Fermentation process for improving production level of recombinant collagen

Abstract

The invention discloses a fermentation process for improving the production level of recombinant collagen, and relates to the field of fermentation devices. A fermentation process for improving the production level of recombinant collagen comprises a fermentation tank, and further comprises a plurality of groups of transmission shafts which are rotationally connected in the fermentation tank; the limiting groove is formed in the transmission shaft; the first mounting ring is slidably connected to the limiting groove of the transmission shaft, an inflatable air bag is fixedly connected to the first mounting ring and filled with gas, the inflatable air bag is affected by the weight of the first mounting ring, the inflatable air bag and the first mounting ring are located at the bottom of the transmission shaft in daily use, and the inflatable air bag can expand under the influence of external temperature. When the volume is increased, the drainage performance is improved; according to the present invention, with the inflation air bag filled with helium gas, the high temperature at the bottom can be transferred to the upper side through the inflation air bag during the use, such that the accumulation and the concentration of the heat are avoided, the temperature in the fermentation tank is balanced, the growth of the strain is convenient, and the subsequent collagen preparation is convenient.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation devices, and in particular relates to a fermentation process for improving the production level of recombinant collagen. Background Art

[0002] Collagen is the most abundant protein in the body and the main component of the extracellular matrix (ECM). It plays an important role in maintaining the normal physiological functions of cells, tissues, and organs and repairing injuries. Because of its unique structure, collagen has excellent biocompatibility and low immunogenicity, and has been widely used in the pharmaceutical, health care and cosmetic industries.

[0003] In the existing technology, the main source of collagen is animal tissue extraction. With the development of biotechnology, the recombinant collagen obtained by microbial fermentation using genetic recombination technology has achieved great results. Compared with natural collagen, the recombinant collagen produced using this technology solves the viral risks of traditional extraction methods, while significantly improving the stability, hydrophilicity and biocompatibility of collagen.

[0004] The existing fermentation process for collagen production level, such as the invention patent with patent application number CN202110999056.2 and the name of a fermentation process for improving the production level of recombinant collagen, provides a fermentation process for improving the production level of recombinant collagen, and relates to the field of genetic recombinant engineering bacteria fermentation technology. A fermentation process for improving the production level of recombinant collagen, comprising the following steps: S1: inoculate the Pichia pastoris engineered bacteria in a seed tank for culture, add glycerol and feed medium after the dissolved oxygen rises significantly, and when the wet weight increases to more than 100g / L, transplant to a fermentation tank to start fermentation culture; S2: culture the Pichia pastoris engineered bacteria in the fermentation tank until the dissolved oxygen rises significantly, use carbon source medium and feed medium to mix carbon source feed, and stop mixing carbon source feed when the wet weight of the material increases to more than 150g / L; S3: after the carbon source is exhausted, add methanol and feed medium to induce expression until the fermentation ends. The invention can increase the expression amount of recombinant collagen and shorten the fermentation cycle, thereby improving the fermentation production level of recombinant collagen and is suitable for stable industrial production.

[0005] When the above-mentioned device is in use, a fermentation tank is required to mix the bacteria with other culture medium liquids, and then ferment in a constant temperature aerobic environment. In actual use, since the heating method of the fermentation tank is generally to heat the outer wall, the temperature inside the fermentation tank reaches the requirement through heat transfer. In this process, the liquid temperature at the heating end is higher than the liquid in the center area of ​​the fermentation tank, resulting in heat imbalance. There is an imbalance in the growth of bacteria in the fermentation tank in different areas, which in turn affects the yield of collagen finally extracted, and there are certain shortcomings. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a fermentation device that can overcome the above problems or at least partially solve the above problems.

[0007] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is: a fermentation process for improving the production level of recombinant collagen, comprising a fermentation tank, and also comprising:

[0008] A plurality of transmission shafts are rotatably connected in the fermentation tank;

[0009] A limiting groove is arranged on the transmission shaft;

[0010] The mounting ring 1 is slidably connected to the limiting groove of the transmission shaft, and the mounting ring 1 is fixedly connected to an inflatable airbag, which is filled with gas. Affected by the weight of the mounting ring 1, the inflatable airbag and the mounting ring 1 are located at the bottom of the transmission shaft in daily use. Affected by the external temperature, the inflatable airbag will expand, and its volume will increase while its own drainage, thereby offsetting the influence of the weight of the mounting ring 1, and driving the mounting ring 1 to move upward along the limiting groove;

[0011] A plurality of groups of spoilers, all fixedly connected to the inflatable airbag, are used to disrupt the flow of the liquid;

[0012] The second mounting ring is slidably connected to the limiting groove of the transmission shaft. The second mounting ring is connected to multiple groups of floating rods that can float on water. The multiple groups of floating rods can overcome the weight of the second mounting ring and drive the second mounting ring to float on the water surface. When the transmission shaft rotates, under the influence of the limiting groove, the floating rods will play the role of a stirring shaft, stirring the liquid surface, thereby forming turbulence and increasing the mixing rate between the liquids.

