the supernatant until 60% saturation, centrifuge to collect the precipitate, dissolve the precipitate in 50mM sodium acetate buffer at pH 5.5, dialyze to remove salt for 24 hours, load the dialyzed enzyme solution onto a Sepharose 6B column, elute with 50mM sodium acetate buffer at pH 5.5 at a flow rate of 0.5 mL / min, and collect the activity peak.

Images