[0013] Furthermore, the float rod is provided with a plurality of groups of conical holes, and the conical holes are arranged at an angle. When the float rod rotates, liquid is sucked into the large end of the conical holes, and the liquid is discharged from the smaller end by pressurization. When the large end of the conical hole is close to the side of the float rod close to the water surface and the small end is close to the side away from the water surface, the liquid will be sprayed into the air.

[0014] Furthermore, a motor is fixedly connected to the fermentation tank, a rotating shaft is rotatably connected to the output end of the motor inside the fermentation tank, the rotating shaft is located on the axis of the fermentation tank, a gear part 1 is fixedly connected to the output end of the rotating shaft, and a gear part 2 meshing with the gear part 1 is fixedly connected to the transmission shaft.

[0015] In order to facilitate the generation of more bubbles and increase the contact between the bacteria and oxygen, further, multiple groups of arcuate walls are arranged on the inner wall of the top of the fermentation tank, and multiple groups of air outlets are arranged at the bottom of the fermentation tank. Multiple groups of piston members are fixedly connected to the rotating shaft, and the telescopic end of the piston member is against the arcuate wall. Through the rotation of the rotating shaft, the piston member will suck the gas at the top of the fermentation tank, and then discharge the gas through the air outlet end.

[0016] In order to facilitate the dispersion of bubbles, further, the air outlets are arranged in a ring shape at equal intervals on the transmission shaft.

[0017] Furthermore, the piston member includes a piston cylinder, a piston rod, and a spring member. The multiple groups of piston cylinders are fixedly connected to the rotating shaft in a ring shape at equal intervals. The piston cylinder is provided with an air inlet and an air outlet with a one-way valve. The piston rod is slidably connected in the piston cylinder. The piston cylinder is provided with a spring member whose two ends are respectively connected to the piston cylinder and the piston rod. The piston rod is the telescopic end of the piston member. The spring member can make the piston rod always abut against the arc wall. Through the rotation of the rotating shaft, the piston cylinder, under the cooperation of the spring member and the piston rod, continuously sucks in the outside gas through the air inlet, and finally discharges it to the outlet through the air outlet.

[0018] Furthermore, an air pipe is arranged in the rotating shaft, and two ends of the air pipe are respectively connected to the air outlet and the air outlet hole of the piston cylinder.

[0019] Further, the following steps are included:

[0020] S1: Inoculate the Pichia pastoris engineered bacteria into a seed tank for cultivation, add glycerol and feed medium after the dissolved oxygen rises significantly, and transfer to a fermentation tank to start fermentation culture when the wet weight increases to more than 100 g / L;

[0021] S2: After the Pichia pastoris engineered bacteria in the fermentation tank are cultured until the dissolved oxygen rises significantly, a carbon source medium and a feed medium are used to mix the carbon source feed, and when the wet weight of the material increases to more than 150 g / L, the mixed carbon source feed is stopped;

[0022] S3: After the carbon source is exhausted, methanol and feed medium are added to induce expression until the end of fermentation.

[0023] Furthermore, the Pichia yeast engineered bacteria fermentation medium used in the seed tank and the fermentation tank in step S1 includes components with the following concentrations: CaSO4·2H2O is 0.52-1.125 g / L, K2SO4 is 7.42-17.8 g / L, MgSO4·7H2O is 6.12-13.5 g / L, KOH is 1.715-3.92 g / L, glycerol is 21-37 g / L; glucose is 20-40 g / L; (NaPO3)6 is 6.5-13 g / L, and white corn syrup dry powder is 37 g / L.

[0024] Further, the feed medium includes components with the following concentrations: CaSO4·2H2O is 0.1-0.705g / L; K2SO4 is 0.15-10.92g / L; MgSO4·7H2O is 0.25-8.94g / L; KOH is 0.12-2.478g / L; white corn syrup dry powder is 0.3-4g / L; the carbon source medium in step S2 is glycerol, glucose or a mixed medium of glycerol and glucose; the fermentation culture conditions are: fermentation temperature 28-32°C, ammonia water adjusts the pH to 4.8-5.5, dissolved oxygen before induction expression is not less than 30%, tank pressure is 0.04-0.06MPa, dissolved oxygen after induction expression is not less than 5%, and tank pressure is 0.08-0.1MPa.

[0025] After adopting the above technical scheme, the present invention has the following beneficial effects compared with the prior art: the present invention uses an inflatable airbag filled with helium, which can transfer the higher temperature at the bottom to the top through the inflatable airbag when in use, thereby avoiding the accumulation and concentration of heat, promoting the temperature in the fermentation tank to be constant, facilitating the growth of bacteria, and facilitating the subsequent preparation of collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In the attached picture:

[0027] Figure 1 A schematic diagram of a fermentation process for improving the production level of recombinant collagen proposed by the present invention Figure 1 ;

[0028] Figure 2 A schematic diagram of a fermentation process for improving the production level of recombinant collagen proposed by the present invention Figure 2 ;

[0029] Figure 3 A fermentation process for improving the production level of recombinant collagen proposed by the present invention Figure 2 The structural diagram of part A in the figure;

[0030] Figure 4 A schematic diagram of a fermentation process for improving the production level of recombinant collagen proposed by the present invention Figure 3 ;

[0031] Figure 5 A top view of a fermentation process for improving the production level of recombinant collagen proposed by the present invention;

[0032] Figure 6 A cross-sectional schematic diagram of a fermentation process for improving the production level of recombinant collagen proposed by the present invention;

[0033] Figure 7 A schematic diagram of the structure of an inflatable airbag and a transmission shaft in a fermentation process for improving the production level of recombinant collagen proposed by the present invention.

[0034] In the figure: 1, fermentation tank; 101, motor; 102, rotating shaft; 103, arc wall; 104, gear part 1; 105, gear part 2; 2, transmission shaft; 201, limit groove; 202, mounting ring 1; 203, inflatable airbag; 204, spoiler; 205, mounting ring 2; 206, floating rod; 207, tapered hole; 301, piston cylinder; 302, piston rod; 303, spring part; 304, air pipe; 305, air outlet. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0036] Embodiment 1:

[0037] Reference Figure 1-7 , a fermentation process for improving the production level of recombinant collagen, comprising the following steps:

[0038] S1: Inoculate the Pichia pastoris engineered bacteria in the seed tank for cultivation, add glycerol and feed medium after the dissolved oxygen rises significantly, and transfer to fermentation tank 1 to start fermentation culture when the wet weight increases to more than 100 g / L;

[0039] S2: After the Pichia pastoris engineered bacteria in the fermentation tank are cultured until the dissolved oxygen rises significantly, a carbon source medium and a feed medium are used to mix the carbon source feed, and when the wet weight of the material increases to more than 150 g / L, the mixed carbon source feed is stopped;

[0040] S3: After the carbon source is exhausted, methanol and feed medium are added to induce expression until the end of fermentation.

[0041] The Pichia yeast engineered bacteria fermentation medium used in the seed tank and the fermentation tank in step S1 includes the following components in concentration: CaSO4·2H2O is 0.52-1.125g / L, K2SO4 is 7.42-17.8g / L, MgSO4·7H2O is 6.12-13.5g / L, KOH is 1.715-3.92g / L, glycerol is 21-37g / L; glucose is 20-40g / L; (NaPO3)6 is 6.5-13g / L, and white corn syrup dry powder is 37g / L.

[0042] The feed culture medium comprises the following components in concentrations: CaSO4·2H2O is 0.1-0.705 g / L; K2SO4 is 0.15-10.92 g / L; MgSO4·7H2O is 0.25-8.94 g / L; KOH is 0.12-2.478 g / L; white corn steep liquor dry powder is 0.3-4 g / L; the carbon source culture medium in step S2 is glycerol, glucose or a mixed culture medium of glycerol and glucose; the fermentation culture conditions are: fermentation temperature is 28-32 DEG C, pH is adjusted to 4.8-5.5 with ammonia water, dissolved oxygen is not less than 30% before induction expression, tank pressure is 0.04-0.06 MPa, dissolved oxygen is not less than 5% after induction expression, and tank pressure is 0.08-0.1 MPa.

[0043] The Pichia yeast engineered bacteria fermentation medium used in the seed tank and the fermentation tank in step S1 includes the following components in concentration: CaSO4·2H2O is 0.5875-0.8225 g / L, K2SO4 is 9.1-12.74 g / L, MgSO4·7H2O is 7.45-10.43 g / L, KOH is 2.07-2.891 g / L, glycerol is 20.0-30.0 g / L; glucose is 20.0-30.0 g / L; (NaPO3) is 66.5-9.1 g / L, and white corn steep liquor powder is 3-5 g / L. The feed medium includes the following components in concentration: CaSO4·2H2O is 0.25-0.5875 g / L; K2SO4 is 5.46-9.1 g / L; MgSO4·7H2O is 4.47-7.45 g / L; KOH is 0.75-2.065 g / L; white corn syrup powder is 2.1-3.5 g / L. The flow rate of the feed medium in step S2 is 5-8 mL / h / L, and the flow rate of the carbon source medium is 8-15 mL / h / L; the flow rate of the feed medium in step S3 is 3-5 mL / h / L, and the flow rate of methanol is 3-7 mL / h / L. The flow rate of the feed medium in step S1 is 5-8 mL / h / L, and the flow rate of glycerol is 10-18 mL / h / L. In step S1, the Pichia pastoris engineered bacteria are cultured until the wet weight increases to 160-180 g / L, and then the bacteria are transferred to the fermentation tank; in step S2, the Pichia pastoris engineered bacteria are cultured until the wet weight increases to 180-200 g / L, and then the carbon source mixing and feeding are stopped.

[0044] Also includes:

[0045] A plurality of transmission shafts 2 are rotatably connected in the fermentation tank 1; a limiting groove 201 is arranged on the transmission shaft 2; a mounting ring 1 202 is slidably connected to the limiting groove 201 of the transmission shaft 2, and an inflatable airbag 203 is fixedly connected to the mounting ring 1 202, and the inflatable airbag 203 is filled with helium. Affected by the weight of the mounting ring 1 202, the inflatable airbag 203 and the mounting ring 1 202 are located at the bottom of the transmission shaft 2 in daily use. Affected by the external temperature, the inflatable airbag 203 will expand, and its own drainage will increase as its volume increases, thereby offsetting the influence of the weight of the mounting ring 1 202, and driving the mounting ring 1 202 to move upward along the limiting groove 201; a plurality of spoilers 204 are fixedly connected to the inflatable airbag 203, and are used to disrupt the flow of liquid; a mounting ring 205 is slidably connected to the limiting groove 201 of the transmission shaft 2 The positioning groove 201 is connected to a plurality of groups of floating rods 206 that can float on water on the mounting ring 205. The plurality of groups of floating rods 206 can overcome the weight of the mounting ring 205 and drive the mounting ring 205 to float on the water surface. The liquid does not exceed the top of the transmission shaft 2. When the transmission shaft 2 rotates, under the influence of the positioning groove 201, the floating rods 206 will play the role of a stirring shaft to stir the surface of the liquid, thereby forming turbulence and increasing the mixing rate between the liquids. The fermentation tank 1 is fixedly connected with a motor 101, and a rotating shaft 102 fixedly connected to the output end of the motor 101 is rotatably connected in the fermentation tank 1. The rotating shaft 102 is located on the axis of the fermentation tank 1, and a gear member 1 104 is fixedly connected to the output end of the rotating shaft 102. The transmission shaft 2 is fixedly connected with a gear member 2 105 meshing with the gear member 104.

[0046] When the device is in use, the Pichia pastoris in the prior art is used, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 31, 2020, with the deposit number CGMCC No. 20458.

[0047] Specific operations:

[0048] S1: Inoculate the Pichia pastoris engineering bacteria at an inoculation rate of 10% in a 100L seed tank containing 70L fermentation medium for cultivation at a temperature of 28°C, adjust the pH to 4.8 with ammonia water, tank pressure of 0.06MPa, and dissolved oxygen of not less than 30%. After the dissolved oxygen rises significantly, add 50% glycerol and feed medium, the flow rate of 50% glycerol is 10mL / h / L, and the flow rate of feed medium is 5mL / h / L; when the wet weight increases to 160g / L, transplant to a 1500L fermentation tank containing 700L fermentation medium to start fermentation culture at a temperature of 28°C, adjust the pH to 4.8 with ammonia water, tank pressure of 0.06MPa, and dissolved oxygen of not less than 30%; S2: Inoculate the Pichia pastoris engineering bacteria in the fermentation tank After culturing until the dissolved oxygen rises significantly, use 50% glycerol and feed medium for mixed carbon source feeding, the flow rate of 50% glycerol is 8mL / h / L, and the flow rate of feed medium is 5mL / h / L; when the wet weight of the material increases to 185g / L, stop mixing the carbon source feeding; S3: after the glycerol is exhausted, add methanol and feed medium to induce expression until the fermentation is completed, the flow rate of methanol is dynamically adjusted within the range of 3-7mL / h / L, the flow rate of feed medium is 3mL / h / L, the temperature is 28°C, the pH is adjusted to 4.8 with ammonia water, the tank pressure is 0.08MPa, the dissolved oxygen is not less than 5%, and the entire fermentation process is completed when the wet weight of Pichia pastoris or the protein expression amount no longer increases. The Pichia yeast fermentation medium used in the seed tank and fermentation tank includes the following components: CaSO4·2H2O is 0.5875g / L, K2SO4 is 8.05g / L, MgSO4·7H2O is 7.45g / L, KOH is 3.59g / L, glycerol is 20.0g / L; glucose is 30.0g / L; (NaPO3) is 67.1g / L, and white corn steep liquor powder is 3g / L. The feed medium includes the following components: CaSO4·2H2O is 0.1g / L; KK2SO4 is 5.46g / L; MgSO4·7H2O is 7.45g / L; KOH is 0.23g / L; white corn steep liquor powder is 3.5g / L.

[0049] Another way to operate:

[0050] S1: Inoculate the Pichia pastoris engineering bacteria at an inoculation rate of 6% in a 100L seed tank containing 50L fermentation medium for cultivation at a temperature of 30°C, adjust the pH to 5.5 with ammonia water, the tank pressure of 0.06MPa, and the dissolved oxygen of not less than 30%. After the dissolved oxygen rises substantially, add 50% glycerol and feed medium, the flow rate of 50% glycerol is 16mL / h / L, and the flow rate of feed medium is 6mL / h / L; when the wet weight increases to 180g / L, transplant to a 1500L fermentation tank containing 600L fermentation medium to start fermentation culture at a temperature of 30°C, adjust the pH to 5.5 with ammonia water, the tank pressure of 0.06MPa, and the dissolved oxygen of not less than 30%; S2: Inoculate the Pichia pastoris engineering bacteria in the fermentation tank After the bacteria are cultured until the dissolved oxygen rises significantly, glucose and feed medium are used for mixed carbon source feeding, the flow rate of glucose is 9mL / h / L, and the flow rate of feed medium is 8mL / h / L; when the wet weight of the material increases to 200g / L, the mixed carbon source feeding is stopped; S3: after the glucose is exhausted, methanol and feed medium are added to induce expression until the fermentation is completed, the flow rate of methanol is dynamically adjusted within the range of 3-7mL / h / L, the flow rate of feed medium is 5mL / h / L, the temperature is 30°C, the pH is adjusted to 5.5 with ammonia water, the tank pressure is 0.09MPa, the dissolved oxygen is not less than 5%, and the entire fermentation process is completed when the wet weight of Pichia pastoris or the protein expression amount no longer increases. The Pichia yeast fermentation medium used in the seed tank and fermentation tank includes the following components: CaSO4·2H2O is 0.47g / L, K2SO4 is 18.2g / L, MgSO4·7H2O is 6.21g / L, KOH is 2.07g / L, glycerol is 21.0g / L; glucose is 22.0g / L; (NaPO3) is 66.5g / L, and white corn steep liquor powder is 3.5g / L. The feed medium includes the following components: CaSO4·2H2O is 0.6217g / L; K2SO4 is 0.15g / L; MgSO4·7H2O is 6.55g / L; KOH is 0.12g / L; white corn steep liquor powder is 2.1g / L.

[0051] Another way to operate:

[0052] S1: Inoculate the Pichia pastoris engineering bacteria at an inoculation rate of 8% in a 100L seed tank containing 60L fermentation medium for cultivation at a temperature of 32°C, adjust the pH to 5.0 with ammonia water, tank pressure of 0.05MPa, and dissolved oxygen of not less than 30%. After the dissolved oxygen rises significantly, add 50% glycerol and feed medium, the flow rate of 50% glycerol is 13mL / h / L, and the flow rate of feed medium is 6mL / h / L; when the wet weight increases to 180g / L, transplant to a 1500L fermentation tank containing 650L fermentation medium to start fermentation culture at a temperature of 32°C, adjust the pH to 5.0 with ammonia water, tank pressure of 0.05MPa, and dissolved oxygen of not less than 30%; S2: Inoculate the Pichia pastoris engineering bacteria culture in the fermentation tank After the dissolved oxygen rises significantly, 50% glycerol and feed medium are used for mixed carbon source feeding, the flow rate of 50% glycerol is 15 mL / h / L, and the flow rate of feed medium is 7 mL / h / L; when the wet weight of the material increases to 190 g / L, the mixed carbon source feeding is stopped; S3: after the glycerol is exhausted, methanol and feed medium are added to induce expression until the fermentation is completed, the methanol flow rate is dynamically adjusted within the range of 3 to 7 mL / h / L, the flow rate of feed medium is 4 mL / h / L, the temperature is 32°C, the pH is adjusted to 5.0 with ammonia water, the tank pressure is 0.1 MPa, the dissolved oxygen is not less than 5%, and the entire fermentation process is completed when the wet weight of Pichia pastoris or the protein expression amount no longer increases. The Pichia yeast fermentation medium used in the seed tank and fermentation tank includes the following components: CaSO4·2H2O is 1.175g / L, K2SO4 is 16.15g / L, MgSO4·7H2O is 5.96g / L, KOH is 3.92g / L, glycerol is 30.0g / L; glucose is 20.0g / L; (NaPO3) is 68.8g / L, and white corn steep liquor powder is 5g / L. The feed medium includes the following components: CaSO4·2H2O is 0.19g / L; K2SO4 is 8.56g / L; MgSO4·7H2O is 0.25g / L; KOH is 2.065g / L; white corn steep liquor powder is 0.4g / L.

[0053] Specifically, during fermentation, the motor 101 is started, and the transmission shaft 2 is rotated with the cooperation of the gear parts. At this time, the spoiler 204 and the floating rod 206 will rotate accordingly due to the restriction of the positioning groove 201, thereby achieving a similar stirring effect. At the same time, as the temperature of the inner wall of the tank body is heated, it will be higher than the temperature of the liquid, especially at the bottom of the fermentation tank 1. At this time, the inflatable airbag 203 will continue to absorb heat. As the heat rises, it will expand, and then its volume will increase, and its own drainage capacity will increase. The inflatable airbag 203 will rise accordingly and come to the place where the temperature is relatively low above. At this time, through heat exchange, the temperature of the inflatable airbag 203 will gradually decrease, and the temperature of the liquid nearby will increase. As the temperature of the inflatable airbag 203 decreases, it will gradually shrink, and then sink to the bottom of the fermentation tank 1 again to absorb heat again. This cycle is repeated until the fermentation is completed. At the same time, in the process of the inflatable airbag 203 moving up and down, some liquid will be moved to make the liquid flow, and the temperature of the liquid at different positions will be further balanced.

[0054] Embodiment 2:

[0055] Reference Figure 1-7 , a fermentation process for improving the production level of recombinant collagen, which is basically the same as Example 1, and further: a plurality of groups of arc walls 103 are arranged on the inner wall at the top of the fermenter 1, a plurality of groups of gas outlets 305 are arranged at the bottom of the fermenter 1, a plurality of groups of piston members are fixedly connected to the rotating shaft 102, and the telescopic end of the piston member is against the arc wall 103. Through the rotation of the rotating shaft 102, the piston member will inhale the gas at the top of the fermenter 1, and then the gas will be discharged through the gas outlet 305 through the gas outlet end. The gas outlet 305 is arranged on the transmission shaft 2 in an annular shape with equal intervals. The piston member includes a piston cylinder 301, a piston rod 302, and a spring member 303. The plurality of groups of piston cylinders 301 are fixedly connected to the rotating shaft 102 in an annular shape with equal intervals. The cylinder 301 is provided with an air inlet and an air outlet with a one-way valve, and the piston rod 302 is slidably connected in the piston cylinder 301. A spring member 303 is provided in the piston cylinder 301, and its two ends are respectively connected to the piston cylinder 301 and the piston rod 302. The piston rod 302 is the telescopic end of the piston member. The spring member 303 can make the piston rod 302 always abut against the arc wall 103. Through the rotation of the rotating shaft 102, the piston cylinder 301 continuously inhales the outside gas through the air inlet with the cooperation of the spring member 303 and the piston rod 302, and finally discharges it to the air outlet 305 through the air outlet. An air pipe 304 is provided in the rotating shaft 102, and its two ends are respectively connected to the air outlet 305 and the air outlet of the piston cylinder 301.

[0056] When the output end of the motor 101 rotates, the piston will continuously inhale the air at the top of the fermentation tank 1, and then discharge it through the air outlet 305, so that the gas comes to the bottom, and then the flow of the liquid at this time makes the liquid with higher temperature at the bottom come to the top along with the gas. At the same time, these bubbles will bring a large amount of oxygen. In the process of these bubbles moving upward, there is a possibility that they will be broken up by the spoiler 204, so that the large bubbles will become small bubbles, increasing the contact area between the liquid and the gas, injecting oxygen into the liquid, increasing the oxygen content in the liquid, and promoting the growth of the bacteria.

[0057] Embodiment 3:

[0058] Reference Figure 1-7 , a fermentation process for improving the production level of recombinant collagen, which is basically the same as Example 2, and further: a plurality of groups of conical holes 207 are arranged on the floating rod 206, and the conical holes 207 are arranged at an angle. When the floating rod 206 rotates, liquid is sucked into the large end of the conical hole 207, and the liquid is discharged from the smaller end by pressurization. When the large end of the conical hole 207 is close to the side of the floating rod 206 close to the water surface and the small end is close to the side away from the water surface, the liquid will be sprayed into the air.

[0059] Through the setting of the conical hole 207, when the transmission shaft 2 rotates, some liquid will be thrown into the air, and then the liquid will come into contact with the gas. The liquid will absorb a certain amount of oxygen. At the same time, when it falls above the water surface, it will produce shocks, allowing some oxygen to enter the liquid again, further facilitating the growth of the bacteria and the subsequent preparation of collagen.

[0060] The present invention uses an inflatable airbag 203 filled with helium. When in use, the higher temperature at the bottom can be transferred to the upper part through the inflatable airbag 203, avoiding the accumulation and concentration of heat, promoting the temperature in the fermentation tank 1 to be constant, facilitating the growth of bacteria and the subsequent preparation of collagen.

[0061] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A fermentation process for improving the production level of recombinant collagen, comprising a fermentation tank (1), characterized in that: Also includes: A plurality of transmission shafts (2) are rotatably connected to the fermentation tank (1); A limiting groove (201) is arranged on the transmission shaft (2); The mounting ring (202) is slidably connected to the limiting groove (201) of the transmission shaft (2). The mounting ring (202) is fixedly connected with an inflatable airbag (203). The inflatable airbag (203) is filled with helium. Affected by the weight of the mounting ring (202), the inflatable airbag (203) and the mounting ring (202) are located at the bottom of the transmission shaft (2) during daily use. Affected by the external temperature, the inflatable airbag (203) will expand, and its own drainage will increase while its volume increases, thereby offsetting the influence of the weight of the mounting ring (202) and driving the mounting ring (202) to move upward along the limiting groove (201); A plurality of groups of spoilers (204), all fixedly connected to the inflatable airbag (203), for disrupting the flow of the liquid; The second mounting ring (205) is slidably connected to the limiting groove (201) of the transmission shaft (2). The second mounting ring (205) is connected to a plurality of groups of floating rods (206) capable of floating on water. The plurality of groups of floating rods (206) can overcome the weight of the second mounting ring (205) and drive the second mounting ring (205) to float on the water surface. When the transmission shaft (2) rotates, under the influence of the limiting groove (201), the floating rods (206) will play the role of a stirring shaft, stirring the surface of the liquid, thereby forming turbulence and increasing the mixing rate between the liquids.

2. A fermentation process for improving the production level of recombinant collagen according to claim 1, characterized in that: The floating rod (206) is provided with a plurality of groups of tapered holes (207), and the tapered holes (207) are arranged obliquely. When the floating rod (206) rotates, liquid is sucked into the large end of the tapered hole (207), and the liquid is discharged from the smaller end by pressurization. When the large end of the tapered hole (207) is close to the side of the floating rod (206) close to the water surface, and the small end is close to the side away from the water surface, the liquid is sprayed into the air.

3. A fermentation process for improving the production level of recombinant collagen according to claim 2, characterized in that: The fermentation tank (1) is fixedly connected to a motor (101), and a rotating shaft (102) fixedly connected to an output end of the motor (101) is rotatably connected inside the fermentation tank (1), the rotating shaft (102) is located on the axis of the fermentation tank (1), a gear member 1 (104) is fixedly connected to the output end of the rotating shaft (102), and a gear member 2 (105) meshingly connected to the gear member 1 (104) is fixedly connected to the transmission shaft (2).

4. A fermentation process for improving the production level of recombinant collagen according to claim 3, characterized in that: The inner wall at the top of the fermentation tank (1) is provided with a plurality of arc-shaped walls (103), and the bottom of the fermentation tank (1) is provided with a plurality of gas outlets (305). The rotating shaft (102) is fixedly connected with a plurality of piston members, and the telescopic ends of the piston members are against the arc-shaped walls (103). When the rotating shaft (102) rotates, the piston members will inhale the gas at the top of the fermentation tank (1), and then discharge the gas through the gas outlet (305) through the gas outlet end.

5. A fermentation process for improving the production level of recombinant collagen according to claim 4, characterized in that: The air outlets (305) are arranged in a ring shape at equal intervals on the transmission shaft (2).

6. A fermentation process for improving the production level of recombinant collagen according to claim 5, characterized in that: The piston member comprises a piston cylinder (301), a piston rod (302), and a spring member (303). The multiple groups of piston cylinders (301) are fixedly connected to the rotating shaft (102) in an annular shape at equal intervals. The piston cylinder (301) is provided with an air inlet and an air outlet with a one-way valve. The piston rod (302) is slidably connected in the piston cylinder (301). The piston cylinder (301) is provided with a spring member (303) at both ends of which are respectively connected to the piston cylinder (301) and the piston rod (302). The piston rod (302) is the telescopic end of the piston member. The spring member (303) can make the piston rod (302) always abut against the arc wall (103). Through the rotation of the rotating shaft (102), the piston cylinder (301) continuously sucks in external gas through the air inlet under the cooperation of the spring member (303) and the piston rod (302), and finally discharges the gas to the outlet (305) through the air outlet.

7. A fermentation process for improving the production level of recombinant collagen according to claim 6, characterized in that: An air pipe (304) is arranged inside the rotating shaft (102), and two ends of the air pipe (304) are respectively connected to the air outlet (305) and the air outlet hole of the piston cylinder (301).

8. A fermentation process for improving the production level of recombinant collagen according to claim 6, characterized in that: The following steps are involved: S1: Inoculate the engineered Pichia pastoris in a seed tank for cultivation, add glycerol and feed medium after the dissolved oxygen rises significantly, and transfer to a fermentation tank (1) to start fermentation culture when the wet weight increases to more than 100 g / L; S2: After the Pichia pastoris engineered bacteria in the fermentation tank are cultured until the dissolved oxygen rises significantly, a carbon source medium and a feed medium are used to mix the carbon source feed, and when the wet weight of the material increases to more than 150 g / L, the mixed carbon source feed is stopped; S3: After the carbon source is exhausted, methanol and feed medium are added to induce expression until the end of fermentation.

9. A fermentation process for improving the production level of recombinant collagen according to claim 8, characterized in that: The Pichia yeast engineered bacteria fermentation medium used in the seed tank and the fermentation tank in step S1 includes the following components in concentration: CaSO4·2H2O is 0.52-1.125g / L, K2SO4 is 7.42-17.8g / L, MgSO4·7H2O is 6.12-13.5g / L, KOH is 1.715-3.92g / L, glycerol is 21-37g / L; glucose is 20-40g / L; (NaPO3)6 is 6.5-13g / L, and white corn syrup dry powder is 37g / L.

10. A fermentation process for improving the production level of recombinant collagen according to claim 9, characterized in that: The feed culture medium comprises the following components in concentrations: CaSO4·2H2O is 0.1-0.705 g / L; K2SO4 is 0.15-10.92 g / L; MgSO4·7H2O is 0.25-8.94 g / L; KOH is 0.12-2.478 g / L; white corn steep liquor dry powder is 0.3-4 g / L; the carbon source culture medium in step S2 is glycerol, glucose or a mixed culture medium of glycerol and glucose; the fermentation culture conditions are: fermentation temperature is 28-32°C, pH is adjusted to 4.8-5.5 with ammonia water, dissolved oxygen is not less than 30% before induction expression, tank pressure is 0.04-0.06 MPa, dissolved oxygen is not less than 5% after induction expression, and tank pressure is 0.08-0.1 MPa.

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